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PMC503393 | Background
==========
Give sorrow words; the grief that does not speak
Whispers the o\'er fraught heart and bids it break.
Shakespeare, *Macbeth*IV, iii, 209
Grieving the death of a loved one has an ancient history: from time immemorial, cultures have provided the bereaved with advice and rituals to address -- and express -- the experience of grief \[[@B1]\]. Over the past several decades, efforts to aid the bereaved have increasingly focused on the physical and psychological morbidity, and the spiritual suffering and social isolation associated with bereavement. The resulting plethora of intervention options, ranging from mutual-help support groups to prescribed pharmacotherapy and professionally led psychotherapy, is striking, as is the panoply of settings in which bereavement care can be found: hospitals, hospices, churches, palliative care units, community-based services, and bereavement-specific foundations all provide an array of bereavement care interventions. This welter of activity testifies to the broadly valued goal of decreasing the severity of bereavement-related symptoms.
Given the abundance of care options, what is the best way to care for a bereaved person? Numerous studies measuring the impact of bereavement interventions have been published in diverse journals, yet no consensus has emerged in the medical, mental health, or social work communities regarding whether one form of treatment is preferable to another \[[@B2]-[@B5]\]. We therefore have conducted a systematic review of bereavement care interventions. Our goal is to present a comprehensive yet coherent synthesis of the current literature that will promote the advancement in the quality of care and research on behalf of bereaved individuals.
Methods
=======
Data sources
------------
To identify studies in the traditional medical literature as well as the complementary and alternative medicine literature, we searched the following databases: MEDLINE; PsychINFO; Cumulative Index to Nursing and Allied Health (CINAHL); BIOSIS Previews; ISI Science Citation Index Expanded and Social Sciences Index; Evidence Based Medicine Reviews (EBMR), including the Cochrane Database of Systematic Reviews (DSR), the Cochrane Controlled Trial Registry (CCTR), Database of Abstracts of Reviews of Effectiveness (DARE), and the American College of Physicians\' (ACP) Journal Club Review; Sociological Abstracts; Alt HealthWatch; and Wilson Web from 1966 to 2003. We identified all relevant articles on bereavement care interventions by using the primary search terms of \"*bereaved*or *bereavement*\" and \"*grief*\", combined with secondary descriptors of \"*intervention*or *support*or *counselling*or *therapy*\" and \"*controlled*or *trial*or *design*\".
Study selection
---------------
Our inclusion criteria specified that each study: 1) addressed the treatment of bereaved individuals, and 2) included an evaluation of a selected method of therapy aimed at reducing the grief reaction due to bereavement. We considered only articles written in the English language. We then reviewed the titles and abstracts of all articles we retrieved through our initial database search, and obtained the full texts of all applicable studies. We also reviewed the references in all applicable studies for additional pertinent studies.
Data extraction
---------------
The full articles of all studies that met inclusion criteria and passed subsequent title and abstract reviews were retrieved and examined independently by two of the authors. Each article was reviewed for measured outcomes, patient and decedent characteristics, and intervention characteristics. These measures included sample size, type of intervention, length of intervention, patient\'s relationship to the deceased, time since the bereaved death, and patient demographics. Data was extracted and any disagreements were resolved through discussion, clarification, and consensus within the research team.
Characteristics of reviewed studies
-----------------------------------
The initial literature search generated 737 citations. Elimination of duplicate citations yielded 340 references. 2 studies, written in Chinese and Spanish, were excluded. Reviewing the titles culled the sample to 243 citations, and a review of the abstracts found 87 of these to be potentially relevant. Of these, 9 were dissertations, 2 were irretrievable, 2 were duplicate publications of the same study, and 15 were ineligible because they did not meet our inclusion criteria. The resulting set of 74 articles was subject to review for data extraction. A list of all citations found, including those excluded from this analysis, is available \[see [Additional file 1](#S1){ref-type="supplementary-material"}\].
Of the 74 studies that met inclusion criteria, almost 6,000 participants within these studies experienced a multitude of losses -- of parents, spouses, children, and other loved ones who had died from a wide range of causes, both sudden and protracted. The therapies utilized and outcomes evaluated varied widely. Heterogeneity among both the outcomes and the measures used to assess similar outcomes precluded an effort to summarize data across studies, even in the form of generic effect-size measures. Furthermore, for a significant portion of the studies, concerns regarding the internal or external validity of the reported results cautioned against making quantitative summary statements regarding treatment effects.
Results
=======
The 74 studies selected for detailed review evaluated diverse types of interventions designed to ameliorate the adverse physical and psychological outcomes associated with bereavement. These interventions can be classified according to various schemes, including their underlying theoretical framework (ranging from Freudian psychoanalysis to neurotransmitter imbalances), the format of the intervention (individual, group, family, marital), the timing of the intervention (acute, intermittent crisis, chronic), the tasks assigned to the bereaved (ranging from verbalizing feelings to taking medication), or the population targeted for the intervention (children, adults, seniors). We chose to organize this review on the basis of the social framework used to implement the intervention (that is, either personalized structured therapeutic relationships or less personal systems-level interventions), as this attribute of the interventions emerged as the most verifiable and salient measure.
Structured therapeutic relationship
-----------------------------------
Eight studies feature ***pharmacotherapy***, but only four compared active therapy to non-pharmacotherapy controls, and only one study clearly reported their random allocation method (Table [1](#T1){ref-type="table"}) \[[@B6]-[@B13]\]. These studies targeted adults and seniors, ranged in sample size from 10--80 subjects, and used a variety of drugs, including tricyclic antidepressants (TCA), selective serotonin reuptake inhibitors (SSRI), buprioion, and benzodiazepines. Overall, these studies demonstrated a statistically significant beneficial effect of pharmacotherapy on ameliorating symptoms of depression and improving subjective sleep quality \[[@B6]-[@B11],[@B13]\]. These benefits persisted only as long as the subjects continued to receive pharmacotherapy. Pharmacotherapy was found, however, to have a mixed effect on bereavement intensity as measured by symptoms of grief (i.e., Texas Revised Inventory of Grief, Inventory of Complicated Grief). For example, Warner and colleagues (2001) did not find evidence of an effect of benzodiazepines (diazepam) on bereavement-related grief intensity\[[@B12]\]. One study combined pharmacotherapy with psychotherapy in a 16-week double-blinded factorial design trial of nortriptyline (NT) and interpersonal psychotherapy \[[@B6]\]. The 80 patients were randomly assigned to one of four treatment conditions: NT plus interpersonal psychotherapy, NT plus medication clinic (i.e., no interpersonal psychotherapy), placebo pill plus interpersonal psychotherapy, and placebo pill plus medication clinic (i.e., no interpersonal psychotherapy conditions). Details of the psychotherapy were not described. While the results displayed a statistically significant benefit of nortriptyline over placebo regarding remission of depression, none of the treatment conditions were associated with diminishment of grief.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Pharmacotherapy Interventions
:::
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Medication** **Pop** **CG** **RA** **Num**\* **TSL (days)** **Dose** **DT (days)** **Key Outcome Measures** **Article**
------------------------------ --------- -------- -------- ----------- ---------------- ----------------------------------------------- --------------- ---------------------------------------------------------------- -----------------------------------
Nortriptyline Senior Y Y-NE 80/66 216--279 Steady-state plasma level: 50--120 ng/mL 112 Depression (HAM-D); Grief (TRIG) Reynolds, Miller, et al, 1999\*\*
Senior Y Y-NE 27/27 210 (mean) Steady-state plasma level: 79.9+/-28.3 ng/mL\ \<180 Sleep (PSQI); Depression (HAM-D, BDI) Taylor, Reynolds, et al, 1999
Daily dose: 70.8+/-22.2 mg
Senior Y NR 30/24 276 Steady-state plasma level: 72.7 ng/mL\ 112 Sleep (PSQI) Pasternak, Reynolds, et al, 1994
Daily dose: 53.0 mg
Senior N NA 13/13 150--750 Daily dose: 49.2 mg 9--184 Depression (HAM-D, BDI, BSI); Grief (TRIG, JGI); Sleep (PSQI) Pasternak, Reynolds, et al, 1991
Nortriptyline and Paroxetine Adult N NA 21/15 183--4158 PT Daily dose: 20--50 mg\ 120 Depression (HAM-D); Grief (ICG); Sleep (PSQI) Zygmont, Prigerson, et al, 1998
NT Daily dose: 50--160 mg
Desipramine Adult N NA 10/9 NR Daily dose: 75--150 mg 28 Depression (HDRS, CGI, Raskin DS); Grief (Separation Distress) Jacobs, Nelson, et al, 1987
Bupropion Adult N NA 22/14 42--56 Daily dose: 150--300 mg 56 Grief (TRIG, ICG); Depression (HAM-D) Zisook, Schuchter, et al, 2001
Diazepam Senior Y Y 35/30 \<14 2 mg/pill, self-administered \<42 Bereavement (BPQ) Warner, Metcalfe, et al, 2001
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Notes: \* All Ns are reported as (starting population of bereaved individuals/bereaved population completing all follow-ups), unless only study included only one assessment. \*\* Study also included psychotherapy condition. Legend: Pop, Target Population; CG, Control Group; RA, Random Assignment; Num, Number of subjects; TSL, Time Since Loss; DT, Duration of Trial; NA, Not Applicable; NR, Not Reported; UC, Unclear; Y, Yes; N, No; Y-NE, Randomization mentioned, but allocation method not explicitly stated; RS, Randomization Subverted.
:::
***Support groups or counselling***constituted the intervention in 39 studies, of which 23 had control groups and 15 claimed random allocation, yet only three of these included clearly described allocation methods (Table [2](#T2){ref-type="table"}) \[[@B14]-[@B52]\]. Ten of these were mutual/self-help, with the majority taking the form of informal group therapy. The remaining 29 studies were professionally led support groups targeting select subgroups including parentally bereaved children, college students, and seniors, as well as many specific adult populations. Program implementation across studies varied even further. This variation was found in terms of number of sessions (one to 25), whether the sessions proceeded with full-fledged patient-driven discussion or highly structured protocols, whether attendance was mandatory or individually motivated, as well as in the nature of the group leadership and the format (individual, group, or marital). Perhaps due to these or other differences in the interventions, some studies documented study treatment effects \[[@B22],[@B26],[@B29]-[@B31],[@B33],[@B34],[@B52]\] while other studies showed no effect \[[@B15],[@B17],[@B27],[@B37],[@B46],[@B51]\].
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Support/Counselling Interventions
:::
**Type** **Format** **Pop** **CG** **RA** **Num** **TSL (days)** **DT** **Key Outcome Measures** **Article**
-------------------------------------------------------- ----------------- --------- -------- -------- ----------- ---------------- --------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------
Mutual/Self-help Individual Adult Y Y-NE 162/62 \~30 NR Psychiatric Functioning (GHQ); Social Support/psychological and psychophysiological variables (author-created) Vachon, Lyall, et al, 1980
Mutual/Self-help (included professionally-lead groups) Group Senior Y RS 339/295 30--60 56, 365 days Self-Esteem (Rosenberg\'s Self-Esteem Scale); Life Satisfaction (LSI-A); Depression (GDS); Grief (TRIG) Caserta & Lund, 1983
Mutual/Self-help Group Senior Y N 23 34--474 21 days; 7 sessions Domain Specific State Locus of Control (Zeigler-Reid State Locus of Control Measure); Trait Locus of Control (I-E); Distress (BSI, GSI) McKibbin, Guarnaccia, et al, 1997
Mutual/Self-Help Group Adult Y Y 113/67 90--365 63 days; 9 sessions Depression (GHQ, BDI); Anxiety (STAI); Social Functioning (SAS); Social Support (SSQ) Tudiver, Hilditch, et al, 1992
Mutual/Self-help Group Adult Y Y-NE 113/112 90--365 63 days Healthcare visit rates (Family Physician, Specialist, Psychiatrist) Tudiver, Permaul-Woods, et al, 1995
Mutual/Self-help Group Adult Y N 38/21 90--750 70 days; 10 sessions Treatment Expectancy (Expectancy Scale); Depression (BDI); Avoidance, Anxiety (Social Anxiety and Distress Scale); Enjoyability (Pleasant Events Scale); Life Satisfaction (Life Satisfaction Scale) Walls & Meyers, 1985
Mutual/Self-help Group Adult Y N 721/502 \~1290 365 days; \>3 sessions Depression, Anxiety, Somatization (Hopkins Symptom Checklist); Self Esteem, Well-being, Mastery (Not reported) Lieberman & Videka-Sherman, 1986
Mutual/Self-help Group Adult Y N 667/391 365--1095 365 days Depression, Anxiety, Somatization (Not reported); Self Esteem (Rosenberg 1965); Life Satisfaction, Mastery, Medication (Not reported); Social Functioning Parental Functioning Attitudes (BPQ) Videka-Sherman & Lieberman, 1985
Mutual/Self-help Group Adult N Y-NE 61/55 120--1095 84 days; 12 sessions Avoidance/Intrusion (IES); Stress Symptoms (SRRS); Depression (BDI); Mental Distress (BPRS, SCL-90); Social Functioning (SAS-SR); Overall Functioning (GAS) Marmar, Horowitz, et al, 1988\*\*
Mutual/Self-help Group Adult N NA 53/33 \<730 8 sessions, optional 4 Psychosomatic Symptoms (SCL-90 subscales: somatization, obsessive-compulsive, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, psychoticism, GSI) Rogers, Sheldon, et al, 1982
Professionally Lead Individual Adult Y NR 493/225 120 1 day; 1 session Grief (HGRC) Kaunonen, Tarkka, et al, 2000
Professionally Lead Family Adult Y Y-NE 50/30 1--2 1--120 days; up to 8 sessions General Health Questionnaire (self-rated); Anxiety, Depression (Leeds Scale) Forrest, Standish, & Baum, 1982
Professionally Lead Family Adult Y UC 334/161\* \<1--180 7--70 days; up to 10 sessions Medical Illness (CMI, MMPI); Psychiatric Illness (Boston Bereavement, Mood Inventory); Family Functioning (Ferriera-Winter, Bodin Drawing); Crisis Coping (Intrapersonal, Family, Job/Financial, Social); Social Cost (Gross Income, Living Expenses, Absenteeism, Economic Loss) Williams, Lee, & Polak, 1976 Polak, Egan, et al, 1975
Professionally Lead Family Adult Y UC 176/86\* \<1--180 7--70 days; up to 10 sessions Neurotic Symptoms Scale; Bodin Family Closeness; Crisis Coping Scale; Religious helping of others; Authoritarian Family Functioning; Depression; Monthly Income; Monthly Expenses; Social Costs; Bereavement Adjustment Williams & Polak, 1979
Professionally Lead Family Adult N NA 77/37\* \<1 \>360 days Personal and social phenomena of death (structured interview) Oliver, Sturtevant, et al, 2001
Professionally Lead Family Child Y Y-NE 72/55 \<730 15 sessions Depression (CDI, CBCL, PERI Demoralization Scale); Parental Warmth (CRPBI); Family Cohesion (Family Environment Scale); Parent perception of support (author-created scale); Family Coping (F-COPES) Sandler, West, et al, 1992
Professionally Lead Group Senior N NR 28/11 90--7300 \<140 days; up to 20 sessions Social Support (ASSIS); Affect/Mood (PANAS); Emotional/Social Loneliness (ESLI) Stewart, Craig, et al, 2001
Professionally Lead Group Adult Y Y 197/166 \<180 70 days; 10 sessions Grief (TRIG, GRI); Distress (POMS); Depression and Anxiety (SIGH-AD) Goodkin, Blaney, et al, 1999
Professionally Lead Group Adult Y Y-NE 242/185 0--35 77 days; 3 sessions Distress (POMS-TMD, Anxiety-tension, Depression-dejection, Anger-hostility, Confusion-bewilderment, Overall emotional disturbance); Self-Esteem (Rosenberg 1965 scale) Swanson, 1999
Professionally Lead Group Adult Y Y-NE 150/120 30--240 270 days Depression (CES-D, BDI); Anxiety (A-Sta); Somatic Symptoms (SOM); Emotional Symptoms (EMOT); Life Satisfaction (Lsat, SelfAnch) Kay, Guernsey de Zapien, et al, 1993
Professionally Lead Group Adult Y Y-NE 119/119 \<180 70 days; up to 10 sessions Immunological measures (CD3+CD4+ cell count, CD3+CD8+ cell count, CD4/CD8 ratio, CD3+ cell count, CD4 cell count, Lymphocyte count, T-lymphocyte count); Neuroendocrine measure (Plasma cortisol level) Goodkin, Feaster, et al, 1998
Professionally Lead Group Adult Y Y-NE 110/80 \~730 28 days; 8 sessions Coping and Adaptation (TAT) Balk, Lampe, et al, 1998
Professionally Lead Group Adult Y Y-NE 36/36 \<180 70 days; up to 10 sessions Plasma Viral Load (HIV-1 RNA copy number) Goodkin, Baldewicz, et al, 2001
Professionally Lead Group Adult Y N 159/127 42--140 Up to 25 sessions Social Support (SSES); Group Involvement (Liberman & Videka-Sherman, 1986); Depression (CES-D, POMS-D); Anger (POMS-A); Anxiety (POMS-T); Stress (IES) Levy, Derby, et al, 1993
Professionally Lead Group Adult Y N 121 30--4745 30--365 days Grief (HGRC subscales: Despair, Panic behavior, Personal growth, Blame and Anger, Detachment, Disorganization) DiMarco, Menke, & McNamara, 2001
Professionally Lead Group Adult N Y 139/107 90--17155 84 days; 12 sessions Avoidance/Intrusion (IES); Grief (TRIG); Interpersonal Distress (IIP); Social Functioning (SAS-SR); Depression (BDI); Anxiety (STAI); Mental Distress (BSI, GSI); Self-Esteem (SES); Physical Functioning (SF-36); Symptomatic Distress (SCL-90) Piper, McCallum, et al, 2001\*\*
Professionally Lead Group Adult N N 83/70 \<30--8030 49 days; 7 sessions Physical, Emotional, and Social Functioning (author created measures); Self Esteem (Rosenberg, 1962); Locus of Control (I-E); Life satisfaction (Neugarten, Havighurdt, & Tobin, 1961); Attitude Toward Women (Spence & Helmreich, 1972, Gump 1972) Barrett, 1978
Professionally Lead Group Adult N NA 392/77 NR 56 days Distress (BSI); Group Process and Satisfaction (author created questionnaire) Glajchen & Magen, 1995
Professionally Lead Group Adult N NA 174/138 780 730 days Motives for joining (Lieberman 1979); Interpersonal relations (Porat 1987); Group leadership style (Porat 1987); Perceived contribution of treatment on recovery Geron, Ginsberg, & Solomon, 2003
Professionally Lead Group Adult N NA 21/21 NR 70 days; up to 10 sessions Perceived Social Support (PRQ); Perceived Stress (PSS) Davis, Hoshiko, et al, 1992
Professionally Lead Group Adult N NA 21/21 365--3650 52 days; up to 8 sessions Depression (CDI); Anxiety (HSC-25); Knowledge of Death and Bereavement (KDBQ) Stoddart, Burke, & Temple, 2002
Professionally Lead Group Adult N NA 20 90--1095 \<1095 days; unlimited sessions Grief (TRIG); Social Network (SNM, SNG) Forte, Barrett, & Campbell, 1996
Professionally Lead Group Adult N NA 12/5 60--780 28 days; 8 sessions Emotional Distress (EPI); Family Adjustment (FACES-III); Social Adjustment (SAS-SR) Heiney, Ruffin, & Goon-Johnson, 1995
Professionally Lead Group Child Y Y-NE 17/17 \>730 42 days; 6 sessions Self-Esteem (PH); Depression (CDI); Behavior (CBCL-TRF, CBCL-YSR) Huss & Ritchie, 1999
Professionally Lead Group Child N NA 38/29 \<900 300 days; 12 sessions Depression (BID); Attitude/ Conception of Death (ATCD) Shilling, Koh, et al, 1992
Professionally Lead Group Child N NA 18/18 \<730 52 days; 8 sessions Bereavement Survey (author created); Loss Resolution (LRS-Modified); Distress and Somatic Complaints (ALAC) Opie, Goodwin, Finke, et al, 1992
Professionally Lead Group Child N NA 6/6 240--1020 42 days; 6 sessions Psychological measures (Lewis Counselling Inventory, IPAT) Quarmby, 1993
Professionally Lead Group Child N NA 4/4 \<90 77 days; 11 sessions Self-Esteem (Piers-Harris Self-Concept Scale); Descriptive (Risk Impact, Negative Chain Events, Opening Up Opportunities) Zambelli & DeRosa, 1992
Professionally Lead Couple/ Marital Adult Y Y-NE 57/31 NR Mean of 6 sessions Grief (TRIG); Irritability, Depression, Anger (IDA) Lilford, Stratton, et al, 1994
Notes: \* Families, not individuals. \*\* Study also included psychotherapy condition. Legend: Type, Type of Intervention; Format, Format of Intervention; Pop, Target Population; CG, Control Group; RA, Random Assignment; Num, Number of subjects; TSL, Time Since Loss; DT, Duration of Trial; NA, Not Applicable; NR, Not Reported; UC, Unclear; Y, Yes; N, No; Y-NE, Randomization mentioned, but allocation method not explicitly stated; RS, Randomization Subverted.
:::
Several studies documented substantial spontaneous improvements in bereavement symptomology in the control groups. Kay and others (1993) report a bereavement intervention for Mexican-American widows \[[@B33]\]. They found that all widows improved on all depression scales, state anxiety, life satisfaction, and emotional and somatic symptom scales over the course of two years. However, those widows in the experimental support group exhibit significantly improved changes in these scores. Tudiver and colleagues (1992) conducted a mutual-help support group for recently bereaved widowers \[[@B17]\] that can be compared to Vachon and colleagues\' (1980) and Barrett\'s (1978) widow studies \[[@B14],[@B39]\]. Tudiver and others found significant improvement over time (baseline to eight months) for all widowers, but found no significant differences between those who received treatment and a comparison group of windowers who were on the wait list to receive treatment but had not.
***Psychotherapy-based treatments***, another form of psychological interventions, can be done in different formats (family, group, or individual), and via different approaches. Of the 25 studies that use psychotherapy as an intervention, approaches included cognitive-behavioral, psychodynamic, psychoanalytical, and interpersonal approaches, as well as combinations of these and modality and social support (Table [3](#T3){ref-type="table"})\[[@B6],[@B19],[@B22],[@B35],[@B38],[@B53]-[@B72]\]. Seventeen of these studies utilized control groups, only 13 claimed randomization, and only five of these clearly stated their method of allocation.
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Psychotherapy Interventions
:::
**Type** **Format** **Pop** **CG** **RA** **Num** **TSL (days)** **DT** **Key Outcome Measures** **Article**
----------------------------------- ------------ --------- -------- -------- --------- ---------------- -------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ -------------------------------------------
**Cognitive-behavioral** Individual Senior Y N 58/NR 120--180 70 days; 4 sessions Mastery (Personal Mastery Scale); Well-being (MHI subscales, ABS Subscale, PERI self-esteem); Distress (PERI Demoralization Scales, MHI subscales) Reich & Zautra, 1989
Individual Senior N NA 4/4 540--730 98 days; 14--18 sessions Distress (SUDS); Grief (ICG); Depression (BDI); Anxiety (BAI) Harkness, Shear, et al, 2002\*\*
Individual Adult Y Y 30/25 \>90 35 days; 10 sessions Avoidance/Intrusion (IES); Anxiety (SCL-90); Depression (SCL-90); Mood (POMS) Lange, van de Ven, et al, 2001
Individual Adult Y Y-NE 26/14 180--7300 70 days; 6 sessions Depression (Wakefield, BDI); Physical Symptoms (Mawson et al, 1981); Fear (FQ); Grief (TRIG); Avoidance (Bereavement Avoidance Tasks) Sireling, Cohen, & Marks, 1988
Group Adult Y Y-NE 261/147 46--229 84 days; 8 sessions Mental Distress (BSI, GSI); PTS Symptoms (TES); Grief (GES); Physical Health (HHB); Marital Strain (DAS) Murphy, Johnson, et al, 1998 Murphy, 1997
Group Adult Y Y-NE 110/80 \~730 28 days; 8 sessions Coping and Adaptation (TAT) Balk, Lampe, et al, 1998
Group Adult Y N 38/21 90--750 70 days; 10 sessions Treatment Expectancy (Expectancy Scale); Depression (BDI); Avoidance, Anxiety (Social Anxiety and Distress Scale); Enjoyability (Pleasant Events Scale); Life Satisfaction (Life Satisfaction Scale) Walls & Meyers, 1985
Group Adult N NA 8/8 \>30 56 days; 8 sessions Avoidance/Intrusion (IES); Depression (BDI, SCL-90-R); Anxiety (SCL-90-R, STAI); Grief (GRI); Distress (PERI Demoralization) Sikkema, Kalichman, et al, 1995\*\*\*\*
Group Child Y UC 19/18 \<730 NR Behavior (BRIC-S, BRIC-H); Depression (DSRS); Grief (BP) Hilliard, 2001
**Psycho-dynamic** Individual Senior Y Y 228 \~60 \<180 days; Unlimited sessions Number of Office Visits, Types of Illnesses Gerber, Wiener, Battin, et al, 1975
Individual Senior Y Y-NE 33/30 90--1170 14 days; 4 sessions Mental Distress (BSI); Depression (GDS); Hopelessness (GHS); Avoidance/Intrusion (IES); Mood (PANAS) Segal, Bogaards, et al, 1999
Individual Adult Y Y 66/56 \<49 90 days; up to 9 sessions General Health(general health questionnaire) Raphael, 1977
Individual Adult Y N 72/63 60--462 12--20 sessions Avoidance/Intrusion (IES-A, IES-I); Depression (SCL-90); Anxiety (SCL-90); Total Pathology (SCL-90); Stress-Intrusion (SRRS); Neurotic Symptoms (BPRS) Horowitz, Weiss, et al, 1984
Individual Adult N Y-NE 12/6 365--3650 196 days Depression (Wakefield); Grief (TRIG) Phobic Avoidance (FQ); Hostility/Anger/Guilt (HAG); Attitude to self and deceased (author-created scales); Avoidance (Bereavement Avoidance Tasks); Physical Symptoms (Maddison & Viola, 1968); Compulsive Behavior (Compulsive Activity Checklist); Social Adjustment (Watson & Marks, 1971) Mawson, Marks, et al, 1981
Individual Adult N NA 1/1 \<180 112 days; 10 sessions Grief (Grief Scale); Coping (CRI) Orton, 1994
Group Senior Y Y 150/117 \<365--7300 540 days; 6 sessions Depression (BDI); Socialization (RSAS) Constantino, 1988\*\*\*\*\*
**Psycho-dynamic** Group Adult Y Y-NE 56/53 120--330 8 sessions Depression, Anxiety, Somatization (Hopkins Symptom Checklist); Grief Intensity, Preoccupation, Guilt, Anger (Lieberman & Videka-Sherman, 1986); Psychological Distress (Bradburn Affect Balance Scale); Locus of Control (Pearlman et al, 1981); Self-Esteem (Rosenberg scale, 1965); Social Adjustment (Pearlman et al, 1981, Lieberman & Videka-Sherman, 1986) Lieberman & Yalom, 1992
Group Adult Y N 50/50 NR 90 days; 14 sessions Grief (TRIG) Sabatini, 1988--89
Group Adult N Y 139/107 90--17155 84 days; 12 sessions Avoidance/Intrusion (IES); Grief (TRIG); Interpersonal Distress (IIP); Social Functioning (SAS-SR); Depression (BDI); Anxiety (STAI); Mental Distress (BSI, GSI); Self-Esteem (SES); Physical Functioning (SF-36); Symptomatic Distress (SCL-90) Piper, McCallum, et al, 2001\*
Group Adult N Y-NE 61/55 120--1095 84 days; 12 sessions Avoidance/Intrusion (IES); Stress Symptoms (SRRS); Depression (BDI); Mental Distress (BPRS, SCL-90); Social Functioning (SAS-SR); Overall Functioning (GAS) Marmar, Horowitz, et al, 1988\*\*
Group Child Y N 16/16 30--365 56 days Depression (CDI); Behavior (AP, AT); Grief (BP); Family alliance (TP); Grief, family relationship (TC) Tonkins & Lambert, 1996
Group Child N NA 45/37 30--3650 70 days Trauma (CPTSRI) Salloum, Avery, & McClain, 2001
**Psycho-analytic** Group Adult N N 154/59 NR 84 days; 12 sessions Affect (author created); Psychodynamic Work (PWORS); Severity of objectives (author created) McCallum, Piper, & Morin, 1993
**Inter-personal** Individual Senior Y Y-NE 80/66 216--279 112 days; up to 16 sessions Grief (TRIG) Reynolds, Miller, et al, 1999\*\*\*
**Behavioral and Psycho-dynamic** Individual Adult Y N 83/83 \<1825 15--20 sessions Anger (State Trait Anger Inventory); Anxiety (STAI); Avoidance/Intrusion (IES); Somatic/psychoneurotic symptoms (SCL-90); (Locus of Control Scale) Kleber & Brom, 1987
Notes: \* Study also included support/counselling condition. \*\* Treatment also included aspects of interpersonal psychotherapy. \*\*\* Study also included pharmacotherapy condition. \*\*\*\* Treatment also included aspects of social support. \*\*\*\*\* Study also included social activities condition. Legend: Type, Type of Intervention; Format, Format of Intervention; Pop, Target Population; CG, Control Group; RA, Random Assignment; Num, Number of subjects; TSL, Time Since Loss; DT, Duration of Trial; NA, Not Applicable; NR, Not Reported; UC, Unclear; Y, Yes; N, No; Y-NE Randomization mentioned, but allocation method not explicitly stated.
:::
***Cognitive-behavioral therapy***was employed in nine trials, four of which used individual sessions while five studies used group sessions. Murphy and colleagues (1998) studied an intervention for parents bereaved by the violent death of their children \[[@B57]\]. The results show no treatment effect between intervention and control groups over the five main tested outcome variables. The authors then proceeded with a post-hoc subgroup analysis, which identified mothers with high Global Severity Index scores and grief at baseline as potentially benefiting from intervention during the period, while fathers who received the intervention appeared to have more posttraumatic stress disorder (PTSD) symptoms at six-month follow-up.
Kleber and Brom (1987) conducted a comparative outcome study of three forms of short-term psychotherapy \[[@B69]\]. They compared the results of 83 patients suffering from a major loss who had been randomized into hypnotherapy (behavioral), trauma desensitization (behavioral), psychodynamic therapy, and a delayed-treatment control group. They found all three therapies successful in improving patients\' conditions, but did not find any particular therapy to be significantly more effective than another. While the control group showed slight recovery, over time the three therapies were more effective in reducing symptoms of the bereavement response.
Studies of ***psychodynamic***therapy, which strives for the patient to understand and cope better with feelings by re-experiencing them and talking them through with the aid of the therapist, was found to be quite prevalent in the bereavement care literature. Overall, the results are mixed, with more support found in the group format of psychodynamic therapy than in individual therapy. Of the studies we evaluated as psychodynamic therapy, six were individual in format, seven had a group format, and eight employed control groups; five of these claimed random allocation (one additional study randomly assigned subjects to two experimental conditions but lacked a control group).
***Psychoanalysis***, as exemplified by Freud, proceeds with an inward investigation of unconscious mental processes and childhood experiences as the principal therapeutic procedure. Problematic measurement methodology beset the one study that utilized a group format to provide a psychoanalytic-based intervention (with no details regarding the tasks assigned to the patients)\[[@B68]\]. This study focused primarily on the relationship between the patient\'s personal affect (measured by an unvalidated affect assessment scale) and a favorable treatment outcome (measured again by an ad-hoc unvalidated measure).
***Behavioral therapy***uses learning principles (such as behavior modification, systematic desensitization, and aversion) to eliminate or reduce unwanted reactions to either external situations, one\'s thoughts and feelings, and bodily sensations or functions. Behavioral therapy was used in only one study, which compared traumatic desensitization to hypnotherapy and psychodynamic therapies \[[@B69]\]. As described in the section on cognitive-behavioral therapies above, all three therapies resulted in significant improvements from pre- to post-treatment as compared to controls, and no one therapy was found to be more effective than the others in treating bereavement-related symptoms.
***Interpersonal therapy***aims to improve communication skills and increase self-esteem during a short time period by focusing on a patient\'s behaviors and social interactions with family and friends, directly teaching how better to relate to others. Only one study used interpersonal therapy as a bereavement care intervention, and this study found no effect on grief as the only measured outcome \[[@B6]\].
Systems-oriented interventions
------------------------------
Seven studies featured interventions that altered the manner in which the healthcare system interacted with patients, family, and friends prior to death, guided by an underlying (yet not fully explicated) notion that interactions experienced by loved ones prior to death can influence the subsequent bereavement process (Table [4](#T4){ref-type="table"}) \[[@B73]-[@B79]\]. Six of the seven interventions provided enhanced or augmented care, in the form of palliative care, hospice care, or care coordination. One intervention gave family members the option of witnessing resuscitation efforts \[[@B79]\]. Overall, the studies that reported systems-oriented interventions produced mixed results of efficacy, with only three of the seven studies showing any treatment effect, mostly in long-term follow-up ranging from 60--365 days post-death. In fact, no study found significant treatment effects when measured during the intervention.
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
Systems-Oriented Interventions
:::
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Intervention** **Pop** **CG** **RA** **Num** **Time of Evaluation** **Key Outcome Measures** **Article**
------------------------- ---------------------------------------- -------- -------- --------- --------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------ --------------------------------------
Care Coordination Relative of cancer death Y Y 94 365 days pre-death\ Anxiety (HADA, Leeds Depression and Anxiety Scale); Depression (HADD, Leeds Depression and Anxiety Scale); Social Support (Family Apgar Scale) Addington, MacDonald, et al, 1992
56 days post-death
Emergency Room Relative of Emergency Room Death Y N 100/66 180--365 days post-death Changes in satisfaction of care, information received (author-created questionnaire) Adamowski, Dickinson, et al, 1993
Hospice Care Relative of cancer death Y Y-NE 96 42 days post-death\ Depression (CES-D); Anxiety (Rand Health Insurance Study); General Health (Rand Health Insurance Study); Social Functioning Kane, Klein, et al, 1986
540 days post-death
Palliative Care Relative of cancer death Y Y 183 60--270 days pre-death\ Grief (TRIG2) Ringdal, Jordhoy, et al, 2001
390 days post-death
Relative of cancer death Y NR 119/49 0--60 days post-death Anxiety, Depression, Mental Exhaustion (\"observations and ratings\") Haggmark & Theorell, 1988
Relative of cancer death Y N 49/37 365 days post-death Health, Anger, Mental State, Depression (Holland & Segroi\'s instrument) Haggmark, Bachner, & Theorell, 1991
Witnessed Resuscitation Relative of unsuccessful resuscitation Y Y 18 30 days post-death 90 days post-death Grief (TRIG1, TRIG2); Avoidance/Intrusion (IESA, IESI); Depression (BDI, HADD); Anxiety (HADA, BAI) Robinson, Makenzie-Ross, et al, 1998
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Legend: Pop, Target Population; CG, Control Group; RA, Random Assignment; Num, Number of subjects; NR, Not Reported; Y, Yes; N, No; Y-NE Randomization mentioned, but allocation method not explicitly stated.
:::
Ringdal and colleagues (2001) found no significant differences between those family members whose relative received palliative care and those who received traditional care \[[@B76]\]. This intervention, however, was not directed to the bereaved relatives, but rather to their terminally ill relatives. The bereaved relatives did show an overall significant decline in TRIG grief scores over one year post-bereavement for both palliative and traditional care groups.
Robinson (1998) examined the psychological effect of witnessing resuscitation efforts of patients in the emergency room on bereaved relatives \[[@B79]\]. They found no psychological differences between the control group who did not witness the resuscitation attempt and the experimental group who had the option of viewing the resuscitation effort. In fact, at the three- and nine-month follow up, the experimental group exhibited median scores lower (that is, better) than the controls on five of the eight measured scales. At nine months, the authors found the difference in TRIG2 scores approaching the 5% significance level with a reported p = 0.08. These findings provide no evidence to support the popular belief that relatives should be excluded from the resuscitation room, and provide only weak evidence of possible psychological benefit of witnessed resuscitation; they do not, however, suggest that having witnessed an unsuccessful resuscitation attempt alleviates the grief reaction of the bereaved.
Discussion
==========
When reviewed systematically, the current bereavement intervention literature -- notwithstanding the existence of many intriguing reports -- yields few reliable conclusions to guide treatment. Good evidence supports the pharmacological treatment of depression occurring in the context of bereavement. For all other forms of intervention, however, and for all attempts to diminish grief *per se*, no consistent pattern of treatment benefit has been established across well-designed experimental studies.
Why -- despite prevalence of bereavement, the intense dedication on the part of the bereavement research community, and the multitude of peer-reviewed published bereavement studies -- does the field of bereavement care lack a formidable evidence base? In order to improve the effectiveness and quality of bereavement care, this question begs to be addressed. On the basis of our systematic review of the literature, we postulate the following five factors as hindering methodical scientific progress regarding bereavement care interventions.
Excessive theoretical heterogeneity
-----------------------------------
As the history of science and medicine suggests, successful scientific inquiry into a topic is typically a cumulative process undertaken by a community of investigators working within a shared scientific paradigm \[[@B80],[@B81]\]. The field of bereavement care intervention studies does not appear to be organized in such a manner, but instead consists of distinct groups of investigators working within disparate theoretical frameworks: pharmacologic, psychodynamic, psychoanalytic, behavioral, cognitive-behavioral, interpersonal, and social supportive theories each vie for attention. Indeed, although specification of an underlying treatment-theory conceptual model may improve causal inference \[[@B82]\], the bereavement care literature may be too invested in and reliant on theoretical justifications of treatments. Consequently, the compiled published reports demonstrate a cumulative \'Tower of Babel\' phenomenon, with the different theory-dominated perspectives failing to engage each other meaningfully: the sum is no greater than the parts, and perhaps less.
Stultifying between-study variation
-----------------------------------
Treatments featured in published studies vary almost as much as the authors who tested them. One can observe substantial variation across studies regarding the type of intervention generally or regarding the specific implementation of a specific type of intervention (such as different doses of pharmaceuticals); regarding characteristics of targeted patient populations; regarding outcome measurements and study design methodology. Scrutinizing the key outcome measures listed in the accompanying tables illustrates this remarkable heterogeneity. Although these differences have been due in part to diverse treatment-theory paradigms, even studies conducted within the same theoretical paradigm often differed markedly in terms of what potential benefit was being tested, and how it was being measured. Such substantial variation between studies stymies comparison or confirmation of treatment effects.
Inadequate reporting of intervention procedures and implementation
------------------------------------------------------------------
Aside from the pharmacological studies, which reported the dosing of the intervention medication, very few reported studies describe the intervention procedures in sufficient detail for readers to envision clearly what tasks or activities intervention subjects were asked to perform. This under-specification prevents sensible analysis, within a class of treatments (such as cognitive-behavioral therapy), of observed differences in treatment effects (since the implementation of cognitive-behavioral therapy, for instance, may have been quite different in seemingly similar intervention studies).
Few published \"replication\" studies
-------------------------------------
Inadequate specification of intervention procedures, combined with other factors at work within the community of bereavement care investigators, may have resulted in the dearth of published replication studies. This lack of replication prevents the accumulation of a body of evidence that would confirm, refute, or refine prior estimates of treatment effects.
Methodological study-design and data-analysis flaws
---------------------------------------------------
A final factor inhibiting research progress in the realm of bereavement care interventions encompasses a number of recurring methodological flaws that greatly limit inferences regarding treatment effects. First and foremost is the omission of control groups. Control groups are essential for the valid evaluation of a bereavement intervention, particularly because of the typically self-limited course of grief: even absent any treatment, most bereaved people show \"diminished pathological symptoms and fewer signs of disturbance within two years of the loss\"\[[@B65]\]. Purported beneficial treatment effects observed in an intervention group without a suitable control group therefore may in fact be simply the natural grief remission process. A second common study design feature is the non-random assignment of study subjects into treatment and control groups, which again limits the strength of inference regarding observed \'treatment\' effects, as these differences between treatment and control groups may be due to selection or assignment bias. Third, many studies measured subject outcomes using untried assessment tools that had been created on an *ad hoc*basis, and which may therefore have compromised measurement accuracy and inference validity. Lastly, studies that failed to demonstrate a statistically-significant difference for the main outcome measure often performed numerous *post hoc*subgroup analyses, a practice that negates the rigor of statistical hypothesis testing.
If these five factors are indeed hampering progress towards improving bereavement care interventions and quality of care for bereaved individuals, then concrete actions could facilitate progress within the field of bereavement care, specifically: 1) Convening a consensus-building conference among key stakeholders and investigators to define a specific research agenda that would draw on a limited number of theoretical paradigms and delineate key elements of treatment theory \[[@B82]\]; 2) Focusing on interventions to improve key outcomes that are valued by bereaved individuals; 3) Targeting well-defined patient populations at well-defined phases of bereavement; 4) Conducting high-quality randomized controlled trial research designs, employing rigorous tests of hypotheses defined prior to the conduct of the study, and eschewing unplanned subgroup analyses; 5) Weighing the ethical arguments for and against the use of randomized control subjects in such research; 6) Increasing incentive to conduct and publish highly-comparable replication studies; and 7) Enforcing the adoption of uniform standards regarding clinical trial study reporting (such as outlined in the CONSORT statement \[[@B83]\]) by journal editors and the bereavement research community.
Competing interests
===================
None declared.
Abbreviations
=============
CG Control group
NT Nortriptyline
SSRI Selective serotonin reuptake inhibitors
TCA Tricyclic antidepressants
TRIG Texas Revised Inventory of Grief
Authors\' contributions
=======================
AF assisted in the design of the review, conducted data collection, data abstraction, and drafted the manuscript. MH assisted in the design of the review and critically reviewed several drafts of the manuscript. RP assisted in data collection and abstraction. CF conceived of and designed the review, assisted in drafting and revision of the manuscript, and provided support and mentorship through the process. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1472-684X/3/3/prepub>
Supplementary Material
======================
::: {.caption}
###### Additional File 1
All citations. This file contains citations to all of the studies identified by our literature search and screened for inclusion in this review, as well as other scholarly works consulted during the conduct of this review.
:::
::: {.caption}
######
Click here for file
:::
Acknowledgements
================
The conduct of this review was supported in part by funds from The Children\'s Hospital of Philadelphia and by grant KO8 HS00002 from the Agency for Health Care Research and Quality.
| PubMed Central | 2024-06-05T03:55:46.580652 | 2004-7-26 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC503393/",
"journal": "BMC Palliat Care. 2004 Jul 26; 3:3",
"authors": [
{
"first": "Amanda L",
"last": "Forte"
},
{
"first": "Malinda",
"last": "Hill"
},
{
"first": "Rachel",
"last": "Pazder"
},
{
"first": "Chris",
"last": "Feudtner"
}
]
} |
PMC503394 | Background
==========
Back pain is one of the most common and costly health problems in developed countries, where more than half of adults suffer from this condition each year \[[@B1]\] and 70% to 80% suffer from it at some time in their lives \[[@B2]\]. Patients with back pain are often dissatisfied with standard medical care \[[@B3]\], especially in comparison to care provided by alternative providers \[[@B4]-[@B6]\]. In fact, back/neck pain is the number one condition for which Americans seek complementary or alternative medical (CAM) care. During 1997, 30% of Americans with back problems visited CAM practitioners, especially chiropractors and massage therapists, for this condition and another 18% used CAM self-care \[[@B7]\]. Yet, to date, few of these therapies have been adequately evaluated for effectiveness, in part because of methodological challenges including recruiting sufficient numbers of participants, designing reasonable interventions and selecting appropriate control groups.
Prior to designing and conducting two pilot clinical trials evaluating the effectiveness of five different CAM therapies for chronic low back pain (LBP) in older (65+ years) and younger (20 through 64 years) adults, we sought to refine recruitment strategies. As part of this effort, we surveyed chronic low back pain patients about their interest in trying each of these five CAM therapies if included in their health plan benefits and as part of two separate clinical trials. In this descriptive and exploratory study, we also collected information about their knowledge of, experience with, and perceptions of each of these therapies.
Methods
=======
Study design
------------
From April to October 2001, we conducted telephone interviews with 249 patients who were currently suffering from non-specific low back pain that had persisted at least three months. The patients were members of a non-profit managed health care system (Group Health Cooperative in the Puget Sound region of Washington State) and a large multi-specialty group practice (Harvard Vanguard Medical Associates, Boston, Massachusetts). Our goal was to interview 150 patients from Group Health and 100 patients from Harvard Vanguard who were otherwise healthy and spoke English, with 50% of the interviews from adults 65 years or older. The Institutional Review Boards of Group Health Cooperative, Seattle WA and Harvard Pilgrim Health Care, Boston, MA approved the study.
Sample
------
Using automated visit data, we identified and mailed letters to 787 patients with working phone numbers who visited a Group Health (n = 422) or a Harvard Vanguard (n = 365) primary care provider and had a diagnosis consistent with non-specific low back pain between 12 and 52 weeks previously. Because we were planning to use the results of the survey to help us refine recruitment strategies for two pilot randomized trials, our exclusion criteria for this study were those we planned to use in the subsequent trials. We therefore used the automated visit data to exclude the following individuals from our sample:
• those whose back pain may have been due to a specific disease or condition (i.e., sciatica, herniated disc, spondylolisthesis, spine fracture, vertebral fracture, cancer);
• those who had other pain conditions that could complicate the interpretation of trial results (rheumatoid arthritis, ankylosing spondylitis, fibromyalgia);
• those who were inappropriate candidates for one or more of the clinical trial treatments (aneurism, coagulation disorders, osteoporosis) or who were unlikely to be able to give informed consent or to participate in the baseline and follow-up assessments of the trial (Alzheimers disease, dementia, major psychoses, blindness, deafness).
Before administering the in-person survey, we asked eight screening questions and then excluded individuals who did not have back pain at the time of the interview or who had not had back pain for at least 12 weeks, who had previously had low back surgery, who reported having fractured a vertebrae, who were pregnant, who were involved in back-pain related litigation, who had serious health problems, or who could not speak English.
We tried to contact all mailees, but could not reach 28 (7%) persons from Group Health and 73 (20%) from Harvard Vanguard despite at least seven phone calls. Among the 394 Group Health and 292 Harvard Vanguard patients who were contacted, 57 from Group Health and 81 from Harvard Vanguard refused the interview and we could not determine their eligibility status, 195 from GHC and 104 from Harvard Vanguard were ineligible upon screening, and 142 from Group Health and 107 from Harvard Vanguard were eligible and interviewed. Thus, we were able to screen 70% of all mailees for eligibility (80% from Group Health and 58% from Harvard Vanguard). All screened and eligible mailees were interviewed.
In both areas, individuals aged 65 and older were more likely to refuse screening (in Seattle: 19% of 210 older adults vs. 8% of 212 younger; in Boston: 31% of 184 older adults vs. 13% of 181 younger) and those under 65 were less likely to be contacted by phone (in Seattle: 12% younger vs. 1% older could not be contacted; in Boston: 31% younger vs. 9% older could not be contacted).
Most patients were ineligible because they were not experiencing back pain at the time of the interview (n = 82 from Group Health and n = 68 from Harvard Vanguard) or their pain had not persisted for three months (n = 39 from Group Health and n = 11 from Harvard Vanguard).
Survey questionnaire
--------------------
We conducted a phone interview that lasted an average of 17.7 minutes (SD = 6.1; range = 8 to 50 minutes). It included questions about demographic characteristics (age, race/ethnicity, education); back pain characteristics (years since first episode of back pain lasting more than two weeks, number of days of pain in the last six months, bothersomeness of pain on a 0 to 10 scale, expectations of pain level one year from the time of interview, and use of medications in the past week); self-reported knowledge (measured on a five point scale) of five CAM treatments or self-care methods (acupuncture, chiropractic, massage, meditation, t\'ai chi); previous use of these therapies for any reason and for back pain specifically (and helpfulness of the therapy for back pain relief); perceived harm from previous use of these therapies; expectations of helpfulness of these therapies for current back pain; willingness to try these therapies if offered by the health plan for no additional cost and for a \$10 per visit co-pay; willingness to participate in two hypothetical clinical trials, one evaluating acupuncture, chiropractic, and massage and another involving massage, meditation, and t\'ai chi. (Respondents were told that the control group in both trials would receive a book about self-management of back pain.) Finally, respondents were asked about which treatment they most preferred among those offered in each trial. Gender and geographic location were obtained for respondents from the enrollment files of each healthplan. General definitions of each therapy were provided only for respondents who informed the interviewers they did not know what a particular therapy was. Acupuncture was described as a system of healing that involved inserting hair thin needles into acupuncture points just beneath the skin or using other methods, such as heat, to stimulate these points, whereas chiropractic was defined as a system of therapy that uses manipulation to adjusts the spine and other body parts to \"promote normal nerve functions\". Massage was described as the systematic rubbing and manipulation of muscle and other tissues to relieve bodily infirmities, while meditation was defined as a \"self-directed practice for relaxing the body and calming the mind\". Finally, tai chi was described as a Chinese martial art that uses slow and smooth body movements and is often practiced for its purported health benefits. The survey was pre-tested on a convenience sample of 15 people (both older and younger) in Seattle and 5 people in Boston.
Statistical analyses
--------------------
We analyzed the data using the SAS statistical software version 6.12 (SAS Institute, Cary, NC). Descriptive data were characterized using percentages or medians. For each of the five CAM therapies, we performed separate exploratory logistic regressions to identify specific characteristics associated with 1) high degree of knowledge (4 or 5 on a 5-point scale) (five separate models), 2) prior use (for any reason and for back pain) (10 separate models), 3) high expectations of helpfulness for current back pain (7 to 10 on a 0 to 10 scale) (five separate models), 4) greatest likelihood of trying therapy for no additional cost (all five therapies) and for a \$10 per visit co-pay (acupuncture, chiropractic, massage only) (eight separate models), and 6) greatest likelihood of participating in each of the two hypothetical clinical trials (two separate models). Thus, a total of 30 separate logistic models were created.
For each of the 30 dependent variables, we identified potential predictor variables in advance and evaluated them in preliminary models. In Table [1](#T1){ref-type="table"}, the potential predictor variables evaluated in the preliminary models for each of the 28 therapy-specific dependent variables are indicated by an X. In addition, we modeled the likelihood of being \"definitely willing\" to participate in a hypothetical clinical trial of acupuncture, chiropractic, and massage and of being \"definitely willing\" to participate in a hypothetical clinical trial of massage, meditation, and t\'ai chi. In both models, we evaluated the following 22 variables as potential predictor variables of being \"definitely willing\" to participate in the hypothetical clinical trial: demographic characteristics (age, gender, race, education, geographic location), prior use of each of the therapies included in the trial (i.e., acupuncture, chiropractic, and massage for one trial and massage, meditation, and t\'ai chi for the other trial) for any reason (and for back pain), knowledge of these three included therapies, prior perceived harm from these three included therapies, years since first back pain, symptom bothersomeness, high expectations of each included CAM therapy for current back pain, number of days of back pain in last six months, and medication usage in the week prior to the interview.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Potential Predictor Variables Evaluated in 28 Therapy-Specific Logistic Regression Models
:::
Dependent Variables for Logistic Regressions
----------------------------------------------------------- ---------------------------------------------- ------------------------ -------------------------------------- ------------------------------------------- ------------------------------------------- --------------------------------------------------
Potential Predictor Variable High Knowledge of Therapy\* Prior Use of Therapy\* Prior Use of Therapy for Back Pain\* High Expectations of Success of Therapy\* Likelihood of Trying Therapy at No Cost\* Likelihood of Trying Therapy for \$10 Co-pay\*\*
Geographic location (Boston vs. Seattle) X^¶^ X X X X X
Age (65+ vs. \< 65) X X X X X X
Gender (female vs. male) X X X X X X
Race (white, non-white) X X X X X X
Education (no college vs. some college) X X X X X X
≥ 5 years since first back pain X X X
≥ 90 days of LBP in last 6 mo. X X X
High symptom bothersomeness (7 -- 10) on a 0 -- 10 scale X X X
High knowledge of therapy (4 or 5) on a 1 -- 5 scale X X X
Prior use of therapy X X X
Prior use of therapy for back pain X X X
High expectations of therapy (7 -- 10) on a 0 -- 10 scale X X
Medication usage in past week X X
Prior harm from therapy X X
\* Separate models were done for each of the five therapies (acupuncture, chiropractic, massage, meditation, t\'ai chi) \*\* Separate models were done for acupuncture, chiropractic, and massage. ^¶^An X indicates that a particular potential predictor variable was evaluated in a model with the specific dependent variable.
:::
Initially, we evaluated potential predictor variables in preliminary models containing five or fewer independent variables. Any independent variable associated with the dependent variable at a p value of 0.15 or less in a preliminary model was a candidate for the appropriate final model. We used a backwards elimination procedure to evaluate candidate predictor variables and to determine the final models \[[@B8]\]. All variables with a p value of 0.01 or less were retained in the final model. Odds ratios (OR) are presented along with 95% confidence intervals (95% CI). Table [4](#T4){ref-type="table"} presents the odds ratios that describe the significant associations (p \< 0.01) for each of the 28 therapy -- specific dependent variables.
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
Predictors of Knowledge of, Experience with, Expectations about, and Willingness to Try Five Complementary and Alternative Medical (CAM) Therapies
:::
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Odds ratios\***(95% CI) for the independent predictor variables used in the final models for each CAM therapy
------------------------------------------------------------ ----------------------------------------------------------------------------------------------------------------- ---------------------- --------------------- --------------------- ------------------------
High Knowledge of specific therapy (4--5) Tried acupuncture:\ Tried chiropractic:\ Tried massage:\ Tried meditation:\ LOGISTIC NOT VALID\*\*
43.6 (16.7--113.6) 12.8 (6.2--26.7) 7.6 (4.0 -- 14.7) 11.6 (4.6--29.3)
Bostonian:\
4.8 (1.9--12.3)
Previously tried specific therapy No associations Bostonian:\ 65+ yrs:\ Female:\ No associations
0.5 (0.3 -- 0.8) 0.4 (0.2 -- 0.7) 2.5 (1.3 -- 4.8)
Previously tried specific therapy for back pain No associations None 65+ yrs:\ No associations LOGISTIC NOT VALID\*\*
0.3 (0.2 -- 0.6)
High expectations of specific therapy (7 -- 10) 65+ yrs:\ Knowledge:\ 65+ yrs:\ No associations No associations
0.4(0.2 -- 0.8) 2.9 (1.6 -- 5.2) 0.3 (0.2 -- 0.5)
Tried acupuncture:\
4.3 (2.1 -- 9.0)
Very likely to try specific therapy for free High expectations:\ High expectations:\ High expectations:\ High expectations:\ High expectations:\
15.4 (3.6 -- 66.1) 27.4 (9.5 -- 79.3) 16.4 (7.4 -- 36.5) 3.6 (1.7 -- 7.7) 14.3 (5.4 -- 38.3)
Tried meditation:\
2.4 (1.3 -- 4.5)
Very likely to try specific therapy for \$10 /visit co-pay High expectations:\ High expectations:\ High expectations:\ NOT QUERIED NOT QUERIED
6.8 (3.0 -- 15.5) 8.1 (4.2 -- 15.7) 6.4 (3.5 -- 11.4)
Bostonian:\ Bostonian:\
2.3 (1.4 -- 4.0) 1.8 (1.001 -- 3.2)
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
\*These odds ratios describe the significant associations (p \< 0.01) for each of the 28 therapy -- specific dependent variables. For example, we found that those who had tried acupuncture were 43.6 times more likely to have high knowledge of acupuncture. No other variables were related to high knowledge of acupuncture. \*\* These logistic regression models did not converge. Categorization for independent variables: Age (\<65; 65+) Knowledge of therapy (1--3; 4--5) Gender (M; F) Expectations of therapy (missing through 6; 7+) Geography (Seattle; Boston) Prior Use of therapy (no; yes)
:::
Results
=======
Characteristics of respondents
------------------------------
Most study participants were white, women, and had attended college (Table [2](#T2){ref-type="table"}). Most had had back problems for at least five years, had experienced back pain at least 90 days in last six months, had used medications in the prior week, and expected little change in their pain in a year.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Demographic and Back Pain Characteristics of 249 Survey Respondents
:::
Characteristic Percent
--------------------------------------------------------------------- ---------
Location (Boston) 43
Age (\< 65) 52
Women 60
White 80
Attended some college 57
At least 5 years since first back pain lasting longer than 2 weeks 60
90+ days of LBP in last 6 mo. 61
High symptom bothersomeness in the past week (≥ 7) on 0 -- 10 scale 42
Used medication for LBP in the past week 56
Expect pain to be similar in a year 72
Missing data -- last variable has 10 missing values (4% of all observations), 1 variable has 5 (2%), all others have 3 or fewer.
:::
Knowledge of, experience with, perceptions of, and willingness to try CAM therapies
-----------------------------------------------------------------------------------
Except for chiropractic, most participants reported little or no knowledge of these therapies (Table [3](#T3){ref-type="table"}). In logistic regressions, prior use of a therapy consistently predicted high knowledge of that therapy (Table [4](#T4){ref-type="table"}).
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Knowledge of, Experience with, Expectations about, and Willingness to Try Five CAM Therapies\*
:::
--------------------------------------------------------------------------------------------------------------------------------------------------------------------
Acupuncture\ Chiropractic\ Massage\ Meditation\ T\'ai Chi\
(N = 249) (N = 249) (N = 249) (N = 249) (N = 249)
---------------------------------------------------------------------------------------------- -------------- --------------- ----------- ------------- ------------
Knowledge about Therapy (%)
1 -- 2 (1=\"no knowledge\") 69 44 52 72 91
3 17 22 24 15 6
4 -- 5 (5=\"a lot of knowledge\") 14 34 24 13 3
Ever tried therapy (%) 18 54 38 27 8
Ever tried therapy for LBP (%) 11 45 24 7 0.4
Median helpfulness for LBP among prior users (0 to 10 scale) 5 6 7 5 \*\*
Pain or harm reported by prior users (%) 13 23 13 5 16
Median expectation of helpfulness for current LBP (0 to 10 scale) 5 5 7 3 5
Did not provide expectation rating (%) 25 10 9 12 24
High expectations of helpfulness for current LBP (7 to 10 on 0 to 10 scale) (%) 19 28 48 15 16
Very likely to try therapy if primary care provider thought reasonable and no extra cost (%) 64 51 69 27 41
Very likely to try therapy if primary care provider thought reasonable and \$10 co-pay (%) 51 42 56 NA NA
--------------------------------------------------------------------------------------------------------------------------------------------------------------------
NA = Not Asked. \* Each column refers to a specific therapy and the specific question about the therapy is shown in the first column. \*\* Only 1 person had tried t\'ai chi for low back pain previously. All variables, except expectations of helpfulness of current LBP (where % are given in the table) have missing values for \< 5% of respondents.
:::
More than half of the participants had tried chiropractic compared with 38% who had tried massage and substantially fewer who had tried the other therapies (Table [3](#T3){ref-type="table"}). No demographic characteristics were related consistently to use of these therapies (Table [4](#T4){ref-type="table"}). Chiropractic and massage were also the most commonly used of the therapies specifically for low back pain. Users of massage rated treatment helpfulness higher than did users of other therapies (Table [3](#T3){ref-type="table"}). Reports of harm or increased pain were highest for chiropractic (23%) and lowest for meditation (5%).
Respondents believed that massage would be most helpful for their current back pain (median rating of 7) and that meditation would be least helpful (median rating of 3) (Table [3](#T3){ref-type="table"}). One quarter of all respondents were unable to rate their expectation of acupuncture or t\'ai chi, compared to about 10% for the other therapies. Respondents 65 years of age or older were less optimistic than younger respondents about the helpfulness of acupuncture and massage (Table [4](#T4){ref-type="table"}). High expectations of helpfulness of chiropractic were more common in those with high knowledge of this therapy and high expectations of helpfulness of acupuncture were more common among those who had tried it (Table [4](#T4){ref-type="table"}).
More than half of the respondents said they would be \"very likely\" to try acupuncture, chiropractic, or massage if provided by their health plan for no additional cost and their physician felt it was reasonable. Fewer respondents said they would be \"very likely\" to try meditation training (27%) or t\'ai chi (41%) under those circumstances (Table [3](#T3){ref-type="table"}). In logistic regression models, the strongest predictors of being very likely to try a particular therapy were high expectations of a therapy and, for meditation, prior use of the therapy (Table [4](#T4){ref-type="table"}). About 80% of those very likely to try acupuncture, chiropractic, or massage for no additional cost were also very likely to try it for a \$10 per visit co-pay (Table [3](#T3){ref-type="table"}). Paralleling the finding for free care, the strongest predictor of willingness to try a therapy for a \$10 per visit co-pay was high expectations of success for that therapy. Respondents from Boston were more willing to try acupuncture. Those reporting harm or pain from chiropractic were less willing to try this therapy again.
Willingness to participate in a clinical trial
----------------------------------------------
More than half of respondents were \"definitely willing\" to participate in each of two hypothetical clinical trials about which they were asked and less than 5% were definitely unwilling to participate (Table [5](#T5){ref-type="table"}). When asked which of the treatments in each trial they would most prefer, respondents preferred massage and acupuncture in the trial of acupuncture, chiropractic, and massage and, in the second trial, strongly preferred massage to meditation. However, a significant fraction (24%) expressed a preference for t\'ai chi. We found no demographic, back pain, or CAM characteristics associated with being \"definitely willing\" to participate in the hypothetical trial of acupuncture, chiropractic, and massage. People who were \"definitely willing\" to participate in the hypothetical trial of massage, meditation, and t\'ai chi were more likely to have high expectations of meditation (OR = 3.1, 95% CI = 1.4 -- 7.0).
::: {#T5 .table-wrap}
Table 5
::: {.caption}
######
Willingness to Participate in Clinical Trials of CAM Therapies for Low Back Pain and Preference for Therapies
:::
---------------------------------------------------------------------------------------------------------------------------------------------
Percent\
(N = 249)
--------------------------------------------------------------------------------------------------------------------------------- -----------
Definitely willing to participate in clinical trial of acupuncture, chiropractic, massage, and a self-help back pain book (%)\* 62
Preferred treatment among above:
Massage 43
Acupuncture 35
Chiropractic 18
None or Other 3
Book 1
Definitely willing to participate in clinical trial of massage, meditation, t\'ai chi, and a self-help back pain book (%)\*\* 53
Preferred treatment among above:
Massage 63
T\'ai Chi training 24
Book 5
Meditation training 4
None or Other 4
---------------------------------------------------------------------------------------------------------------------------------------------
Missing values -- \< 4% of responses for each variable are missing. \*Your healthplan is thinking about conducting a study evaluating several treatments for people with chronic low back pain. In this study, participants would have a one in four chance of being assigned to one of the following treatments: acupuncture, chiropractic, massage, or a book designed to help patients better understand their low back pain. Participants would be expected to try the treatment they were assigned to at least once. Participants would still retain access to their usual care and participation in this study would be free. If you were asked to take part in a study like this would you be willing to participate? \*\* Same question was asked, but the treatments were massage, meditation training, and t\'ai chi training.
:::
Discussion
==========
Our findings suggest that many patients would be willing to try specific CAM therapies for back pain, especially if they had high expectations for their helpfulness. Interestingly, we found no consistent relationships between high expectations for a particular therapy and either previous use of that therapy or high self-perceived knowledge of that therapy.
Our findings regarding knowledge, previous use, and expectations for these therapies were largely similar for Seattle and Boston and for older and younger adults. However, those over 65 years old were less likely to have high expectations of acupuncture and massage and to have tried massage previously.
Since we conducted the study in two metropolitan areas where CAM use is fairly common, our results might not represent the CAM views of patients with back pain in more rural areas or in other regions of the country. Another limitation of our study was that 30% of people we attempted to contact could not be assessed for eligibility, leading to the possibility of a high non-response rate. Because we have almost no information on the characteristics of the individuals with unknown eligibility, we do not know if they differ from those included in the study, and cannot adequately estimate the magnitude and direction of potential biases regarding interest in CAM that might exist in our sample. However, the fractions of individuals who were unable to be assessed for eligibility were similar among those less than 65 years of age and those 65 and older in each metropolitan area (45% vs. 40% in Boston, respectively; 20% vs. 20% in Seattle).
Respondents showed a clear preference for receiving hands-on treatments delivered by a practitioner compared to attending classes that teach self-care techniques. Whether this reflects a preference for provider-oriented, more passive, therapies or the belief that classes teaching these specific self-care therapies would be less effective is not clear. Unfortunately, our interview did not include questions about yoga, which has recently received more popular press than meditation or t\'ai chi as a self-care therapy for back pain \[[@B9],[@B10]\].
Survey respondents were not enthusiastic about \"meditation training\" as a treatment for back pain. Relatively few of those who indicated prior use of meditation for physical or mental health problems had used the forms of meditation most commonly taught in a medical setting (e.g., mindfulness meditation). Consequently, studies recruiting patients to participate in interventions including meditation training may need to carefully describe the treatment in terms of a concrete goal (e.g., stress reduction).
There is still relatively little knowledge about and experience with acupuncture and t\'ai chi even in Boston and Seattle where use of CAM therapies is generally high. In fact, about one -- quarter of respondents were unable to provide an expectation of the helpfulness of acupuncture or t\'ai chi. Nevertheless, substantial fractions of participants were willing to try acupuncture and t\'ai chi as a treatment if their primary care provider thought it reasonable, and in the case of acupuncture, even if they had to pay a \$10 co-pay each visit. Our finding that people in our sample reported being almost as willing to try acupuncture as massage, despite less knowledge of, expectations about and experience with it, is intriguing and requires further inquiry.
Although participants in this study reported more knowledge of and experience with chiropractic, they were more enthusiastic about massage. A recent survey \[[@B11]\]of 46,000 Consumer Reports subscribers found that among those who had experienced back pain, the relatively few who had tried deep tissue massage rated it more favorably than those who had tried medications or physical therapy. The use of massage in this country has been growing steadily since the 1960\'s, with the largest increases in the 1990\'s \[[@B12]\]. In fact, in surveys of CAM use in the US population conducted in 1990 and 1997, Eisenberg et al. \[[@B7]\] found that massage as a treatment for various medical conditions had increased 61% over the seven-year period, while chiropractic remained fairly stable. By 1997, the estimated percentage of US adults who had used chiropractic was similar to that who had used massage, 11%. The relative popularity of massage may result from the more positive experiences of those who have tried it compared with chiropractic or acupuncture, and higher expectations that massage would be helpful for their current pain. Moreover, chiropractic users were more likely to report treatment related \"harm\" or \"pain\" than were users of massage.
Implications for clinical trials
--------------------------------
Most survey respondents indicated they were \"very willing\" to participate in our two hypothetical clinical trials evaluating different CAM treatments for chronic back pain. Massage was the preferred treatment in both trials, but more than one in five survey respondents stated a preference for acupuncture and t\'ai chi. In view of the long-standing popularity of chiropractic, surprisingly few respondents reported chiropractic as their top choice. Nonetheless the finding that massage was substantially more popular than chiropractic mirrors the results among acute low back pain patients in a clinical trial who were randomized to a choice of acupuncture, chiropractic, massage, or usual care or to usual care alone \[[@B13]\]. In that study, 52% of the participants said they would choose massage if given a choice, compared with only 24% who said they would choose chiropractic if given a choice. This finding could reflect the fact that many people have access to chiropractic as part of their current health care coverage \[[@B14]\].
Despite low levels of knowledge about t\'ai chi and acupuncture, the finding that over 40% of respondents indicated they were very likely to try these therapies suggests that recruiting enough subjects for clinical trials involving these therapies may be feasible if moderate to large patient populations are available. Recruiting patients for meditation trials, however, is likely to be difficult. Consequently, when we recruited patients for a pilot trial that included a stress reduction intervention based on the principles of mindfulness meditation, we chose to describe it as \"Mindfulness Based Stress Reduction\" rather than mindfulness meditation.
We believe that clinical trials evaluating obviously different treatments for chronic low back pain, such as massage and meditation, may have problems retaining subjects who do not receive the treatment (e.g., massage) that attracted them to the study. This problem may be exacerbated if patients have an exceptionally strong preference (or dislike) for one treatment. Inclusion of multiple CAM modalities in a single study risks tempting potential participants to sign up for the study in the hope of receiving a desired treatment, and then dropping out if they receive a different treatment.
In addition, if one treatment is vastly more popular than another, it could be difficult to disentangle the effects of patient expectations and treatment efficacy per se, leading to difficulties in interpreting positive study outcomes. This problem is compounded by concerns about the subjective nature of back pain outcomes, the difficulty in masking participants to study treatment, and the strong skepticism of some researchers that CAM treatments can be effective, even when results are impressive. Masking patients to treatment is quite difficult in studies of many types of conventional as well as CAM treatments if the treatments involve a physical modality, such as massage, or active participation of the patient in the treatment, as in t\'ai chi. In such circumstances, using masked outcomes assessors is important to minimize bias. We also suggest that patient (and provider) expectations for treatment and prior experience with each treatment, be measured and, if appropriate, controlled for in the analyses. Finally, if a particular therapy is shown effective in clinical trials in different populations, mechanistic studies will be important for determining how these therapies achieve their effects. Such studies are especially important to convince skeptics that CAM therapies actually have specific effects. In the meantime, the high and rising public interest in CAM therapies, especially for musculoskeletal conditions \[[@B12]\], highlights the importance of evaluating the effectiveness of various CAM treatments for back pain and our findings suggest that recruiting for these efforts may not be difficult.
Conclusions
===========
Most patients in our sample were interested in trying options for treating chronic back pain that lie outside the conventional medical spectrum, even within the context of a clinical trial. This was true even among patients who had relatively little knowledge of or experience with the therapy. Given our limited knowledge about the effectiveness of most CAM therapies, there is a clear need for additional studies evaluating their effectiveness. Fortunately, our results suggest that researchers will not find it difficult to recruit patients interested in participating in clinical trials of many of the CAM therapies.
Competing interests
===================
None declared.
Authors\' contributions
=======================
KJS participated in the development of the questionnaire and took primary responsibility for the analysis of the data and for drafting the manuscript. DCC was the PI on one of the grants funding the study, participated in the development of the questionnaire and the analysis of the data and played a major role in drafting the manuscript. MTC participated in the development of the questionnaire and the analysis of the data. JE and JBS coordinated the project and oversaw the data collection. RBD participated in the analysis of the data and provided statistical oversight. DME was the Principal Investigator on one of the grants funding the study, participated in the development of the questionnaire and the analysis of the data. All authors read and approved the manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1472-6882/4/9/prepub>
Acknowledgements
================
This project was supported by grants AT00606 and AT00622 from the National Center for Complementary and Alternative Medicine. We thank the following people for their contribution to the successful completion of this study: Claire Canning, Kristin Delaney, John Ewing, Amy Fields, Christel Kratohvil, and Rene Talenti.
| PubMed Central | 2024-06-05T03:55:46.588137 | 2004-7-19 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC503394/",
"journal": "BMC Complement Altern Med. 2004 Jul 19; 4:9",
"authors": [
{
"first": "Karen J",
"last": "Sherman"
},
{
"first": "Daniel C",
"last": "Cherkin"
},
{
"first": "Maureen T",
"last": "Connelly"
},
{
"first": "Janet",
"last": "Erro"
},
{
"first": "Jacqueline B",
"last": "Savetsky"
},
{
"first": "Roger B",
"last": "Davis"
},
{
"first": "David M",
"last": "Eisenberg"
}
]
} |
PMC503395 | Background
==========
Sepsis is a common cause of morbidity and mortality following surgery or trauma, and is characterized by activation of a systemic inflammatory response, severe hypotension, and major damage to multiple organs \[[@B1]\]. Although neutrophil migration into the tissue sites is crucial for effective elimination of infection, it also plays an important role in inflammatory tissue injury. The selectins, β2 integrins (i.e., CD18: Mac-1, LFA-1) and members of the immunoglobulin gene superfamily adhesion molecules, such as ICAM-1, play a significant role in neutrophil adhesion and transendothelial migration \[[@B2]-[@B4]\]. The expression and activation of these adhesion molecules on neutrophils and the endothelium, as well as the presence of a chemotactic gradient (eg. chemokines) appear to be important factors in neutrophil transmigration \[[@B3]\].
The selectins mediate neutrophil rolling while the β-2 integrins are important for firm adhesion and transendothelial migration \[[@B2]-[@B5]\]. The selectin family consists of three closely related cell surface molecules with differential expression by leukocytes (L-selectin), platelets (P-selectin), and vascular [e]{.underline}ndothelium (P-selectin and E-selectin) \[[@B6]\]. ICAM-1 is one of the major ligands that binds to β-2 integrins (i.e., Mac-1 LFA-1) and is involved in neutrophil firm adhesion to endothelial and transendothelial migration \[[@B3]\]. ICAM-1 is constitutively expressed at a low concentration; however, under inflammatory conditions it is highly inducible in many cell types \[[@B7]\].
*[In vivo]{.underline}*studies have shown that administration of small molecule ligands, and/or neutralizing antibodies to the selectins, ICAM-1 or β2-integrins can protect tissues from injury following endotoxin exposure, bacterial infection, or ischemia \[[@B8]-[@B10]\]. However, blocking reagents have the potential to stimulate or inhibit other receptors thus confounding the results. For example, a study by Kyriakides *et al*. has shown that soluble P-selectin attenuated skeletal muscle reperfusion injury by inhibition of the classical complement pathway \[[@B11]\]. Genetically altered mice deficient in adhesion molecules have been developed, which provide an alternative approach to study the role of the adhesion molecules in neutrophil recruitment and tissue injury. Recent studies using the transgenic mice have shown results indicating that neutrophils can use different adhesion pathways to emigrate from the systemic vasculature into the tissues and that the inflammatory responses may be site specific and stimulus dependent. For example, Mizgerd *et al*. have demonstrated a significant reduction in neutrophil infiltration into the peritoneal cavity at 4 hours of streptoccocal peritonitis in ICAM-1 mutant mice \[[@B8]\]. Another study by Kamochi *et al*. has shown that P-selectin and ICAM-1 significantly contributed to liver and lung injury at 4 hours of systemic endotoxemia in ICAM-1 and P-selectin/ICAM-1 double mutant mice \[[@B12]\]. Further, Bullard *et al*. have shown that P-selectin and ICAM-1 double mutant mice exhibited complete loss of neutrophil migration into the peritoneum during *S. pneumoniae*-induced peritonitis \[[@B13]\]. In contrast to these studies, Serman *et al*. have shown no differences in survival between wild type and ICAM-1-deficient mice following an intra-peritoneal injection with *E. coli*, *S. auerus*, or *P. aeruginosa*\[[@B14]\]. More importantly, recent clinical trials of anti-adhesion therapy in an attempt to reduce injury associated with traumatic shock and reperfusion injury failed to show a significant benefit despite strong preclinical data \[[@B15]\]. In an attempt to understand the disparity between the preclinical and clinical trial studies, it was noted that the lengths of injury in the clinical setting were longer than those of the preclinical studies. It appears that the underlying mechanism of neutrophil infiltration with a short period of insult is different from those of injury associated with a longer period of insult \[[@B15]\]. Additionally, adhesion-dependent and -independent neutrophil activation and migration can differentially be regulated by target tissue and mode of stimuli. Therefore, the goal of the study presented here was to investigate the role of ICAM-1 and P-selectin in peritonitis induced by CLP under a short and longer period of injury. The model of CLP-induced peritonitis used in this study is a clinically relevant model of sepsis \[[@B16]\]. This model was chosen to simulate the critical polymicrobial bacteremia-induced tissue injury that occurs in septic patients. The data of this study suggest that neutrophil infiltration into the peritoneal cavity following CLP can utilize an ICAM-1 and P-selectin independent pathway.
Methods
=======
All chemicals were purchased from Sigma Chemical (St. Louis, MO), unless otherwise noted.
Animals
-------
Only adult male mice (i.e., 8--10 wk) were used in this study. All animals received humane care in compliance with the *Guide for the Care and Use of Laboratory Animals*(National Institutes of Health Publication No. 85-23, revised 1985). Experimental protocols were reviewed and approved by the Michigan State University Animal Use and Care Committee.
Gene-targeted double mutant mice deficient in P-selectin and ICAM-1 (P/I double mutant), C57BL/6-Icam1^tm1Bay^Selp^tm1Bay^, backcrossed to C57BL/6, were used in this study. Breeding pairs of double-knockout mice were purchased directly from Jackson Laboratory (Bar Harbor, ME) and bred under the guidance of University Laboratory Animal Resources at Michigan State University. The wild-type (WT) mice were male C57BL/6, which were acclimated to the animal laboratory environment for one week before the start of experimentation. Before and after surgery, all the animals had unlimited access to food and water.
Induction of peritonitis
------------------------
### Experimental model of polymicrobial sepsis
Polymicrobial sepsis was induced by CLP as previously described by Chaudry, *et al*. \[[@B16]\]. Surgical utensils were sterilized and all experimental procedures were performed under aseptic conditions. Adult male mice weighing 23 to 28 gm were anesthetized with inhaled methoxyflurane (Baxter Caribe Inc., Guayama, PR). The abdominal hair was shaved, the skin was cleaned with 75% ethanol using a sterile gauze and scrubbed with betadine solution. After drying, a 2-cm midline incision was made, the cecum was identified and ligated below the ileocecal valve using 0--5 silk suture with care being taken not to occlude the cecal valves. The cecum was punctured on both sides with a 21 G needle and gently squeezed to extrude a small amount of fecal material. The cecum was then restored to its normal position and the abdomen was closed in two layers using 5.0 nylon suture. Sham animals underwent the same procedures excluding ligation and puncture of the cecum.
### Experimental model of peritonitis induced by 2% glycogen
Adult male mice (i.e., 8--10 wk) were lightly anesthetized by inhalation anesthesia with methoxyflurane. A volume of 2% sterile glycogen solution, equal to 10% of body weight, was injected intraperitoneally (i.p.). Six hours after glycogen inoculation, mice were euthanized and peritoneal lavage and blood samples were collected. The collected samples were identified with ID numbers to assure a blind fashion performance of the tests and data analysis. Total leukocyte and differential counts were performed as described below.
Peripheral blood and tissue procurement
---------------------------------------
Blood samples were obtained from the right ventricle via a left anterior thoracotomy at the time of sacrifice, using a sterile heparinized syringe containing 50 μl of heparin (100 USP Units/ml). Blood smears were prepared and stained with Wright-stain (LeukoStat, Fisher Scientific, Pittsburgh, PA) for differential cell counts. The total number of peripheral blood leukocytes was determined by lysing the red blood cells using 3% acetic acid solution with the aid of a Neubauer hemocytometer. The remaining blood was centrifuged, and plasma was collected and stored at -70°C. A portion of the liver was fixed in buffered 10% formalin and embedded in paraffin, and a second portion was snap frozen in liquid nitrogen and stored at -70°C until used for immunohistochemistry staining.
Collection of peritoneal lavage fluid
-------------------------------------
The peritoneal fluids were collected using repetitive (2 times) instillation and withdrawal of 2 and 5 ml respectively of sterile saline solution using a syringe with a 22 G needle. The peritoneal lavage sample was placed on ice, immediately processed for centrifugation at 4°C, and the supernatant and the cell pellet were collected separately. The cell pellet was used for total and differential cell counts. Differential cell counts were determined on cytospin preparations of peritoneal lavage stained with Wright-stain (LeukoStat). The total number of peritoneal leukocytes was determined as described above for the peripheral blood leukocyte count. Additionally, the presence of neutrophils in peritoneal lavage was confirmed by immunohistochemical staining of cytospin preparation using a primary antibody (IgG2a) specific to mouse neutrophil as described below. Further, the neutrophil content was quantified by measuring the myeloperoxidase (MPO) level of peritoneal lavage as described below.
Demonstration of neutrophil recruitment by myeloperoxidase assay
----------------------------------------------------------------
The MPO contents of the peritoneal lavage supernatant and the cell pellet were quantified as previously published by our laboratory \[[@B17]\]. Briefly, the peritoneal cell pellet was resuspended in a potassium phosphate buffer, froze at -70°C, thawed, and sonicated for 40 seconds for two cycles (Ultrasonic Convertor, Model CL4, Misonix, Farmingdale, NY). The sample was then incubated at 60°C for 2 hours followed by centrifugation at 10,000 rpm for 5 minutes at 4°C. The supernatant was collected and used for the MPO assay. The MPO activity was determined using a tetramethylbenzidine substrate kit (ImmunoPure, Pierce, Rockford, IL) and read at 450 nm using a human leukocyte MPO as the standard. One unit of MPO activity was defined as the quantity of enzyme degrading 1 μmol peroxide/minute at 25°C. Similarly, the MPO content of peritoneal supernatant was measured.
Determination of neutrophil infiltration and ICAM-1 expression by Immunohistochemistry
--------------------------------------------------------------------------------------
To confirm the identity of neutrophils in peritoneal lavage, immunohistochemical staining was performed using acetone-fixed cytospin cell preps. The primary antibody (clone 7/4, IgG2a) specific to mouse neutrophil (Cedarlane, Westbury, NY), the biotin-conjugated secondary antibody (PharMingen, San Diego, CA), and a Vectastain avidin-biotin complex reagent and 3,3\'-diaminobenzidine chromogen kits (Vector Laboratories, Inc., Burlingame, CA) were used as previously described in detail \[[@B17]\]. The expression of ICAM-1 on endothelial cells was examined using acetone-fixed cryosections of the tissue. The liver was used in this study. Similarly, ICAM-1 expression was identified using a specific monoclonal antibody to mouse ICAM-1 (3E~2~clone, PharmMingen, San Diego, CA) and a biotin-conjugated secondary antibody. Tissue sections were counter stained with hematoxylin (Gill\'s formula, Vector Laboratories) and mounted with DAKO Mounting Media (DAKO Corp, Carpinteria, CA). The samples were examined using a Nikon light microscope interfaced with a spot 24-Bit Digital Color Camera.
Statistical analysis
--------------------
All data were expressed as means ± standard error of the mean. Comparisons between two groups were performed using an unpaired *t*-test by way of *StatView version 5.0.1 software©*for Windows. Comparisons between multiple groups and various time points were analyzed using ANOVA with subsequent Fisher\'s PLSD test. *P*≤ 0.05 was considered significant.
Results
=======
Verification of ICAM-1 and P-selectin deficiency in P/I null mice
-----------------------------------------------------------------
The Jackson Laboratory, where the P/I null breeding pairs were purchased, had initially tested the double knockout of the P/I null mice. In addition, the ICAM-1/P-selectin deficiency was confirmed in our laboratory in randomly selected litter mice tissue samples using RT-PCR and immunohistochemical staining of the liver tissue, as previously published by our laboratory (18). The ICAM-1 expression was determined in all the animals used in this study. Figure [1](#F1){ref-type="fig"} shows ICAM-1 expression in liver tissues from the wild-type and P/I null mice by immunohistochemical staining technique. The ICAM-1 expression was constitutively present in wild-type control mice as indicated by light brown staining along the endothelium of the central vein, sinusoids, and portal vasculature (Figure [1](#F1){ref-type="fig"}, WT CT). The ICAM-1 expression was markedly increased in wild-type mice following CLP and continued to be evident at 24 hours of CLP (Figure [1](#F1){ref-type="fig"}, WT 6 h and WT 24 h). In contrast, ICAM-1 expression was absent in the tissues of P/I null mice before and after CLP treatment (Figure [1](#F1){ref-type="fig"}, P/I CT, 6 h and 24 h). The intestinal tissue ICAM-1 expression has also been examined, which was similar to that of the liver tissue. However, liver, due to its large endothelial cell content, serves as an excellent tissue source for ICAM-1 expression. For this reason, liver is routinely used in our studies to confirm the ICAM-1 expression.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Immunostaining of ICAM-1 expression in WT and P/I null mice.**Tissues ICAM-1 expression was determined by staining the liver sections with an anti-ICAM-1 antibody specific to mouse by applying the immunoperoxidase technique, and examined under a light NIKON microscope. The top row represents wild-type (WT) control (CT), CLP 6 h and CLP 24 h, respectively. Note the increased intensity of ICAM-1 staining of central veins (arrow heads) and sinusoids (arrows) with progression of sepsis. In contrast, ICAM-1 expression in P/I null mice liver sections was completely absent in controls, 6 h after CLP and 24 h after CLP (lower row). Control group represents mice that were not subjected to sham or CLP experimental treatment.
:::
:::
Clinical signs of sepsis
------------------------
Clinical signs of sepsis were manifested as quietness, lack of response to stimulus, absence of socializing behavior, ruffled hair coat and lack of appetite. All mice subjected to CLP displayed these signs that became significant at 24 hours of CLP, and no striking differences were observed between the wild-type and P/I null mice.
Effect of sepsis on leukocyte count in wild-type and P/I null mice
------------------------------------------------------------------
### Peripheral blood leukocytes
To establish the blood cell parameters under normal physiological conditions in wild-type and P/I null mice, peripheral blood samples were collected from randomly selected mice. In this article these mice are denoted as \"Control\", which were not subjected to the sham or CLP operation. The leukocyte and differential counts of peripheral blood were measured. As shown in Table [1](#T1){ref-type="table"}, the circulating leukocyte counts were not different between the wild-type and P/I null mice. However, the P/I null mice showed a significantly higher number of neutrophils and lower lymphocytes when compared to their wild-type counterparts (Table [1](#T1){ref-type="table"}). There was no significant difference in monocyte count between the wild-type and P/I null mice.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Leukocyte and differential counts in WT and P/I null mice. Randomly selected normal mice not subjected to the sham or CLP treatment, were anesthetized and the peripheral blood and peritoneal lavage collected for leukocyte content and differential analysis. Data are expressed as mean ± SEM. Total leukocytes for peripheral blood represents the absolute number of cells X 10^6^/ ml and for peritoneal lavage represent the absolute number of cells X 10^6^/ lavage. The values in parenthesis denote absolute number of the cells X 10^6^. \* *p ≤ 0.05*absolute number of cells of the wild-type compared to respective P/I null mice. *n*represents the number of mice per each group.
:::
**Source** **Total Leukocytes**(× 10^6^) **Differential (%)**
------------------------ ------------------------------- ----------------------- ----------------------- --------------------
**Peripheral blood**
**WT**(*n = 4*) 10.4 ± 1.5 26 ± 2 (2.9 ± 0.5) \* 72 ± 3 (7.2 ± 0.6) \* 2 ± 1 (0.3 ± 0.1)
**P/I null**(*n = 5*) 10.3 ± 1.4 69 ± 7 (6.8 ± 0.9) 32 ± 6 (3.4 ± 0.5) 2 ± 1 (0.2 ± 0.1)
**Peritoneal Lavage**
**WT**(*n = 6*) 3.8 ± 0.9 16 ± 1 (0.6 ± 0.2) 19 ± 3 (0.8 ± 0.3) 65 ± 5 (2.4 ± 0.7)
**P/I null**(*n = 6*) 3.6 ± 1.2 3 ± 1 (0.2 ± 0.1) 54 ± 5 (1.9 ± 0.4) 43 ± 6 (1.5 ± 0.7)
:::
The induction of CLP-induced peritonitis resulted in a significant leukopenia in wild-type as well as P/I null mice as compared to their control counterparts (Table [2](#T2){ref-type="table"}). It is interesting to note that although not statistically significant, leukopenia in the P/I null mice was less severe than those of the corresponding wild-type counterparts. This difference was mainly reflected by the presence of a higher number of neutrophils in the P/I null mice blood (Table [2](#T2){ref-type="table"}). Similarly, CLP induced a significant neutropenia in both wild-type and P/I null mice when compared to the respective control mice. The most severe neutropenia occurred in the wild-type mice at 24 hours of CLP. In our studies peritonitis was also induced in response to 2% glycogen, which served as a positive control stimuli to induce an acute peritonitis as have previously been reported by many other investigators. As shown in Table [2](#T2){ref-type="table"}, both wild-type and P/I null mice presented significant leukopenia at 6 hours of *i.p.*inoculation of 2% glycogen. Further, the inflammatory response to the sham operation, which elicited a trauma-induced peritonitis, caused leukopenia in both wild-type and P/I null mice. As shown in Table [2](#T2){ref-type="table"}, there was a significant drop in the circulating blood leukocytes at 6 and 24 hours after the sham operation when compared to the respective control mice. However, a significant neutropenia was only present at 24 hours of the sham operation.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Peripheral blood leukocyte and neutrophil counts in WT and P/I null mice subjected to CLP and 2% glycogen-induced peritonitis. *Control*group represents normal mice that were not subjected to sham or peritonitis treatment. *Sham (CLP)*represents the CLP respective sham group. Data are expressed as mean ± SEM, representing the absolute number of cells X 10^6^. \* *p ≤ 0.05*compared to respective control group; *\# p ≤ 0.05*wild-type compared to P/I null mice at the same time point and treatment. *n*represents the number of mice per each group.
:::
**Experimental design** **WT mice Total Leukocytes (× 10^6^/mL)** **P/I null mice Total Leukocytes**(× 10^6^/mL) **WT mice Neutrophils**(× 10^6^/mL) **P/I null mice Neutrophils**(× 10^6^/mL)
------------------------------- ------------------------------------------- ------------------------------------------------ ------------------------------------- -------------------------------------------
**Control**(*n = 4*) 10.1 ± 1.3 10.3 ± 2.1 2.8 ± 0.7 **\#** 6.7 ± 1.3
**Sham (CLP) 6 hr**(*n = 4*) 3.6 ± 0.6 \* 4.5 ± 0.3 \* 2.2 ± 0.1 **\#** 3.8 ± 0.5
**Sham (CLP) 24 hr**(*n = 6*) 2.9 ± 0.7 \* 4.4 ± 0.8 \* 1.4 ± 0.3 \* 2.3 ± 0.1 \*
**CLP 6 hr**(*n = 6*) 2.0 ± 0.4 \* 3.4 ± 0.9 \* 1.2 ± 0.3 \* 2.8 ± 0.9 \*
**CLP 24 hr**(*n = 6*) 1.1 ± 0.3 \* 3.8 ± 1.1 \* 0.6 ± 0.2 \* 2.8 ± 0.8 \*
**2% Glycogen 6 hr**(*n = 4*) 3.4 ± 0.1 \* 4.4 ± 0.8 \* 2.5 ± 0.1 3.6± 0.6
:::
### Peritoneal leukocytes
Similar to peripheral blood, the cell parameters of peritoneal fluid under normal physiological conditions in the wild-type and P/I null mice were determined in randomly selected mice. The mice were not subjected to the sham or CLP operation. As Table [1](#T1){ref-type="table"} shows, no significant difference was found in total peritoneal leukocyte counts between the wild-type and P/I null groups (i.e., WT = 3.8 ± 0.9 × 10^6^vs. P/I = 3.6 ± 1.2 × 10^6^). However, it is interesting to note that monocytes/macrophages were the predominant cell type present in the peritoneal cavities of the wild-type mice, whereas in the P/I null mice lymphocytes were the predominant cell type (Table [1](#T1){ref-type="table"}). Neutrophils constituted a smaller percentage of the peritoneal leukocytes in both the wild-type and P/I null mice (Table [1](#T1){ref-type="table"}). Although the P/I null mice peritoneal lavage presented a lower percentage of neutrophils than those of the wild-type counterparts, the differences between the absolute numbers of neutrophil counts did not reach statistically significance (i.e., *p*= 0.08).
Sepsis induced by CLP (i.e., 6 and 24 hr) caused a significant leukocyte infiltration into the peritoneal cavities of both wild-type and P/I null mice when compared to their corresponding sham group (Figure [2A](#F2){ref-type="fig"}). The peritoneal leukocyte influx consisted predominantly of neutrophils as identified by Wright staining as well as in situ immunohistochemical staining using specific monoclonal antibody to mouse neutrophil (Figure [3](#F3){ref-type="fig"}). As shown in Figure [2B](#F2){ref-type="fig"}, a significantly greater number of neutrophils infiltrated into the peritoneal cavities of wild-type mice than those of the P/I null mice at 6 hours after CLP, which reached to comparable levels at 24 hours of CLP. Although a fewer number of neutrophils were present in the peritoneal cavities of P/I null mice, the ratio of neutrophil infiltration (i.e. ratio = infiltrated peritoneal neutrophils in response to CLP/neutrophils normally present in peritoneal cavity of the control mouse) was significantly higher in the P/I null mice than those of wild-type mice. There was a 16-fold and a 33-fold increase in the ratio of peritoneal neutrophils in wild-type at 6 and 24 hours after CLP, respectively. However, in P/I null mice, the peritoneal neutrophil infiltration ratio increased 54-fold and 204-fold at 6 and 24 hours after CLP, respectively.
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**Total peritoneal leukocyte and neutrophil counts in WT and P/I null mice subjected to CLP or 2% glycogen-induced peritonitis.**Wild-type (WT) and P/I null were subjected either to no treatment (*Control*), sham (*Sham CLP*), CLP, or 2% glycogen. The mice were then euthanized at 6, and 24 hours of treatment, peritoneal lavages were collected and analyzed for leukocyte and neutrophil contents. Data are expressed as mean ± SEM, representing the absolute number of cells X 10^6^per each lavage. Significant differences existed between sham, CLP and 2% glycogen as compared to their respective control group (\* *p \< 0.05)*, and as P/I null mice compared to respective wild-type at the same time period and treatment (*\# p ≤ 0.05*). Data from six independent experiments with total sample size of 4 mice per each control and 2% glycogen group, 6 to 8 mice per each sham and 8 to 11 mice per each CLP treatment group.
:::
:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**Staining of cytospin preparations from peritoneal lavage.**The top three rows show the Wright staining of the cellular components of peritoneal lavages. Cellular preps from the control (CT) mice of both WT and P/I null groups demonstrating mononuclear cells as the predominant cell types (left column: WT CT, P/I CT). Control group represents mice that were not subjected to sham or CLP experimental procedures. CLP induced a significant neutrophil infiltration (arrows) at 6 and 24 hours in both WT (top row: WT 6 h, WT 24 h) and P/I null mice (second row: P/I 6 h, P/I 24 h). Neutrophils were also the predominant cell type at 6 hours of 2% glycogen-induced peritonitis in WT and P/I null mice (third row: WT 6 h, P/I 6 h). The lower row represents immunoperoxidase staining of the cells (CLP mice) using an anti-neutrophil antibody specific to mouse, verifying the neutrophils as stained in brown color (WT 6 h, P/I 6 h).
:::
:::
Peritonitis was also examined in response to 2% glycogen. A significant leukocyte infiltration into the peritoneal cavity occurred in both wild-type and P/I groups at 6 hours after 2% glycogen injection (Figure [2A](#F2){ref-type="fig"}). Similar to the CLP response, the leukocyte influx into the peritoneal cavities in response to glycogen consisted predominantly of neutrophils (Figures [2B](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}). Further, sham operation caused a significant leukocyte infiltration into the peritoneal cavity of both wild-type and P/I null mice at 24 hours (Figures [2A](#F2){ref-type="fig"}, and [2B](#F2){ref-type="fig"}). Similarly, this peritoneal leukocyte influx consisted predominantly of neutrophils.
Demonstration of peritoneal neutrophil recruitment by myeloperoxidase assay
---------------------------------------------------------------------------
To further quantify the degree of neutrophil infiltration into the peritoneal cavity, the MPO levels of both the peritoneal lavage cell pellets and supernatants were measured. Although the MPO levels of the cell pellets indicated the presence of a significant number of neutrophils in the peritoneal cavity after CLP, the MPO values did not always correlate with the absolute number of infiltrated neutrophils. The Wright\'s staining of cytospin preparations of the peritoneal cells demonstrated highly activated phagocytes with vacuolized cytoplasm often containing numerous intracellular bacteria and in most cases with loss of cellular integrity (Figure [3](#F3){ref-type="fig"}). Thus, it was of concern to find out whether the MPO contents of neutrophils were released into the extracellular environment due to activation and loss of cellular integrity. To accomplish this objective, the MPO levels of the peritoneal lavage supernatants were also measured. As figure [4B](#F4){ref-type="fig"} shows, at 6 hours of CLP, significant levels of MPO were detected in the peritoneal lavage supernatants of both P/I null mice and wild-type animals when compared to those of the control mice. At 24 hours of CLP, further increases of MPO levels were present in the peritoneal supernatants of both mice groups. It is interesting to note that P/I null mice demonstrated a higher MPO levels in their peritoneal lavage supernatants than the wild-type counterparts. This increase may reflect a greater degree of neutrophils activation and/or loss of cell membrane integrity of the P/I null mice, and thereby the release of their MPO into the supernatant. The differences were not statistically significant.
::: {#F4 .fig}
Figure 4
::: {.caption}
######
**Neutrophil infiltration as determined by MPO contents of peritoneal cell pellet and peritoneal supernatant.**Wild-type (WT) and P/I null were subjected to CLP, euthanized at specific time points, the peritoneal lavages harvested, centrifuged, and the supernatant and the cell pellet were collected separately and assayed for MPO contents. **(A)**Peritoneal lavage cell pellet. **(B)**Peritoneal lavage supernatant. Note that at 6 h after CLP, although a significant neutrophil infiltration into the peritoneal cavity of the P/I null group is present, it is significantly impaired compared to the corresponding WT group. Values are expressed as the mean ± SEM. \* *p \< 0.05*compared to respective control group. + *p ≤ 0.05*P/I null mice compared to respective wild-type at the same time period of CLP. Data from 6 independent experiments with total sample size of 8 mice per each treatment.
:::
:::
Demonstration of neutrophil recruitment by immunohistochemistry
---------------------------------------------------------------
Previous studies have indicated that tissue MPO activity can be affected by other factors (19, 20). and that MPO values may not indicate the true presence of neutrophils. Additionally, recent studies have identified MPO as the cellular component of macrophages (i.e., Kupffer cells) (21), as well as histiocytes (22). Therefore, an immunohistochemical staining technique was used to confirm the presence of neutrophils in the peritoneal cavity. The cytospin preparations of the peritoneal lavages were immunostained with a specific antibody to mouse neutrophil. Figure [3](#F3){ref-type="fig"} (lower row) shows the Immunostaining of the peritoneal lavages obtained from wild-type and P/I null mice, in which neutrophils are stained in brown color.
Discussion
==========
Neutrophil infiltration is coordinated by the interplay of the adhesion molecules and the chemoattractants, which plays an important role in inflammatory tissue injury. Recent clinical trials of anti-adhesion therapy did not demonstrate a protective effect in trauma-induce shock despite very strong pre-clinical data (15). Further studies to clear this disparity suggested that the model systems applied and the length of injury are important factors that might modulate the underlying mechanism of neutrophil activation and migration (15). Therefore, this study was undertaken to examine the role of P-selectin and ICAM-1 molecules in the wild-type and P/I null mice subjected to a short and longer periods of peritonitis induce by CLP. The CLP model, chosen to mimic the normal course of sepsis in humans and animals, induced the classic signs of sepsis and a significant systemic inflammatory response. This response was associated with increased leukocyte infiltration into the peritoneal cavities of both the wild-type and P/I null mice. The leukocyte influx into the peritoneal cavity consisted predominantly of neutrophils (i.e., 62--80%), which significantly increased in both wild-type and P/I null mice at 6 and 24 hours of CLP. At the early phase of CLP (i.e., 6 h), the total number of neutrophils infiltrated into the peritoneal cavities of P/I null mice were significantly lower than those of the corresponding wild-type mice, which reached to a comparable level at 24 hours of CLP. In contrast to CLP-induced peritonitis, peritonitis induced by 2% glycogen exhibited no significant differences in the number of neutrophils infiltrated into the peritoneal cavities between the wild-type and those of the P/I null mice at 6 hours (Figure [2](#F2){ref-type="fig"}). The results of this study suggest differential regulation of the inflammatory response and neutrophil response by mode and length of the injury.
The data of this study showed that under normal physiologic environment the number of leukocytes present in peripheral blood and peritoneal lavage was comparable in the P/I null and wild-type mice. However, the ratio of neutrophils and lymphocytes were reversed between these two groups. In peripheral blood of P/I mice, neutrophils constituted a larger percentage of the leukocytes, which were significantly higher than those of the wild-type mice. In a striking contrast to the peripheral blood, the P/I null mice had a smaller number of neutrophils in their peritoneal cavities, when compared to those of the wild-type mice. Previous reports have also noted an increase in peripheral blood neutrophils in mutant mice \[[@B13]\]. However, to our knowledge, the decrease in peritoneal neutrophils has not been previously reported. It appears that within the normal physiologic environment, neutrophil trafficking in the peritoneal cavity is low in the P/I null mice and that neutrophil transmigration is regulated through adhesion pathways that utilize P-selectin and ICAM-1 molecules. The reason for increased circulating peripheral blood neutrophil counts in the P/I null is not known. However, this phenomenon has also been previously reported in CD18, P-sel/ICAM-1 mutant mice, and in patients with moderate or severe leukocyte adhesion deficiency \[[@B13],[@B23],[@B24]\]. Further investigation is needed to determine the following: whether hematopoiesis of neutrophils production is enhanced in the P/I null mice; removal of the neutrophils from blood circulation is reduced/delayed; and/or the neutrophil\'s life span has been increased.
In the present study, P-selectin/ICAM-1 appears to be partially involved in neutrophil migration into the peritoneal cavity but only during the early stages of the response to CLP-induced peritonitis. Conversely, these adhesion molecules were not required for maximal neutrophil migration after trauma (i.e. sham operation) or chemically-induced peritonitis (i.e 2% glycogen) and during the late phase of CLP (i.e, 24 hours). The data suggests that there might be a functional role for these adhesion molecules during the initial stages of the inflammatory response, and as the inflammatory process progresses, deficient adhesion mechanisms are bypassed. This notion has also been proposed in leukocyte recruitment in an *[in vivo]{.underline}*experimental model of autoimmune encephalomyelitis, which is a T-cell-mediated disease. Kerfoot and Kubes have shown that during the early phase of encephalitis the leukocyte rolling was P-selectin dependent; however, with the progression of disease α4-integrin pathway was important in leukocyte rolling and adhesion \[[@B25]\].
Interaction of ICAM-1 expressed on the surface of vascular endothelial cells with the β~2~-integrins (eg., CD11b and CD18) expressed on neutrophils has shown to be a critical event mediating stable neutrophil adhesion and migration across the vascular endothelial barrier \[[@B26]-[@B28]\]. Although the study presented in this article suggests a non-role of ICAM-1 in neutrophil infiltration into the peritoneal cavity in response to peritonitis, it has to be noted that the P/I null mice are not a true ICAM-1 knockout. The P/I null mice may have a low level of alternatively spliced forms of ICAM-1 that could have been up-regulated on the vascular endothelium, and thereby promoting neutrophil migration \[[@B29]\]. Further, the lack of ICAM-1, *per se*, is not a critical factor that results in dysfunctional β~2~-integrin-mediated migration. Other adhesion molecule(s), ligand(s), and/or yet unknown counter-receptor(s) could mediate neutrophil infiltration. For example, ICAM-2, a ligand for β~2~-integrins, could be a potential candidate \[[@B32]\]. Other studies have shown a critical role of vascular cell adhesion molecule-1 (VCAM-1) in mediating neutrophil transendothelial migration and inflammatory tissue injury \[[@B30],[@B31]\]. This adhesion molecule interacts with α4-integrin (α4β1, VLA-4). There is a body of evidence that α4-integrin can mediate several steps of leukocyte recruitment cascade (i.e., rolling and adhesion) through α4-integrin/MADCAM-1 (mucosal addressin cell adhesion molecule-1) and α4-integrin/VCAM-1 pathways. Vajkoczy et al., have shown that T-cells can adhere without rolling in spinal cord microvessels via α4-integrin \[[@B32]\]. Neutrophils express α4-integrin, and studies by Bowden et al. have demonstrated an important role of α4-integrin/VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium \[[@B33],[@B34]\]. Additionally, the α4-integrin/VCAM-1-dependent neutrophil adhesion under flow conditions has been shown in neutrophils isolated from critically ill septic patients \[[@B35]\]. Neutrophil also express CD11d/CD18 and α9-integrin, which both bind VCAM-1, and could possibly, play an important role in neutrophil extravasation at sites of inflammation \[[@B36]\]. The importance of α4- and α9-integrin/VCAM-1 pathways in neutrophil infiltration in CLP-induced peritonitis remains unclear.
It is possible that neutrophil infiltration utilizes other secondary or tertiary adhesion pathways, and/or is facilitated by proteins that mediate the function(s) of the adhesion molecules. For example, a novel glycosylphosphatidyl inositol-anchored protein (GPI-80) that may regulate β~2~-integrin-mediated cell adhesion and motility of neutrophils has been described \[[@B38]\]. Additionally, other proteins are recognized to act as ligands for β~2~integrins, such as those produced during coagulation as well as complement activation and tissue factor, which could facilitate neutrophil adhesion and infiltration into the peritoneal cavity \[[@B39],[@B40]\]. Moreover, the local concentrations of chemokines appear to be critical factors in dictating the local neutrophil recruitment in an acute inflammatory response \[[@B41]\]. It has been shown that chemokines and their receptors are involved in leukocytes migration not only by inducing chemotaxis but also by regulating integrins to trigger cell arrest in shear flow \[[@B42]\].
Contrary to several other studies that have demonstrated the functional importance of P-selectin in models of myocardial infarction and inflammatory lung and liver injury, the data presented in this study indicated that P-selectin is not essential for neutrophil migration into the peritoneal cavity \[[@B43]-[@B45]\]. In support of our data, several studies have shown that blocking of P-selectin with monoclonal antibody or deletion of P-selectin and ICAM-1 did not inhibit neutrophil infiltration and tissue injury caused by hepatic reperfusion \[[@B18]\], endotoxin shock (46), i.p injection of 2% glycogen \[[@B47]\], and intestinal inflammation \[[@B48],[@B49]\]. In contrast to our results of the P/I null mouse study presented in this article, Bullard et al., have previously reported a complete loss of neutrophil migration into the peritoneum of P/I null mice during peritonitis induced by *i.p.*inoculation of *Streptococcus pneumonia*\[[@B13]\]. The difference could due to the length of the experimental setting. In the Bullard *et al*. study, the inflammatory response and neutrophil infiltration into the peritoneal cavity was studied at 4 hours of the induction of peritonitis, while, in our study neutrophil infiltration was evaluated at 6 and 24 hours of peritonitis. In support of this reasoning is the study reported by Mizgerd *et al*. who have shown a compromised neutrophil migration into the peritoneal cavity of E-/P-selectin and ICAM-1 mutant mice in response to the injection of *Streptococcus pneumoniae*at 4 hours after injection, with no impairment present at 24 hours of bacteria injection \[[@B8]\]. Additionallt, Mizgerd *et al*. demonstrated that ICAM-1 is not necessary for neutrophil migration during glycogen-induced peritonitis, as shown otherwise by other authors \[[@B9]\]. Further, the difference could due to the possibility that in Bullard\'s study the infiltrated neutrophils into the peritoneal cavity of P/I null mice became necrotic and disintegrated upon challenges with *Streptococcus pneumonia*organisms. In our study, the data of peritoneal supernatant MPO levels have demonstrated a significant level of MPO released into the peritoneal cavity in mice subjected to CLP (Figure [4B](#F4){ref-type="fig"}). In Bullard\'s study the MPO levels of the peritoneal lavages were not measured. Further, in our study the Wright staining as well as the *in situ*immunohistochemical staining of the peritoneal cells clearly confirms the presence of neutrophils in the cytospin preparations (Figure [3](#F3){ref-type="fig"}). The data collectively indicate that P-selectin and ICAM-1 each may have a role in neutrophil migration during early stages of acute bacterial peritonitis, but at later stages alternative pathways are recruited to mediate neutrophil migration. Functional redundancy of adhesion molecules and the cytokine production may be sufficient to compensate for the absence of P-selectin and ICAM-1 in mediating neutrophil infiltration into peritoneal cavity in response to CLP in P/I null mice.
Another important difference between the results of the study presented in this article and those of previous studies may relate to the employment of essentially different models of sepsis and inflammation. While previous studies employed hemorrhagic shock-, *Streptococcus-*or LPS-induced peritonitis, the present study employed polymicrobial septic peritonitis as well as chemical and trauma-induced peritonitis. Published studies have shown quantitative and qualitative differences in the inflammatory response induced by gram-positive, gram-negative, polymicrobial sepsis and purified endotoxin \[[@B50]-[@B52]\]. These differences include: the species of bacteria employed and their respective antigens, the magnitude of leukocytic response, the magnitude and kinetics of cytokine production, and finally, mortality. Thus, the collective data suggest that the model of septic insult used to induce an inflammatory response is an important consideration. One major advantage of the model used in our study is that the relative magnitude of the inflammatory response in animals subjected to CLP-induced peritonitis is similar to those observed in septic patients \[[@B53],[@B54]\].
Conclusions
===========
The data presented in this study have shown a clear, time-dependent neutrophil migration and infiltration into peritoneal cavity in responses to peritonitis, which is independent of P-selectin and ICAM-1 adhesion molecules. The negative nature of the data presented here and the failure of the anti-adhesion clinical trials, are of great importance. These findings demand innovative alternative approaches to neutrophil transmigration in inflammatory response and suggest that targets other than these adhesion molecules need to be identified. A better understanding of the mechanisms leading to neutrophil migration is critical for the development of new therapeutic strategies for treating inflammatory disease without compromising the host\'s immune response mechanisms.
Competing interests
===================
None declared.
Authors\' contribution
======================
CR participated in the technical procedures for CLP, collection of blood and tissue samples, and MPO assay. KH and HA participated in preparation of tissue sample for immunohistochemistry and special staining. KH also assisted in preparation of the manuscript. EC conceived the study, and participated in its design, coordination, and preparation of the manuscript. All authors read and approved the final manuscript.
Abbreviations
=============
CLP, cecal ligation-puncture; ICAM-1, Intercellular adhesion molecule-1; mAbs, monoclonal antibodies; MPO, Myeloperoxidase; P/I null mice, P-selectin/ICAM-1-deficient mice
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1472-6890/4/2/prepub>
Acknowledgments
===============
The authors wish to thank Dr. Wayne Smith, M.D., Head, Section of Leukocyte Biology, Departments of Pediatrics and Immunology, at Baylor College of Medicine, for the critical review and comments to this manuscript. This research study was supported by the Department of Physiology, and the Division of Human Pathology, College of Human Medicine at Michigan State University.
| PubMed Central | 2024-06-05T03:55:46.593438 | 2004-7-26 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC503395/",
"journal": "BMC Clin Pathol. 2004 Jul 26; 4:2",
"authors": [
{
"first": "Elahé T",
"last": "Crockett"
},
{
"first": "Crystal",
"last": "Remelius"
},
{
"first": "Karen",
"last": "Hess"
},
{
"first": "Hayma",
"last": "Al-Ghawi"
}
]
} |
PMC503396 | Background
==========
The specialty of pediatric pulmonology is relatively new, having been recognized as a pediatric sub-specialty by the American Board of Medical Sub-specialties in 1984. In 1997, there were approximately 500 board certified pediatric pulmonologists in the United States and Canada \[[@B1]\]. This number has increased in recent years with 708 board certified pulmonologists being identified in 2003 \[[@B2]\]. It has been estimated that there is one pediatric pulmonologist for every 280,000 children in the United States \[[@B1]\]. There are over 50 pediatric pulmonary fellowship programs in North America with approximately 30--35 fellows graduating each year. The demand for pediatric pulmonologists has increased during the past decade, with many academic centers looking for two or more pulmonologists simultaneously \[[@B3]\]. A recent national survey of medical directors at children\'s hospitals across the country found that vacancy rates for faculty in pediatric pulmonology (25 of 136 positions vacant = 18.4%) was ranked second highest, behind only pediatric endocrinology, among over 40 pediatric subspecialties \[[@B3]\]. Despite this demand, little is known about overall interest in this field among pediatric residents. For this reason, this study was completed to examine interest among pediatric residents in entering a pulmonary fellowship. The specific aim of this study, therefore, was to determine at our institution: 1) the level of pediatric resident interest in pursuing a pulmonary fellowship; 2) potential factors involved in development of such interest; 3) whether the presence of an active fellowship program affected resident interest in such a program.
Methods
=======
This study involved the distribution of a questionnaire to all pediatric residents. The questionnaire was initially distributed in 1992 prior to institution of a pulmonary fellowship. The questionnaire was placed in the hospital mailbox of each resident. The study was repeated (and the questionnaire redistributed) in 2002 after the fellowship, which began in 1994, had been functioning for several years. To improve the response rate, the questionnaire was distributed twice, one month apart, during each time period. The questionnaire was a three-page, 18 question form that took approximately 15 minutes to complete (copy enclosed in Appendix \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]). The questionnaire asked several epidemiological questions (e.g. year of residency, whether medical school was attended at this institution), several questions that pertained to an individual resident\'s interest in entering any specialty fellowship, and approximately 12 questions specifically dealing with interest in doing a pulmonary fellowship and factors that may be either positively or negatively related to such interest. The survey utilized a flow diagram, and consequently, slightly different questions were asked depending on a resident\'s interest or lack of interest in a pulmonary fellowship. The study was analyzed after dividing the respondents into 2 groups: those that had considered a pulmonary fellowship during their residency (\"+PF\") and those that had not considered a pulmonary fellowship during their residency (\"-PF\"). Neither Human Subjects approval nor specific resident consent was obtained. However, all questionnaires were answered anonymously and residents were in no way coerced or forced to complete the survey. Residents who completed the form were given a certificate for a free meal at Children\'s Hospital of Wisconsin. Several questions were based on a 4-point Likert scale and ranked from 0 (\"not at all important\") to 3 (\"very important\") in a resident\'s decision to consider or not consider doing a pulmonary fellowship. Numerical responses for each survey period were averaged (mean ± SD) and ranked. Fisher\'s exact test was used to compare categorical variables and the Student\'s t-test was used to compare the means of measured variables in 2 independent samples. A p value of ≤ 0.05 was considered significant. Medical Education records were also reviewed anonymously (data collated by year of residency) in 2003 to determine residents\' actual career decisions.
Results
=======
The questionnaire was distributed to 52 pediatric residents (including 2 chief residents) in 1992 and to 59 pediatric residents (including 3 chief residents) and 14 medicine/pediatric residents (including 1 chief resident) in 2002. A medicine-pediatric residency program did not exist during the initial distribution period. To avoid compromising confidentiality in the relatively small medicine/pediatric group, residents were not asked to list their residency program in 2002 and consequently the 2 resident groups during that period were combined. Forty-three of the 52 residents completed the survey in 1992 (79%) compared to 63 of 73 (86%) during 2002 (p = NS).
Of the 43 respondents in 1992, 30 (70%) had considered doing a fellowship in any pediatric subspecialty and of those, 15 (35% of all respondents) believed they were \"very likely\" to do a fellowship. Of the 63 respondents in 2002, 40 (63%) had considered doing a pediatric fellowship and 16 of those (25% of all respondents) were \"very likely\" to continue with fellowship training. These numbers compare with 9 of the 52 residents in 1992 (17%) who actually completed fellowship training compared to 16 of the 47 graduating residents from the 2002 survey (34%) who began fellowship training in 2003 or 2004.
Eight residents (19%) in 1992 had considered a pulmonary fellowship compared to 7 residents (11%) in 2002 (p = NS). Table [1](#T1){ref-type="table"} lists these responses based on survey period and year of residency (internal medicine-pediatric residency is 4 years long). Although all residents have significant exposure to pulmonary patients during their residency, no correlation existed between interest in a pulmonary fellowship and having previously taken a one-month elective in pediatric pulmonology during residency. Three of the 15 residents who had considered a pulmonary fellowship (\"+PF\") had taken a pulmonary elective compared to 7 of the 91 residents who had not considered such a fellowship (\"-PF\"; p = 0.15). Of the 10 residents who had taken a pulmonary elective, only 2 thought that the elective experience affected their decision in considering a pulmonary fellowship (one from the +PF and one from the -PF group).
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Resident response based on year of survey (1992 or 2002) and year of residency (1--5)
:::
1992: Residency Year Resident Number Number Responded \% Response +PF\* -PF\*\*
---------------------- ----------------- ------------------ ------------- ------- ---------
1 21 20 95% 5 15
2 19 12 63% 2 10
3 10 9 90% 0 9
4 2 2 100% 1 1
1992 totals 52 43 79% 8 35
2002: Residency Year Resident Number Number Responded \% Response +PF\* -PF\*\*
1 24 22 92% 3 19
2 21 20 95% 3 17
3 22 17 77% 1 16
4 5 3 60% 0 3
5 1 1 100% 0 1
2002 totals 73 63 86% 7 56
\* +PF refers to those residents who had considered doing a pulmonary fellowship during their residency. \*\* -PF refers to those residents who had not considered doing a pulmonary fellowship during their residency.
:::
Table [2](#T2){ref-type="table"} lists and ranks the reasons given by residents who had considered doing a pulmonary fellowship. The highest ranked factors given by +PF residents included wanting to continue one\'s education after residency, enjoying caring for pulmonary patients in general, and enjoying both pulmonary physiology and the pulmonary faculty. The only factor that approached a statistically significant difference for +PF between the 2 survey periods was the statement \"I enjoy the tracheostomy-ventilator population,\" with a score of 0.6 ± 0.5 in 1992 vs. 1.6 ± 1.1 in 2002 (p = 0.057), suggesting that this factor became somewhat more important to the 2002 +PF residents in their consideration of a pulmonary fellowship. Table [3](#T3){ref-type="table"} lists and ranks reasons given by residents who had not considered doing a pulmonary fellowship. The scores given by -PF residents were generally not as high as those given by +PF residents, i.e. the factors listed were not as important to the -PF vs. the +PF residents. Major factors listed by -PF residents included not enjoying the tracheostomy/ventilator population and certain pulmonary patients in general, including chronic patients, as well as a desire to enter general pediatrics or another fellowship. The significant differences for -PF between the 2 survey periods are listed in Table [3](#T3){ref-type="table"}.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Mean (± SD) scores for factors given by +PF residents (those who considered a pulmonary fellowship)
:::
Factor\* 1992 Score 1992 Rank 2002 Score 2002 Rank
----------------------------------------------------- ------------ ----------- ------------- -----------
I want to continue my education after residency 2.8 ± 0.4 1 2.4 ± 0.8 1
I enjoy pulmonary-related procedures 2.5 ± 0.8 2 2.0 ± 1.0 10
I enjoy caring for pulmonary patients 2.4 ± 0.5 3 2.4 ± 0.5 1
I like pulmonary physiology 2.4 ± 0.8 3 2.3 ± 0.8 4
I like the pulmonary faculty 2.4 ± 0.5 3 2.4 ± 0.8 1
I enjoy pulmonary inpatient coverage 2.3 ± 0.5 6 2.3 ± 0.5 4
I enjoyed working with a particular faculty member 2.1 ± 0.9 7 2.1 ± 0.7 7
I might enjoy pulmonary-related research 2.0 ± 1.2 8 1.4 ± 0.8 12
I enjoy pulmonary clinics 2.0 ± 1.0 8 2.2 ± 0.8 6
I enjoy cystic fibrosis patients 1.9 ± 1.1 10 2.1 ± 1.1 7
I enjoyed caring for a particular pulmonary patient 1.7 ± 1.3 11 2.1 ± 0.7 7
The salary would be attractive 1.1 ± 1.2 12 1.0 ± 0.8 13
I enjoy the tracheostomy/ventilator population 0.6 ± 0.5 13 1.6 ± 1.1\# 11
\*Each factor was scored from 0 (\"not at all important\") to 3 (\"very important\") relating to a resident\'s decision to consider doing a pulmonary fellowship. \#2002 score approached statistically significant difference vs. 1992 score (p = 0.057).
:::
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Mean (± SD) scores for factors given by -PF residents (those who did not consider a pulmonary fellowship)
:::
Factor\* 1992 Score 1992 Rank 2002 Score 2002 Rank
----------------------------------------------- ------------ ----------- ------------- -----------
I don\'t enjoy the trach/vent population 2.0 ± 1.1 1 1.9 ± 1.3 1
I want to enter general pediatrics 1.9 ± 1.2 2 1.8 ± 1.3 2
I don\'t enjoy certain pulmonary patients 1.6 ± 1.1 3 1.2 ± 1.1 4
There are too many chronic patients 1.5 ± 1.1 4 1.5 ± 1.1 3
I want to enter another fellowship 1.5 ± 1.2 4 1.2 ± 1.3 4
I don\'t know enough about it to decide 1.5 ± 1.1 4 1.0 ± 1.1 7
Not enough pulmonary patient experience 1.4 ± 1.1 7 0.6 ± 0.9\# 9
I don\'t enjoy the BPD^+^population 1.2 ± 1.0 8 1.2 ± 1.1 4
I don\'t think pulmonary is very interesting 0.9 ± 0.9 9 0.6 ± 0.8 9
Some of the pulmonary patients scare me 0.7 ± 0.8 10 0.8 ± 1.0 8
I can\'t afford being a fellow 0.7 ± 1.1 10 0.4 ± 0.8 12
The pulmonologists work too hard 0.4 ± 0.6 12 0.4 ± 0.7 12
I don\'t enjoy the cystic fibrosis population 0.3 ± 0.6 13 0.6 ± 0.9 9
Too few pulmonary job openings 0.3 ± 0.5 13 0.0 ± 0.2\# 17
I don\'t enjoy the asthma population 0.2 ± 0.5 15 0.4 ± 0.8 12
Pulmonologists don\'t earn enough money 0.2 ± 0.5 15 0.1 ± 0.4 16
Poor experiences with pulmonary faculty 0.2 ± 0.4 15 0.3 ± 0.8 15
\*Each factor was scored from 0 (\"not at all important\") to 3 (\"very important\") relating to a resident\'s decision to not consider doing a pulmonary fellowship. \#Indicates 2002 score significantly different than 1992 score, p \< 0.001. ^+^bronchopulmonary dysplasia
:::
The last question in the survey asked residents what they thought they would be doing 5 years in the future. These responses are shown in Table [4](#T4){ref-type="table"}. The majority of residents during both survey periods believed that they would be in either non-academic or academic general pediatrics in 5 years. However, when grouped together during the 2 periods, the +PF residents were less likely to see themselves in the future as general pediatricians compared to the -PF residents (p \< 0.05). Interestingly, when the actual career decisions of the 1992 residents were reviewed, 35 of the 52 residents (67%) went into non-academic general pediatrics, 5 (10%) entered academic general pediatrics, 9 (17%) completed pediatric subspecialty training, 2 (4%) began a non-pediatric medical specialty, and in 1 case (2%) the eventual career decision could not be determined. Only 1 resident (from 1992 survey) in the entire group of 106 survey responders (\~1%) believed they would be in the field of pediatric pulmonology in the future. This resident did complete a pulmonary fellowship at another institution and is currently in an academic pulmonary practice. Additionally, a second resident (from 1992 survey) is currently an academic pediatric pulmonologist after having initially completed one year of a different subspecialty fellowship following residency. That resident then completed a pulmonary fellowship at this institution. Lastly, there was no significant difference in the overall level of interest in a pulmonary fellowship from 1992 compared to 2002.
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
Anticipated professional plans of resident 5 years following survey completion
:::
Category 1992 2002
------------------------------------------------ ------ -------- ----- -----
+PF -PF +PF -PF
General pediatrics, non-academic 1 14.5\* 2 29
Academic general pediatrics 1 5 1 8.5
Academic non-pulmonary pediatric specialty 3 12 2 16
Academic pediatric pulmonology 1 0 0 0
Non-academic non-pulmonary pediatric specialty 1 1.5 1 1.5
Non-academic pediatric pulmonology 0 0 0 0
Non-pediatric medical specialty 0 0 0 1
Non-medical vocation 0 0 0 0
Unknown 1 2 1 0
See text and footnote to Table 1 for explanation of +PF and -PF. The numbers represent actual number of residents responding. \*Some residents listed 2 categories, hence their score was divided between them.
:::
Discussion
==========
This study found that a significant percentage of pediatric residents considered doing a pulmonary fellowship after their residency training, ranging from 11% in the 2002 group to 19% in the 1992 group. However, these residents also viewed themselves as less likely to enter general pediatrics perhaps suggesting that they were simply considering several pediatric subspecialties at some time during their residency training. This seems likely, as the highest scored factor by the +PF residents was the desire to continue their education after residency. Despite fairly high percentages of residents considering a pulmonary fellowship, only 1 resident in the entire group (\~1%) actually believed that they would be a pediatric pulmonologist 5 years after the survey was completed. This percentage is very similar to that of first-time takers of the 1995 General Pediatrics Certifying Examination who believed they would be a pediatric pulmonologist in the future (1.1%) \[[@B4]\]. If one were to extrapolate this number to the entire class of graduating pediatric residents per year, about 2600 residents, approximately 30 residents would be entering the field of pediatric pulmonology each year \[[@B5]\]. This number is very similar to the actual number of graduating residents who enter a pediatric pulmonary fellowship each year \[[@B2]\].
The majority of residents who considered doing a pulmonary fellowship (8 of 15) were in their first year of residency. From personal experience, residents often consider various practice options early on in their training and frequently do not tend to narrow their choices until their second or third year of residency. This observation should be kept in mind when trying to recruit residents for pulmonary fellowship positions by seeking out those residents potentially interested in Pulmonology early on in residency rather than later.
The ranking of factors that may contribute to an interest in a pulmonary fellowship were remarkably similar during the 2 time periods. The only score that approached a statistically significant difference was the statement \"I enjoy the tracheostomy/ventilator population\" and this score tended to increase in 2002. However, a dislike for the tracheostomy/ventilator population also received the highest score among those residents not interested in a pulmonary fellowship and was even higher than both the desire to enter general pediatrics and another fellowship program. These data may simply be a center phenomenon but might suggest a more global \"disinterest\" in this patient population that may need to be further studied. Two scores in the -PF resident group decreased from 1992 to 2002: not enough pulmonary patient experience to decide on a pulmonary fellowship and too few perceived pulmonary job openings. The first may be a positive reflection on the local resident experience with pulmonary patients in recent years or on the fellowship program itself though this is only speculative. The second received very low scores during both periods and may not be very relevant. However, there has been some evidence of a reversal in the prior trend of residents entering general pediatrics in recent years, suggesting greater interest among residents in fellowships \[[@B6]\]. On the other hand, a recent survey of practicing pediatric pulmonologists noted that 69% of respondents did not believe that there was need for additional pulmonologists in their locale \[[@B7]\]. Interestingly, despite recent articles relating high post-residency debt with a disinterest in entering a fellowship, this was not noted in this study with mean scores during both survey periods of less than 1.0 for the statement \"I can\'t afford being a fellow.\" \[[@B8],[@B9]\]. This study did not ask residents to state their current level of indebtedness. Other \"job concern\" factors including job availability and the workload of pulmonologists did not appear to be significant negative factors for the -PF residents.
This study has certain shortcomings. This study asked residents to score specific factors that may or may not have been relevant to an individual resident. Although residents were given the opportunity to add personal comments, few did. In addition, certain patient populations, e.g those with respiratory infections, were not included as options in the survey and these omissions could cause study bias. Other factors that may contribute to residents\' decisions regarding fellowship training were not addressed in this study. These factors include resident teaching by the faculty, resident gender, spouse occupation, mentor encouragement or other personal reasons \[[@B10]-[@B12]\]. In addition, this study involved residents in only one program and cannot be generalized to all residency programs. Although this study did not find that the presence of a pulmonary fellowship significantly affected resident interest in such a fellowship, a significant difference may have been found with a larger sample size. In fact, the study appears to be underpowered despite the high response rate. More than 250 individuals would need to have been included in the study to detect a significant difference in residents\' initial interest in a pulmonary fellowship (assuming a power of 80%). In addition, other changes may have occurred within the residency program including higher loan repayments, changes in department philosophy (e.g. new department chairman), and changes in local pediatric job opportunities, which may have affected the results. Despite these limitations, this study may prove useful to those who are recruiting pediatric residents as potential pulmonary fellows. Further larger studies looking at multiple residency programs may provide more insight in the future. Lastly, studies like these help to reiterate the need for the specialty of pediatric pulmonology to \"prospectively and objectively determine realistic future training needs\" \[[@B13]\].
Conclusions
===========
Although many pediatric residents consider enrolling in a PF (\~10--20% here), few (\~1% here) will actually pursue a career in pediatric pulmonology. The presence of a PF program did not significantly alter resident interest, though other confounding factors may be involved.
Abbreviations
=============
BPD: bronchopulmonary dysplasia
PF: pulmonary fellowship
+PF: those residents who considered taking a pulmonary fellowship
-PF: those residents who had not considered taking a pulmonary fellowship
Competing interests
===================
None declared.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1472-6920/4/11/prepub>
Supplementary Material
======================
::: {.caption}
###### Additional File 1
Appendix. Resident questionnaire
:::
::: {.caption}
######
Click here for file
:::
Acknowledgments
===============
The author would like to thank Gloria Larsen, the Pediatric Residency Program Coordinator, for her assistance in supplying medical education residency records and also thank Dr. Robert Kliegman for his review of the manuscript.
| PubMed Central | 2024-06-05T03:55:46.597394 | 2004-7-26 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC503396/",
"journal": "BMC Med Educ. 2004 Jul 26; 4:11",
"authors": [
{
"first": "William M",
"last": "Gershan"
}
]
} |
PMC503397 | Background
==========
Quantitative studies provide answers or insights for many important questions or issues in health care and clinical research. Other important questions dealing with why, how, contexts, and experiences of individuals or groups, can be best addressed using qualitative methods. Other issues benefit from interleaving or integration of both research traditions. Miller and Crabtree \[[@B1]\], describe their experiences working in family medicine, a clinical domain where balancing qualitative and quantitative research styles benefits both patients and their families and health care professionals. They embrace holding \"quantitative objectivism in one hand and qualitative revelations in another\" and encourage others to use findings from both paradigms in understanding and practicing effective health care. Creswell and colleagues expand on this theme by stating that \"When used in combination, both quantitative and qualitative data yield a more complete analysis, and they complement each other\" \[[@B2]\].
Most studies in the major clinical journals have been quantitative studies. Very few qualitative studies and even fewer that combine both qualitative and quantitative approaches are published. An example of the breadth of qualitative studies and how findings and results can be combined across paradigms is a study by Jolly and Wiles \[[@B3],[@B4]\] who used mixed methods to study a nurse-led intervention for 422 adults after myocardial infarction and 175 adults with new-onset angina in 67 general practices in the United Kingdom. Their study showed statistically insignificant results at 1 month for eating healthy food, participating in exercise programs, and successful smoking cessation. Although patients in the nurse-led group were more likely to attend a rehabilitation program (37% vs. 22%, P = 0.001) attendance was disappointingly low. The researchers interviewed a group of patients using qualitative methods and found that people felt survival after a myocardial infarction indicated that the event had not been all that serious. Health care professionals often communicated simplified data about recurrence and being \"back to normal\" in 6 weeks. Because of these two issues, patients felt that their cardiac problems had probably been mild and therefore were not sufficiently motivated to implement major lifestyle changes.
Another example of the use of mixed methods was research done by Willms and Wilson and their colleagues \[[@B5]-[@B7]\] on smoking cessation. They found the meanings that patients who smoked attributed to their cigarettes (peer acceptance, coping during a time of stress and feeling out of control, feeling more like an adult, and smoking as more glamorous, tough, and rebellious) had more influence on cessation than did such external conditions as nicotine gum or counseling. Until the complex issues of why individuals smoke were dealt with, few were motivated to change their attitudes towards smoking and thus stop smoking.
Another effective example of integrated qualitative (ethnography) and quantitative (epidemiology) methods was a study done by Borkan and colleagues \[[@B8]\] to determine predictors of recovery after hip fracture in elderly patients. Traditional predictors such as age, type of break, and comorbidity, were collected by using standard questionnaires. In-depth interviews were used to collect injury narratives focusing on internal explanations of the fracture, sense of disability, and view of the future after hip fracture. None of the epidemiology factors predicted successful outcomes but those who perceived their fracture as more external or mechanical as opposed to an internal or organic problem (e.g., related to chronic disease) were more likely to have good recovery. Persons who perceived their disability in the context of autonomy, independence, and connection with the outside world also showed better ambulation at 3 and 6 months than persons with a more narrow and confined view of the fracture and its resulting disability.
Donovan and colleagues \[[@B9]\] used mixed methods to study prostate cancer screening and treatment choices to determine why study recruitment was lower than expected. Rousseau and Eccles and their colleagues \[[@B10],[@B11]\] used qualitative methods (case interviews) to explain the limited use of computerized guidelines for asthma and angina in a primary care study done in the United Kingdom. Many other examples exist; Creswell and colleagues describe 5 additional mixed methods studies in primary care as well as provide criteria for evaluating mixed methods studies \[[@B2]\].
We postulate that qualitative studies, either stand-alone reports or studies with mixed methods, are occurring more frequently in health care. This paper was done to describe the publishing of qualitative studies in 1 year of clinical literature, document and present the range of content and techniques in these studies, and establish a baseline for subsequent studies. We defined our sample to include all articles published in a set of major general medical, mental health, or nursing journals during 2000. We determined how many qualitative studies were published and in which journals, and extracted design methods and healthcare content, and how often studies used mixed methods and analyses. Because the nursing literature published a higher proportion of qualitative studies in our sample we also compared studies published in nursing journals with other journals to ascertain if quantitative differences exist across disciplines in the use of qualitative methods. Our analysis is a quantitative review of qualitative studies in health care in 2000.
Methods
=======
The Health Information Research Unit of the Department of Clinical Epidemiology and Biostatistics, Faculty of Health Sciences at McMaster University in Hamilton, Ontario, Canada was the editorial office for four evidence-based summary journals in 2000: *ACP Journal Club*(internal medicine content), *Evidence-Based Medicine*(family/general practice content), *Evidence-Based Nursing*(general care nursing content), and *Evidence-Based Mental Health*(mental health care content). Their purpose is to provide enhanced abstracts and commentaries on important high-quality original studies and review articles for their respective clinical audiences. To identify these studies and review articles, 6 research staff read major clinical journals to ascertain if articles were in 1 or more categories of therapy, diagnosis, prognosis, etiology, economics, clinical prediction guides, differential diagnosis, and qualitative studies and if so, did each meet predefined methodology criteria for study quality\[[@B12]\]. For 2000 we intensified our data collection to provide data to update and develop new clinical retrieval searching hedges for MEDLINE, PsycINFO, CINAHL, and EMBASE using methods described by Haynes and colleagues\[[@B13]\]. One hundred and seventy journals provided data for this article.
The staff of the Health Information Research Unit has established quality criteria for the 8 categories of clinical literature that must be met before articles are judged appropriate for clinical application and publication in an abstract journal. Qualitative studies have 3 criteria:
• content relates to how people feel or experience certain situations, specifically those that relate to health care
• data collection methods and analyses are appropriate (primary analytical mode is inductive rather than deductive)
• units of collection and analysis are ideas, thoughts, concepts, phrases, incidents, or stories that become categories or themes.
The reading methods have been developed during the past 13 years and inter-rater reliability kappa (chance adjusted agreement) for identifying categories and applying criteria is consistently \> 80%.
For this paper, KAM, one of the readers, analyzed the qualitative studies. Qualitative systematic reviews were excluded leaving only reports of original studies. These were assessed to extract journal title, qualitative study type, data collection methods, research question, persons studied, setting, and disease or health condition considered. In addition, studies with mixed methods were further analyzed although we did not use stringent criteria for assessing the quality \[[@B2]\] of the combination of methods. We identified mixed methods articles using a loose criterion of \"some numerical or statistical analysis of quantitative data or qualitative data that had been turned into quantitative data\". (An example of quantifying qualitative data is the study done by Borkan and colleagues \[[@B8]\] on hip fracture.) The analysis had to be fairly substantial--for example, a simple descriptive analysis of baseline demographics of the participants was not sufficient to be included as a mixed methods article.
In addition, Giacomini and Cook \[[@B14],[@B15]\], as part of the Evidence-Based Working Group in the Users\' Guides to the Medical Literature, describe attributes that they have identified as belonging to high-quality qualitative studies: participant selection, data collection, and analysis methods. These aspects were also extracted for analysis in this report.
Data were taken from article abstracts and if needed, the full text was reviewed. Methodologies assessed were phenomenology, grounded theory, ethnography, case studies, narrative analysis, participant action, critical incident techniques, and discourse analysis. Author descriptions were used and if an additional methodology was found it was added to the list of types using definitions and descriptions from the Handbook of Qualitative Analysis, 2^nd^edition by Denzin and Lincoln \[[@B16]\]. Data collection and sampling procedures were also extracted. Multiple designations were allowed. To assess the reproducibility a random 10% (n = 35) sample of citations was reviewed using predefined decision rules by another researcher trained in research methods.
Results
=======
The 170 journals included 60 330 articles of which 31 496 (52%) contained original data or were review articles. 3830 of these (6%) passed criteria for being high-quality and clinically relevant in 1 of the 8 categories. 367 articles met quality criteria for original studies or reviews of qualitative studies. Table [1](#T1){ref-type="table"} lists the journals that published at least 1 qualitative study. Twelve systematic reviews were excluded leaving 355 qualitative studies for assessment. Approximately 0.6% of all articles in the 170 journals and 9% of all high-quality, clinically relevant studies were qualitative studies.
The reproducibility of the categorization was measured by kappa (chance adjusted agreement): 0.92 for disease/condition, 0.83 for groups studied, 0.81 for setting, 0.73 for data collection, and 0.63 for data analysis type. The agreement for data analysis type was disappointing but not surprising in that 20% of the studies did not label their analyses necessitating assignment of analysis type by the data extractors. Agreement was low for participant selection methods (kappa 0.5) and therefore data on participant selection methods are not reported.
The 355 qualitative studies appeared in 48 journals (mean 7.4 articles per journal, range 1 to 86). These 48 journals were only 28% of the 170 clinical journals being read. Most of the qualitative studies were published in nursing journals: The 17 nursing titles included 214 qualitative studies (61% of all of the qualitative studies).
Few qualitative studies were published in the high-circulation, general healthcare journals. Using SCI Impact Factor ranking for 2000, only 4 of the top 20 journals (Table [2](#T2){ref-type="table"}) published qualitative studies. These 4 journals published 15 qualitative studies with *BMJ*publishing 12. The highest-ranking journal with qualitative studies was *Annals of Internal Medicine*, ranked number 6. *JAMA*, ranked number 2, published articles about qualitative studies in 2000 \[[@B14],[@B15]\] but did not publish any qualitative studies.
Mixed qualitative and quantitative studies
------------------------------------------
37 qualitative studies (11%) included qualitative and quantitative methods and analyses. These were published in 17 journals with only 1 article in *BMJ*from the top 20 journal titles in Table [1](#T1){ref-type="table"}. *Social Science and Medicine*published 10 of these mixed methodology studies--the most of any title studied.
Content
-------
Content of the studies is shown in Table [3](#T3){ref-type="table"}. Many studies dealt with a range of participants and settings. Patients (56%), family (22%), and other non-health care professionals (14%) were studied more often than health professionals (nurses (21%), physicians (11%), and others (5%)). Non health care settings occurred more often with home or similar settings being studied in 44% of studies and other community settings in 16%. Health care settings were the hospital (25%), clinic (17%), nursing home (5%), and the emergency department (2%).
Disease/condition breakdowns represented common health care situations: cancer (11%), mental health (10%), pregnancy and childbirth (9%), cerebrovascular disease (10%), general issues such as vaccinations or Internet use, and nonspecific spectrum of diseases (e.g., all patients in a clinic) (12%). Many uncommon issues were also assessed. For example, Tongprateep \[[@B17]\] reports a phenomenology study designed to help nurses better understand essential elements of spirituality and health among rural Thai elders.
Analysis of the 37 articles with mixed methods showed similar patterns for settings, persons studied, and disease/condition evaluated except that more physicians were studied (P \< 0.025) and more situations dealing with injury (P \< 0.001) were evaluated. For the 211 articles in Nursing journals, very little difference was also seen except that fewer physicians were studied (P \< 0.001) and more studies were done outside clinical settings (P \< 0.001).
Phenomenology (37%), grounded theory (35%), and ethnography (18%) were used most often with some case studies (7%), narrative analysis (6%), participant action (3%) research, critical incident techniques (1%), and discourse analysis (1%) (Table [4](#T4){ref-type="table"}). More than one qualitative method was used in 8% of studies. This pattern of methodology choice was similar for the 37 mixed methods studies and the 211 Nursing articles except that mixed studies methods used relatively more case studies and the Nursing studies used fewer of them (P \< 0.025). The mixed methods studies did not included participatory action research, critical incident technique, or discourse analysis studies, methods that could be difficult to combine with quantitative studies.
Semi-structured interviews were used (77%) with some focus groups (18%) and observation (14%). These methods are major data gathering techniques in qualitative studies. Questionnaires (7%), document analysis (6%), and structured (4%) and unstructured interviews (1%) were used less often. For mixed methods studies, patterns were similar although questionnaires were used more frequently (24% vs. 7%, P \< 0.01). Nursing studies did not differ for data gathering techniques.
Sampling is important in all studies--often no single right way exists for a study question. Purposive, snowball, and theoretical sampling are often used in qualitative studies and random and consecutive sampling for quantitative studies. All methods were represented in this analysis but the breakdowns are not reported because of low inter-rater agreements for categorization and missing author information.
Discussion
==========
In 2000 the major clinical journals published many qualitative studies--approximately 9% of all high-quality, clinically relevant articles. Most of the qualitative studies were reports of original research although 12 (3%) were systematic reviews. Most of the qualitative studies were in nursing journals although some medical journals such as *BMJ*and *Annals of Internal Medicine*also published several. Three of the high circulation medical journals (*New England Journal of Medicine, Lancet,*and *JAMA*) and 16 of the top 20 clinical journals, based on SCI Impact Factors, did not publish any qualitative studies. This is likely a reflection on the emphasis on a positivist, numerical approach that many of these journals embrace. The difference in proportion of qualitative studiers published in nursing journals is probably because of two historical, but linked factors. Qualitative studies have roots in women\'s studies and the nursing profession has always dealt with the patient as much more of a whole person rather than basic sciences facts and numbers. Both of these factors lead to more emphasis on understanding and embracing qualitative methods for research and practice. This view is substantiated by the fact that MEDLINE indexes most of the qualitative studies under the term Nursing Methodology Research until 2003.
A substantial proportion of the qualitative studies (11%) included both qualitative and quantitative (mixed) data. In general, these mixed methods studies were similar to the single methodology studies except they did more assessments of physicians and relied more on questionnaires to gather data for analysis. The presence of these mixed methods or multipardigmatic studies as described by Miller and Crabtree \[[@B1]\] and Creswell \[[@B18]\] is encouraging for those who espouse harnessing methodologies appropriate for exploring, explaining, and interpreting the complexities and ranges of issues in health care practice and research.
It is also interesting comparing qualitative studies in Nursing and non-Nursing journals. Regardless of the differences in proportion of qualitative studies published, from a content point of view few differences exist between the Nursing and non-Nursing journals except that more physicians were studied in the non-Nursing journals and fewer studies were done in clinical settings--not unsurprising findings. This indicates that the content and methods of qualitative studies seem to be similar across disciplines or if the methods are combined with quantitative methods.
This review of the publication of qualitative studies is limited in several ways. The proportion of journals studied was very low in relation to the total number of journals published. MEDLINE indexes over 4000 journals and this number is still a relatively small proportion of all journals that deal with health care. In addition, all of the journals searched were published in English so we do not know about qualitative studies in other languages. Although our criteria were relatively strict for including qualitative studies, our criteria for mixed methods studies could certainly have been stronger. We did not count the number of high-quality quantitative studies that could have included some qualitative analyses. We studied only 1 year of publishing; much could have changed since 2002.
Qualitative studies provide insight into social, emotional, and experiential aspects of health and health care and as such, they have an important place in understanding health and health care. Hopefully more studies will be published and more will be published in the high impact (high circulation) journals. This paper provides a basis for measuring increases.
Conclusion
==========
Qualitative studies are being done and are published in a wide range of healthcare journals. These journals however are not the highest impact journals. It is encouraging to see that the number of qualitative studies that were published in 2000 and also the number of studies that combined qualitative and quantitative methods. More can be done however to complete and publish qualitative studies, and where appropriate, integrate the best of both methodologies. Both qualitative and quantitative researchers and clinicians need to work together to make this happen. Journal editors can also encourage submission of qualitative and mixed methods studies and facilitate publication of those they do receive.
List of Abbreviations
=====================
SCI ISI Science Citation Index
Competing interests
===================
None declared.
Author Contributions
====================
This work was done in partial fulfillment of PhD requirements for KAM. Both authors have supplied intellectual input in designing and implanting the survey. KAM has collected and analyzed the data and both authors have contributed to writing the paper and agree on its content.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1472-6947/4/11/prepub>
Acknowledgement
===============
This research was funded by the U.S. National Library of Medicine.
The Hedges Team who did the data collection, entry, and verification included Nancy Bordignon, Angela Eady, Brian Haynes, Susan Marks, Ann McKibbon, Doug Morgan, Cindy Walker Dilks, Stephen Walter, Nancy Wilczynski, and Sharon Wong. Catherine Arnot Smith provided the second review methods for inter-rater agreement.
Figures and Tables
==================
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Journal Title with number of Original and Review Qualitative Studies for 2000
:::
**Journal Title** **Number of Qualitative Studies**
------------------------------------------------------ -----------------------------------
Journal of Advanced Nursing 88
**Social Science and Medicine** **34**
Qualitative Health Research 30
**Journal of Clinical Nursing** **22**
Western Journal of Nursing Research 15
**Cancer Nursing** **14**
BMJ 12
**Research in Nursing and Health** **12**
Midwifery 10
**Public Health Nursing** **10**
Family Practice 9
**ANS Advances in Nursing Science** **8**
British Journal of General Practice 8
**Heart and Lung** **8**
Journal of Nursing Scholarship 8
**Patient Education and Counseling** **8**
Journal of Pediatric Nursing 6
**Clinical Nursing Research** **5**
Journal of Family Practice 5
**Journal of General Internal Medicine** **5**
Birth 4
**Health Education and Behavior** **4**
Archives of Pediatrics and Adolescent Medicine 3
**Canadian Journal of Nursing Research** **3**
Canadian Journal of Public Health 3
**Journal of Pediatric and Oncology Nursing** **3**
Neonatology Review 3
**Pediatrics** **3**
Qualitative Health Care 3
**Applied Nursing Research** **2**
Psychiatric Services 2
**Addiction** **1**
American Journal of Psychiatry 1
**American Journal of Public Health** **1**
Annals of Emergency Medicine 1
**Annals of Internal Medicine** **1**
Archives of Physical Medicine and Rehabilitation 1
**Australian and New Zealand Journal of Psychiatry** **1**
BJOG (British Journal of Obstetrics and Gynaecology) 1
**British Journal of Psychiatry** **1**
CMAJ (Canadian Medical Association Journal) 1
**Diabetes Care** **1**
Fertility and Sterility 1
**International Journal of Geriatric Psychiatry** **1**
Journal of Epidemiology and Community Health 1
**Journal of Psychosomatic Medicine** **1**
Nursing Research 1
**Western Journal of Medicine** **1**
:::
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Top Health Care Journals with SCI Impact Factor for 2000 and Number of Qualitative Studies
:::
**Journal Title** **SCI Impact Factor** **Qualitative Studies**
---------------------------------------------------------------- ----------------------- -------------------------
New England Journal of Medicine 29.521 0
**JAMA** **15.402** **0**
Archives of General Psychiatry 11.778 0
**Circulation** **10.893** **0**
Lancet 10.232 0
**Annals of Internal Medicine** **9.833** **1**
Annals of Neurology 8.480 0
**Journal of the American College of Cardiology** **7.082** **0**
Psychological Bulletin 6.913 0
**Arthritis and Rheumatism** **6.841** **0**
American Journal of Psychiatry 6.577 1
**Archives of Internal Medicine** **6.055** **0**
American Journal of Medicine 5.960 0
**American Journal of Respiratory and Critical Care Medicine** **5.443** **0**
Gut 5.386 0
**BMJ** **5.331** **12**
Hypertension 5.311 0
**Diabetes Care** **4.992** **1**
Journal of Infectious Diseases 4.988 0
:::
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Content for Qualitative Studies (n = 355), Mixed Methodology Studies (n = 37), and Studies from Nursing Journals (n = 211)
:::
**Category** **All studies** **% All studies** **\# Mixed Methods** **% Mixed Methods** **\# Nursing Journals** **% Nursing Journals**
------------------------------------- ----------------- ------------------- ---------------------- --------------------- ------------------------- ------------------------
**Groups studied**
Patients 198 56 17 45 114 54.0
**Family** **78** **22** **9** **24** **52** **24.6**
Nurses 73 21 6 16 55 26.1
**Other People** **49** **14** **0** **0** **48** **22.7**
Physicians 38 11 8\*\* 21 9\* 4.3\*
**Other Health Care Professionals** **20** **6** **1** **3** **12** **5.7**
Settings
Home or non institution 155 44 16 42 100 47.4
**Hospital** **87** **25** **10** **26** **65** **30.1**
Clinic 62 17 7 18 16\* 7.6\*
**Community** **48** **14** **7** **18** **36** **17.1**
Nursing Home 19 5 2 5 13 6.2
**Emergency Dept** **6** **2** **1** **3** **2** **0.9**
Church, Jail, Other 5 1 2 5 2 0.9
**Disease/Condition**
Various 38 11 3 8 19 9.0
**Cancer** **40** **11** **5** **13** **28** **13.2**
Mental Health 38 10 3 8 26 12.3
**Pregnancy/birth** **33** **9** **3** **8** **16** **7.6**
Cerebrovascular Disease 34 10 2 5 22 10.4
**General Health** **33** **9** **2** **5** **17** **8.1**
Frail Elderly 29 8 3 8 21 10.0
**HIV/AIDS** **21** **6** **3** **8** **11** **5.2**
Drugs/Sex Trade 13 4 2 5 7 3.3
**Death and Dying** **13** **3** **0** **0** **7** **3.3**
Diabetes 9 3 2 5 4 1.9
**Critical Care** **8** **2** **1** **3** **8** **3.8**
Injury 7 2 5\* 13 5 2.4
**Asthma** **6** **2** **1** **3** **0** **0**
Pain 7 2 1 3 3 1.4
**Smoking** **4** **1** **1** **3** **2** **0.9**
Miscellaneous disease/conditions 41 12 5 13 26 12.3
\*P \< 0.001 \*\*P \< 0.025. Note that totals do not add to 100% because studies often included several methods, settings, diseases, or populations.
:::
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
Methodologic Analysis of the Qualitative Studies (n = 355), Mixed Methods (n = 37), and Nursing Journals (n = 211)
:::
**Category** **\# All studies** **% All studies** **\# Mixed Methods** **% for Mixed Methods** **\# Nursing Journals** **% Nursing Journals**
----------------------------- -------------------- ------------------- ---------------------- ------------------------- ------------------------- ------------------------
Analysis Type
Phenomenology 131 37 14 37 87 41.2
**Grounded Theory** **124** **35** **10** **26** **74** **35.1**
Ethnography 64 18 8 21 33 15.6
**Case Studies** **24** **7** **5** **13\*** **9** **4.3\*\***
Narratives 21 6 2 5 12 5.7
**Participatory** **13** **4** **0** **0** **10** **4.7**
Critical Incident Technique 4 1 0 0 3 1.4
**Discourse Analysis** **4** **1** **0** **0** **3** **1.4**
Data Collection
Semi-structured Interviews 272 77 27 71 167 79.1
**Observation** **64** **18** **6** **16** **37** **17.5**
Focus Groups 50 14 5 13 31 14.7
**Questionnaires** **25** **7** **9\*\*\*** **24** **14** **6.6**
Documents 23 6 2 5 11 5.2
**Unstructured Interviews** **13** **4** **1** **3** **10** **4.7**
Structured Interviews 2 1 0 0 2 0.9
\* P \< 0.001 \*\*P \< 0.025 \*\*\* P \< 0.01
:::
| PubMed Central | 2024-06-05T03:55:46.600691 | 2004-7-22 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC503397/",
"journal": "BMC Med Inform Decis Mak. 2004 Jul 22; 4:11",
"authors": [
{
"first": "Kathleen Ann",
"last": "McKibbon"
},
{
"first": "Cynthia S",
"last": "Gadd"
}
]
} |
PMC503398 | Background
==========
Hypertriglyceridemia in combination with abnormally low concentrations of HDL cholesterol (High Density Lipoprotein Cholesterol) is one of the most common and atherogenic profiles of lipid metabolism \[[@B1]\]. In the PROCAM study \[[@B2]\], the 6-year incidence of coronary events in men aged between 40 and 60 years, was twelve times higher than in the control group. The prevalence of this abnormality varies among ethnic groups \[[@B3]\]. It is found in 13% of the Mexican adults living in urban areas \[[@B4]\]. It is more common in men than in women (20.9% vs 7.2%) and in some age groups (i.e. men aged 30 to 39 years) this prevalence is as high as 30%. This lipid profile is the most frequent form of dyslipidemia in the metabolic syndrome \[[@B5]\]. However it is also found in subjects affected by primary dyslipidemias (e.g familial combined hyperlipidemia).
In the Veteran Affairs HDL Intervention Trial (VAHIT), the use of a fibrate (gemfibrozil) resulted in a 22% reduction in the incidence of cardiovascular events in subjects with low HDL cholesterol and a broad range of triglyceride values \[[@B6]\]. The benefit was accounted for by the positive effects obtained in cases with insulin resistance \[[@B7]\]. In spite of these positive results, there are few studies assessing the efficacy of other fibrates in the treatment of this form of dyslipidemia \[[@B8]\]. Relevant data such as the percentage of cases that achieve treatment goals are not described in the majority of these reports. Also, variables predicting a greater likelihood of achieving treatment goals remain to be identified. Our objective was to assess the efficacy and safety of ciprofibrate (100 mg/day) for the treatment of cases with hypertriglyceridemia / hypoalphalipoproteinemia in an open label, multicenter, international study. A clinically oriented approach is used for the description of the results.
Materials and Methods
=====================
The trial included men and post-menopausal or non-pregnant women aged between 30 and 70 years who had hypertriglyceridemia (fasting concentrations between 1.68--3.9 mM/l (150 -- 350 mg/dl) and hypoalphalipoproteinemia (HDL cholesterol ≤ 1.05 mM/l (40 mg/dl) for women and ≤ 0.92 mM/l (35 mg/dl) for men). The LDL cholesterol had to be lower than 4.2 mM/l (160 mg/dl). Patients were excluded if they had an acute coronary event during the three months preceding the study, type 1 diabetes, uncontrolled hypertension, severe renal dysfunction, nephrotic syndrome or aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels \> 1.5 × the upper limit of normal (ULN), or if their creatine phosphokinase (CPK) levels were \> 3 × ULN. Consumption of any lipid-altering drug within the previous 4 weeks (6 months for probucol) prevented entry into the study. Patients could be receiving other concomitant medication as long as the dosage was not modified during the study.
The Ethics Committee in each institution approved the protocol and every patient provided witnessed, written informed consent prior to entering the study.
This was a multicenter, international, open-label study. Patients were recruited from 25 lipid clinics from México (n = 152), Brazil (n = 129), Chile (n = 78) and Colombia (n = 78). Cases fulfilling the inclusion criteria were invited to participate. The initial visit included a medical evaluation, a physical examination and the prescription of an isocaloric diet consisting of 50% carbohydrate, 30% fat, 20% protein with a cholesterol content of 200 mg \[[@B9]\]. Blood samples were obtained after a 9--12 h fasting period. All patients were assigned to receive ciprofibrate 100 mg at bedtime. The second and final visit was scheduled 4 months later. During this visit drug compliance and safety profile were assessed and body weight as well as laboratory parameters were measured. Drug compliance was measured by counting the returned pills. Adherence to the diet was not assessed during the study
The primary efficacy parameter was the percentage change in triglycerides from baseline. Secondary efficacy parameters included the percentage change in total cholesterol, HDL cholesterol and non-HDL cholesterol from baseline. Non-HDL cholesterol was calculated by subtracting the HDL cholesterol from the total cholesterol levels. In a post hoc analysis, the percentage of cases that achieved the treatment goals proposed by the ATP-III recommendations \[[@B10]\] on the final visit was also estimated.
At each visit, AST, ALT, fasting plasma glucose and CPK levels were measured. Clinically relevant complications were defined as either CPK \> 5 × ULN accompanied by muscle pain, tenderness or weakness or ALT or AST \> 3 × ULN. Patients were excluded from the study if they developed severe hyperglycemia or any other significant complication to treatment. Other reasons for premature withdrawal were lack of compliance to the medication. Cases were instructed to contact their physician in case of any side effect.
All samples were analyzed in a central laboratory (Quest laboratories). In Brazil, the local laboratory of every center was used instead. Blood samples were taken after an overnight fast (≥ 9 hours). Measurements were performed during the first 24 hours after the blood was drawn; blood samples were kept at 4°C until the analysis. All laboratory analyses were performed with commercially available standardized methods. Glucose was measured using the glucose oxidase method. Total serum cholesterol and triglycerides levels were measured using an enzymatic method. HDL cholesterol levels were assessed using phosphotungstic acid and Mg2+.
Statistical analysis was performed using SPSS for Windows version 10. An intention to treat analysis was used. Two sided ANOVA tests were used for assessing differences between groups for continuous variables. All categorical variables were analyzed using the chi squared test. Multiple logistic regression models were constructed for the identification of variables associated with the achievement of treatment goals.
Results
=======
Four hundred thirty seven patients were included. The clinical characteristics of the study subjects are shown in table [1](#T1){ref-type="table"}. Almost half of the population had a body mass index between 25 and 30 kg/m^2^(n = 221); an additional 32.5% were obese (n = 142) Diabetes was present in 125 subjects (28.7%).
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Baseline characteristics of the patients included in study (n = 437)
:::
**Variable** **N = 437**
--------------------------------------- -------------
Sex
Male (n(%)) 239 (54.7)
Female (n(%)) 198 (45.3)
Age (years)\* 54.7 ± 12.1
Body mass index (kg/m^2^) 28.8 ± 4.6
Diabetes (n(%)) 125 (28.7)
Fasting plasma glucose (mM/l)\* 5.2 ± 0.6
Family history of dyslipidemia (n(%)) 171 (39.2)
Coronary heart disease (n(%)) 196 (45)
Aspartate aminotranferase (mU/l) 23 ± 11
Alanine aminotransferase (mU/l) 26 ± 14
Data expressed as mean ± standard deviation. \*To convert to mg/dl, multiply by 18
:::
Both evaluations were completed in every case. The medication was stopped before the trial was completed by 117 subjects (26.7%). In the majority of cases this was not related to side effects (see below). In addition, 46 cases (10.5%) had poor compliance to the medication. The body weight remained constant in all patients. The alcohol and tobacco consumption was not modified during the study.
Ciprofibrate treatment and diet had a significant beneficial effect on the lipid profile, as shown in table [2](#T2){ref-type="table"}. After 4 months of treatment, plasma triglyceride concentrations were decreased by 44% (p \< 0.001). HDL cholesterol concentrations were increased by 10.1% (p \< 0.001). Non-HDL cholesterol was decreased by 19.2% (p \< 0.001). Total cholesterol was also favorably modified (-14.9%, p \< 0.001). In contrast, LDL cholesterol had a minor modification (-5.4%, p \< 0.001). A significant decrease in fasting glycemia was observed in both obese and diabetic cases. This change was not found in lean subjects.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Changes in the lipid profile and clinical characteristics between baseline and post-treatment values
:::
**Intention to treat analysis N = 437**
------------------------------- ----------------------------------------- ------------ --------------------
**Baseline** **Final** **Percent change**
Triglycerides (mM/l) ^†^ 3.01 ± 0.7 1.61 ± 0.8 -44 ± 33\*
HDL Cholesterol (mM/l)^‡^ 0.91 ± 0.1 0.98 ± 0.4 10 ± 52\*
Non-HDL cholesterol (mM/l)^‡^ 4.57 ± 0.9 3.61 ± 1.5 -19 ± 36\*
Cholesterol (mM/l)^‡^ 5.5 ± 0.9 4.57 ± 1.8 -14.9 ± 35\*
LDL Cholesterol (mM/l)^‡^ 3.1 ± 0.9 2.8 ± 1.3 -5.4 ± 59\*
Mean ± standard deviation are presented. \*p \< 0.001 ^†^To convert to mg/dl multiple by 89 ^‡^To convert to mg/dl multiple by 38
:::
The achievement of treatment goals is the ultimate aim of lipid-lowering therapy. Almost half of the cases had reduced their triglyceride concentrations below 1.68 mM/l (150 mg/dl) (n= 191(43.7%). HDL cholesterol levels above 1.05 mM/l (40 mg/dl) were found in 51% of the cases (n = 223). Also, a significant proportion of the subjects (63.15%, n = 276) achieved the non-HDL cholesterol goal 4.2 mM/l (\< 160 mg/dl). The LDL cholesterol goal \< 3.4 mM/l (\< 130 mg/dl) was attained by 56.2 % (n = 246). A full correction of the hypertrigliceridemia / low HDL cholesterol occurred in 129 subjects (29.5%). Many of them also had a non-HDL cholesterol level below 4.2 mM/l (160 mg/dl) (n = 101, 23.1%)
The lipid response during treatment differed between cases with a body mass index above or below 25 kg/m^2^. As is shown in figure [1](#F1){ref-type="fig"}, the percentage change in HDL cholesterol was higher in lean subjects. In contrast, the non-HDL cholesterol concentration had a significantly greater reduction among subjects with excess body weight. Both differences remained significant even after adjusting for age and gender. The lipid profile did not differ between these groups during the initial visit.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
The percentage of change in the lipid parameters is different in cases with a body mass index above 25 kg/m^2^compared to the response observed in lean individuals during treatment with ciprofibrate and diet.
:::
:::
Patients with diabetes had moderate hyperglycemia during the study. Their fasting glycemia was 7.7 ± 2.6 mM/l (139 ± 47 mg/dl) at baseline. A small but statistically significant decrease in glucose concentration was observed at the end of the trial (5.7 ± 3 mM/l, 104 ± 54 mg/dl p \< 0.001). At baseline, their lipid profile differed, from the non-diabetic subjects, only with regards to higher triglyceride concentrations (3.13 ± 0.97 vs 2.95 ± 0.66 mM/l, p \< 0.001). The lipid response to ciprofibrate and dietary treatment did not differ from that observed in the whole group. Individuals with a family history of dyslipidemia (n = 171) had higher cholesterol, triglycerides and LDL cholesterol at baseline compared to the rest of the population. The lipid response to treatment was similar to that reported in the whole group.
Multiple regression models were constructed to identify variables associated with the achievement of the treatment goals. For every target value, the main determinants were the baseline value and the percentage change after treatment. No other parameter provided additional information in any of the models. Thus, we analyzed the variables associated with the percentage change during treatment. The non-HDL cholesterol model provided more information compared to models derived from the other lipid parameters. In the non-HDL cholesterol model, a body mass index greater than 25 kg/m^2^, the triglyceride response and the coexistence of cholesterol above 5.2 mM/l (200 mg/dl) (mixed hyperlipidemia) were predictors for a greater non-HDL cholesterol response (table [3](#T3){ref-type="table"}). For both, the HDL cholesterol and the triglycerides models, the inclusion of a body mass index \< 25 kg/m^2^added little information and had only borderline statistical significance. No other clinically relevant variable was associated with the percentage change of any other lipid parameter.
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Multiple regression model using as dependent variable percent of change in non-HDL cholesterol concentration
:::
**Variable** **Beta coefficient ± standard error** **p value**
------------------------------------ --------------------------------------- -------------
Constant 14.4 ± 2.6 \< 0.001
BMI \< 25 kg/m^2^ 7.6 ± 3.6 0.036
Mixed hyperlipidemia 0.82 ± 0.2 0.003
Percent of change of triglycerides 0.68 ± 0.4 \< 0.001
Correlation coefficient = 0.647, r^2^= 0.419, p = 0.001 BMI, Body mass index
:::
Liver function tests were not modified by the treatment. No patient had a significant alteration of any of the laboratory tests. There were no incidents of either myopathy or liver dysfunction. No persistent elevations in ALT, AST or CPK, defined as clinically relevant, were reported during the course of the study.
Discussion
==========
The combination of hypertriglyceridemia / low HDL cholesterol is a common abnormality of lipoprotein metabolism and is associated with increased cardiovascular risk. Our data show that ciprofibrate and an isocaloric diet are an effective treatment for this dyslipidemia. However, there was significant variation in response to treatment between individuals. Excess body weight may be an important determinant of the lipid response. It is associated with a greater degree of non-HDL cholesterol reduction and a relatively smaller elevation in HDL cholesterol.
A significant improvement in plasma triglycerides and HDL cholesterol concentrations resulted from the administration of ciprofibrate and dietary modifications. Our results are in agreement with previous studies \[[@B11]-[@B15]\]. This study differs from previous reports due to its design. We wanted to assess the lipid response to ciprofibrate in a real life environment. Hence, the highly controlled conditions of a randomized, double blind study were avoided. Also, we limited our inclusion criteria to subjects with hypertriglyceridemia / low HDL cholesterol, instead of including subjects with a wide variety of lipid profiles. The results are in accordance with the uncontrolled design of the study. By the use of ciprofibrate and isocaloric diet, almost half of the cases achieved the triglyceride goal (1.68 mM/l, 150 mg/dl). HDL cholesterol levels above 40 mg/dl were found in 51% of the cases. The full correction of the combination of hypertriglyceridemia / low HDL cholesterol occurred in a third of the population. This rate is similar to that reported for the LDL cholesterol goals achieved by the use of statins. Thus, our results reflect the strengths and limitations of treating this lipid abnormality in an uncontrolled setting.
A large range of lipid responses was observed between individuals. There are few reports designed to analyse the determinants of the lipid response to fibrates. Robins reported a lower HDL cholesterol elevation with a fibrate when insulin resistance is present \[[@B7]\]. In this report, a lower HDL cholesterol response was observed in patients with excess body weight (body mass index above 25 kg/m^2^) in comparison to that found in lean individuals. Since excess body weight is strongly associated with insulin resistance \[[@B16],[@B17]\], our observations may be in agreement with the findings of Robins. Interestingly, the presence of insulin resistance was associated with the lowest incidence of coronary events in the VAHIT study. In our report, obese individual had a larger decrease in non-HDL cholesterol levels. The greater response in non-HDL cholesterol observed in our obese (and possibly insulin resistant) subjects may be one possible explanation for the greater benefit found in insulin resistant subjects during the VAHIT study. Our data suggest that the mechanism of action of ciprofibrate may be altered by the pathophysiology of the disorder being treated. The same phenomenon has been observed with the use of statins \[[@B18],[@B19]\]. The greater reduction in non-HDL cholesterol in subjects with excess body weight could be explained by an increased clearance of remnants, IDL and VLDL particles. Ciprofibrate may enhance their clearance either by decreasing the concentration of the apolipoprotein CIII (an inhibitor of the lipolytic activity of the lipoprotein lipase) \[[@B20]-[@B22]\] or by increasing the mass and activity of lipoprotein lipase \[[@B23]\]. Genes encoding apolipoprotein CIII and lipoprotein lipase contain a PPAR-alpha response element; hence their expression may be modified during treatment with a fibrate \[[@B24]\]. Additional studies are needed to identify other possible determinants of the lipid response to treatment with a fibrate.
Several limitations of the study must be recognized. The uncontrolled design resulted in a relatively high rate of drug discontinuation. However, this phenomenon is a common finding in studies done in open populations assessing the adherence to different lipid-lowering medications \[[@B25]\]. To overcome this limitation, an intention to treat analysis was used. Also, the lack of a run-in period in which the effect of diet could be measured and the absence of information about the adherence to the diet prevented us from discerning to what extent the observed result was due to fibrate alone. Finally, some of the conclusions, like the identification of the determinants of the lipid response, came from a post hoc analysis.
In conclusion, ciprofibrate is effective in the treatment of patients with hypertriglyceridemia / low HDL cholesterol. Significant reductions in triglycerides and non-HDL cholesterol resulted from ciprofibrate therapy. In addition, higher HDL cholesterol levels were found at the end of the treatment. Excess body weight alters the lipid response to ciprofibrate. A greater non-HDL cholesterol lowering is achieved in subjects with excess body weight compared to that found in lean individuals. Controlled trials are needed to compare the lipid-lowering effects of ciprofibrate in groups of subjects defined by their adiposity or other markers of insulin resistance.
Competing interests
===================
This study was supported by an educational grant provided by Sanofi-Synthelabo. It included the study expenses and it will cover the article processing charge. No other competing interest need to be declared
Author contributions
====================
CAAS participated in the design of the study, performed the statistical analysis and drafted the manuscript. AALV participated in the design of the study and in the preparation of the manuscript. FJGP participated in the design of the study and in the preparation of the manuscript. All other authors were responsible of the inclusion and follow-up of the study subjects. All authors read and approved the final manuscript.
Acknowledgments
===============
This study was supported by an unrestricted grant from SANOFI-Synthelabo
| PubMed Central | 2024-06-05T03:55:46.605268 | 2004-7-23 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC503398/",
"journal": "Cardiovasc Diabetol. 2004 Jul 23; 3:8",
"authors": [
{
"first": "Carlos A",
"last": "Aguilar-Salinas"
},
{
"first": "Andréia",
"last": "Assis-Luores-Vale"
},
{
"first": "Benjamín",
"last": "Stockins"
},
{
"first": "Hector Mario",
"last": "Rengifo"
},
{
"first": "José Dondici",
"last": "Filho"
},
{
"first": "Abrahão Afiune",
"last": "Neto"
},
{
"first": "Lísia Marcílio",
"last": "Rabelo"
},
{
"first": "Kerginaldo Paulo",
"last": "Torres"
},
{
"first": "José Egídio Paulo de",
"last": "Oliveira"
},
{
"first": "Carlos Alberto",
"last": "Machado"
},
{
"first": "Eliana",
"last": "Reyes"
},
{
"first": "Victor",
"last": "Saavedra"
},
{
"first": "Fernando",
"last": "Florenzano"
},
{
"first": "Ma Victoria",
"last": "Hernández"
},
{
"first": "Sergio Hernandez",
"last": "Jiménez"
},
{
"first": "Erika",
"last": "Ramírez"
},
{
"first": "Cuauhtémoc",
"last": "Vazquez"
},
{
"first": "Saul",
"last": "Salinas"
},
{
"first": "Ismael",
"last": "Hernández"
},
{
"first": "Octavio",
"last": "Medel"
},
{
"first": "Ricardo",
"last": "Moreno"
},
{
"first": "Paula",
"last": "Lugo"
},
{
"first": "Ricardo",
"last": "Alvarado"
},
{
"first": "Roopa",
"last": "Mehta"
},
{
"first": "Victor",
"last": "Gutierrez"
},
{
"first": "Francisco J",
"last": "Gómez Pérez"
}
]
} |
PMC503399 | Background
==========
Sarcomas arising from the retroperitoneum are rare tumors, accounting for 10--15% of all soft tissue sarcomas \[[@B1]\]. Liposarcoma is the single most common soft tissue sarcoma and accounts for at least 20% of all sarcomas in adults \[[@B1]\]. Classification of liposarcoma into four types, based on morphologic features and cytogenic aberrations, is now widely accepted \[[@B2]\]. These four types are (a) well differentiated; (b) dedifferentiated; (c) myxoid/round cell and (d) pleomorphic. The extent of differentiation, as reflected by histological grade, remains the most important determinant of clinical course and of ultimate prognosis for patients with liposarcoma after resection. The following case illustrates the great morphological and biological heterogeneity of these tumors. A very rapid recurrence was observed, and this recurrence was considerably less heterogeneous than the primary tumor, consisting mainly of the calcific, nonlipomatous component.
Case presentation
=================
A 65-year-old male presented with a three-week history of progressively worsening abdominal distension. He denied any abdominal pain but stated that he noticed an increased frequency of bowel movements. His past medical history was unremarkable. On examination, he was afebrile and had a hugely distended abdomen with an immobile, nontender mass occupying all four quadrants of the abdomen. Computed tomographic (CT) scan revealed a large, heterogeneous lobulated mass occupying most of the abdomen (Figure [1](#F1){ref-type="fig"}). The peripheral component appeared lipomatous and the margins of this component were difficult to estimate accurately. There was also a heterogeneous nonlipomatous component that contained areas of lesser density, as well as a central stellate region of calcifications. The preoperative differential diagnosis included a retroperitoneal sarcoma (especially dedifferentiated liposarcoma), desmoid tumor, undifferentiated carcinoma, carcinoid or sclerosing mesenteritis; lymphoma was also considered.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
CT appearance at multiple cuts (A -- E) of a huge retroperitoneal mass with lipomatous and nonlipomatous components. The nonlipomatous component (arrow X, Panel D) contains calcific elements. Note the posterior extension of the lipomatous component (arrow Y, Panel D), which extends superiorly (Panels A -- C). The exact boundaries of this component are difficult to appreciate on CT.
:::
:::
While neoadjuvant chemotherapy and radiation comprise a frequent approach for retroperitoneal sarcomas at our institution, the extent of the tumor made this approach unfeasible. Resection was therefore planned unless an intraoperative biopsy revealed lymphoma. Resection was accomplished through a T-type incision (Figure [2](#F2){ref-type="fig"}), and entailed removal of the right kidney, terminal ileum, ascending colon, sigmoid colon and the left spermatic cord structures, all of which were intimately attached to the mass. Encasement of the external iliac artery and vein was also encountered near the end of the procedure. This was not fully appreciated preoperatively, as that component of the tumor was so much less conspicuous on CT than the rest of the tumor, given its fatty consistency (Figure [1E](#F1){ref-type="fig"}). The mass was split in half to facilitate dissection from the iliac vessels. An anastomosis was constructed from descending colon to rectum. A transverse colon mucous fistula and an ileostomy were brought out, as the ileum was dusky at the end of the procedure.
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Operative exposure of the tumor through a T-type abdominal incision.
:::
:::
The tumor was submitted for histological examination in two parts measuring, 30.0 × 10.0 × 6.5 cm and 32.0 × 20.0 × 10.0 cm, weighing 19.8 kg in total (Figure [3](#F3){ref-type="fig"}). On gross examination the mass was variegated, with fleshy and solid cystic degeneration containing areas of osseous consistency. Microscopic examination revealed a juxtaposition of well differentiated liposarcoma and a spindle cell sarcoma with heterologous chondrosarcomatous elements consistent with a dedifferentiated liposarcoma (Figure [4](#F4){ref-type="fig"}).
::: {#F3 .fig}
Figure 3
::: {.caption}
######
*En bloc*resection specimen of heterogeneous tumor with attached organs. Note the lipomatous regions (A), the calcified areas (B), and the remaining nonlipomatous component (C).
:::
:::
::: {#F4 .fig}
Figure 4
::: {.caption}
######
Histologic appearance of various elements of the tumor as sampled in different regions. A. Well differentiated liposarcoma. B. Low grade spindle cell component. C. Cellular spindle cell component. D. Chondrosarcomatous component.
:::
:::
The patient was discharged from hospital on the seventh postoperative day. He was followed in the surgical oncology outpatient clinic monthly. Four months after resection the patient had a follow-up CT scan, which demonstrated an intra-abdominal recurrence consisting almost completely of a calcified, nonlipomatous tumor (Figure [5](#F5){ref-type="fig"}). The patient died one month later.
::: {#F5 .fig}
Figure 5
::: {.caption}
######
CT appearance at multiple cuts (A -- D) of the recurrent tumor. The recurrence was more homogeneous than the primary tumor, consisting almost completely of the calcific component of the nonlipomatous portion of the tumor.
:::
:::
Discussion
==========
The patient in our case manifested the dedifferentiated variant of liposarcoma. The term \"dedifferentiated liposarcoma\" refers to the development of a high grade nonlipogenic sarcoma juxtaposed to a well differentiated liposarcoma \[[@B3]\]. The majority (80 -- 90%) occur primarily *de novo*, although secondary dedifferentiation can occur with multiple recurrences of a well differentiated liposarcoma \[[@B4]\]. CT and Magnetic resonance imaging scans typically reveal well defined nonlipomatous masses associated with fatty tumor; the transition between the two components is characteristically abrupt, although blended transitions are seen in about 20% of cases \[[@B5]\]. Calcifications appear in about 30% and usually correspond to osseus metaplasia, although they may represent osteosarcomatous or chondrosarcomatous elements. The most frequent phenotype of dedifferentiation is that of a high grade pleomorphic malignant fibrous histiocytoma-like sarcoma \[[@B4],[@B6]\]. Other phenotypes observed include leiomyosarcomatous, rhabdomyosarcomatous, osteosarcomatous and angiosarcomatous elements, as well as other nonlipogenic elements \[[@B3],[@B7]\]. A further distinctive pattern in some cases is the presence of micronodular spindle cell whorls, often associated with ossification \[[@B8]\].
Among liposarcomas, the presence of features of the dedifferentiated variant strongly portends a worse prognosis. The overall 5-year survival of dedifferentiated liposarcomas is 20%; the 5-year survival of well differentiated liposarcomas is 83% \[[@B9]\]. Dedifferentiated liposarcomas recur locally in 40 -- 83% and distant metastases appear in 15 -- 30% \[[@B4],[@B7],[@B9]\]. Therefore, histomorphologic features impact outcomes related to retroperitoneal liposarcomas.
While generally the phenotype of the nonlipogenic component does not impact prognosis of dedifferentiated liposarcomas, the presence of calcifications has been identified as an adverse prognostic factor \[[@B5]\]. In the present situation, it is obvious that the biologically most aggressive component consisted of the calcified (chondrosarcomatous) component. That is, the recurrence was less heterogeneous than the primary tumor, as it had widespread and dense calcifications, but no obvious lipomatous elements.
Complete resection of the tumor is perhaps the most important factor determining long-term survival. Unfortunately, the rate of complete respectability is only about 53% \[[@B10]\]. As illustrated in the present case, in addition to the limitations imposed by the retroperitoneal anatomy, another obstacle to successfully obtaining margins is the difficulty in distinguishing normal retroperitoneal fat from the lipogenic component of the tumor \[[@B9]\]. This was illustrated by the underestimation of the extent of the tumor around the iliac vessels. Moreover, the intraoperative decision to remove the kidney was made in view of the difficulty in distinguishing normal perinephric fat and neoplastic fat; kidney was not involved with tumor, once examined microscopically. Indeed, in a series of retroperitoneal liposarcomas from Memorial Sloan-Kettering Cancer Center, nephrectomy was performed in 38% of patients, although the number in which kidney was actually involved on pathology was not reported \[[@B9]\]. Thus, anatomical constraints and difficulty distinguishing more differentiated fatty tumor from normal fat limit the surgeon\'s ability to confidently and completely remove all neoplastic elements.
Conclusions
===========
Dedifferentiated liposarcomas represent aggressive variants of liposarcomas. Each morphological element of these heterogeneous tumors may manifest completely different biology. The overall biological behavior of dedifferentiated liposarcomas is likely dictated by the most aggressive element, which typically resides in the nonlipomatous portion of the tumor.
Competing Interests
===================
None declared.
Authors Contributions
=====================
SK, HB, WT, and OB made substantial contributions to the intellectual content of the paper, in the interpretation of data, and in drafting the manuscript. All authors read and approved the final manuscript.
Acknowledgements
================
Written consent was obtained from the patient\'s relatives for publication of this study.
| PubMed Central | 2024-06-05T03:55:46.607064 | 2004-7-23 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC503399/",
"journal": "World J Surg Oncol. 2004 Jul 23; 2:25",
"authors": [
{
"first": "Shahzeer",
"last": "Karmali"
},
{
"first": "Halligrimur",
"last": "Benediktson"
},
{
"first": "Walley",
"last": "Temple"
},
{
"first": "Oliver F",
"last": "Bathe"
}
]
} |
PMC503400 | Background
==========
The growing concern for the benefits from health research to be studied
-----------------------------------------------------------------------
Health research funding bodies are under increasing pressure to demonstrate the outcomes, or benefits, of the research that they fund \[[@B1]-[@B6]\]. Traditional peer review of research focussed on the outputs in terms of journal articles, the training of future researchers and the development of careers. These are still seen as important, but in some analyses they have been merged into broader multi-dimensional categorisations of the benefits from health research \[[@B7],[@B8]\].
The onus hitherto has been on public sector funding bodies. There is a general recognition in the UK, however, of the importance of the role of the medical charities: they fund approximately one third of UK medical research -- a level \'unparalleled elsewhere in the world and nor is it found in other areas of science\' \[[@B9]\]. Therefore, in an era of accountability, public involvement in research issues and growing competition for contributions, some medical charities see the virtue in being able to demonstrate the outcomes of the research they fund. Not all the pressures, however, are external and some funding bodies, including the Wellcome Trust, which is an endowment and not collection-based charity, are being pro-active in their attempts to identify and track the outcomes of the research they fund. One factor relevant for both public sector and charity funding bodies is the recognition that assessing the benefits from their research may assist in identifying research strategies most likely to produce benefits \[[@B2],[@B7],[@B10],[@B11]\]. Concerns such as those above led a UK medical charity, the Arthritis Research Campaign (ARC), to approach RAND Europe with the idea of conducting an assessment of the long-term outcomes from research that they have funded.
The purpose of this paper is to set out the aims of the study and the methods being adopted. In particular, it will show how an existing generic approach to the assessment of benefits from health research \[[@B7],[@B12]\] has been adapted to meet the needs of this specific study. After providing the background to the study, the paper describes the methods to be adopted. These were initially agreed after a consultative phase in which the evaluative framework was refined on the basis of interviews with six key actors who have played various roles within ARC and advice from ARC\'s Development Committee, which acts as the steering group for this project. They were then confirmed following a pilot stage in which three case studies were conducted; some examples from the pilot studies, which endorsed the feasibility of the proposed approach, will be given to illustrate the account of the methods.
The increasing attention on musculoskeletal conditions and the role of the Arthritis Research Campaign (ARC)
------------------------------------------------------------------------------------------------------------
Attention is being drawn to the increasing scale of the burden of musculoskeletal conditions, and the associated costs, in various ways including through the establishment of the Bone and Joint Decade 2000--2010 and the recent collaborative report with the World Health Organization (WHO) \[[@B13]\]. At the same time, there is a realisation that the benefits of research in this area are sometimes less immediately apparent than in some other fields. For example, two classic studies of the economic benefits from biomedical research \[[@B14],[@B15]\] both highlighted arthritis as an area where research and higher medical care expenditure may have comparatively little impact on mortality. Furthermore, one recent attempt to put a monetary value on the benefits from health research in Australia \[[@B16]\] adapted a method developed in the USA \[[@B17]\] and again demonstrates the difficulties of undertaking such analysis in the musculoskeletal field. These observations might suggest that a more careful and wide-reaching assessment of benefits from research is particularly needed in the field of arthritis.
ARC is the leading medical charity in this field in the UK and one of the largest collection-based medical charities in the UK. The Research Outputs Database (ROD) records the funding acknowledgements on all UK biomedical papers contained on the citations indices of the Institute for Scientific Information \[[@B18]\]. Analysis conducted on ROD reveals ARC to be, \'in a dominant position within the UK in the arthritis subfield\' \[[@B19]\]. ARC\'s funding is associated with more arthritis publications than that from either the Medical Research Council (MRC) or the Wellcome Trust. It spent almost £22 million in the year 2001--2002; its major aim \'is to support the highest quality research into the cause, cure and treatment of arthritis and musculoskeletal diseases\' \[[@B20]\]. It adopts a variety of funding modes for a range of types of research including its support of two research centres. The preliminary interviews, described above, highlighted the importance of the work by Marc Feldmann and Sir Ravinder Maini at one of these, the Kennedy Institute of Rheumatology in London, in developing anti-Tumour Necrosis Factor (anti-TNF) therapy as an effective treatment for rheumatoid arthritis and other autoimmune diseases. The pair won the 2003 Albert Lasker Award for Clinical Medical Research for this discovery.
Objectives of the evaluation/research questions
-----------------------------------------------
Within the general climate of an increased emphasis on the outcomes from research, four main objectives were specified for the particular study described in this article:
• Review and document outcomes for ARC research grants
• Illustrate the strengths and weaknesses of different modes of research funding
• Identify factors associated with translation of research, and attempt to develop \'early indicators\' of likely successful translation
• Identify \'good news stories\' and vignettes of the research process for use by ARC in public engagement and fund raising activities.
Methods
=======
Rationale for using a case study approach
-----------------------------------------
Traditional methods of peer review have long been favoured by medical research funding bodies for evaluating research, but bibliometric methods have had a variable history. An early move by the National Institutes of Health (NIH) to establish a publications\' database was cancelled by the 1980s \'as too expensive for the management information it produced\' \[[@B21]\]. The MRC in the UK reviewed the possibility of making greater use of bibliometric measures to inform per review of major long term programmes, but the steering group established to oversee the review concluded that, \'bibliometric analysis would not add sufficient value to peer review to be worthwhile routinely and should not be introduced into MRC procedures\' \[[@B22]\]. Nevertheless, there are circumstances where bibliometric analysis can provide research funding bodies with useful information \[[@B19]\] and, as discussed below, they can be incorporated into broader case studies. On its own, however, it is unlikely to provide much information about the longer-term outcomes from research funding.
The Economic and Social Research Council (ESRC) in the UK commissioned a project to identify the impact of their research on non-academic audiences. It involved tracing the activity of the participating researchers after their projects ended and mapping the networks of researchers and relevant non-academic users and potential beneficiaries \[[@B23]\]. The study concluded that the preferable way to determine and assess the existence of impacts of socio-economic research on non-academic audiences \'is through detailed, project-by-project qualitative analysis\' \[[@B23]\]. Such an approach probably entails adopting a case study approach, and there is a long history of applying the case study approach to examine the utilisation of research \[[@B24]\]. Indeed, where the emphasis is on demonstrating the outcomes from health research, a case study approach has mainly been used \[[@B7],[@B25]\] and been recommended for use in future studies \[[@B4],[@B26],[@B27]\].
Case studies will enable narratives or stories to be told to illuminate how the research funded in the early 1990s was translated (or not) into practice; each case, therefore, could potentially provide an illustrative example of the outcomes from ARC research. Furthermore, the planned 16 case studies will be based on a variety of modes of ARC research funding and types of research. They will also be organised using a common structure. This should enable cross-case study analysis to demonstrate (via illustrative case studies) the strengths and weaknesses of different modes of funding and categories of research. It should also facilitate the identification of factors associated with the translation of research, perhaps through various phases, into policies, products, and clinical practice that produce a health gain.
The evaluation framework described below was developed in a way that incorporates previous experience and knowledge on these issues \[[@B7],[@B12],[@B24],[@B26],[@B28]\]. This should ensure that questions are asked about a range of factors that previous experience suggests are likely to be related to the translation of research. Additionally, because the evaluation framework includes a multi-dimensional categorisation of benefits from research, the full range of outputs and outcomes relevant to different types of research, and modes of funding, will be looked for in the studies. The case studies will, in part, be conducted to see if they produce evidence consistent with existing hypotheses about factors linked to the translation of research and the role of different modes of research funding and types of research. But they will also be exploratory and should allow the generation of new hypotheses, particularly ones specifically relevant for research funding from a medical charity.
Timescale
---------
In deciding the time window to use for selecting case studies, a compromise usually has to be made between the quality of records/likely ability of researchers to recall their activities and the selection of grants whose outputs have had sufficiently long to develop \[[@B29]\]. The latter point was important in this study because the aim was to move beyond considering traditional outputs and also examine outcomes such as health gains. ARC instituted a new computerised database during the early 1990s and all their grants awarded since 1990 are held on this database. Prior to this, only paper records of unknown completeness were available. As an appropriate compromise between the various factors, we therefore decided to select grants that were awarded between 1990 and 1994.
Selection of cases
------------------
Within a case study approach it is unlikely that the selection of cases will follow a straight-forward sampling logic in which those selected are assumed to be representative of a larger group \[[@B30]\]. Nevertheless, in adopting a multi-case approach the project aims to ensure not only that the benefits from the full range of modes of funding and types of research can be illustrated, but also that there is scope for some cross-case analysis. The selection of cases will, therefore, be somewhat purposive. Case studies based on four modes of funding will be included: institute grant, programme grant, project grant and fellowships. ARC-funded researchers will also be divided into three groups on the basis of their qualifications: basic researchers, clinical researchers, and Allied Health Professionals (AHP) such as physiotherapists. In their classic case study analysis of research utilisation, Yin and Moore \[[@B24]\] went to considerable lengths to ensure that they were including only studies where it was thought there had been utilisation. We do not propose to go that far, but, given that the idea is to illuminate the outcomes, it is considered desirable to concentrate on studies where it is thought there is a reasonable chance that there will be something to show. When examining the outcomes of research, even a stratified sampling approach is not thought to be sufficient because most impact usually comes from a small number of studies \[[@B23]\].
As a first step, we shall identify all publications in the relevant period from the principal investigators awarded ARC funds. Then, the researchers will be classified according to the journal impact factors (see below) of the journals in which their articles appear. The aim will be to draw up shortlists of possible researchers to include in the study: those in the top decile and those the middle of the range, with the final selection made on the basis of advice from ARC\'s Development Committee.
Organisation of data collection
-------------------------------
For case studies it is appropriate to use multiple sources of evidence converging on the same issues \[[@B4]\] and adopt a process of triangulation \[[@B27],[@B30]\]. Three main interdependent methods will be used: documentary and literature review; semi-structured interviews with key informants; and bibliometric analysis. They will be applied in a partially overlapping way.
### Documentary and literature review
We will read key project documents including the original research grant proposals, referees\' reports and end of project reports. On the basis of the end of year reports from researchers, and the interviews (see below), we will also identify and read the core publications attributed to the research grant and any subsequent publications such as key citing papers, relevant clinical guidelines etc.
### Semi-structured interviews with key informants
There will be about three interviews per case study. They will be based on a semi-structured interview schedule informed by the evaluation framework described below. They will, therefore, explore the origins of the research and the primary outputs such as the publications. In this way the initial list of publications identified as being related to the project will be refined. Furthermore, there will be a full exploration not only of the contribution to research training and career development, but also of any translation of the research findings into product development, policy and practice. In each case study the initial interviews will be with members of the relevant research team. Then snowballing techniques will be used to identify the people who might be able to provide most information about how the research has influenced subsequent research or been translated into product development, policy and practice.
### Bibliometric analysis
Bibliometric approaches can play a useful role in the analysis of the research funded by specific biomedical research-funding bodies \[[@B19],[@B31]\]. In the current analysis, the list of research papers published as a result of the project will first be refined as described above. Following that, bibliometric analysis will be conducted to record various matters including: the full funding acknowledgements; number of authors; citation counts; and comparison of number of citations with the journal impact factor of the publishing journals. This analysis will be conducted by a further part of the research team: those responsible for maintaining the ROD described above.
### Clearance and validation
In every case a draft copy of the case study report will be sent to the principal investigator for comment. Such a step is an important part of the validation process and not just a matter of professional courtesy \[[@B24]\].
Evaluation framework for ARC case studies
-----------------------------------------
There are two elements in the evaluation framework adopted to organise the case studies being conducted in the assessment of the outcomes from ARC-funded research. Building on the framework developed by Buxton and Hanney \[[@B7],[@B12]\], the two elements consist of a multi-dimensional categorisation of benefits from health research, and a model of how best to assess them. A logic model such as this helps facilitate assessment rather than pretending to be a precise model of how research utilisation occurs. The framework has been developed in various ways to meet the particular circumstances of ARC-funded research, which is often basic and investigator-led.
There are many steps involved in assessing outcomes from research. One of the key advantages in taking a detailed approach, such as that described below, is that it enables the issue of the counter-factual to be addressed. In other words, what would the world have looked like without the specific research being examined?
The categories of payback
-------------------------
The multi-dimensional category of payback provides the evaluation criteria for the outputs and outcomes from ARC funding. The 5 main categories are:
a\) Knowledge production
b\) Research targeting, capacity building and absorption
c\) Informing policy and product development
d\) Health benefits
e\) Broader economic benefits.
Each can be considered in turn, with various sub-categories explored and possible measures described.
### Knowledge production
The knowledge produced by research is the first output and is contained in various publications and patent applications. Any type of publication can be considered, but it is generally thought that peer reviewed articles are the most important and, at least for biomedical research in industrialised countries, it is thought reasonable to assume that the overall output of research publications is fairly represented by peer-reviewed papers in international journals \[[@B19]\]. In addition to counting the number of publications, their quality and their impact can be assessed in various ways. The quality of knowledge production has traditionally been assessed by peer review, but various other methods can be applied. Papers that are accompanied by an editorial are often seen as being of particular significance. For those studies that are included in a systematic review there are now formal quality assessment techniques \[[@B32]\], as there are for reviews appearing in an overview \[[@B28]\].
Citation analysis can be applied to assess the impact the specific article is having within the research community \[[@B33],[@B34]\]. Previous experience suggests that knowledge production will be particularly important for basic research, and certainly, on average, papers in basic research journals tend to be cited more frequently than ones in clinical journals \[[@B19],[@B35]\].
A journal\'s \'impact factor\' is based on the average number of times an article in the journal is cited; it can provide a short-hand version of citation analysis by giving some indication of the importance of the journal in which an article appears. The use of impact factors in analysis of biomedical research has been criticised \[[@B36]\] but, provided care is taken \[[@B37]\], it has been shown to be of some value \[[@B19]\].
Particularly when considering research that might be aimed at potential users outside the research community, it is often desirable to use a range of publication outlets including those journals with the highest readership among the groups at whom the research is targeted. In some fields these might well be journals that do not have an impact factor but are, nevertheless, significant as vehicles for dissemination of the knowledge produced \[[@B38]-[@B40]\].
### Research targeting, capacity building and absorption
The better targeting of future research is frequently a key benefit from research, especially from research that is more basic and/or methodologically oriented. An indication of this comes from citation analysis. The enhanced targeting can be of the research conducted both by others and by the original researcher(s). Where follow-on research, especially by members of the original research team, is clearly associated with the original research it can be useful to obtain information on the source and amount of such funding \[[@B39]\]. As is developed in the paragraph below, one of the key roles of a medical charity can be to fund research in its field that will help to open up questions/issues that will then attract further funding from the general research funders such as the MRC and the Wellcome Trust.
Research training can be provided both as a result of the employment of staff on research projects and programmes, and through explicit funding for research training and career development \[[@B1]\]. One measure of research training, which may appear crude but has nevertheless been used in previous studies, is the number and level of higher or research degrees resulting, either totally or in part, from the research funding \[[@B1],[@B14],[@B39],[@B41]\]. The career development of arthritis researchers goes much wider than specific training and is of considerable importance to ARC which aims to ensure that the pool of researchers in this field is a strong as possible. The reasoning is that this, in turn, should help ensure that arthritis as a topic is able to gain an appropriate share of the research funding available from general medical research funders. Some of ARC\'s funding schemes aim explicitly to provide career development, and for other researchers the receipt of a project grant from ARC can be important in advancing their career in research. Interviews can address this. Furthermore, they may also enable us to consider how far career development based on ARC funding helps propel some researchers into positions within the health-care system where they can play a role in ensuring that the later stages of translating research findings into outcomes are achieved.
### Informing policy and product development
Research can be used to inform policymaking in a wide range of circumstances and the key issue is that policymaking involves those in positions of authority making choices that have a special status within the group to which they apply \[[@B27]\]. Policymaking is interpreted very broadly here and refers not just to national policies of the government, but also includes: policies made by managers at many levels within a health service; policies agreed at national or local level by groups of health-care practitioners in the form of clinical or local guidelines; policies developed by those responsible for training/education/inspection in various forms including training packages, curricula and audit and evaluative criteria \[[@B3]\]; and policies about media campaigns run by health-care providers. Basic research is less likely than that from clinical researchers or AHP to be used to inform policy. Various methods have been proposed for analysing the impact of research on health policymaking, including documentary review and interviews \[[@B26],[@B27]\].
The position of systematic reviews is a little complex. They are themselves a form of research, but inclusion of a study in a systematic review is a form of secondary output and might lead on to further use.
At a similar level, although involving very different processes, research can also be used to inform product development \[[@B38]\]. Informing policies and product development are conceptually similar in that there generally has to be some subsequent adoption of the policy, or product, before the health and economic benefits can accrue \[[@B7]\].
### Health benefits
Benefits in terms of health gains might be viewed as the \'real\' payback or outcomes from health research. Greater effectiveness of health-care resulting from research-informed drugs or procedures should lead to increased health. Various measures of health gain exist, but for arthritis the emphasis, in most cases, is likely to be on those that assess reduction in pain or disability, and increase in mobility. While the benefits from arthritis research will not generally be measured in terms of life years gained, in some circumstances they might be captured by using Quality Adjusted Life Years (QALYs). This is often seen, in countries such as the UK, as a more appropriate approach than using Disability Adjusted Life Years (DALYs) \[[@B42]\]. There have been recent attempts to put a monetary valuation on the reduction in mortality and morbidity as a result of health research \[[@B16],[@B43]\], but that is not being proposed for this study. At an overall level, it is possible that figures for the potential population who could benefit from the new drug or procedure could be identified, along with information about the level of benefit that individual patients might receive. If knowledge about adoption levels was then also taken into consideration it might be possible to indicate overall levels of benefit.
This category of benefits can be thought of as going wider than health gain, and some aspects can be seen as benefits to the health sector more generally. Cost savings in the provision of health-care may result from research-informed changes in the organisation of services or in the particular therapies delivered. It might be necessary to consider various issues here. These include whether potential savings have in practice been realised -- either as cash savings or as the release of resources for other valuable uses \[[@B44]\]. Furthermore, it would be important to check whether costs are not simply being transferred elsewhere. Improvements could also arise in the process of health-care delivery and these could be measured by techniques such as patient satisfaction surveys \[[@B7]\].
### Broader economic benefits
A range of benefits can accrue to the national economy from the commercial exploitation of research. These can take the form of employment and profits resulting from the manufacture and sale of drugs and devices \[[@B45]\]. The national economy could also benefit from exports and/or import substitution \[[@B46],[@B47]\].
Whilst there is a danger of double counting, it is probably also important to adopt a human capital approach and focus on the value of production gained from having a healthy workforce. This can be measured by examining the reduction in days off work. Typically, in a human capital approach, potential future earnings are calculated for people who, as a result of advances in medical research, can continue to contribute to national production \[[@B14],[@B15],[@B48]\]. Those who use it, however, share the concerns that such an approach to assessing the benefits from research could have equity implications in that it would seem to favour research relevant for those of working age. This concern might be relevant here, in that many who suffer most from arthritis are retired, but reducing the days off work caused, for example, by low back pain, could be important. The economic burden of low back pain has been identified \[[@B49]\] and the potential role of research in reducing it was recently highlighted in a wide-ranging discussion of the benefits from medical research in the USA \[[@B50]\].
Model for assessing the outputs and outcomes
--------------------------------------------
The second element of the evaluation framework is the logic model. Its various stages are shown on Figure [1](#F1){ref-type="fig"} and provide a way of organising the case studies. At least seven stages and two interfaces are identified and although they are presented in a linear form, the reality is much more complicated and there is also considerable feedback \[[@B7],[@B12]\].
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Model for Organising the Assessment of the Outcomes of Health Research.**Sources: Adapted from previous versions of the Buxton/Hanney model for assessing the payback from health research \[12,27\].
:::
:::
Stage 0: Topic/issue identification
Interface A: Project specification and selection
Stage 1: Inputs to research
Stage 2: Research processes
Stage 3: Primary outputs from research
Interface B: Dissemination
Stage 4: Secondary outputs -- policymaking and product development
Stage 5: Adoption by practitioners and public
Stage 6: Final outcomes
While it is not possible totally to tie the categories of benefits to certain stages of the model, it is possible to identify broad correlations: categories a) and b) (knowledge and research benefits respectively) are together considered to be the primary outputs from research; category c) (informing policy and product development) relates to the secondary outputs; and categories d) and e) (health and broader economic benefits respectively) are the final outcomes. This approach can be incorporated into the analysis of each stage in turn as is set out below, where a few examples, drawn from the pilot studies, are used to illustrate how the framework seemed to be working in practice but could be refined in certain ways.
### Stage 0: Topic/issue identification
The topic or issue identification stage covers the generation of the original ideas for the research. Its nature can vary considerably depending on whether the main driving force is internally generated by the researcher, or externally generated \[[@B27]\]. Most ARC funding falls into the former category: for many researchers the topics will be curiosity-driven and based on examination of the existing stock or pool of knowledge and opinions about where gaps, and/or opportunities, exist and further research could advance understanding. Such factors will also inform more clinical and AHP researchers, but here consideration of clinical needs could also be a factor and might be based on personal experience of treating patients, as became clear in the interview with the principal investigator in one of the case studies. Where research topics are externally generated, the identification of the issue comes from a process of needs assessment that could involve analysis either just within the scientific community or more widely. In the latter case, many groups could be involved. These include not only members of the wider research community and representatives of research funding bodies, but also potential users and beneficiaries of the research drawn from some combination of the wider political, professional, industrial and societal environment.
### Interface A: Project specification and selection
The nature of the activities at Interface A will vary depending on the type of issue identification. Where the topics are externally generated, there are potential difficulties in ensuring both that the research community is actively engaged with the priorities that have been identified and that the project specification meets the needs as identified \[[@B27]\]. Where the issues are internally generated, the interface involves traditional processes of the researcher developing a detailed proposal and submitting it for peer review. Most of the issues are internal to the scientific world, but there is still a key interface between individual researchers and ARC as the research-funding body. Documentary analysis of ARC files provided information in the pilots that sometimes highlighted issues about how far the proposal was subject to changes as a result of the review process. It also proved useful, however, to supplement this with questions in the interviews.
### Stage 1: Inputs to research
It can be important to consider not only the financial inputs, including any beyond the specific ARC funding, but also the experience of the research team and the knowledge base on which they built. Part of the idea behind examining any other funding brought in to support ARC research is again to see how far ARC funding is helping to facilitate the funding of arthritis research by general funders of health research: is ARC funding studies that produce findings that others believe are worth further investigation? The pilot studies confirmed that the complexities of identifying the exact funding streams behind any piece of research were best addressed by using a case study approach involving initial documentary review and following up issues in interviews. The pilots involved a case where other contributory funding contributed to what was clearly an ARC project, and therefore little attempt was made to portion out credit for outcomes to any funder other than ARC. In another case, however, the research was part of a stream of ARC-funded work and an effort was made to try to draw boundaries around what would be appropriate to include in the case study.
### Stage 2: Research processes
Consideration can be given to how appropriate the proposed methods for a study turned out to be, and whether any difficulties were encountered. In some cases it could be relevant to explore how far potential users were involved at this stage. It is possible that difficulties identified at this stage could explain later problems with translation or uptake of the research findings.
### Stage 3: Primary outputs from research
Knowledge production, as represented by the various types of publications, is a major primary output from the research. Various ways of measuring this were discussed above. The pilots also showed that the interviews used to refine the lists of publications from the specific funding in question, could also sometimes help to identify where non-conventional sources were being used as outlets for publications. Most of the primary outputs will feed into the stock of knowledge.
The research benefits in terms of targeting future research represent either feedbacks to further research conducted by team members, or findings that feed into the stock of knowledge and help target future research of others. An example from one pilot study showed not only how the principal investigator used her project to inform her own further work, but was also able to contribute to a much larger collaborative project. Interviews in another study showed that the research had informed considerable further work in industry, but as yet this had not led to any product development. Under the framework being used, it is possible to give that ARC-funded work considerable credit for informing the further research, but record its limited impact at the subsequent stages.
Capacity building can also be seen as a primary output. Accounts were given, in pilot study interviews, of the research training and higher degrees that resulted from the research.
### Interface B: Dissemination
Dissemination is usually seen as being somewhat more active than the mere production of academic publications containing the knowledge. There are, however, clear overlaps between some activities. Sometimes it is possible to record not just dissemination activities but also the successful transfer of research findings to potential users in the political, industrial, professional environment and wider society. Previous analyses of how to increase the implementation of research findings \[[@B28]\] will help inform the issues being examined in the case studies at the dissemination and later stages. Presentations to potential academic and user groups, and media activities, are major ways of disseminating findings, as are the production of brief summaries of findings targeted at specific user groups. In previous case studies, attention has also focused on the way some researchers conduct study days, or training, based on the approach developed by their research and these can be highly effective dissemination mechanisms \[[@B51]\]. The pilots provided an example of the importance of this and, indeed, of the role of individual researchers in networking and disseminating information.
### Stage 4: Secondary outputs--policymaking and product development
As noted above, policymaking and product development activities can result in a wide range of secondary outputs, and various methods are needed to identify research-informed policies. In one case study, a review of a database revealed that one project had been cited in a clinical guideline unbeknown to the research team, whereas in another pilot it took interviews to identify that the research was informing local guidelines and care pathways. The use of the research in systematic reviews was also revealed in various ways in the pilot studies. Where the research seems to have resulted in secondary outputs it is useful to explore the factors that have led to this.
In relation to product development, if research findings are incorporated into the process of developing a product, for example a new drug for arthritis, this can be seen as an important secondary output. In the preliminary set of interviews, most people referred to how ARC-funded research had played a key role in the production of anti-TNF therapy for arthritis. In a pilot study, interviews revealed the extent to which industry\'s attempts to use one stream of research for product development had not, so far, been successful.
### Stage 5: Adoption by practitioners and public
For the research findings incorporated into secondary outputs to result in final outcomes there usually has to be some behavioural change by practitioners, and/or the public. This may involve take-up of new drugs or procedures as set out in a secondary output such as a guideline from the National Institute for Clinical Excellence (NICE). Sometimes the adoption comes as a direct result of the primary outputs, as when clinicians -- often at the cutting edge -- decide to implement research findings even prior to the development of clinical guidelines. Either way, it is important to try to establish the adoption or take-up rates and to explore how far the behavioural change can be attributed to the specific research findings, as opposed to other factors such as a more general change in climate of opinion in relation to, for example, the importance of exercise. In one pilot study where interventions based on research filtered into practice, a series of interviews was used to attempt to identify both the precise role of the specific ARC-funded project and possible levels of uptake.
The role of the public in responding to informed advice -- often research-based -- is seen as increasingly important, especially in a field such as arthritis \[[@B52]\]. Various factors can be explored here. These include the extent to which patient behaviour might change as a result of interactions with health-care providers who promote research-based messages, and how far the public might respond directly to publicity about research findings when they are used, for example, in media campaigns encouraging participation in preventative activities \[[@B28]\].
### Stage 6: Final outcomes
The final outcomes are the health and broader economic benefits identified in categories d) and e) above. These are increasingly seen as being the ultimate goal of health research funding, but their precise estimate in practice often remains difficult \[[@B5]-[@B7]\]. In one pilot study, it was possible to produce audit figures from one area where there is known to have been local implementation of the research findings.
Planned analysis and synthesis
------------------------------
Each of the 16 cases will be written up as a narrative organised according to a common structure based on the various stages of the logic model. Each study should potentially, therefore, provide illumination as to the processes that could lead to outcomes and illustrations of such outcomes. In addition, the common structure of each case should facilitate some cross-case analysis that will not only look for common factors associated with research that has led to outcomes, but also see how far such outcomes are associated with different modes of funding and types of research. Some of this analysis should be based on the previous findings that are embedded into the evaluation framework: for example, basic research might be expected to produce a reasonable number of knowledge outputs but be less likely than clinical or AHP research to inform policies. Some other aspects of the analysis, however, are likely to be exploratory: detailed analysis of factors related to the role of medical charity research in contributing to outcomes appear, as yet, not to be well established.
Conclusions
===========
This paper sets out the aims and methods to be adopted in an innovative study to review the outcomes of the research funded by the Arthritis Research Campaign, one of the leading medical charities in the UK. At a time of growing emphasis on both accountability and evidence-based policy making, it is important for research-funding bodies to be able to show the results of their funding and base their policies on analyses of the processes involved in producing outcomes \[[@B53]\]. Based on the results of the piloting, a decision was made to go ahead with the full study.
Finally, one of the challenges for the future will be to operationalise such analysis on a regular, and therefore less resource intensive, manner. It is hoped that the study will also shed light on these practical considerations, and do so in a way that will enable a system to be developed that meets the specific needs of the particular research funding body \[[@B4]\], in this case ARC.
Competing interests
===================
The work was funded by ARC.
Authors\' contributions
=======================
JG led the design of the study, with all authors making contributions. SH drafted the article with contributions from all authors.
Acknowledgements
================
The research team are grateful to ARC, not only for funding the project but also for the considerable support and constructive comments made by the members of ARC\'s Development Committee who act as the steering group for this project. We also gratefully acknowledge the assistance given by those people associated with ARC who agreed to be interviewed at the preliminary stages when the evaluative framework was being refined, and those who participated in the pilot studies. We are grateful, too, for the helpful comments from the reviewers.
| PubMed Central | 2024-06-05T03:55:46.607888 | 2004-7-23 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC503400/",
"journal": "Health Res Policy Syst. 2004 Jul 23; 2:4",
"authors": [
{
"first": "Stephen R",
"last": "Hanney"
},
{
"first": "Jonathan",
"last": "Grant"
},
{
"first": "Steven",
"last": "Wooding"
},
{
"first": "Martin J",
"last": "Buxton"
}
]
} |
PMC506778 | Background
==========
Cystic fibrosis (CF) is a progressive disease primarily affecting the intestines, lungs, and pancreas. The gene responsible for CF was identified in 1989 \[[@B1]\] as coding for the cystic fibrosis transmembrane conductance regulator (*cftr*), a membrane chloride channel. CF is one of the most common autosomal recessive diseases in Caucasians with a carrier rate of 3--4% \[[@B2]\], and is characterized by recurrent infection and chronic inflammation. Recently it was found that infants with CF demonstrate changes in forced expiratory volume in 1 second (FEV~1~), functional residual capacity (FRC), and other parameters of lung function prior to the onset of recurrent infection \[[@B3]-[@B5]\].
Soon after the CF gene was discovered, a knockout mouse was developed. This mouse demonstrates subtle changes in epithelial cell phenotype, including alterations in secretory glycoconjugates and changes in secretory vesicles \[[@B6]\]. Monocytic infiltrates and altered lung mechanics have also been found \[[@B7]\]. Unfortunately, the *cftr* knockout mouse does not develop overt lung disease, which has severely limited its usefulness. However, the availability of new methods for pulmonary testing in rodents \[[@B8],[@B9]\] now presents the opportunity to re-examine the *cftr* knockout mouse for functional lung changes. In the present study, therefore, we examined pulmonary function in young adult *cftr* -/-, *cftr* +/-, and *cftr*+/+ S489x mice in an effort to establish a lung phenotype.
Results
=======
Effect of *cftr* gene dosage on pressure-volume (PV) curves
-----------------------------------------------------------
Routine evaluation of dynamic lung function employs the stepwise variation in air volume on both the inflation and deflation phases of a single breath. Measurement of airway pressures at each step results in the classic pressure-volume (*PV*) curve which is dependent upon both lung structure and interfering pathology. *PV* curves were measured in triplicate, starting from positive end-expiratory pressure (PEEP) values of 0, 3, and 6 cmH~2~O in S489X mice at 30--60 days of age following genotyping for the normal and mutant *cftr* alleles. The 3 cmH~2~O PEEP curves obtained for each genotype are presented in Figure [1A](#F1){ref-type="fig"}. Note that these *PV* curves all begin at *V* = 0 ml, which is the FRC defined by the 3 cmH~2~O PEEP. The *PV* curves obtained at PEEP levels of 0 and 6 cmH~2~O were similar.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Relationship between *cftr* genotypes and *PV* curves**. Littermates from 30--60 days of age were genotyped and individuals with *cftr*+/+ (Black), *cftr*+/- (Green), and *cftr*-/-(Red) were evaluated using pressure volume curve analysis at Peeps of 0, 3, and 6. A: *PV* curve at PEEP 3; B: Calculated *Cst* for all *PV* curves; C: Calculated hysteresis for PV curves in A. All measures were corrected individually for lung weight. Error bars are +/- standard deviation. \*p \< 0.05 when compared to *cftr*+/+ and \*\*p \< 0.05 when compared to cftr+/-.
:::
:::
The static compliance (*Cst*) of the lungs, which reflects elastic recoil at a given pressure, was calculated from the slopes of the *PV* curves. As shown in Figure [1B](#F1){ref-type="fig"}, the static compliance of the homozygous knockout lung was significantly decreased compared to the homozygous normal lung (p \< 0.01)). Furthermore, *Cst* was significantly reduced in both *cftr*+/+ (p \< 0.001) and *cftr*-/- (p \< 0.001) as compared to age-matched *cftr*+/- mice. Hysteresis was altered among the 3 genotypes (Figure [1C](#F1){ref-type="fig"}). A statistically significant increase in hysteresis was observed in both *cftr*+/- (P \< 0.0001) and *cftr*+/+ mice (p \< 0.05) compared to *cftr*-/- mice. These data suggest the presence of a gene dosage effect in which an altered lung structure in the heterozygous animals leads to an elevated compliance relative to the two homozygous animals. Lung weights were measured and no statistically significant differences were observed among the three genotypes (Figure [1D](#F1){ref-type="fig"}).
Airway mechanics of *cftr* deficient lungs
------------------------------------------
We applied the forced oscillation method to the mice and determined respiratory mechanical input impedance \[[@B10],[@B11]\]. We fit the constant-phase model of respiratory mechanics \[[@B12]\] to impedance (*Zrs*) and determined values for airway resistance (*Raw*), tissue damping (*G*), and tissue elastance (*H*). Figure [2](#F2){ref-type="fig"} shows that *Raw*, *G*, and *H* were significantly reduced in the *cftr*+/- mice as compared to both *cftr*+/+ and *cftr*-/- animals (Panels A, C, & D) at all PEEP levels. *Raw* also decreased with PEEP in a similar fashion in all three genotypes. In contrast, *Raw*, *H* and *G* were significantly increased in the *cftr*-/- mice compared with *cftr*+/+ and *cftr*+/-, and showed a greater dependence on PEEP (Panels A, C & D). The ratio *G*/*H*, termed hysteresivity (both the low and high frequency), was not significantly affected by either genotype or PEEP (Panel E). Thus, the absence of either 1 or 2 copies of the *cftr* gene had significantly different effects on the phenotype of the lung. Paradoxically, the absence of only one *cftr* copy resulted in a greater lung compliance (lower elastance) than if neither or both copies were present.
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**Variation in respiratory mechanic among *cftr* genotypes.** Values for *Raw*(A), *G*(B), *H*(C), and *η*(D) were determined by fitting the constant-phase model to measurements of *Zrs* from *cftr*+/+ (Black), *cftr*+/- (Green) and cftr-/- (red) genotypes. All measures were normalized by multiplication by lung weight. Error bars are +/- standard deviation. \*p \< 0.05 when compared to *cftr*+/+ and \*\*p \< 0.05 when compared to cftr+/-.
:::
:::
Discussion
==========
The usefulness of the *cftr* knockout mouse as a model of cystic fibrosis has been severely limited by its failure to demonstrate readily measurable lung disease, the primary cause of morbidity and mortality in humans \[[@B13]\]. However, in the present study use of sophisticated measurements of lung function revealed a functional lung phenotype in the knockout mouse (Table [1](#T1){ref-type="table"}); the complete absence of *cftr* in the lung of young adult animals resulted in decreased *Cst* and *η* and increased *Raw*, *G* and *H* as compared to normal littermate controls.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Relative effect of genotype on lung function compared to that observed in the *cftr*+/+ mouse.
:::
**Genotype** ***PV* curve position** ***Cst*** ***PV* Hysteresis** ***Raw*** ***G*** ***H*** ***η***
--------------- ------------------------- ----------- --------------------- ----------- --------- --------- ---------
***cftr*+/-** ← ↑ ↑ ↓ ↓ ↓ ↔
*cftr*-/- → ↓ ↓ ↑ ↑ ↑ ↔
:::
A particularly intriguing further observation was that *Cst* and hysteresis in *cftr*+/- mice was significantly higher than in *cftr*+/+ animals while *G* and *H* were decreased. As this was not associated with any pathology such as emphysema, we conclude that it represents a functionally different lung from that of the *cftr*+/+. Our data thus reveal a remarkable inverse correlation between the effect of one and two non-functional copies of the *cftr* gene.
What do these data mean in terms of lung structure? The knockout animal has cystic fibrosis by definition, and our data now show it to also have lung disease manifest as a reduced compliance and increased resistance. These changes could reflect changes in the intrinsic mechanical properties of the parenchyma, or simply a reduction in lung volume. The former effect could include alterations in the biophysical properties of the air-liquid interface in the lungs, and would be expected to result in a change in *η*\[[@B14]\]. Indeed, because *cftr* is a chloride channel and is thought to be involved in water balance, a change in surface tension in the lung, and consequently in *η*, might be expected. However, as shown in Figure [2](#F2){ref-type="fig"} and Table [1](#T1){ref-type="table"}, although *G* and *H* both increase, they do so in the same proportion so there is no significant change in *η* between the three *cftr* genotypes. On the other hand, lung weights were not different among the different groups of mice, so the decreased compliance and increased resistance of the *cftr*-/- animals was not simply due to their having smaller lungs than control animals. This suggests that the parenchymal structure in the lungs of the homozygous and heterozygous animals were organized differently, in a manner that affected *G* and *H* similarly.
As documented in numerous publications, the mouse strain used in the present study does not develop chronic inflammatory disease up to the age (30--60 days) used in this study (for review see \[[@B15]\]). On the other hand, Broaches-Carter et al. \[[@B16]\] have shown that *cftr* levels are highest in the developing lung and decrease 75-fold at birth. *In utero* over-expression of *cftr* has also been shown to affect lung growth and development\[[@B17]\], and the severity of disease in the knockout mouse has been shown to be influenced by genetic background \[[@B18]\]. These data thus suggest that *cftr* may affect the early development of the lung in a manner that is affected by the interaction of other genes.
Are there any functional consequences for increased lung compliance in the heterozygous *cftr* animals? Interestingly, there is no decrement in lung function in human heterozygotes \[[@B19]-[@B21]\]. Also, the heterozygote frequency for CF in humans is higher than expected, likely reflecting a selective advantage because there is no evidence for genetic drift \[[@B22],[@B23]\]. Indeed, selective advantage in CF has been proposed to reflect resistance to tuberculosis, influenza and cholera \[[@B24]\]. When one looks in nature for other examples of heterozygous advantage, the sickle cell trait which confers resistance to malaria \[[@B25]\] is perhaps the only such recognized genetic trait in humans. In Norway rats, a single Mendelian gene controls resistance to Warfarin, an anticoagulant used to control rat populations; homozygous wild-type rats are killed by Warfarin and homozygous mutant allele animals are highly susceptible to vitamin K deficiency \[[@B26]\]. The results of the present study indicate that a similar selective advantage may pertain to *cftr*, something we term a \"Goldilocks Effect\". That is, while two defective copies of the gene are detrimental and two normal copies are satisfactory, one normal and one defective gene may results in an optimal dosage for lung development.
Further studies of pulmonary mechanics in *cftr* knockout mice should reveal additional genetic loci that modulate the influence of *cftr* on lung growth and development. Corresponding studies in humans should be useful in evaluating the effect of therapies on reversing altered pulmonary function in the CF patient.
Conclusions
===========
Using sophisticated techniques to a evaluate rodent pulmonary function; a distinct, readily quantifiable lung phenotype was identified in the *cftr* knockout mouse. In addition, the *cftr*+/- mouse had a distinguishable pulmonary function phenotype from that observed in either the homozygous normal or mutant genotype mice. These data are consistent with CFTR-dependent, physiologic changes in the structure and function of the lung.
Methods
=======
Mouse strain
------------
The S489X mouse 5^th^ generation backcross to C57Bl/6 has been maintained by random mating for the past 8 years. This colony has a 100% mortality rate among *cftr* knockouts by 45 days of age unless the animals are placed on an elemental liquid diet and corncob bedding upon weaning \[[@B27]\]. Mice 30--60 days of age from our S489X mouse colony were genotyped for the normal and mutant *cftr* alleles. Age and litter matched *cftr*+/+ and *cftr*+/- were used for each *cftr*-/- mouse examined. Six animals were included in each group. All experiments were approved by the animal care and use committee.
Pulmonary function tests
------------------------
The mice were anesthetized with intra-peritoneal pentobarbital (90 mg/kg) and the trachea was dissected free of surrounding tissue and cannulated with a 20-gauge cannula. The animals were then connected to a small animal ventilator (*flexiVent*, SCIREQ Inc. Montreal, PQ, Canada) and ventilated with a tidal volume of 10 ml/kg; inspiratory:expiratory ratio of 66.67%, respiratory rate of 150 breaths/minute, and maximum pressure of 30 cmH~2~0. PEEP was controlled by submerging the expiratory limb from the ventilator into a water trap. Each animal was paralyzed with pancuronium bromide (0.5 mg/kg) and allowed to equilibrate on the ventilator until spontaneous breathing ceased (5 minutes).
### Respiratory mechanics
To measure *Zrs*, mechanical ventilation was interrupted and the animal was allowed to expire against the set level of PEEP for 1 s. We then applied an 8 s broad-band volume perturbation signal to the lungs with the *flexiVent*, after which ventilated was resumed. This was repeated at PEEP levels of 0, 3 and 6 cmH~2~O. The volume perturbation signal consisted of the superposition of 18 sine waves having frequency spaced roughly evenly over the range 0.25 Hz to 19.625 Hz. *Zrs* was calculated from the displacement of the ventilator\'s piston and the pressure in its cylinder as described previously \[[@B10],[@B11]\]. Correction for gas compressibility as well as resistive and accelerative losses in the *flexiVent*, connecting tubing and the tracheal cannula were performed as described previously \[[@B28]\] using dynamic calibration data obtained by applying volume perturbations through the tubing and tracheal cannula first when it was completely closed and then when it was open to the atmosphere.
We interpreted the measurement of *Zrs* in terms of the constant phase model \[[@B12]\]
where *Raw* is a frequency independent Newtonian resistance reflecting that of the conducting airways\[[@B29]\], *Iaw* is airway gas inertance, *G* characterizes tissue damping, *H* characterizes tissue stiffness (elastance), *i* is the imaginary unit, *α* links *G* and *H*, and *f* is frequency. We also calculated a quantity known as hysteresivity (*η* = *G/H)*, which is believed to increase when regional heterogeneities develop in the lung \[[@B30]\]. *Raw*, *G* and *H* were all normalized by multiplication by lung weight.
### PV curves
Starting at the *FRC* defined by the PEEP, the *flexiVent* was programmed to deliver seven inspiratory volume steps for a total volume of 0.8 ml followed by seven expiratory steps, pausing at each step for 1 s. Plateau pressure (*P*) at each step was recorded and related to the total volume (*V*) delivered to produce a quasi-static *PV* curve. *Cst* was calculated from the slope of each curve \[[@B31]\], and was normalized by division by lung weight.
*Zrs* measurements at each PEEP level and *PV* curves were obtained in triplicate. Data were statistically evaluated using paired t-test with p \< 0.05 being taken as significant
Acknowledgement
===============
This work was supported by the Ochsner Clinic Foundation and NIH grants R01 HL-67273 and NCRR COBRE P20 RR-15557. The authors thank Dr. Conrad Hornick for his assistance.
| PubMed Central | 2024-06-05T03:55:46.612352 | 2004-7-27 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC506778/",
"journal": "BMC Genet. 2004 Jul 27; 5:21",
"authors": [
{
"first": "J",
"last": "Craig Cohen"
},
{
"first": "Lennart KA",
"last": "Lundblad"
},
{
"first": "Jason HT",
"last": "Bates"
},
{
"first": "Michael",
"last": "Levitzky"
},
{
"first": "Janet E",
"last": "Larson"
}
]
} |
PMC506779 | Background
==========
With the inclusion of Taekwondo as a medal sport in the 2000 Olympic Games interest has gained momentum from participants, national governments and scientists alike. With increased participation, the issue of safety becomes very important and timely. The rules and regulations with regards to these Championships were that of the World Taekwondo Federation \[[@B1]\]. All athletes were sixteen years of age and older and held black belt first degree as a minimum requirement to compete. At the time of data collection, punches were allowed to the front of the torso in the area covered by the chest protector worn by the athletes. No punches were allowed to the head or other parts of the body. Kicks were allowed to the torso and head, the latter of which was covered by a helmet similar to the one worn in amateur boxing. Regardless of the area of contact, only one point was granted by the referees for a successful blow. One could win the match by means of a knockout, therefore, contact was encouraged. There was a change of rules introduced in 2003, which included granting two points for head shots and an additional point for an eight-count knockout \[[@B1]\].
Prospective studies on Taekwondo injuries sustained at single tournaments have been conducted before \[[@B1]-[@B3]\]. For instance, Zemper and Pieter \[[@B2]\] found injury rates for American elite male Taekwondo athletes to be 127.4/1,000 athlete-exposures and for females, 90.1/1,000 athlete-exposures. One athlete-exposure (A-E) refers to one athlete being exposed to the possibility of being injured. Since there are always two athletes competing during any one bout, there are two athlete-exposures per bout \[[@B2],[@B3]\]. In a later study, Pieter et al. \[[@B3]\] reported injury rates of 139.5/1,000 A-E and 96.5/1,000 A-E for European men and women, respectively. In neither study were the differences tested for statistical significance. However, at a recreational tournament in the United Kingdom, the men (51.3/1,000 A-E) sustained statistically significantly more injuries than the women (47.6/1,000 A-E) \[[@B4]\].
It is not clear why the men in the studies mentioned above sustained more injuries. Sample size may be a factor. For instance, in a prospective study covering multiple Taekwondo tournaments, Pieter and Zemper \[[@B5]\] reported a statistically significantly higher injury rate for the women (105.5/1,000 A-E versus 95.1/1,000). In the largest prospective judo injury study to date, young and adult females (130.6/1,000 A-E) incurred a higher injury rate than their male counterparts (122.6/1,000 A-E) \[[@B6]\]. In addition to sample size, the number of female competitors is also lower \[[@B5],[@B6]\].
As expected in a collision sport, the contusion was the most frequently occurring injury type in both male and female Taekwondo athletes \[[@B2]-[@B4]\]. At the elite level, more serious injuries such as fractures and cerebral concussions also occur \[[@B3],[@B7]\]. Men seem to incur more of these serious injuries \[[@B2],[@B3]\]. Less information is available in karate, for the authors have not consistently reported the injuries by gender. Based on what is known from prospective studies on elite karate athletes, the men also seem to incur more serious injuries, some of which have led to time loss \[[@B8],[@B9]\].
The body region most frequently affected in single tournaments involving both recreational and elite Taekwondo athletes is the lower extremities, especially the (instep of the) foot \[[@B2]-[@B4]\]. Since full-contact or Olympic Taekwondo is characterized by kicking, this should come as no surprise.
There is a lack of data on gender differences in injuries to body region and body part in karate. Most prospective studies combined the injury rates for males and females \[e.g., \[[@B8]\]\]. Using a prospective design, Pieter \[[@B10]\] reported that the head and neck sustained most of the injuries in both elite male and female karate athletes.
There is also scarce information on male-female comparisons on body regions injured in judo athletes. Our own studies in judo seem to indicate that in the women, the upper extremities are mostly affected, while in the men, the head and neck as well as the lower extremities are injured most often \[[@B11],[@B12]\].
In line with the frequent use of the legs in Taekwondo, the main injury mechanism was found to be delivering or receiving a kick \[[@B2],[@B5]\]. Further analysis revealed that the roundhouse kick was most often implicated, especially in men \[[@B3],[@B4],[@B13]\]. The fact that injuries occur as a result of receiving a kick may be partially related to unblocked attacks, which has led to the recommendation for the coaches to work on improving the blocking skills or evasive maneuvers of their athletes \[[@B2]\].
The purpose of this study was to identify and compare the rates of injury in Canadian male and female Taekwondo competitors relative to total number of injuries, type, body part injured and mechanism.
Methods
=======
Subjects (219 males and 99 females) participated in the 1997 Canadian National Taekwondo Championships in Toronto, Canada. Injuries were recorded on an injury form to document any injury seen and treatment provided by the health care team as it was required by law. The first author was the only person who kept the injury forms and entered the data, therefore, keeping the identity of athletes confidential. Oral consent was obtained from the athletes for assessment and providing therapy. Data describe the athlete and nature, site, severity and mechanism of the injury. No reliability and validity information for the instrument is available and this study was carried out to pre-test the injury data collection form (Figure [1](#F1){ref-type="fig"}).
Injuries were diagnosed by the tournament physician (MK), who has been the national team chiropractic physician for several years and is an experienced (black belt) Taekwondo athlete himself. One injury form was filled out by the attending physician for each time the athlete reported a new injury. However, at each presentation there could be more than one injury reported on the same Injury Report form. For the purposes of this study, an athlete was considered injured if any of the following conditions applied \[[@B14]\]: 1) any circumstance that forced the Taekwondo athlete to leave the competition; 2) any circumstance for which the referee or athlete had to stop competition; 3) any circumstance for which the athlete requested medical attention. In other words, the definition included so-called time-loss injuries (stoppage of a bout) as used in the NCAA Injury Surveillance System \[[@B15]\].
Injury rates were calculated from matches fought using the basic rate formula: (\# injuries / \# athlete-exposures) × 1,000 = \# injuries per 1,000 athlete-exposures (A-E). The Colorado concussion classification was utilized in management of the concussions \[[@B16],[@B17]\]. According to this classification, a first degree concussion is identified by confusion, no loss of memory and no loss of consciousness (LOC). A second degree involves confusion, loss of memory but no LOC and the third degree is when there is LOC \[[@B16],[@B17]\].
Results
=======
The age range for the males was 17--34 years with a mean of 24.2 years and for the females, 16--26 years with a mean of 21 years. The age was not recorded for 3 males and 1 female. Table [1](#T1){ref-type="table"} displays the injury data and rates for the Canadian Taekwondo athletes.
The lower extremities were the most commonly injured body region in the men (32.0 /1,000 A-E), followed by the face (eyes, nose, cheek, lips, jaw; 18.3/1,000 A-E), and the spine (neck, upper back, low back and coccyx) (13.8/1,000 A-E). If the head and neck (which includes the face area) are combined, as was done in other studies \[[@B4],[@B7]\], this body region incurred the second highest injury rate: 24.9/1,000 A-E. All injuries to the women were sustained to the lower extremities with the foot incurring most of the injuries (15.2/1,000 A-E; Table [2](#T2){ref-type="table"}).
The top five injuries in the males include the sprain (22.8/1,000 A-E), followed by the joint dysfunction (13.7/1,000 A-E), contusion and laceration (11.4/1,000 A-E each), and strain (9.1/1,000 A-E). The cerebral concussion is ranked sixth (6.9/1,000 A-E). There were one third degree and two first degree concussions. In the women, the contusion was the most often occurring injury (15.2/1,000 A-E), followed by the sprain and strain (5.1/1,000 A-E each; Table [3](#T3){ref-type="table"}).
Table [4](#T4){ref-type="table"} displays the rates of the injury mechanisms by gender. Receiving a kick by the men included those connecting with the head/face (18.3/1,000 A-E), trunk (6.9/1,000 A-E), and thigh (2.3/1,000 A-E). Delivering a kick as an injury mechanism in the men included, among others, kicking to the elbow (6.9/1,000 A-E), kicking with the toes, to the trunk and with the knee (2.3/1,000 A-E each). In the women, delivering a kick (10.1/1,000 A-E) was the main injury mechanism and comprised kicks to the elbow, while the kick that was received involved a knee kick.
Discussion
==========
The injuries incurred by the Canadian Taekwondo athletes compare favourably to those found by others. As mentioned above, American elite athletes recorded injury rates of 127.4/1,000 A-E (men) and 90.1/1,000 A-E (women) \[[@B2]\], while European colleagues had rates of 139.5/1,000 A-E (men) and 96.5/1,000 A-E (women) \[[@B3]\]. At one Greek national championship, the men (20.6/1,000 A-E) sustained statistically significantly fewer injuries than the women (36.4/1,000 A-E) \[[@B13]\]. The total injury rate of the Canadian male and female Taekwondo athletes combined, was also lower than that of their African counterparts (86.6/1,000 A-E) \[[@B7]\]. At an Open British tournament, injury rates of 51.3/1,000 A-E (men) and 47.6/1,000 A-E (women) were reported \[[@B4]\]. However, the athletes competing at this particular tournament were of sub-elite level. It is hypothesised that injuries may be related to level of skill and experience, although confirmatory research still needs to be carried out \[[@B18]\]. All competitors were black belts, but no information is available on their experience in Taekwondo and in competition. Future research should include general Taekwondo as well as competition-specific experience in addition to belt level. Comparative data gleaned from prospective studies on other martial arts injuries incurred at single tournaments are depicted in table [5](#T5){ref-type="table"}.
In view of the nature of the sport, as alluded to above, it is not surprising to find the lower extremities to sustain most of the injuries as was found in previous studies as well \[[@B2],[@B3],[@B13]\]. Within the lower extremities, the foot (i.e., instep) was the most often injured body part, as was the case with the females in the present study, which led to the suggestion for the Taekwondo governing bodies to recommend padding to help decrease injuries to this site \[[@B22]\]. In karate, on the other hand, the head and neck incur most of the injuries \[[@B8],[@B10]\], while in judo, the upper extremities are more at risk \[[@B6],[@B11]\].
As expected, the contusion was found to be the most frequently occurring injury type in other studies on Taekwondo injuries \[e.g., \[[@B2],[@B3],[@B13]\]\]. The sprain ranked in the top three of most frequently occurring injuries across several tournaments \[[@B5]\]. The contusion was also the most often occurring injury in karate \[[@B9],[@B10]\], while the epistaxis ranked second in Dutch men and women \[[@B10]\]. In Finnish elite male karate athletes the laceration was ranked second and the epistaxis in women \[[@B9]\]. In judo, the strain in men and the abrasion in women were sustained most often \[[@B11],[@B12]\]. Since the sample sizes in the current investigation as well as in the aforementioned karate and judo studies are rather small, more research is needed to arrive at more definitive conclusions. However, differences between the martial arts in terms of techniques used and competition rules undoubtedly play a major role.
Of more concern, however, is the occurrence of cerebral concussions. The Canadian males recorded a higher rate than found in American (4.7/1,000 A-E) \[[@B2]\] and Greek (1.0/1,000 A-E) \[[@B13]\] elite Taekwondo athletes, but lower than those competing in the 1993 European Cup (15.5/1,000 A-E) \[[@B3]\] and the 1991 World Championships (15.3/1,000 A-E) \[[@B23]\]. The Canadians also recorded lower rates than elite Dutch karate athletes (13.2/1,000 A-E) competing under semi-contact rules \[[@B10]\]. Given the serious implications of these injuries, preventive measures, testing of equipment and follow-up research are urgently needed \[e.g., \[[@B18]\]\].
In accordance with what was found previously, the injury mechanisms included both receiving and delivering kicks for men and women alike \[[@B2],[@B5]\]. The men, more than the women, tended to get injured as a result of receiving a kick \[[@B2],[@B3]\]. It is suggested that the technique most likely implicated is the roundhouse kick \[[@B3],[@B4],[@B13]\]. Kicking the elbow typically leads to injury, especially if the kick is executed with the instep of the foot, such as when using the roundhouse kick to attack or counter-attack. Yet another reason to implement foot padding, as already mentioned above. In karate, punching is the main injury mechanism for both men and women \[[@B8],[@B10]\], which may be related to the head and neck region being most frequently injured.
More research on Canadian Taekwondo athletes of different age groups and skill levels is needed. Age in the present study is not believed to have played a role in the injuries sustained. The injury profile of the Canadians is quite similar to those found in other studies with Taekwondo athletes in their early twenties \[[@B2],[@B3],[@B5]\]. Children and juniors in Taekwondo were reported as incurring higher injury rates than adults \[[@B4],[@B13]\]. Future studies should also include time lost due to injury.
Joint dysfunction was identified as the second most common injury sustained by male athletes (13.7/1,000 A-E). Haldeman \[[@B24]\] defines joint dysfunction quoting Drum (1973) as,\"Joint mechanics showing area disturbances of function without structural change; subtle joint dysfunctions affecting quality and range of joint motion. They are diagnosed with the aid of motion palpation, as well as stress and motion radiography investigation\" \[p. 623\]. Greenman \[[@B25]\] states: \"Joint dysfunction is characterized by findings of misalignment, relative fixation, loss of normal range-of-motion and end-play, tenderness, and tissue texture abnormality\" \[p. 13--14\]. Although controversial, the term has been used widely in the literature, mostly by chiropractors, physical therapists and occasionally by biomechanists and medical doctors \[[@B24]-[@B33]\]. Further studies are required to validate the current finding.
Conclusion
==========
The total injury rates for the Canadian Taekwondo athletes compare favourably to those reported in the literature, which is contrary to what was expected based on such a small sample size. It is hypothesized that recent rule changes may have contributed to these relatively low rates when compared to those found for other single tournaments, although more research is indicated before a definitive conclusion may be drawn. Interestingly, joint dysfunction was identified for the first time, which warrants more study. The injury data collection form should also include the technique used as a specification of the injury mechanism. General Taekwondo and competition-specific experience in addition to belt rank should also be recorded.
Competing interest
==================
None declared.
Authors\' contributions
=======================
MK collected the data, designed the Injury Report form, and wrote the initial draft of the manuscript. WP did the result part, revised and proof read the manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2474/5/22/prepub>
Acknowledgements
================
We would like to thank Canadian Memorial Chiropractic College for funding this study.
Figures and Tables
==================
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Injury data collection form.
:::
:::
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Injury rates (95%CI) in adult Canadian Taekwondo athletes.
:::
Men Women Total
---------------------------------- -------------------- ------------------ -------------------
Number of athletes 219 99 318
Number of reported injuries 35 5 40
Number of athlete-exposures (AE) 438 198 636
Injury rates
\-- per 100 athletes 16.0 (10.7--21.3) 5.1 (0.7--9.5) 12.6 (8.7--16.5)
\-- per 1,000 AE 79.9 (53.4--106.4) 25.3 (3.2--47.4) 62.9 (43.4--82.4)
:::
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Distribution of injuries by body part per 1,000 athlete-exposures.
:::
Men Women
------------ -------- ------ ------------ -------- ------
Body part Number Rate Body part Number Rate
Head 3 6.9 Hamstrings 1 5.1
Eyes 2 4.6 Ankle 1 5.1
Nose 3 6.9 Foot 3 15.2
Cheek 1 2.3
Lip 1 2.3
Jaw 1 2.3
Neck 1 2.3
Hands 3 6.9
Upper back 1 2.3
Low back 2 4.6
Pelvis 1 2.3
Coccyx 2 4.6
Hamstrings 3 6.9
Leg 2 4.6
Ankle 5 11.4
Foot 2 4.6
Toes 2 4.6
Total 35 79.9 Total 5 25.3
:::
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Distribution of injuries by injury type per 1,000 athlete-exposures.
:::
Men Women
------------------- -------- ------ ------------- -------- ------
Injury type Number Rate Injury type Number Rate
Sprain 10 22.8 Contusion 3 15.2
Joint dysfunction 6 13.7 Sprain 1 5.1
Contusion 5 11.4 Strain 1 5.1
Laceration 5 11.4
Strain 4 9.1
Concussion 3 6.9
Abrasion 1 2.3
Epistaxis 1 2.3
Total 35 79.9 Total 5 25.3
:::
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
Distribution of injuries by mechanism per 1,000 athlete-exposures.
:::
Men Women
-------------------- ------- ------ ------------------- ---- ------
Injury mechanism No Rate Injury mechanism No Rate
Receiving a kick 12 27.4 Delivering a kick 2 10.1
Delivering a kick 7 16.0 Receiving a kick 1 5.1
Simultaneous kicks 1 2.3 Not recorded 2 10.1
Other 3 6.9
Not recorded 12 27.4
Total 35 79.9 Total 5 25.3
:::
::: {#T5 .table-wrap}
Table 5
::: {.caption}
######
Comparative injury rates per 1,000 athlete-exposures (95%CI) in adult martial arts athletes\*.
:::
Sport/Study Men Women
------------------------ ---------------------- ----------------------
Taekwondo (this study) 79.9 (53.4--106.4) 25.3 (3.2--82.4)
Judo \[12\] 48.5 (18.5--78.6) 34.3 (4.2--64.3)
Judo \[19\] 115.1 (90.9--139.3) \--
Judo \[20\] 51.3 (1.0--101.6) 125.0 (107.7--142.3)
Judo \[11\] 25.2 (6.5--43.8) 41.3 (14.3--68.3)
Karate \[10\] 168.9 (144.1--193.6) 158.5 (120.0--197.1)
Karate \[19\] 65.5 (43.1--87.8) \--
Karate \[21\] 135.6 (105.9--165.3) \--
Karate \[9\] 157.7 (123.6--191.8) 80.4 (27.9--132.9)
\*Except for our own studies, injury rates are estimated based on the information provided by the authors
:::
| PubMed Central | 2024-06-05T03:55:46.613951 | 2004-7-27 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC506779/",
"journal": "BMC Musculoskelet Disord. 2004 Jul 27; 5:22",
"authors": [
{
"first": "Mohsen",
"last": "Kazemi"
},
{
"first": "Willy",
"last": "Pieter"
}
]
} |
PMC506780 | Background
==========
The World Health Organization recently published a performance ranking of the health systems of its 191 member countries, and intends to update it at regular intervals \[[@B1]-[@B4]\]. It was based on a framework outlining a set of social goals to which health systems should contribute \[[@B5]\]. It was argued that systems should contribute to improving population health, be responsive to the people they serve and be financed fairly. Five outcome indicators were defined -- the level of population health, inequalities in health, the level of responsiveness, inequalities in responsiveness and fairness in financial contributions. Estimates of attainment on these five indicators were made for the 191 countries that were members of WHO at that time, and a composite (overall) attainment indicator was constructed for each country as a weighted average of attainment on the five individual outcome indicators.
Publication of the analytical framework and the resulting ranking provoked considerable comment, and a variety of issues concerning the methodology and country positions in the ranking have been raised. A central component of the methodology for measuring overall attainment was the use of fixed weights, common to all countries, to aggregate the five indicators. This feature has been controversial, with some arguing that people in different cultural and social settings value individual health system goals in different ways \[[@B6]-[@B13]\]. The fixed weights had the virtue of being based on expert opinion, having been derived from the valuations of 1,007 respondents -- largely health system professionals -- to a WHO survey \[[@B14]\]. However the weights were common to all countries, regardless of their development status and cultural traditions.
In this paper we examine the sensitivity of the attainment scores to alternative weighting schemes that allow weights to vary across countries. These country-specific weights may reveal varying objectives of policy makers or constraints under which they operate. Melyn and Moesen \[[@B15]\] have referred to such weights as \'benefit of the doubt\' weights.
Methods
=======
WHO used fixed weights (0.25, 0.25, 0.125, 0.125, 0.25) to aggregate five health system outputs (respectively, the level of population health, inequality in the distribution of health, the level of health system responsiveness, inequality in the distribution of responsiveness, and fairness in financial contributions) into a scalar health system attainment index. Overall attainment ranged from 35.7 (Sierra Leone) to 93.4 (Japan) on a \[0--100\] scale.
We propose here an analytical framework that reduces to the WHO fixed-weight methodology as a special case, but that allows varying degrees of freedom for weights to be defined that -- in the sense of Melyn and Moesen -- implicitly take into account individual country circumstances. For shorthand, we say countries \"choose\" such weights, which in reality are determined as solution values of a linear program. If the linear program is a fair representation of the objective function of and constraints faced by decision makers, the weights are indeed \"chosen\", but even if this condition is not necessarily met, the resulting weights may still be of interest.
The extent of freedom to choose weights in a linear program can be set by the analyst. The analyst can enforce fixed weights common to all countries, allow countries complete freedom to choose their own weights, or adopt a middle ground in which countries are granted limited freedom to choose weights within bounds thought to be sensible by experts.
A generic statement of the performance evaluation problem in primal-dual linear programming format is:
In these programs y is a country\'s output vector, x is its input vector, Y is the sample output matrix and X is the sample input matrix. In the present context y is (5 × 1), Y is (5 × 191), x is (n × 1) and X is (n × 191), with n to be specified below.
The primal program seeks the maximum radial expansion of a country\'s outputs, provided that it not exceed the standards established by a convex combination (λ ≥ 0, ∑λ = 1) of best-practice countries in the sample. The optimal value of φ provides a distance measure (i.e. an indication of how far a country has to go) to match best practice as observed in the sample. Since φ ≥ 1, the attainment of a country is evaluated as φ^-1^≤ 1. Best practice countries have φ^-1^= 1, other countries have φ^-1^\< 1, so φ provides a basis for acomplete ranking of countries on their relative ability to deliver five health system outputs. The dual program seeks a set of nonnegative weights μ,υ attached to a country\'s outputs and inputs that maximize its attainment. Each country\'s output weights are normalized by μy = 1, but each is free to select its own set of nonnegative weights.
In constructing the overall attainment index, WHO identified five output indicators and no inputs, preferring to treat each country\'s health system as a \"health output management unit\". Consequently each country\'s input vector is represented as a scalar with unit value. Under these circumstances the performance evaluation problem simplifies to:
where ω = υ + υ~0~. The modified primal program seeks the maximum feasible radial expansion of a country\'s outputs consistent with best practice observed in the sample. The modified dual program seeks a set of nonnegative weights μ for a country\'s outputs that put it in the best light. A country can be expected to assign relatively large weights to those outputs at which it excels relative to best practice, and relatively low, possibly zero, weights to those outputs at which it lags behind best practice, subject to the normalization μy = 1. (See Annex for a graphical explanation.)
By complementary slackness, μ~m~(Y~m~λ - φy~m~) = 0, m = 1, \..., M, so slack in any element of a country\'s projected output vector φy implies that the country assigns a zero weight to that output. Since it is unreasonable to allow a country to assign a zero weight to any output deemed sufficiently important to have been included in the WHO performance evaluation exercise, it is desirable to restrict weights in some way. This can be accomplished most easily by appending constraints to the dual side of (2) of the form:
γ~m~≥ μ~m~y~m~/μy ≥ β~m~, m = 1, \..., M. (3)
Restrictions (3) place lower and upper bounds on the relative importance of each output (as measured by μ~m~) in total output.
Implementation of the weight-restricted linear program requires specifying the 2M = 10 parameters γ~m~, β~m~. One procedure is to ignore the upper bounds γ~m~and set the lower bounds β~m~\> 0. This eliminates the possibility of a country assigning zero weights to those outputs at which it lags behind best practice. A less arbitrary procedure is to follow Takamura and Tone \[[@B16]\] by adapting Saaty\'s Analytical Hierarchy Process (AHP) \[[@B17]\]. This procedure exploits expert judgment, that could be provided for example by the above-mentioned survey of health system professionals, to set lower bounds β~m~\> 0 and upper bounds 1 \> γ~m~. Although these bounds are common to all countries, they allow countries limited freedom to select weights appropriate to their circumstances.
Results
=======
The fixed weights used by WHO)) \[[@B4]\] to aggregate the five health system indicators gave countries no freedom to choose weights appropriate to their circumstances. We compare the WHO attainment index with three alternative indexes allowing countries varying degrees of freedom to choose weights. The first index is based on the solution to program (2), without weight restrictions, thereby allowing complete freedom to choose. The second index is based on (2), with lower bounds in (3) of β~m~= 0.10 on all weights, allowing substantial freedom to choose. The third index is based on (2), with lower and upper bounds in (3) set by a modified AHP procedure.
In the modified procedure, \"expert opinion\" was taken to mean the average values of weights arising from population-representative country surveys, each of which included a module on health system goals \[[@B9]\]. Respondents were queried about their individual preferences on the five stated health system goals in a total of 51 countries, in some of which multiple surveys were performed, and country means were calculated on the basis of these individual responses \[[@B18]\]. The survey methods, reliability, validity, representativeness, sample size and respondent characteristics are extensively described in Ustün et al. \[[@B9]\], and are also reported in summary form in Sadana et al. \[[@B19]\], Mathers et al. \[[@B20]\], Mathers et al. \[[@B21]\] and Sadana et al. \[[@B22]\]. The survey instruments are available at <http://www.who.int/evidence/hhsr-survey/>.
For each output, the lower bound for calculation of the third index was taken as the minimum of the country average weights, and the upper bound the maximum \[[@B23]\]. Country mean weights and survey types, as well as survey wide maximum and minimum weights are shown in Table [1](#T1){ref-type="table"}. The lower bounds are accordingly β~m~= (0.19, 0.17, 0.12, 0.11, 0.22) and the upper bounds γ~m~= (0.29, 0.25, 0.18, 0.17, 0.30). This specification allows limited freedom to choose. We refer to the four indexes as WHO, LP1, LP2 and LP3, respectively.
Summary statistics of the four attainment indexes appear in Table [2](#T2){ref-type="table"}. The three LP distributions have higher means than the WHO distribution, and two of them have lower dispersion. However increasing restrictions on freedom to choose reduce the mean, and increase the dispersion, of the LP attainment indexes toward the mean and dispersion of the WHO attainment index.
Rank correlations between pairs of attainment rankings appear in Table [3](#T3){ref-type="table"}. Despite the distributional changes due to freedom to choose, rank correlations are positive, high and statistically significant. The lowest correlations involve LP1, the index allowing complete freedom to choose. With the exception of LP1, there is strong agreement about the identity of countries in the top and bottom quartiles of the distribution. Japan is ranked \#1 and Sierra Leone is ranked \#191 on all four indexes.
Figure [1](#F1){ref-type="fig"} shows plots of WHO attainment scores and rankings versus the three LP attainment scores and rankings. The convergence of the distributions of the LP attainment scores and rankings to the WHO scores and rankings is apparent.
Results based on LP1 are unattractive. Over one-third of countries (70 of 191) assign a zero weight to four of the five indicators, and the vast majority of countries assign a weight in excess of 0.9 to either responsiveness distribution or fairness in financial contributions. This means they assign a low or zero weight to population health, the defining goal of the health system, which does not have face validity. In LP1, only Japan, Luxembourg and the United States assign positive weights to all five indicators. Consequently the attainment index is tightly distributed about a very high mean value. The ability to discriminate is sacrificed to freedom to choose, with 75% of countries receiving attainment indexes of 99 or above.
Results from LP2 are somewhat more attractive. Nevertheless, when weights are bounded below by 0.1, over three-quarters (147 of 191) of countries assign the minimum weight to four of the five indicators and a 0.6 = 1 - (4 × 0.1) weight to either responsiveness distribution or fairness in financial contributions. The attainment scores are again compressed about a high mean value, and the ability to discriminate is not much improved, with 75% of countries receiving attainment scores of 93 or above. However despite this dramatic compression, the LP2 ranking is globally very similar to the WHO ranking. Eighteen of the countries ranked in the top 20 by WHO appear in the LP2 top 20, and 14 of the countries ranked in the bottom 20 by WHO appear in the LP2 bottom 20.
Not surprisingly, the distribution of the attainment scores for LP3 looks even more similar to the distribution of the WHO scores, and has a similar mean and standard deviation. Rank correlation is very high, with only one country falling out of the WHO top 20 and only five countries rising out of the WHO bottom 20. Nonetheless, limited freedom to choose has an important impact on individual countries. The USA, given an ability to emphasize the importance of responsiveness level and responsiveness inequality, and to de-emphasize the importance of health level and inequality in the distribution of health, improves its ranking from \#15 to \#3. Australia improves from \#12 to \#7 for similar reasons. Italy, on the other hand, falls from \#11 to \#18, primarily as a result of the movement of other countries. In LP3, the largest positive changes in ranking are for Mauritius (+25) and Saint Vincent (+22), while the largest negative changes in ranking are for Kazakhstan (-39) and Albania (-36).
Discussion
==========
We began by questioning the appropriateness of the fixed weight approach to aggregating indicators adopted by WHO, on the grounds that fixed weights deny countries at varying stages of development the freedom to choose. We then proposed a sequence of linear programming models that allowed countries varying freedom to choose the weights assigned to their indicators. LP1 allows complete freedom to choose, and generates weights we consider unacceptable, particularly because so many countries give a zero weight to improving health. LP2 allows considerable freedom to choose, but generates many country-specific weights falling outside the range of the within-country means used as bounds in LP3. Clearly, the validity of a procedure that routinely assigns weights out of the range of representative cross-population preferences should be questioned, even without a sophisticated theory of empirical ethics or democratic choice.
LP3 applies the AHP procedure to set plausible bounds on weights, and allows limited freedom to choose. It generates a very similar distribution of the attainment index, and a very high linear rank correlation with the WHO ranking. Despite these similarities, we find the LP3 approach intuitively appealing, and are encouraged by its global concordance with the WHO index. However it is fair to ask: if LP3 and WHO generate such similar rankings, why bother? What value does LP3 add? Indeed, individual countries may come to diametrically opposed conclusions about the benefits of LP3 or WHO on the basis of their implied rank changes (e.g. Mauritius vs. Kazakhstan).
Howbeit, our first response to the question \"why bother\" focuses on the distribution of the LP3 weights in comparison to the WHO weights. The WHO weight on responsiveness inequality was 0.125. But the LP3 upper bound of 0.17 is binding on 182 countries, which implicitly want a higher weight on this indicator. The WHO weight on fairness in financial contributions was 0.25. But the LP3 upper bound of 0.30 is binding on 167 countries that want a higher weight. At the other end, the WHO weight on health level is 0.25. But the LP3 lower bound of 0.19 is binding on 170 countries that want a lower weight. The WHO weight on responsiveness level is 0.125. But the LP3 lower bound of 0.12 is binding on 103 countries that want a lower weight. It appears that a majority of countries at all stages of development may implicitly assign greater importance to indicators of health distribution, and less importance to indicators of health level, than the experts whose judgments formed the basis of the original WHO weights.
Our second response is more general. By allowing countries limited freedom to choose their weights, LP3 takes a small but nevertheless significant step toward respecting their varying circumstances. While the narrower the bounds on weights, the smaller the step, even the limited freedom embodied in LP3 makes an important difference to some countries.
Conclusions
===========
Building in the flexibility of varying weights might be a useful way for WHO to respond to the debates and criticisms appearing since publication of the ranking. We conclude by speculating that a variant of LP3 incorporating information regarding which weights are binding, and in which direction, might yield even greater benefits in terms of respecting individual circumstances.
Competing interests
===================
JAL, CJLM and DBE were part of the team at WHO that developed the methods for the world health system performance rankings published by the Organization in 2000.
Abbreviations
=============
AHP (Analytical Hierarchy Process)
DEA (Data envelopment analysis)
LP (Linear program)
LP1 (Linear Program 1)
LP2 (Linear Program 2)
LP3 (Linear Program 3)
WHO (World Health Organization)
Authors\' contributions
=======================
JAL developed methods inspired by the benefit-of-the-doubt concept and drafted an earlier version of the manuscript. CAKL adapted and applied the DEA methods described here to health system performance and drafted the initial version of the current manuscript. CJLM conceived the methods for measuring health system performance. DBE coordinated the research at WHO. All authors revised and approved the manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1472-6963/4/19/prepub>
Supplementary Material
======================
::: {.caption}
###### Additional File 1
Annex.Graphical interpretation of DEA and conceptual links with value theory.doc
:::
::: {.caption}
######
Click here for file
:::
Figures and Tables
==================
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Graphical array showing LP attainment scores vs WHO attainment scores (left column) and LP ranks vs WHO ranks (right column). From top to bottom, WHO vs LP1, LP2, and LP3, respectively.
:::
:::
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Country means and survey types.
:::
Mean survey weight
-------------------------- -------------------- ------ ------ ------ ------ ---------------
Argentina 0.26 0.25 0.13 0.13 0.24 brief^a^
Australia 0.23 0.21 0.15 0.14 0.27 postal^b^
Austria 0.23 0.20 0.15 0.14 0.28 postal
Bahrain 0.26 0.17 0.17 0.13 0.27 brief
Belgium 0.24 0.25 0.13 0.14 0.25 brief
Bulgaria 0.25 0.22 0.13 0.13 0.27 brief
Canada 0.25 0.17 0.16 0.13 0.29 brief
Chile 0.20 0.21 0.15 0.15 0.29 brief
China 0.27 0.21 0.15 0.12 0.25 postal
Costa Rica 0.28 0.25 0.12 0.12 0.22 brief
Croatia 0.25 0.24 0.13 0.13 0.25 brief
Cyprus 0.25 0.23 0.14 0.13 0.25 postal
Czech Republic 0.29 0.22 0.14 0.12 0.24 brief, postal
Denmark 0.26 0.21 0.15 0.12 0.25 postal
Egypt 0.26 0.22 0.14 0.12 0.26 postal
Estonia 0.27 0.22 0.14 0.11 0.25 brief
Finland 0.25 0.21 0.14 0.12 0.27 brief, postal
France 0.24 0.23 0.14 0.13 0.26 brief, postal
Germany 0.24 0.22 0.13 0.14 0.28 brief
Greece 0.25 0.21 0.15 0.13 0.26 postal
Hungary 0.24 0.20 0.16 0.13 0.27 postal
Iceland 0.26 0.20 0.14 0.13 0.26 brief
Indonesia 0.26 0.22 0.15 0.13 0.24 brief
Ireland 0.24 0.25 0.13 0.13 0.26 brief
Italy 0.24 0.23 0.14 0.14 0.25 brief
Jordan 0.26 0.18 0.16 0.12 0.27 brief
Kyrgyzstan 0.25 0.22 0.13 0.13 0.26 postal
Latvia 0.24 0.19 0.15 0.12 0.30 brief
Lithuania 0.24 0.23 0.15 0.13 0.25 postal
Luxembourg 0.25 0.23 0.13 0.13 0.26 telephone^c^
Malta 0.25 0.23 0.13 0.14 0.24 brief
Morocco 0.19 0.18 0.17 0.17 0.29 brief
Netherlands 0.24 0.18 0.17 0.14 0.28 brief, postal
New Zealand 0.24 0.22 0.15 0.13 0.26 brief
Oman 0.25 0.18 0.18 0.14 0.25 brief
Poland 0.24 0.22 0.14 0.12 0.27 brief
Portugal 0.24 0.25 0.13 0.13 0.25 brief
Republic of Korea 0.28 0.21 0.14 0.12 0.25 brief
Romania 0.25 0.22 0.14 0.13 0.26 brief
Russian Federation 0.25 0.17 0.17 0.12 0.29 brief
Spain 0.26 0.24 0.13 0.13 0.24 brief
Sweden 0.23 0.20 0.15 0.14 0.28 brief
Switzerland 0.22 0.21 0.15 0.15 0.27 postal
Thailand 0.21 0.19 0.16 0.16 0.28 brief
Trinidad and Tobago 0.24 0.22 0.14 0.13 0.26 brief
Turkey 0.24 0.23 0.13 0.13 0.27 postal
Ukraine 0.26 0.22 0.14 0.12 0.26 brief
United Arab Emirates 0.23 0.17 0.18 0.14 0.28 brief
United Kingdom 0.26 0.22 0.14 0.12 0.26 postal
United States of America 0.24 0.20 0.15 0.16 0.25 postal
Venezuela 0.28 0.23 0.13 0.11 0.25 brief
Sample minimum 0.19 0.17 0.12 0.11 0.22
Sample maximum 0.29 0.25 0.18 0.17 0.30
**^a^**brief = brief face-to-face survey (Üstün et al. 2001). **^b^**postal = postal survey (Üstün et al. 2001). **^c^**telephone = computer-assisted telephone survey (Üstün et al. 2001).
:::
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Summary Statistics
:::
WHO LP1 LP2 LP3
-------------------------- ------- -------- -------- --------
Mean (%) 73.30 95.18 85.15 79.28
Standard deviation (%) 12.34 4.79 11.34 14.98
Interquartile range (%):
First quartile 63.64 92.99 78.12 69.42
Second quartile 75.39 96.00 87.91 83.37
Third quartile 81.65 99.05 93.25 89.83
Fourth quartile 93.45 100.00 100.00 100.00
:::
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Rank Correlations.
:::
WHO LP1 LP2 LP3
----- ------ ------ ------ ------
WHO 1.00 0.71 0.96 0.97
LP1 1.00 0.75 0.69
LP2 1.00 0.99
LP3 1.00
:::
| PubMed Central | 2024-06-05T03:55:46.616330 | 2004-7-22 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC506780/",
"journal": "BMC Health Serv Res. 2004 Jul 22; 4:19",
"authors": [
{
"first": "Jeremy A",
"last": "Lauer"
},
{
"first": "CA Knox",
"last": "Lovell"
},
{
"first": "Christopher JL",
"last": "Murray"
},
{
"first": "David B",
"last": "Evans"
}
]
} |
PMC506782 | Review
======
The recent awareness of an epidemic of obesity coincides with, and may have contributed to a dramatic increase in the popularity of a variety of low carbohydrate diets. This rapid switch in dietary habits of a significant part of the population, and the virtual revolution in the food industry, is unusual in that it stands in direct opposition to long-standing recommendations of the majority of the nutritional and medical establishment (e.g. \[[@B1],[@B2]\]). Despite isolated examples, such as a recent editorial by Walter Willet pointing to the need to understand low carbohydrate diets \[[@B3]\], there is still little real acceptance by nutrition professionals or health organizations. One aspect of these diets that has been especially controversial is the so-called metabolic advantage -- the idea that more weight may be lost calorie for calorie compared with diets of higher carbohydrate content.
We recently reviewed the literature on metabolic advantage \[[@B4]\]. We showed that there is a sufficient number of reports in the literature to establish the existence of metabolic advantage and we tabulated results from ten or so studies demonstrating that low carbohydrate diets can lead to greater weight loss than isocaloric low fat diets. The reports we cited have frequently been met with the criticism that the data could not be right because they would violate the laws of thermodynamics (\[[@B5],[@B6]\]). An example is the recent demonstration of metabolic advantage in a small, pilot study \[[@B7]\] which, despite its preliminary status, was extremely well controlled. Three groups were studied: A low carbohydrate group (LoCHO = 1800 kcal for men; 1500 kcal for women), a low fat group (LoFat, 1800 and 1500); a third group also consumed a low carbohydrate diet but an additional 300 kcalories were provided (LoCHO+300, 2100 and 1800). The order of average amount of weight lost was LoCHO = 23 lbs, LoCHO+300 = 20 lbs LoFat = 17 lbs. This work received a good deal of attention in the popular press. Media reports, however, included comments of experts that \"It doesn\'t make sense, does it?\" \"It violates the laws of thermodynamics. No one has ever found any miraculous metabolic effects.\" (\[[@B5]\]). If this is an accurate quotation, it is odd indeed. Miraculous, or otherwise, a metabolic effect *was*found. In the absence of an identifiable methodological error, experimental data has to be accepted and numerous investigations, in fact, serve as precedents for Greene et al.\'s findings (Reviews: \[[@B4],[@B8]\]).
In our previous review of metabolic advantage \[[@B4]\] we showed that there is, in fact, no theoretical violation of the laws of thermodynamics, and we provided a plausible mechanism. In general the pathways for gluconeogenesis that are required in order to supply obligate glucose (e.g. to brain and CNS), in combination with increased protein turnover, could account for the missing energy. Here, we simplify the thermodynamic argument and review some of the relevant principles. We show, moreover, that well-established data in the traditional nutritional literature predict metabolic advantage and no one should be surprised. The ironic conclusion is that the principle that weight gain on isocaloric diets must *always*be independent of macronutrient composition would violate the second law of thermodynamics.
What do we mean by \"a calorie is a calorie?\"
==============================================
Because it is a colloquial phrase, it is important to understand exactly what it is meant by \"a calorie is a calorie.\" The most common meaning is that is it impossible for two isocaloric diets to lead to different weight loss. Frequently, the concept is justified by reference to the \"laws of thermodynamics\", but an explicit connection has never been spelled out. More recently, Buchholz & Schoeller \[[@B10]\] appear to identify \"a calorie is a calorie\" with the first law of thermodynamics. They also admit that high protein /low carbohydrate diets can lead to greater weight loss than isocaloric low fat diets in agreement with our assessment \[[@B4]\]. Nonetheless they maintain that \"a calorie is a calorie,\" now justifying it by their connection of the phrase to the concept of energy conservation. It is important to point out that no study of isocaloric diets has ever claimed that the first law of thermodynamics is not true. Buchholz & Schoeller \[[@B10]\] have limited themselves by only including the first law and, therefore, do not understand how the differential weight loss could occur and think it \"deserves further study.\" Our major point here is that there is more than one law of thermodynamics and that a more accurate understanding of the role of the second law shows that differential weight loss is not inconsistent with any physical principle.
Thermodynamics
==============
The idea that \"a calorie is a calorie\" comes from a misunderstanding of the laws of thermodynamics. There are two laws of thermodynamics. (The zeroth law that establishes the concept of temperature and the third law that describes absolute zero are not relevant here). When speaking of \"the law*s*of thermodynamics\" it is important to be sure that one is including the second law. The first law is very different in character from the second law \[[@B9],[@B11],[@B12]\]. The first law is a conservation law: it says that the form of energy may change, but the total is always conserved. The second law is a *dissipation*law: it defines a quantity, the entropy, S, which we traditionally identify with disorder or high probability. The second law says that in any (real) irreversible process, the entropy must increase (ΔS \> 0); balance is *not*expected. Entropy is, in fact, identifiable with irreversibility.
It is important to understand that it is the second law that drives chemical reactions. The first law is a bookkeeping law and tells us that the total energy attributed to work, heat and changes in chemical composition will be constant. It does not tell us whether such a reaction will occur, or if it does, what the relative distributions of the forms of energy will be. To predict the tendency of the reaction to occur, we must employ the second law that says the entropy must increase. In a chemical reaction, at constant temperature and pressure, the entropic and energetic effects are combined into the change in the Gibbs free energy, ΔG, whose sign predicts the direction of reaction, and whose magnitude indicates the maximum amount of work realizable from the reaction.
Application of ΔG\'
===================
To understand the implications of \"a calorie is a calorie,\" that energy yield could be path-independent and the same for all diets consider that it implies that carbohydrate and protein are equivalent fuels as shown in Figure [1](#F1){ref-type="fig"}. The diagram indicates that, because it is a state variable, the free energy (ΔG\') for Path 1 must be equal to that for path 2 + 3. If the ΔG\' values for path 1 and path 2 are taken to be their calorimeter values, they will be approximately equal (\~4 kcal/g, path 1 corrected for ureagenesis). This means that ΔG\' for path 3, the conversion of protein to carbohydrate (also corrected) must be about zero. There exists at least one condition where this is not true, the standard state; it is generally considered that gluconeogenesis from one mole of alanine requires about 6 ATP \[[@B13],[@B14]\]. Of course free energies are concentration dependent, so *in vivo*values will differ from standard state values but they are continuous functions of the concentrations and there will be numerous conditions under which ΔG\' is not zero. In other words, assuming that protein and carbohydrate are energetically equivalent leads to a contradiction.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Pathways for oxidation of macronutrients.
:::
:::
Inefficiency
============
The second law was developed in the context of the industrial revolution and the attempt to understand the efficiency of machines. The law describes the theoretical limits on the efficiency of engines and applies as well to living (irreversible) systems. The second law says that no machine is completely efficient. Some of the available energy is lost as heat and in the internal rearrangement of chemical compounds and other changes in entropy. In other words, although the first law holds even in irreversible processes -- energy is still conserved -- the second law says that something is lost, something *is*unrecoverable. The efficiency of a machine is dependent on how the machine works and, for a biochemical machine, the nature of the fuel and the processes enlisted by the organism. A simple example is the inefficiency of low-test gasoline in high compression gasoline engines. If a \"calorie is a calorie\" were true, nobody would pay extra for high test gasoline. (The calorimeter values of a gasoline will be the same whether or not it contains an antiknock compound). In weight loss diets, of course, inefficiency is desirable and is tied to hormonal levels and enzyme activities
Efficiency and thermogenesis
============================
In nutrition, one component of inefficiency is measured in thermogenesis (thermic effect of feeding), or the heat generated in processing food. There is a large literature on this subject and the general conclusion, as summarized in a recent review by Jéquier \[[@B15]\], is that thermic effects of nutrients is approximately 2--3 % for lipids, 6--8 % for carbohydrates, and 25--30% for proteins. It is interesting that this data itself might be enough to explain metabolic advantage. Here we took the average of Jéquier\'s values (2.5, 7 and 27.5 % for fat, CHO and protein) and calculated the effective energy yield for a 2000 kcal diet. If we assume a diet composition of CHO:fat: protein of 55:30:15, within the range of commonly recommended diets, the calculated effective yield is 1848 kcal. We now consider the effect of reducing carbohydrate progressively and substituting the calories removed equally between fat and protein. Figure [2](#F2){ref-type="fig"} shows that the wasted calories due to thermogenesis increase as carbohydrate is reduced and reach 100 kcal at 21 % carbohydrate. This value of 100 kcal is recommended by several professionals as the goal for daily weight reduction (e.g. \[[@B16]\]). Notably, at 8 % CHO, the value for the early phase of the Atkins \[[@B17]\], South Beach \[[@B18]\] or Protein Power diets \[[@B19]\], 140 kcalories are lost as heat. Now, there will be metabolic accommodations and one can\'t predict that the ratios will stay the same over a long term diet, but the calculations show that the *possibility*of metabolic advantage should not come as a surprise.
::: {#F2 .fig}
Figure 2
::: {.caption}
######
The dependence of effective calories on % carbohydrate in a 2000 kcal diet. Effective calories were determined by subtracting the losses due to thermogenesis as described in the text.
:::
:::
Recommendations for fighting obesity frequently call for small reductions in calories \[[@B16]\]. In fact, given the resistance of steady state systems to small perturbations it is doubtful that this is a promising strategy. Nonetheless, taking the goal at face value, if it could be achieved by a simple change in macronutrient composition, such a method would seem worthy of serious consideration. The arguments above show that such a phenomenon is possible. There are plausible arguments for how it could take place and substantial experimental evidence for its occurrence \[[@B4]\].
Conclusions
===========
A review of simple thermodynamic principles shows that weight change on isocaloric diets is not expected to be independent of path (metabolism of macronutrients) and indeed such a general principle would be a violation of the second law. Homeostatic mechanisms are able to insure that, a good deal of the time, weight does not fluctuate much with changes in diet -- this might be said to be the true \"miraculous metabolic effect\" -- but it is subject to many exceptions. The idea that this is *theoretically*required in all cases is mistakenly based on equilibrium, reversible conditions that do not hold for living organisms and an insufficient appreciation of the second law. The second law of thermodynamics says that variation of efficiency for different metabolic pathways *is to be expected*. Thus, ironically the dictum that a \"calorie is a calorie\" *violates the second law of thermodynamics*, as a matter of principle.
The analysis above might be said to be over-kill although it is important, intellectually, not to invoke the laws of thermodynamics inappropriately. There are also, however, practical consequences. The seriousness of the obesity epidemic suggests that we attack it with all the means at our disposal. Metabolic advantage with low carbohydrate diets is well established in the literature. It does not always occur but the important point is that it can occur. To ignore its possibilities and to not investigate the precise conditions under which it appears would be cutting ourselves off from potential benefit. The extent to which metabolic advantage will have significant impact in treating obesity is unknown and it is widely said in studies of low carbohydrate diets that \"more work needs to be done.\" However, if the misconception is perpetuated that there is a violation of physical laws, that work will not be done, and if done, will go unpublished due to editorial resistance. Attacking the obesity epidemic will involve giving up many old ideas that have not been productive. \"A calorie is a calorie\" might be a good place to start.
Competing interests
===================
None declared.
| PubMed Central | 2024-06-05T03:55:46.619430 | 2004-7-28 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC506782/",
"journal": "Nutr J. 2004 Jul 28; 3:9",
"authors": [
{
"first": "Richard D",
"last": "Feinman"
},
{
"first": "Eugene J",
"last": "Fine"
}
]
} |
PMC506783 | Background
==========
Cancer mortality maps are important tools in health research, allowing the identification of spatial patterns, clusters and disease \'hot spots\' that often stimulate research to elucidate causative relationships \[[@B1],[@B2]\]. In most spatial analysis software a statistical pattern recognition approach has been implemented whereby a statistic (*e.g.*spatial cluster statistic, autocorrelation statistic, *etc*.) quantifying a relevant aspect of spatial pattern is calculated. The value of this statistic is then compared to the distribution of that statistic\'s value under a null spatial model. This provides a probabilistic assessment of how unlikely an observed spatial pattern is under the null hypothesis \[[@B3]\]. Waller and Jacquez \[[@B4]\] formalized this approach by identifying five components of a test for spatial pattern.
1\. The *test statistic*quantifies a relevant aspect of spatial pattern (*e.g*. Moran\'s *I*, Geary\'s *c*, LISA, a spatial clustering metric, *etc*.)
2\. The *alternative hypothesis*describes the spatial pattern that the test is designed to detect. This may be a specific alternative, such as clustering near a focus, or it may be the omnibus \"not the null hypothesis\".
3\. The *null hypothesis*describes the spatial pattern expected when the alternative hypothesis is false (*e.g*. Complete Spatial Randomness, often called CSR).
4\. The *null spatial model*is a mechanism for generating the reference distribution. This may be based on distribution theory, or it may use randomization (*e.g*. Monte Carlo) techniques.
5\. The *reference distribution*is the distribution of the test statistic when the null hypothesis is true.
CSR is the null hypothesis employed by most, if not all, statistical tests for spatial pattern, and is the workhorse of almost all spatial statistical software. Examples of statistics used in these tests include spatial autocorrelation (*e.g*. Moran\'s *I*and Geary\'s c); its local counterpart (*e.g*. LISA); geographic boundary statistics (*e.g*. boundary count and mean length), and a host of techniques for identifying hot spots, cold spots and foci. While CSR is useful in some situations, it often is not a relevant null hypothesis for highly complex and organized systems such as those encountered in the environmental and health sciences \[[@B5],[@B6]\]. For such fields CSR may be not relevant because spatial randomness rarely, if ever, occurs -- some spatial pattern is almost *always*present. Hence in many situations rejecting CSR has little scientific value because CSR does not describe any plausible state of the system. As emphasized by Ord and Getis \[[@B7]\], Type I errors may abound when statistical tests are applied without regard to the global autocorrelation structure. For example, locations would be identified as hot spots simply because they lie in areas of generally high (or low) values, which would lead one to blend together local peaks and clusters of high (low) values. Even when health professionals are interested in identifying areas with generally high (or low) disease rates, it is still important to account for spatial autocorrelation to avoid an over-identification of the number of significant spatial clusters or outliers. In summary what are needed are realistic models that incorporate background pattern -- the spatial and multivariate structure found when the null hypothesis is true.
The term \"*Neutral Model*\" captures the notion of a plausible system state that can be used as a reasonable null hypothesis (*e.g*. \"background variation\"). The problem then is to identify spatial patterns *above and beyond*that incorporated into the neutral model, enabling, for example, the detection of cancer clusters beyond background or regional variation in the risk of developing cancer. Neutral models can be generated using simulation techniques developed in the field of geostatistics \[[@B8]\] which provides a set of statistical tools for analyzing and mapping data distributed in space and time. In particular, sequential Gaussian simulation (SGS) allows one to generate realizations of the spatial distribution of rates that reproduce the sample histogram and spatial patterns displayed by the data, and also account for any auxiliary data or information on the local trend \[[@B9]\].
The objective of this paper is to present geostatistical approaches to generating neutral models that account for the spatial dependence of cancer rates, their regional background and spatially heterogeneous population sizes. These models are then used for the detection of local clusters and anomalies in cancer rates. The new methodology is applied to the analysis of the geographical distribution of lung cancer in three counties of Long Island, New York, which have been investigated under the CSR hypothesis in a previous issue of this journal \[[@B10],[@B11]\]
Methods
=======
Data
----
The use of neutral models in cluster analysis will be illustrated using the lung cancer data analysed in \[[@B10],[@B11]\]. This section briefly summarizes the salient features of this dataset, and readers are referred to the above papers for a detailed description.
The New York State Department of Health (NYSDOH) published the cancer incidence data online as part of their Cancer Surveillance Improvement Initiative, <http://www.health.state.ny.us/nysdoh/cancer/csii/nyscsii.htm>. Data have been released on the following four cancers: breast (female only), colorectal (female and male), lung (female and male), and prostate. These data represent newly diagnosed cancer cases in the period 1993--7 assigned to the patient\'s residence at diagnosis, and they are calculated as the number of cancers for each 100,000 people in the population.
To protect patient privacy, the NYSDOH data provided case counts referenced to ZIP codes rather than individual residences. While ZIP codes are somewhat arbitrary spatial units of analysis with respect to potential health and environmental factors, they provide a convenient way to group the population and preserve confidentiality. The methods presented here do not depend on the spatial unit of aggregation and the reader may use census geography if that is their preference. As in the earlier analysis \[[@B10],[@B11]\], the focus of this study is on the 214 ZIP codes within Nassau, Queens and Suffolk County on Long Island.
Because cancer incidence is related to age, NYSDOH calculated the expected cancer incidence for each ZIP code using the ZIP code\'s age structure and the average incidence by age class for New York State (direct adjustment). We thus are using an external standard (the state average) rather than an internal standard (the average for Long Island), to calculate the expected incidence. A standardized morbidity ratio (SMR) has been calculated by dividing the observed value by the age-adjusted expected incidence. An SMR value of 1.0 indicates that the observed incidence is the same as expected, lower than 1.0 indicates that fewer than expected cases of cancer occurred, and greater than 1.0 indicates that more than expected occurred.
Local cluster analysis under CSR
--------------------------------
Jacquez and Greiling \[[@B10]\] identified significant clustering and spatial outliers in SMR using Anselin\'s local Moran test \[[@B12]\] in the ClusterSeer™ Software <http://www.terraseer.com/products/clusterseer.html>. The local Moran test evaluates local clustering or spatial autocorrelation by computing the contribution of each location to the Moran\'s I statistics for the whole study area. Its null hypothesis is that there is no association between SMR values in neighboring ZIP codes. The working (alternative) hypothesis is that spatial clustering exists. For each ZIP code, referenced geographically by its centroid with the vector of spatial coordinates **u**= (x, y), the LISA (Local Indicator of Spatial Autocorrelation) statistic is computed as:
where z(**u**) is the SMR for the ZIP code being tested, which is referred to as the \"kernel\" hereafter. z(**u**~j~) are the values for the J(**u**) neighboring ZIP codes that are here defined as units sharing a common border or vertex with the kernel **u**(1-st order queen adjacencies). All values are standardized using the mean *m*and standard deviation *s*of the SMR data (here 214 values). Since the standardized values have zero mean, a negative value for the LISA statistics indicates a spatial outlier where the kernel value is much lower or much higher than the surrounding values (e.g. SMR is below the global zero mean while the neighborhood average is above the global zero mean, or conversely). Cluster of low or high values will lead to positive values of the LISA statistics (e.g. both kernel and neighborhood averages are jointly above zero or below zero).
In addition to the sign of the LISA statistics, its magnitude informs on the extent to which kernel and neighborhood values differ. To test whether this difference is significant or not, a Monte Carlo simulation is conducted, which traditionally consists of [sampling randomly]{.underline} and without replacement the global distribution of observed rates (i.e. sample histogram), then computing the corresponding simulated neighborhood averages. This operation is repeated many times (e.g. *L*= 999 draws) and these simulated values are multiplied by the kernel value to produce a set of *L*simulated values of the LISA statistics at location **u**:
with z^(l)^(**u**~j~) = F^-1^\[p^(l)^(**u**~j~)\], F\[.\] is the sample cumulative distribution function (cdf), and p^(l)^(**u**~j~) is a random number uniformly distributed within 0 and 1. This set represents a numerical approximation of the probability distribution of the LISA statistics at **u**, under the assumption of spatial independence. The observed LISA statistics, LISA(**u**), can then be compared to the probability distribution, allowing the computation of the probability of not rejecting the null hypothesis (so-called *p*-value). Following Jacquez and Greiling \[[@B10]\], we used an adjusted significance level *α*= 0.01101 to account for the fact that the multiple tests (i.e. 214 in this study) are not independent since near ZIP codes share similar neighbors. This significance level was obtained using the Bonferroni adjustment which amounts at dividing the significance level *α*= 0.05 by the average number of neighbors in each test. Thus, every ZIP code where the p-value is lower than 0.01101 will be classified as a significant spatial outlier (HL: high value surrounded by low values, and LH: low value surrounded by high values) or cluster (HH: high value surrounded by high values, and LL: low value surrounded by low values).
A typology of neutral models
----------------------------
The use of CSR as the null hypothesis means that the distribution of cancer rates is assumed to be spatially random (no autocorrelation) with uniform risk over the study area. In most cases, however, mortality rates are spatially correlated while the risk of developing cancer varies regionally as a result of changes in environmental exposure or other demographic, social, and economic factors. Another weakness of the above test is that it does not consider whether ratio data are based on many or a few cases, thereby ignoring the instability of rates computed from small population sizes. The basic idea of the proposed approach is to generate neutral models that are more realistic in the sense that they incorporate presence of spatial autocorrelation, non-uniform risk, and account for spatially heterogeneous population sizes.
Table [1](#T1){ref-type="table"} provides a typology of neutral models that could be used for inference regarding numerator and denominator, including incidence and prevalence, as well as mortality rates. Model I corresponds to the CSR case, while model II reproduces the spatial correlation of the cancer rates. Model III reflects the situation where environmental exposures or other factors make the risk non-uniform. Models IV through VI allow one to account for the impact of population size on the stability of observed rates. Unlike Model I these more complex neutral models can not be generated simply by shuffling randomly the SMR data across the 214 ZIP codes, and geostatistical simulation techniques to generate each type of model are described below.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Typology of neutral models. Models differ according to the reproduction of spatial correlation, the incorporation of non-uniform risk, and the filtering of noise caused by spatially varying population sizes.
:::
Risk Accounting for Population size
------------------------------------- -------------------------------- --------
No Yes
Uniform, spatially random **I** **IV**
Uniform, spatially correlated **II** **V**
Heterogeneous, spatially correlated **III** **VI**
:::
Normal score transform of SMR data
----------------------------------
The simulation techniques used in this paper assume a multiGaussian distribution for the variable under study, which requires a prior normal score transform of SMR data to ensure that at least their univariate distribution (histogram) is normal. Normal score transform is a graphical transform that allows one to normalize any distribution, regardless of its shape. It can be seen as a correspondence table between equal *p*-quantiles z~p~and y~p~of the z-cdf F(z) (cumulative histogram) and the standard Gaussian cdf G(y). In practice, the normal score transform proceeds in three steps:
1\. The N original data z(**u**~*α*~) (i.e. SMR data) are first ranked in ascending order. Since the normal score transform must be monotonic, ties in *z*-values must be broken, which has been done randomly as implemented in GSLIB software \[[@B13]\].
2\. The sample cumulative frequency of the datum z(**u**~*α*~) with rank k is then computed as  = k/N - 0.5/N.
3\. The normal score transform of the *z*-datum with rank *k*is matched to the -quantile of the standard normal cdf:
*y*(**u**~*α*~) = *φ*(*z*(**u**~*α*~)) = *G*^-1^\[*F*(*z*(**u**~*α*~))\] = *G*^-1^\[\]
Local cluster analysis under spatial neutral model (Model II)
-------------------------------------------------------------
Model II aims to reproduce the pattern of spatial correlation displayed by the data that is here quantified using the normal score semivariogram \[[@B8],[@B14]\] which plots the average squared difference between normal score transformed SMR data as a function of the separation distance and direction between ZIP codes:
Here \|**h**\| corresponds to the Euclidian distance between two ZIP codes. Note the following discussion can be readily generalized to other distance measures that could be more appropriate to capture contiguity of entities of complex shape: our methodology is general and does not depend on a particular formulation of the distance measures. Following previous simulation studies \[[@B9]\] and in order to account for the noise induced by small population sizes, each pair has been assigned a weight proportional to the square root of the population size, , where n(**u**~*α*~) is the size of the population at risk in the ZIP code with centroid **u**~*α*~. Following an earlier analysis of the data \[[@B10]\], the population in ZIP codes was estimated using 2000 US census numbers.
Spatial neutral models are generated using Sequential Gaussian Simulation (SGS) which proceeds as follows (see \[[@B8]\] p. 380 for more details):
1\. Fit a permissible function \[[@B9]\] to the experimental semivariogram (Equation 3). The modeling was here performed using least-square regression \[[@B15]\]. All semivariogram models were bounded, that is a sill is reached for a given distance referred to as the range of influence. The covariance models were then derived by subtracting the semivariogram model from the sill value.
2\. Define a random path (i.e. using a random number generator) visiting each ZIP code location **u**~*α*~only once.
3\. At each location **u**~*α*~determine the mean and variance of the Gaussian probability distribution of y-values as:
where y^(l)^(**u**~i~) are normal scores simulated at locations previously visited along the random path and located within a search radius from **u**~*α*~, m~Y~is the stationary mean of the variable Y (which is zero following the normal score transform), and C(**u**~i~-**u**~*α*~) is the covariance function of the normal score variable Y for the separation vector **h**~i*α*~= **u**~i~-**u**~*α*~. *λ*~i~are kriging weights obtained by solving the following system of linear equations (simple kriging, SK):
4\. Draw a simulated value from the conditional cumulative distribution function (ccdf) of probability and add it to the data set. In other words, the simulated value at **u**~*α*~is , where p^(l)^is a random number between 0 and 1.
5\. Proceed to the next location along the random path, and repeat the two previous steps.
6\. Loop until all *N*locations (i.e. N = 214 here) are simulated.
7\. Transform the simulated normal scores {y^(l)^(**u**~*α*~); *α*= 1,\..., N} so that the target histogram (in this case the global distribution of observed rates, F\[.\]) is reproduced: z^(l)^(**u**~*α*~) = F^-1^\[p^(l)^(**u**~*α*~)\] with p^(l)^(**u**~*α*~) = G\[y^(l)^(**u**~*α*~)\]
The procedure is repeated using a different random path and set of random numbers to generate another realization. Note that these realizations account for only the histogram and semivariogram model of the SMR data (global conditioning), but they are non-conditional to the SMR data themselves (e.g. location of zones of high or low SMR values).
Once the L sets of N simulated SMR values, {z^(l)^(**u**~*α*~); *α*= 1,\..., N} have been generated, Equation (2) is applied to each member of this set to compute the simulated values of the LISA statistics at each location **u**. The simulated LISA values form the empirical distribution of the LISA statistics, allowing the calculation of the p-value for the test of hypothesis.
Local cluster analysis under a locally constrained spatial neutral model (Model III)
------------------------------------------------------------------------------------
The simulation of neutral model II is conducted using a stationary mean for SMR values, which is unrealistic for situations where environmental exposure or other factors make the risk non-uniform. In this instance the researcher wishes to detect spatial pattern *above and beyond*this non-uniform risk. For example, one might want to detect clusters of melanoma beyond those that are explained by the north-south gradient in solar radiation. Non-uniform risk can easily be accounted for in the simulation procedure by replacing the stationary mean m~*Y*~in Equation 4 by locally varying means m~Y~(**u**~*α*~), that is using the following estimate for the mean and variance of the Gaussian ccdf:
where C~R~(**u**~i~-**u**~*α*~) is the covariance function of the residual normal score variable \[Y(**u**~*α*~) - m~Y~(**u**~*α*~)\] for the separation vector **h**~i*α*~= **u**~i~-**u**~*α*~, and the kriging weights are obtained by solving the following system of linear equations (simple kriging with local means, SKlm):
The first step in the generation of model III is the computation of the local means m~Y~(**u**~*α*~), which define the reference background risk the user wants to consider for the null hypothesis. In this paper a smooth model of background risk values was obtained by using the following kriging estimator of the local means of observed SMR data:
The kriging weights are calculated in two-steps. First, the following \"kriging of the local mean\" system \[[@B8]\] is solved:
Then, to incorporate data reliability due to spatially varying population size directly into the geostatistical filter the kriging weights are rescaled, following \[[@B9]\], as:
This rescaling is applied separately to the negative and positive kriging weights, keeping constant the overall contribution of these two sets of weights; that is the sum of positive (negative) kriging weights is the same before and after rescaling, which ensures that the unbiasedness constraint in system (11) is still satisfied. Note that although the population size is incorporated in the estimation of the local means, it is not accounted for directly into the test of hypothesis, which will be achieved using Models IV through VI introduced below.
Once the local means of the normal score transformed SMR data have been estimated, they are subtracted from the SMR values and the semivariogram of residuals is estimated and modelled. Then, the simulation is performed using SGS and SKlm. Last, the L realizations are used to derive the empirical probability distribution of the LISA statistics and the p-value of the test is computed.
Accounting for population size in local cluster analysis (Models IV to VI)
--------------------------------------------------------------------------
The neutral models introduced so far ignore the fact that cancer rates estimated over small areas, such as United States ZIP code areas or census tracts, tend to be less reliable \[[@B16],[@B17]\], hence larger fluctuations among simulated rates are expected at these locations. If ignored, large differences in population size decrease the ability of Moran\'s I to detect true clustering. There are essentially three approaches to incorporate population sizes in cluster detection: 1) randomly shuffle the cases rather than the rates (i.e. under a heterogeneous Poisson model the cases are allocated to each area using hypergeometric sampling \[[@B18]\]), 2) use a modified version of the test statistics (i.e. Oden\'s I pop \[[@B19]\] or Waldhör\'s I \[[@B20]\]), and 3) transform or standardize the rates first, then compute the LISA statistics on the results (i.e. Empirical Bayes Index \[[@B21]\], Cressie\'s transform \[14 p.385--402\], or any other smoothing algorithm \[[@B17],[@B22]\]). In this paper, the third approach has been adopted and the noise caused by small population sizes was filtered using a variant of the estimator introduced in equation 10:
The kriging weights are calculated in two-steps. First, the following system is solved:
with *g*~0~= *b*~0~× (1-*δ*(***u***~*i*~-***u***~*α*~)) where *b*~0~is the nugget variance in the weighted semivariogram model of SMR data, and *δ*(***u***~*i*~-***u***~*α*~) = *0*if ***u***~*i*~= ***u***~*α*~and 1 otherwise. Then, to incorporate data reliability (i.e. population size) directly into the geostatistical filter the kriging weights are rescaled according to Equation 12. The ability of the proposed approach to reconstruct the underlying disease risk from observed mortality rates has been tested in extensive simulation studies \[[@B9]\].
Results and discussion
======================
Generating spatial neutral models
---------------------------------
Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"} (top graphs) show the geographic distribution of lung cancer in males and females (aggregated to the ZIP code level), in Long Island, New-York. Middle graphs show the experimental weighted semivariograms computed in four directions from the normal score transforms of SMR data. For both males and females SMR normal scores exhibit a range of autocorrelation of about 15 km, with smaller variability (i.e. lower semivariogram values) observed along the NW-SE direction. The spatial anisotropy is less pronounced for female lung cancer and an isotropic model was fitted (solid black line). Regional background is further revealed once the noise and short-range variability of SMR data has been removed using factorial kriging (Equation 10) and the semivariogram model fitted to sampled values (solid line in middle graphs), see Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"} (bottom graph). High SMR values are recorded mainly along the Southern shore of the Island for both genders, while differences between males and females are more striking for low value: the lowest SMR values are observed in the westernmost part of Long Island for females and slightly more to the east for the males. These maps of regional background were subtracted from the original SMR maps, and the spatial autocorrelation of the corresponding residuals was quantified using the experimental semivariograms displayed in Figure [3](#F3){ref-type="fig"}. Since some of the spatially correlated variability is captured by the regional background, the residual semivariograms show lower sills and shorter ranges relatively to the SMR semivariograms of Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Geographic distribution and spatial variability of male lung cancer.**The fill color in each ZIP code represents the SMR, with green indicating relatively low SMR and purple representing relatively high SMR (categories correspond to deciles of the histogram of rates). From these rates, a population-weighted semivariogram is computed in four directions. The semivariogram model (solid line) is used to filter the noise and short-range variability of observed SMR, yielding a smooth map of SMR local means (regional background).
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**Geographic distribution and spatial variability of female lung cancer.**The fill color in each ZIP code represents the SMR, with green indicating relatively low SMR and purple representing relatively high SMR (categories correspond to deciles of the histogram of rates). From these rates, a population-weighted semivariogram is computed in four directions. The semivariogram model (solid line) is used to filter the noise and short-range variability of observed SMR, yielding a smooth map of SMR local means (regional background)
:::
:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**Residual semivariograms for male and female lung cancer.**The regional background displayed at the bottom of Figures 1 and 2 is subtracted from the maps of SMR data, and the spatial variability of these residuals is characterized by population-weighted semivariograms computed in four directions.
:::
:::
One hundred realizations of neutral Models I through III were generated using Sequential Gaussian simulation and the information displayed in Figures [1](#F1){ref-type="fig"} to [3](#F3){ref-type="fig"}. The first two realizations of each model for male lung cancer are displayed in Figure [4](#F4){ref-type="fig"}. The two top maps (model I), which were obtained by shuffling randomly the 214 ZIP code SMR data in Figure [1](#F1){ref-type="fig"} (top map), illustrate the simplistic nature of CSR as null hypothesis in cluster detection. Spatial patterns are reproduced by the middle maps (Model II) where one notices groups of low and high simulated SMR values the position of which changes from one realization to the next since the simulation is not conditioned locally to the observed rates. The regional background displayed in Figure [1](#F1){ref-type="fig"} (bottom graph) is incorporated in Model III, which reduces fluctuations among realizations and led, for example, to high SMR values being consistently simulated in the central part of Long Island.
::: {#F4 .fig}
Figure 4
::: {.caption}
######
**Different neutral models for male lung cancer.**The fill color in each ZIP code represents the simulated SMR, with green indicating relatively low SMR and purple representing relatively high SMR (categories correspond to deciles of the histogram of simulated rates). Simulated maps (realizations) of the spatial distribution of lung cancer SMR data are generated under the assumption of complete spatial randomness (Model I), or created using geostatistical simulation in order to reproduce the spatial autocorrelation displayed by observed rates (Model II) as well as the regional background, i.e. SMR local means (Model III).
:::
:::
Accounting for population size in spatial neutral models
--------------------------------------------------------
Population in Long Island ZIP codes can vary substantially, ranging from 445 to 105,723, with a mean of 23,298, see Figure [5](#F5){ref-type="fig"} (top graph). Population sizes also display a strong spatial pattern, with a gradient from highly populated ZIP codes in the western part of Long Island to the sparsely populated eastern part. The scattergrams in Figure [5](#F5){ref-type="fig"} illustrate how the population size impacts the magnitude of fluctuations among SMR data. As the ZIP codes become less populated the variability among SMR values increases, which reflects the smaller reliability of the rates inferred from small populations at risk and makes problematic the later detection of clusters or spatial outliers.
::: {#F5 .fig}
Figure 5
::: {.caption}
######
**Geographic distribution of population size (male + female) and its impact on stability of SMR values.**The fill color in each ZIP code represents the 2000 population size, with green indicating sparsely populated ZIP codes and purple representing larger population sizes (categories correspond to deciles of the histogram of sizes). The scatterplots illustrate the larger spread of measured SMR for ZIP codes with low population and how the extreme rates recorded in these ZIP codes are smoothed by geostatistical filtering.
:::
:::
Using factorial kriging and the SMR semivariogram models displayed in Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}, the noise caused by small population sizes was geostatistically filtered from SMR maps: compare filtered maps in Figure [6](#F6){ref-type="fig"} with original maps shown at the top of Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}. While the noise filtering does not change the mean of the SMR data, their standard deviation decreases: 0.290 to 0.238 for males and 0.355 to 0.329 for females. The larger decrease observed for male SMR values is caused by the higher amount of noise (i.e. relative nugget effect) reflected as the discontinuity at the origin of the semivariogram. The scattergrams at the bottom of Figure [5](#F5){ref-type="fig"} indicate that the geostatistical filtering changes mainly the extreme rates recorded for sparsely populated ZIP codes. Then, one hundred realizations of neutral Model IV through VI were generated using Sequential Gaussian simulation and the filtered SMR maps statistics.
::: {#F6 .fig}
Figure 6
::: {.caption}
######
**Geostatistical filtering of male and female lung cancer data.**The fill color in each ZIP code represents the noise-filtered SMR, with green indicating relatively low SMR and purple representing relatively high SMR (categories correspond to deciles of the histogram of filtered rates).
:::
:::
Local cluster analysis under various neutral models
---------------------------------------------------
### Female
Figures [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"} show the results of the cluster analysis for female SMR values under the neutral models I through VI, while Table [2](#T2){ref-type="table"} lists the exact number of ZIP codes classified as significant clusters of high values (HH) or low values (LL), and outliers (LH and HL). Table [2](#T2){ref-type="table"} also indicates how the p-value varies among neutral models, highlighting the fact that depending on the assumption being made, the size and locations of clusters/outliers can change.
::: {#F7 .fig}
Figure 7
::: {.caption}
######
**Results of the local cluster analysis conducted for female lung cancer using neutral models I to III.**The fill color in each ZIP code represents the classification into significant low-low or high-high clusters, as well as high-low or low-high outliers. Yellow indicates ZIP codes that have not been found significant using an adjusted significance level *α*= 0.01101.
:::
:::
::: {#F8 .fig}
Figure 8
::: {.caption}
######
**Results of the local cluster analysis conducted for female lung cancer using neutral models IV to VI.**The fill color in each ZIP code represents the classification into significant low-low or high-high clusters, as well as high-low or low-high outliers. Yellow indicates ZIP codes that have not been found significant using an adjusted significance level *α*= 0.01101.
:::
:::
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Number of significant zip codes for the different types of cluster/outliers and neutral models. Results are reported for female lung cancer. Numbers between parentheses indicate zip codes that have similar classification under the reference Model I (CSR). Summary statistics for the p-values are also provided.
:::
Neutral Model Type
------------- -------------------- ---------- ----------- ---------- --------- ----------
Model I Model II Model III Model IV Model V Model VI
High-High 7 0(0) 4(2) 10(5) 0(0) 3(2)
High-Low 1 0(0) 2(0) 0(0) 0(0) 1(0)
Low-High 2 0(0) 4(0) 2(2) 0(0) 1(0)
Low-Low 18 1(0) 2(2) 31(18) 4(4) 6(4)
**P-value**
Mean 0.178 0.230 0.405 0.166 0.237 0.394
CV 85.9% 62.5% 71.3% 95.3% 62.2% 76.9%
:::
Under the CSR model (Model I), results similar to the ones reported in \[[@B11]\] were found. First, the local Moran test identified a single, large cluster of low SMR extending through portions of Flushing in the north and Jamaica in the south. Next to this cluster is the only high-low outlier: Oakland Gardens (11364) with a SMR of 1.116. Sayville (11782) is a significant spatial outlier with low SMR (72% of the New York average), though its SMR has a wide confidence interval resulting from the small number of observed cases there (15,896 habitants). Thus, while statistically distinct from its neighbors, it does not have significantly reduced risk. This is also the case for the second low-high outlier, Manorville (11949, 11,384 habitants), which has a SMR close to one but is located in the western part of Long Island where background rates are higher. Several local clusters of high SMR values occurred in the more central portions of Long Island. There is a cluster in north-mid Long Island, made up of two significant local clusters centered on Bayville (11709) and Mill Neck (11765). This cluster has about 60--70% higher SMR than the New York state average. A large cluster in south central Long Island is composed of four local clusters centered on Ronkonkama (11779), Central Islip (11722), Islip Terrace (11752), and East Islip (11730). This cluster has an SMR about 40% higher than the New York state average. Further east is a third cluster of high female lung cancer incidence centered on Mastic (11950) and including several adjacent ZIP codes. Its SMR is about 60% higher than the New York state average.
Accounting for spatial autocorrelation (i.e. Model II) leads to a substantial reduction in the size of significant clusters compared to the CSR assumption. In fact only one ZIP code is a significant low-low cluster under Model II: Saint Albans (11412) which was the center of the Southern low-low cluster detected under CSR. The scattergram in Figure [9](#F9){ref-type="fig"} (left graph) shows that the use of spatially correlated neutral models leads to larger p-values on average (0.23 vs. 0.18), and those are highly correlated with the ones obtained under CSR (Model I). These larger p-values cause a substantial reduction in the size of significant ZIP codes, since fewer units exceed the adjusted significance level *α*of 0.01101. The reason for that increase in p-values is illustrated for the ZIP code \#11364 (Oakland Gardens) which was the only unit classified as high-low outlier under neutral model I. Figure [10](#F10){ref-type="fig"} (left top graph) shows the distribution of simulated values of the LISA statistics for that ZIP code. Clearly, the variance of the distribution is much larger than the results obtained under CSR, while both means are equal to zero. The spatial autocorrelation of simulated rates increases the likelihood that the J neighboring values are jointly small or high, causing the neighborhood average, hence the LISA value, to exhibit much larger fluctuations among realizations. Consequently, the probability that the observed LISA statistics falls in the tails of the simulated distribution decreases, leading to a larger p-value (0.061 versus 0.003) and a ZIP code that is no longer a significant outlier.
::: {#F9 .fig}
Figure 9
::: {.caption}
######
**Scatterplots of the p-values obtained when conducting the local cluster analysis under CSR assumption (Model I) or more complex neutral models.**Model III reproduces the pattern of spatial correlation as well as the regional background of SMR values, while Model II accounts only for the spatial correlation.
:::
:::
::: {#F10 .fig}
Figure 10
::: {.caption}
######
**Histograms of the values of the LISA statistics simulated for ZIP code \#11364 (Oakland Gardens) under different neutral models.**The black dot denotes the observed LISA statistics which lies inside the 0.95 probability interval for all models except Models I and IV developed under the CSR assumption.
:::
:::
The map of significant ZIP codes at the bottom of Figure [7](#F7){ref-type="fig"} bears little resemblance with the maps obtained under the neutral models I and II. This is expected since Model III addresses a different question, namely the detection of local departures from the regional background. Thus, in general, one would expect HL and LH outliers to be more frequent than spatial clusters HH or LL. Also the local constraining of the neutral models to the regional background causes less variation among realizations, leading to the J neighboring values being consistently either small or large across the realizations. Thus the distribution of 999 simulated LISA values is expected to be narrower than for the two previous models with a shift in the mean. This is illustrated for the ZIP code \#11364 in Figure [10](#F10){ref-type="fig"} (left bottom graph). Because this unit is located in a low-valued area, the use of neutral models reproducing the regional background yields smaller simulated LISA values (average = -0.14 instead of 0.0). In high-valued areas, the shift is expected to be in the opposite way, leading to a larger range of p-values observed across the area, see the scattergram in Figure [9](#F9){ref-type="fig"} (right graph). Table [2](#T2){ref-type="table"} and Figure [9](#F9){ref-type="fig"} indicate that the p-values are of larger magnitude (average: 0.405 versus 0.23 for Model II) and weakly correlated with the ones obtained under CSR.
For female lung cancer, the same numbers of ZIP codes (6) were classified as significant outliers or clusters under neutral model III. The two low-low clusters are Springfield Gardens (11413) and Saint Albans, which was the only significant unit under neutral model II. These ZIP codes are both located in the western part of Long Island with low background SMR values, and in the same area the following three low-high outliers are found: Bellerose (11426), Little Neck (11362), and New Hyde Park (11040) surrounding the high-high cluster Glen Oaks (11004). The last low-high outlier is found in Shelter Island Heights (11965) in the eastern part of Long Island, though its SMR (72% of the New York average) has a wide confidence interval resulting from the small number of observed cases there (1,080 habitants). The two high-low outliers are found in central Long Island characterized by a low SMR background level: Ridge (11961) and Bayport (11705) with SMR values 20 to 40% higher than the New York state average. Three more clusters of high SMR (1.15 to 1.20) are found in the North western part of Long Island, next to the large group of low SMR recorded in Flushing and Jamaica: Bayville (11709), Mill Neck (11765), and Glen Cove (11542).
For all three types of model, accounting for population size through geostatistical filtering leads to a larger number of ZIP codes classified as clusters and fewer outliers, see Figure [8](#F8){ref-type="fig"} and Table [2](#T2){ref-type="table"}. This result can be explained by the smoothing of local fluctuations, in particular the ones recorded in sparsely populated ZIP codes, yielding larger and more compact clusters, such as for Model IV. Figure [10](#F10){ref-type="fig"} (right column) also shows that this smoothing halves the standard deviation of the distributions of simulated LISA statistics. Major differences between Models I and IV include bigger and more compacts clusters of low and high SMR values, the disappearance of two sparsely populated high-high clusters (Bayville and Mill Neck, with 7,134 and 732 habitants, respectively), and the classification of a former high-low cluster into a low-low cluster (Oakland Gardens) since the filtered rate becomes slightly lower than the global mean. A similar trend is observed for spatially correlated neutral models where the filtering increases the number of significant low-low clusters from one to four, all located in the eastern part of Long Island. The comparison of Models III and IV indicates the disappearance of a few sparsely populated ZIP codes which were classified as spatial outliers prior to filtering: HL (Bayport, 8,006 habitants), LH (Shelter Island Heights, Bellerose and Little Neck, with 1,080, 18,726 and 17,502 habitants). The only remaining LH cluster is New Hyde Park which has 39,156 habitants. The HH cluster (Glen Oaks, 14,682 habitants) also disappeared. Conversely, three other ZIP codes with populations ranging from 776 to 21,282 became significant LL clusters under Model IV: East Marion (11939), Woodbury (11797), and Cambria Heights (11411).
Across all six types of neutral models, only one out of 214 ZIP codes is consistently classified into the same category: the low-low cluster at Saint Albans (11412) which has a SMR = 0.82 and a population of 37,452. The stability of this cluster under alternative specifications of the statistical null hypothesis can be used by cancer surveillance and control efforts to quantify the degree of confidence associated with this cancer cluster.
### Male
Results of the cluster analysis for male lung cancer are displayed in Figures [11](#F11){ref-type="fig"} and [12](#F12){ref-type="fig"} and reported in Table [3](#T3){ref-type="table"}. Model I (CSR assumption) yields the same results as the one reported in \[[@B11]\]. Three local clusters of low SMR values were identified, centred on Great Neck (ZIP 11024), Roslyn (11576), and Huntington (11743), all in the northwest portion of Long Island. These clusters are typified by lung cancer SMR values that are 50--75% of the New York State average. A large cluster of lung cancer SMR 20--60% higher than the New York average was identified in central Long Island. Cutchogue (11935) was found a significant high-low outlier although its small population (3,444) impacts the reliability of the morbidity ratio. The two low-high outliers are Moriches (11955) and Rockaway Park (11694).
::: {#F11 .fig}
Figure 11
::: {.caption}
######
**Results of the local cluster analysis conducted for male lung cancer using neutral models I to III.**The fill color in each ZIP code represents the classification into significant low-low or high-high clusters, as well as high-low or low-high outliers. Yellow indicates ZIP codes that have not been found significant using an adjusted significance level *α*= 0.01101.
:::
:::
::: {#F12 .fig}
Figure 12
::: {.caption}
######
**Results of the local cluster analysis conducted for male lung cancer using neutral models IV to VI.**The fill color in each ZIP code represents the classification into significant low-low or high-high clusters, as well as high-low or low-high outliers. Yellow indicates ZIP codes that have not been found significant using an adjusted significance level *α*= 0.01101.
:::
:::
As for female lung cancer, accounting for spatial autocorrelation (i.e. Model II) leads to fewer significant ZIP codes compared to the common CSR assumption. Only two units are now significant: high-high cluster at Shirley (11967) and a low-high outlier at Rockaway Park (11694). Changes are also substantial when looking at results obtained under neutral model III. We found two high-high clusters: Shirley (11967) and Queens Village (11429), one low-low cluster: Corona (11368), and one low-high outlier: Elmont (11003).
Accounting for population size in the cluster analysis (Model IV) enhances the size and compactness of the two major clusters of low and high SMR values, while the classification of three sparsely sampled ZIP codes (Cutchogue, Moriches and Rockaway Park with 3,444, 2,652 and 19,278 habitants respectively) changed from spatial outliers to clusters. A new cluster of high SMR values (SMR = 1.29) is also found in Lindenhurst (11757). Using spatially correlated neutral models the geostatistical filtering (Model V) reveals a new cluster of low SMR values in Port Washington (11050) and Great Neck (11024) with lung cancer SMR values that are 70% of the New York State average. Comparison of Models III and VI indicates that besides increasing the size of clusters identified under Model II geostatistical filtering leads to the identification of a new low-low cluster: Cold Spring Harbor (11724, with a SMR 63% below the New York state average) and one low-high outlier: Springfield Gardens (11413, SMR = 0.80).
Across all six types of neutral models, only one out of 214 ZIP codes is consistently classified into the same category: the high-high cluster at Shirley (11967) which has a SMR = 1.157 and a population of 24,942.
How many realizations are needed?
---------------------------------
The use of randomization in test of hypothesis relies on the assumption that the space of solution is sampled fairly exhaustively and uniformly (equally-probable realizations \[[@B23]\]). It is thus necessary to investigate how conclusions change as a function of the number of neutral models generated. For example, Figure [13](#F13){ref-type="fig"} shows the influence of increasing the sample size from 99 to 999 on the average difference in terms of p-value and classification of ZIP codes into significant outliers and clusters (the reference is the results obtained using 99 realizations). All curves exhibit a plateau within this range of sampling intensity, although the asymptotic behavior depends on the type of neutral models. This result indicates that for this case study enough realizations of neutral models were generated to yield stable classifications of ZIP codes.
::: {#F13 .fig}
Figure 13
::: {.caption}
######
**Impact of the number of realizations and type of neutral models on the stability of local cluster analysis results.**The left graph displays the absolute value of the average change in p-value as the number of realizations increases from 99 to 999. The right graph shows the number of ZIP codes that are classified differently as the number of realizations increases from 99 to 999.
:::
:::
Conclusions
===========
Cancer mortality maps are used by public health officials to identify areas of excess and to guide surveillance and control activities. Maps of incidence as well as mortality are used as input to disease clustering procedures whose purpose is to identify local areas of excess and deficit. While some controversy revolves around the utility of these techniques, it is indisputable that the finding of a confirmed cancer cluster is often of considerable concern. The accurate quantification of local excesses and deficits, as well as regional trends and differences in cancer incidence and mortality, is therefore a problem of considerable practical importance.
Arguably one of the biggest problems facing spatial epidemiology and exposure assessment is that of identifying geographic pattern (*e.g.*hotspots, coldspots, clusters, etc) *above and beyond*background variation. Most, if not all, environmental contaminants and diseases with potential environmental causes occur at a background level in the absence of a pollution- or disease-generating process. Nonetheless, this background pattern is typically ignored in spatial analyses that employ null hypotheses of complete spatial randomness. Because some spatial dependency is expected at background levels, CSR often is an inappropriate null hypothesis.
When should the different types of neutral models be employed? The 6 types of neutral models presented here represent permutations of whether or not population size is accounted for, and 3 types of underlying risk models. As a rule of thumb one should employ that neutral model or those neutral models that most closely correspond to the spatial pattern expected in the absence of the alternative spatial process. So, for a cluster study one would select those neutral models that specify the risk function deemed most likely in the absence of spatial clustering. When working with rates spatial heterogeneity in the size of the at-risk population should always be accounted for, and selections from neutral models of types IV through VI are appropriate. When in doubt about which neutral model to employ, it makes sense to use several in order to determine how sensitive the results are to specification (and misspecification) of the null hypothesis. To the authors\' collective knowledge, CSR is rarely if ever encountered in real-world biological systems. It is an apt descriptor of the \"snow\" that used to appear on late-night television when the programming day was over. It thus seems that neutral model types I and IV will seldom be appropriate. They perhaps will prove most useful for evaluating the extent of bias in past studies that employed CSR.
The approach presented in this paper enables researchers to assess geographic relationships using appropriate null hypotheses that account for the spatial correlation and background variation modeled from the observed rates and any ancillary information (i.e. exposure model). An immediate consequence of using more realistic (i.e. spatially correlated) neutral models are larger p-values, leading to a substantial reduction in the number of ZIP codes declared significant outliers or clusters across Long Island. This result confirms earlier findings that CSR often leads to an over-identification of the number of significant spatial clusters or outliers. These false positives have potentially serious consequences in that it can lead to public alarm and demands for investigation by already stretched state health departments. The drop in the number of significant units is however accentuated by the use of an adjusted significance level (Bonferroni adjustment) to account for the correlation between the tests conducted at neighboring ZIP codes. Further research should investigate the redundancy between the use of spatially correlated neutral models and adjusted significance level, which might lead to an \"under-identification\" of the number of significant spatial clusters or outliers.
When the constraint of local conditioning of neutral models is superimposed to the reproduction of spatial autocorrelation (i.e. model III), the approach allows one to detect local departures from the conditioning background specified by the user. In this paper, this background was identified to the regional variability of SMR data which was estimated geostatistically. Future research will investigate the use of exposure models for local conditioning of neutral models, leading to the detection of clustered or isolated geographical units that depart significantly from the cancer rates expected from exposure data. A similar approach has recently been implemented whereby the regional background observed in the past has been incorporated into the geostatistical simulation of neutral models \[[@B24]\]. This new methodology allowed one to identify geographic pattern *above and beyond*background variation displayed in prior time intervals for cervix cancer mortality rates.
Another issue, which often impacts the results of cluster analysis, is the lack of reliability of rates inferred from small populations. If ignored, large differences in population size decrease the ability of Moran\'s I to detect true clustering/departures from spatial randomness. A geostatistical smoother, which accounts for the spatial pattern of SMR data (i.e. anisotropic variability, range of autocorrelation), has been applied to eliminate the random variability that appeared as a nugget effect on the experimental semivariograms. The smoothing of local fluctuations, in particular the ones recorded in sparsely populated ZIP codes, resulted in the detection of larger and more compact clusters of low or high SMR values as well as the disappearance of some unreliable spatial outliers. Geostatistical filters are very flexible and could be used to filter short-range variability in addition to the noise created by small population sizes. In this case, the focus of the analysis would be on the regional background of the data, allowing the detection of regional clusters.
The neutral models and methods in this paper make possible, for the first time ever, evaluation of the sensitivity of the results of cluster or boundary analyses to specification of the null hypothesis. Within a study, this will provide detailed quantification of the reliability of the results, and will identify those areas that are stable (i.e. always classified as a member of a cluster or not) or whose classification is highly sensitive to specification of the null hypothesis. This end result will be a spatially explicit analysis of potential false positives and false negatives.
Authors\' contributions
=======================
Authors PG and GMJ collaborated intensely on all aspects of the manuscript, from research design to data preparation. PG carried out most of the geostatistical and local cluster analysis and drafted the manuscript. Both authors read and approved the final manuscript.
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Number of significant zip codes for the different types of cluster/outliers and neutral models. Results are reported for male lung cancer. Numbers between parentheses indicate zip codes that have similar classification under the reference Model I (CSR). Summary statistics for the p-values are also provided.
:::
Neutral Model Type
------------- -------------------- ---------- ----------- ---------- --------- ----------
Model I Model II Model III Model IV Model V Model VI
High-High 8 1(1) 2(1) 15(8) 2(1) 4(1)
High-Low 1 0(0) 0(0) 0(0) 0(0) 0(0)
Low-High 2 1(1) 1(0) 1(1) 0(0) 2(0)
Low-Low 14 0(0) 1(1) 24(14) 2(1) 4(2)
**P-value**
Mean 0.185 0.259 0.368 0.166 0.251 0.371
CV 83.4% 54.8% 71.8% 92.9% 58.9% 80.5%
:::
Acknowledgements
================
This research was funded in part by grants R01 CA92669 and 1R43CA105819-01 from the National Cancer Institute and R43CA92807 under the Innovation in Biomedical Information Science and Technology Initiative at the National Institute of Health. The views stated in this publication are those of the authors and do not necessarily represent the official views of the NCI.
| PubMed Central | 2024-06-05T03:55:46.621530 | 2004-7-23 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC506783/",
"journal": "Int J Health Geogr. 2004 Jul 23; 3:14",
"authors": [
{
"first": "Pierre",
"last": "Goovaerts"
},
{
"first": "Geoffrey M",
"last": "Jacquez"
}
]
} |
PMC506784 | Background
==========
Cryptosporidiosis is infection with species of the genus *Cryptosporidium*. Most infections in the UK are due either to *C. parvum*(previously known as *C. parvum*genotype 2 or bovine strain) or *C. hominis*(previously known as *C. parvum*genotype 1 or human strain) \[[@B1]\]. *Cryptosporidium*spp. are protozoan parasites. In otherwise healthy individuals they tend to cause a self-limiting form of gastroenteritis which can last for several days and, sometimes, weeks. In patients with certain forms of immune deficiency, most notable the Acquired Immune Deficiency Syndrome (AIDS), the infection can cause a severe and prolonged diarrhoeal illness which, prior to the widespread use of at highly effective antiretroviral therapy was often fatal \[[@B2]\]
Cryptosporidiosis has now become the most commonly identified protozoal cause of gastroenteritis in the United Kingdom. Most of the epidemiological data to-date has been related to reports of outbreaks. Between the years 1983 to 1997 there were 80 outbreaks cryptosporidiosis in England and Wales affecting 4649 individuals \[[@B3]\]. Of these 80 outbreaks, 25 affecting 3455 cases were associated with drinking water. Indeed large outbreaks of cryptosporidiois have often been associated with drinking water \[[@B4],[@B5]\]. Outbreaks of cryptosporidiosis were a particular problem in the North West Region of England during the 1990s where a single unfiltered surface water source was responsible for several outbreaks \[[@B2]\]. However, outbreak-related cases represent only a small proportion (\<10%) of the total cases reported to national surveillance.
The epidemiology of sporadic (non-outbreak-related) cases is largely unknown. Of three large case-control studies reported in the past few years only one found an association with drinking mains water \[[@B7]-[@B9]\]. The study that did find an association with drinking water was undertaken in a largely rural area that when the study was undertaken received some of its water from systems that did not have modern filtration plants. The other two studies identified contact with another case, contact with cattle and overseas travel as being the main risk factors for sporadic infection.
The question remains what proportion of sporadic cryptosporidiosis infections may be due to the consumption of mains drinking water. This paper reports a study using GIS to investigate the epidemiology of sporadic cryptosporidiosis and particularly address the issues of whether sporadic cases are also associated with consumption of drinking water.
Results
=======
Table [1](#T1){ref-type="table"} shows the number of records from each health authority and the number of exclusions, including reasons for exclusion. Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"} show the geographical distribution of individual cases by indicating a dot on the map of the water supply zones (water quality area for Wales). Figure [3](#F3){ref-type="fig"} indicates the attack rates for each zone in the North West where the shading indicates a range of attack rates. Care should be taken in interpreting the zone rates as the populations covered by each zone/area varied substantially. In some zones high attack rates were seen despite only a single case being identified because of a low denominator population. The area specific attack rates are not shown for Wales as numbers of cases was smaller.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Health authority of reported cases, including reasons for exclusion from analysis
:::
Reasons for excluding post codes
----------------------- ----- ----- ---- ---------------------------------- ---- ---- ---- ------
Bro Taf 2 2 2 100
Bury and Rochdale 51 43 8 4 2 1 1 15.7
Dyfed Powys 49 44 5 5 10.2
East Lancashire 47 45 2 2 4.3
Gwent 12 12 0 0
Iechyd Morgannwg 6 6 6 100
Morecambe Bay 13 10 3 3 23.1
Manchester 69 48 21 3 10 8 30.4
N Cheshire 6 6 0 0
North Wales 121 111 10 9 1 8.3
North West Lancashire 74 59 15 3 2 10 20.3
South Cheshire 63 63 0 0
South Lancashire 20 20 0 0
Salford 34 28 6 4 2 17.6
St Helens & Knowsley 8 8 0 0
Stockport 66 60 6 1 1 4 9.1
West Pennine 32 29 3 1 1 1 9.4
Wigan and Bolton 46 35 11 2 4 4 1 23.9
Wirral 28 28 0 0
TOTAL 747 649 98 37 10 24 27 13.1
:::
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Cryptosporidiosis cases in the North West, January 2001 to February 2002
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Cryptosporidiosis cases in the Welsh Water Area, February 2001 to February 2002
:::
:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**Cryptosporidium attack rates in the North West.**Attack rates calculated for each Water Supply Zone in the North West, January 2001 to February 2002 (per 1000 population).
:::
:::
It can be seen that there is substantial spatial variation in the distribution of reported cases. In part, this variation can be explained by variation in population density. However, much of the variation is unexplained. For example, reports from Liverpool are very uncommon, whilst reports from Greater Manchester are very common.
It was decided to investigate the excess case reporting from Greater Manchester in further detail to look for any possible association with water supplies. Water to the Greater Manchester area comes from five main water treatment works; Lostock (derived from Thirlmere in the Lake District and chlorinated but not filtered), Woodgate Hill (derived from Haweswater and Windermere via the Watchgate Treatment Works near Kendal where the water is treated by rapid gravity sand filtration, though not chemically coagulated before spring 2003), Arnfield-Godley (chemical coagulation, clarification and rapid gravity sand filtration), Buckton Castle (chemical coagulation, dissolved air flotation and rapid gravity sand filtration) and Wybersley (chemical coagulation, dissolved air flotation and rapid gravity sand filtration).
In order to determine whether there was any relationship between attack rate and water supply, all water supply zones in the North West that received any water from one or more of these five supplies were identified (Figure [4](#F4){ref-type="fig"}). For each of these water supply zones, the proportion of the supply from each treatment works were was obtained from United Utilities. The correlation between the attack rate and proportion of water from each treatment works was tested using Kendall\'s rank correlation (Table [2](#T2){ref-type="table"}) \[[@B10]\]. The figure adjusted for ties was used. There was no significant correlation between water source and attack rate.
::: {#F4 .fig}
Figure 4
::: {.caption}
######
**Dominant water treatment works in Greater Manchester during 2001.**Each water supply zone in the Greater Manchester area is identified and colour coded to illustrate which of the five water treatment works supply most of the drinking water. Where there is no single dominant source the zone is left uncoloured
:::
:::
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Water supply and cases of Cryptosporidiosis Correlation between water supply zone specific attack rate and proportion of water received from each of the five main water treatment works supplying Greater Manchester.
:::
Water treatment works Z P value
----------------------- -------- ---------
Lostock -1.084 0.2782
Woodgate Hill 1.713 0.0867
Arnfield -- Godley -1.186 0.2353
Buckton Castle -0.628 0.5294
Wybersley 0.451 0.6517
:::
Discussion
==========
As already mentioned, care should be taken in the interpretation of this analysis. It is notable that the proportion of reports that could not be allocated a correct postcode varied from one health authority to another to some extent. Also variation in attack rate between water supply zones or water quality areas was as likely to be due to differences in population size as to differences in reported cases. This was most obvious in zones/areas with relatively small population sizes where random effects could have a particularly important affect. However, there are a number of obvious features.
The most obvious is the large number of cases from the Greater Manchester conurbation. This covered the Bury and Rochdale, Manchester, Salford, Stockport, West Pennine, and Wigan and Bolton Health Authorities. This excess of cases in Manchester is even more remarkable when compared with the virtual absence of cases from the Liverpool conurbation (Liverpool, Sefton and St Helen\'s & Knowsley Health Authorities). The reason for the excess of cases in Greater Manchester is unclear. Although different reporting habits could play a part, we doubt that it could explain more than a small part of the difference. Reporting practices are not that greatly different across the North West \[[@B10]\]. A sero-epidemiological study, currently underway, may be able to determine whether the low reporting rate from Liverpool is real or not.
An alternate explanation could be that the increase represents different water supplies. Salford, and Wigan and Bolton Health Authorities get much of their water supply from Thirlmere, a supply known to be prone to contamination by *Cryptosporidium*\[[@B6]\], none of the others have been implicated in outbreaks of disease. However, it would appear that the attack rates did not vary in any consistent way in relation to water source and so a waterborne hypothesis for this excess could not be proven. Analysis was restricted to Greater Manchester as analysis of all reports in the North West could be subject to confounding as a result of geographical variation in reporting behaviour, whereas the Health Authorities in Greater Manchester share a very similar notification system. The lack of an association with drinking water source was consistent with the conclusions of the case control study undertaken at the same time which also did not find an association with drinking water \[[@B9]\]. Nevertheless, it will be interesting to see whether the completion of an adequate water filtration plant for the Thirlmere supply which is scheduled for spring 2004 has much, if any, impact on the number of reports from Manchester.
A further explanation could be that the Manchester population experience other risk factors more commonly than the Liverpool population. Possible explanations include contact with contact with animals, visiting swimming pools and overseas travel. We do not have access to data to show whether or not people from Manchester are more exposed to these factors than people from Liverpool. However, Manchester is closer to a major National Park, The Peak District. If people from Manchester use their proximity to the Peak District to spend more time in the countryside and so are more likely to come into contact with farm animals, this could explain the difference. This would be an interesting hypothesis to test in a further study.
In addition to Greater Manchester, there are also areas of increased reporting from North Wales and from North West Lancashire. These hotspots also remain unexplained. North West Lancashire, however, receives much of its water from Thirlmere and a water source cannot be excluded. However, many cases were reported from the Fylde peninsula which only receives a small proportion of its water from Thirlmere.
Conclusions
===========
The use of GIS to study the spatial distribution of cases has been useful in identifying geographical variation, but not necessarily for identifying the reasons for this variation. However, initial analysis does not support the hypothesis that differences in drinking water source is the major reason for this variation. We agree with Dangendorf *et al*. \[[@B12]\] that GIS will contribute substantially to our understanding of the contribution of drinking water to human disease as it aids the identification of possible associations between disease and particular water supplies, provided sufficient information is collected to enable accurate location of cases.
Methods
=======
Consultants in Communicable Disease Control in the North West Region of England and in Wales were asked to forward details of cryptosporidium cases upon notification from the laboratory. A data collection form was completed for each case, giving the following details: name, address, postcode, date of birth, GP name, GP address and date of notification. The form was faxed or e-mailed to Communicable Disease Surveillance Centre (CDSC) -- North West as soon as possible.
Enhanced surveillance for the North West of England and Wales were set up separately, North West England in mid December 2000 and Wales in February 2001. Both ran until February 2002. To check for accuracy, the data were audited every 2 months. Each CCDC was sent a list of the cases they had notified to CDSC North West in the preceding 2 months. Any cases that had not been notified were forwarded to CDSC.
The first stage in the geographical analysis was to check the 747 records for possible duplicate records. These were selected on the criteria of 2 individuals with identical names, dates of birth and postcodes being present in the database. Given that a postcode contains on average only 15 addresses the chances of these being legitimate is highly unlikely. Through this procedure 10 records were deleted from the database. Consequently 737 cases of cryptosporidiosis were identified during the period of enhanced surveillance.
The next step was to assign a grid reference to each postcode and this was achieved using the Royal Mail Postcode Address File. Eighty eight records were excluded as either, an incomplete postcode was entered into the cryptosporidiosis database or a match could not be found in the postcode address file. Therefore, in total the database was reduced to 649 cryptosporidiosis cases. These were plotted as points against a backdrop of the water supply zones for the two main water utilities. The water supply zone and water quality area boundaries were provided by the two main water utilities (United Utilities and Welsh Water). A \"water supply zone\" is an area designated by a water undertaker providing water to the residences of not more than 50,000 people. In general the source is consistent across a particular zone.
Using the GIS each case was also assigned its corresponding water supply zone and the number of cases in each WSZ was divided by the population, based upon data supplied by the two water utilities, to produce the attack rate maps. The analysis was undertaken in ArcGIS 8.1 using point in polygon techniques \[[@B13]\].
Authors\' contributions
=======================
PRH was lead on study design, did the statistical analyses and co-wrote the paper.
SH co-designed the study and co-wrote the paper and undertook most of the data collection.
QS co-designed the study and co-wrote the paper.
SW co-designed the study and co-wrote the paper.
IL undertook the geographical analyses and co-wrote the paper.
KO co-designed the study, co-wrote the paper and obtained data on the water distribution.
RC co-designed the study and co-wrote the paper.
| PubMed Central | 2024-06-05T03:55:46.626065 | 2004-7-23 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC506784/",
"journal": "Int J Health Geogr. 2004 Jul 23; 3:15",
"authors": [
{
"first": "Sara",
"last": "Hughes"
},
{
"first": "Qutub",
"last": "Syed"
},
{
"first": "Sarah",
"last": "Woodhouse"
},
{
"first": "Iain",
"last": "Lake"
},
{
"first": "Keith",
"last": "Osborn"
},
{
"first": "Rachel M",
"last": "Chalmers"
},
{
"first": "Paul R",
"last": "Hunter"
}
]
} |
PMC506785 | Background
==========
On Wednesday 21 July 2004, the Healthcare Commission <http://www.healthcarecommission.org.uk/> released its annual star ratings of the performance of NHS Primary Care Trusts (PCTs) in England for the year ending March 2004. The Healthcare Commission started work on 1 April 2004, taking over all the functions of the former Commission for Health Improvement <http://www.chi.nhs.uk/>, which had released the corresponding PCT ratings for 2002/2003 in July 2003 \[[@B1]\].
A star rating scheme is adopted. PCTs with the highest levels of performance in the measured areas are awarded a rating of three stars. PCTs with mostly high levels of performance, but which are not consistent across all measured areas, are awarded a rating of two stars. PCTs where there is some cause for concern about particular areas of measured performance are awarded a rating of one star. PCTs that have shown the poorest levels of measured performance or little progress in implementing clinical governance receive a rating of zero stars \[[@B2]\].
The performance ratings Web pages of the Healthcare Commission and the former Commission for Health Improvement offer a very limited \"geographic search\" restricted to browsing results by Strategic Health Authority (SHA) <http://ratings2004.healthcarecommission.org.uk/Search/geographic_search.asp> and <http://www.chi.nhs.uk/Ratings/Search%5Cgeographic_search.asp>. This \"geographic search\" does not allow any visual appreciation of PCT performance levels, or any visual comparisons to be made between PCTs or between 2003 and 2004 result sets.
Results
=======
We produced two Web-based interactive map sets of PCT star ratings, one for 2003 and the other for 2004 <http://healthcybermap.org/PCT/ratings/>. The maps use a yellow-green-blue quadri-colour scheme to represent PCT star ratings. Users can switch between the two map sets or themes, \'PCT Ratings 2003\' and \'PCT Ratings 2004\', within the same pane (the two themes are synchronised, so that when users switch themes the corresponding tile from the other theme is always displayed -- Figure [1](#F1){ref-type="fig"}). Map zooming (100% to 800%), panning, MapTips (displaying PCT names), and legends are available. Dynamic overview maps are offered as navigational help (at zoom levels 200%-800%). Map search is also possible (by PCT name or part of it -- Figure [2](#F2){ref-type="fig"}). Clicking a PCT on any of the maps will display the detailed performance report of that PCT for the corresponding year. Printer-friendly versions of the maps can be generated for direct printing from the Web browser.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Screenshots from our Web-based interactive maps of PCT star ratings for 2003 and 2004.**Screenshots from our Web-based interactive maps of PCT star ratings for 2003 and 2004 <http://healthcybermap.org/PCT/ratings>. When users switch between \'PCT Ratings 2003\' and \'PCT Ratings 2004\', the corresponding tile from the other theme is always displayed within the same pane, allowing instant 2003--2004 visual comparisons to be made. In this Figure, Northumberland Care Trust can be seen achieving lower ratings in 2004 (1 star--light green) compared to 2003 (3 stars--dark blue). The detailed 2004 performance report of Northumberland Care Trust (from the Web site of the Healthcare Commission) has been displayed by clicking the Trust shape on the \'PCT Ratings 2004\' map. Note the overview maps displaying the position of the current map tile on a miniature complete map of England.
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**Screenshot of the map search box.**Screenshot of the map search box, which allows users to locate a Trust on the maps by typing the Trust name or part of it. Selecting a PCT from the \'Result\' list and clicking \'show\' will zoom into and display the corresponding map tile for that PCT.
:::
:::
The maps were successfully tested in both Microsoft Internet Explorer and Mozilla Firefox <http://www.mozilla.org/products/firefox/> Web browsers.
Discussion
==========
Many people are more visually oriented and find that spending long hours browsing the flat textual indices of the Internet leaves a lot to be desired, especially when it comes to navigating large online datasets and understanding the relationships, patterns and trends buried in them. Information resources and large textual datasets (like the detailed PCT performance reports in our case--more than 600 PCT reports for 2003 and 2004 combined) can be organised and navigated based on their geographical attributes \[[@B3]\]. These geographical aspects of textual information are sometimes very useful as an index to information, providing an intuitive way of accessing, mining, and understanding it. Some information types like PCT performance ratings lend themselves very well and naturally to geographical indexing and visualisation. In fact, PCT performance ratings describe a geographically differentiated phenomenon, which is the variation in the performance and quality of primary healthcare services between different areas across England.
Using our Web-based interactive maps, users can quickly and intuitively locate any PCT and retrieve detailed performance information about it. They can also visually appreciate at a glance the distribution of PCT performance across England; for example, one can instantly note that there were no three star (dark blue) PCTs in the London region in 2002/2003 and that this has remained unchanged in 2003/2004. Users can visually compare the performance of different PCTs in the same year and also between 2003 and 2004 (by switching between \'PCT Ratings 2003\' and \'PCT Ratings 2004\' themes). The performance of many PCTs has improved in 2004, whereas some PCTs, e.g., Northumberland Care Trust -- Figure [1](#F1){ref-type="fig"}, achieved lower ratings in 2004 compared to 2003.
Conclusions
===========
Web-based interactive geographical interfaces offer an intuitive way of indexing, accessing, mining, and understanding large healthcare information sets describing geographically differentiated phenomena, and can act as an enhanced alternative or supplement to purely textual online interfaces. Geographical interfaces enable instant visual comparisons to be made between different geographical areas and over time (when information sets and maps for successive periods of time are available), thus empowering organisations and decision makers.
Methods
=======
Star ratings of English PCTs for the years 2002/2003 and 2003/2004 were obtained from the Web sites of the former Commission for Health Improvement <http://www.chi.nhs.uk/Ratings/Search/SearchResults.asp?TrustType=PCT> and the Healthcare Commission <http://ratings2004.healthcarecommission.org.uk/Search/SearchResults.asp?TrustType=PCT> respectively. The Internet addresses (URLs) of the corresponding detailed reports of PCT performance were also harvested from the same sources.
The maps were created in ESRI ArcView GIS Version 3.1 <http://www.esri.com/>. We used the 2001 Census PCT (post April 2002 change) boundary dataset, which is the copyright of the Crown/Ordnance Survey <http://www.ordsvy.gov.uk/>, and is freely available to the UK academic community from EDINA UKBORDERS service with the support of the ESRC and JISC <http://edina.ac.uk/ukborders/>. The names/boundaries and labels (codes) of few PCTs changed between 2003 and 2004, but this was properly cared for in our exercise.
We inserted four new fields in the original PCT boundary dataset table to store the 2003 and 2004 star ratings and corresponding detailed report URLs for all English PCTs. The PCTs in the output maps are coloured according to the values in their star rating fields (0, 1, 2, or 3 corresponding to the number of stars awarded), with light colours for low ratings to dark colours for high ratings.
We used ColorBrewer <http://www.colorbrewer.org/> and \[[@B4]\]) to select a suitable colour scheme for our maps. Our chosen scheme is colour blind friendly, black and white photocopy friendly (for printed output), LCD projector friendly, laptop (LCD) friendly, CRT screen friendly, and colour printing friendly--all at the same time (Figure [3](#F3){ref-type="fig"}).
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**Screenshot of ColorBrewer online tool.**Screenshot of ColorBrewer online tool <http://www.colorbrewer.org/> showing the colour scheme we have chosen for our maps. This yellow-green-blue quadri-colour scheme is colour blind friendly, black and white photocopy friendly (for printed output), LCD projector friendly, laptop (LCD) friendly, CRT screen friendly, and colour printing friendly--all at the same time. The corresponding Hue-Saturation-Value numerical triplets for the four colours in our scheme are also shown, ready for using in ArcView 3.x.
:::
:::
The online interactive maps were then produced using the Demo version of alta4 HTML ImageMapper 3.5 extension for ESRI ArcView GIS 3.x <http://www.alta4.com/eng/products_e/im/index.html> and Figure [4](#F4){ref-type="fig"}), and its companion tool alta4 ThemeBrowser 1.0. ThemeBrowser is used to combine separate HTML ImageMapper projects (in our case \'PCT Ratings 2003\' and \'PCT Ratings 2004\' map sets) into a single ThemeBrowser Web page (see <http://www.alta4.com/eng/products_e/im/new_in_im3.html#themebrowser>).
::: {#F4 .fig}
Figure 4
::: {.caption}
######
**Screenshot of the Demo version of HTML ImageMapper 3.5 extension within ArcView GIS 3.1.**Screenshot of the evaluation version of alta4 HTML ImageMapper 3.5 extension within ESRI ArcView GIS 3.1, showing the main settings we have used to generate our Web-based \'PCT Ratings 2004\' interactive map set. The dialogue box on the right shows the \'MapTip Field\' and \'Click Action/URL Field\' settings associated with features (PCTs) on the output map.
:::
:::
It is noteworthy that HTML ImageMapper does not require any server side software installation, and as such is much simpler to use than some other Internet GIS solutions like the client/server version of ALOV Map/TimeMap <http://alov.org/docs/setup.html>.
The standalone versions of ALOV Map/TimeMap <http://alov.org/docs/quickstart.html> and JShape <http://www.jshape.com/> Java applets, which don\'t require any server side setup, are limited by the fact that they need to download the whole map shapefile from the Web server before they can start on the client side, and so are not suitable for large datasets (our PCT boundary dataset in ESRI shapefile format is about 50 MB in size). Other options for generating interactive Web maps from a desktop GIS are discussed in \[[@B5]\].
| PubMed Central | 2024-06-05T03:55:46.628041 | 2004-7-28 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC506785/",
"journal": "Int J Health Geogr. 2004 Jul 28; 3:16",
"authors": [
{
"first": "Maged N",
"last": "Kamel Boulos"
}
]
} |
PMC506786 | Incidence and mortality
=======================
Nearly 170,000 new cases of endometrial carcinomas were estimated worldwide in 1997 \[[@B1]\]. However, incidences throughout different regions of the world vary considerably. Compared to Africa and Asia having the lowest rates of incidence, Western Europe, USA and Canada are shown to have the highest incidence worldwide (Figure [1](#F1){ref-type="fig"}). As shown in figure [2](#F2){ref-type="fig"}, even within Europe the incidence rates are very heterogeneous. In some of these countries, e.g. Germany, endometrial carcinoma is the most common among genital carcinoma \[[@B2]\].
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Estimated endometrial cancer incidences throughout different regions of the world \[1\].
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Estimated endometrial cancer incidences throughout different countries in Europe \[1\].
:::
:::
Endometrial carcinomas occur in advanced age (postmenopausal). The age-related incidence for Germany is shown in Figure [3](#F3){ref-type="fig"}. The overall increase in the incidence of this disease during the last decades is mainly related to higher life expectancy within the developed world.
::: {#F3 .fig}
Figure 3
::: {.caption}
######
Age dependent incidence of endometrial cancer in Germany. Robert-Koch-Institute at: <http://www.rki.de/GBE/KREBS/BROSCHUERE2002/BROSCHUERE2002.HTM>
:::
:::
Etiology and risk factors
=========================
The etiology of the endometrial carcinoma is not fully understood. Most cases appear sporadically whereas about 10% are hereditary. Chief among the latter is the autosomal dominantly inherited hereditary non-polyposis colorectal cancer (HNPCC) \[[@B3]-[@B5]\]. The risk of developing endometrial cancer is believed to be ten times higher for women carrying the gene compared to the general population \[[@B6]\]. The likelihood of a synchronous or metachronous development of endometrial carcinomas is, however, higher for patients with breast, ovarian, and non-hereditary colorectal cancer \[[@B7]\].
Within the current concept of multi-step progression of normal cells to malignancy, recent molecular work has identified several gene alterations important for tumor development. In summary, mutations and amplifications of oncogenes K-ras and HER2/neu, mutations or deletions of tumor suppressor genes p53, p21, p16, and pTEN/MMAC1 as well as impaired DNA repair functions through mutations of hMLH1, hMSH2, and hMSH6 have been connected with the development of endometrial carcinomas \[[@B8]\].
Environmental, dietary and hormonal factors as well as an aging female population have been attributed to an observed increase of endometrial carcinomas over the past few years. Epidemiologic studies have observed correlations between the incidence of endometrial cancer and the usage of estrogens, especially when applied to alleviate perimenopausal and postmenopausal symptoms. Therefore, it appears that estrogen plays a key role in the development and progression of endometrial carcinomas.
There is also convincing evidence that high body mass increases the risk of endometrial carcinomas. Current estimations figure that about 40% of these relate to excess body weight \[[@B9]\]. A plausible biological explanation for obesity influencing the risk of endometrial cancer is found in increased aromatization of androstendione to estrone in adipose tissue \[[@B10]\]. Hyperadrenocorticism, which is more common in obese individuals, also disturbs the estrogen metabolism. The correlation between obesity and hyperadrenocorticism is possibly increased by hyperinsulinism. This also explains the higher frequency of endometrial carcinomas in combination with diabetes mellitus. A high intake of saturated fat may also increase the risk, whereas high consumption of vegetables and fruits may do the opposite \[[@B1],[@B11]\]. Due to the fact that most factors relate to prolonged or intensive exposure to estrogen (hyperestrogenism), this may likely be identified as the underlying concept leading to the development of endometrial cancer. In this process estrogens are believed not to act as carcinogenic agents, but as promoters of carcinogenesis. A state of hyperestrogenism may be caused by exogenous or endogenous factors and may relate to reproductive factors, estrogen exposure or menopausal years \[[@B9],[@B12],[@B13]\]. Several endogenous risk factors are also associated with the increase of endometrial cancer risk e.g. early menarche, late menopause (2.4-fold), nulliparity (2-fold), the polycystic ovarian syndrome, diabetes mellitus (2.8-fold), high blood pressure (1.5-fold), obesity (up to 10-fold), other tumors with estrogen production, atypical endometrial hyperplasia (see Table [1](#T1){ref-type="table"}) and the aforementioned inherited forms of colorectal cancer (10-fold). Basically two hypotheses endeavor to explain the protective effect of high parity: The first one assuming the mechanical removal of premalignant and malignant cells with each delivery, the second describing a protective effect of high progesterone values during pregnancy. It is most likely for both hypotheses to be correct to some extent, whereas a large population based study from Finland favors the second hypothesis \[[@B14]\]. An additional factor may be anovulatory ovarian insufficiency, especially in patients with polycystic ovaries (PCO syndrome). As these patients have a persistent progesterone deficiency due to non-formation of a corpus luteum, they lack this important protective mechanism. Endocrine risk factors also include hormone producing ovarian tumors. This goes for estrogen as well as androgen producing tumors since androgens may be converted to estrone in adipose tissue.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Type of endometrial hyperplasia and rate of progression to cancer \[20\].
:::
Type of hyperplasia Rate of progression
-------------------------------------- ---------------------
Simple (cystic without atypia) 1
Complex (adenomatous without atypia) 3
Atypical
Simple (cystic with atypia) 8
Complex (adenomatous with atypia) 29
:::
Previous irradiation of the pelvis, estrogen replacement therapy (HRT) (especially unopposed HRT) and tamoxifen therapy represent exogenous risk factors. Prolonged and exclusive intake of synthetic estrogens is associated with an up to 15-fold higher incidence of endometrial carcinomas \[[@B15]\]. Similarly, some selective estrogen receptor modulators (SERMs) and especially tamoxifen may have an estrogenic, proliferative effect on the endometrium. Thus tamoxifen therapy is frequently associated with polyps, hyperplasia and carcinomas of the endometrium \[[@B16],[@B17]\]. Tamoxifen seems to induce a 6.4-fold increase in the risk for endometrial cancer \[[@B18]\]. On the other hand, cyclic application of estrogens combined with progestins does not increase endometrial cancer incidence, e.g. hormonal contraception (combined preparations) and may even reduces the risk of endometrial carcinomas by up to 50% (see prevention). In essence, hyperestrogenism will lead to stronger proliferation, thereby causing hyperplasia which may gradually acquire more and more cellular atypia (atypical hyperplasia) and later transform into an endometrioid adenocarcinoma.
Recently a case-control study identified antipsychotic drugs as being a great risk factor for endometrial cancer \[[@B19]\]. These findings may also be explained to some extent by the side effects of the drugs (obesity, insulin resistance, amenorrhea, low gonadal steroids leading to hyperprolactinemia).
Precancerosis
=============
In some cases endometrial cancer develops from atypical endometrial hyperplasia. The likelihood of this happening correlates with the degree of hyperplasia (Table [1](#T1){ref-type="table"}) \[[@B20]\]. According to the World Health Organization (WHO) classification, endometrioid carcinomas are divided into simple and complex forms, each with and without atypia \[[@B21]\]. This classification has also been accepted by the International Society of Gynecological Pathologists.
Endometrial hyperplasia is regarded as a preliminary stage of endometrioid carcinomas. Serous and clear cell carcinomas are on the other hand frequently associated with an atrophic endometrium \[[@B22]\]. A precursor of the serous carcinoma and possibly of some clear cell carcinomas is the endometrial intraepithelial carcinoma (EIC) \[[@B22],[@B23]\].
Prevention
==========
To prevent an outbreak of the disease in menopausal and postmenopausal women long-term estrogen replacement therapy (treatment of menopausal symptoms, osteoporosis etc.) should be supplemented by intermittent application of gestagens. A recent study suggested the use of intrauterine devices (IUD) also possibly reducing the risk for endometrial cancer due to improved elimination or decrease of hyperplastic endometrial cells \[[@B24]\]. Numerous studies have shown that cigarette smoke reduces the risk of an endometrial carcinoma for women after the menopause, although it may increase the risk for premenopausal women \[[@B25]\]. The greatest reduction of risk was found in obese, multiparous women who did not receive hormone treatment \[[@B26]\]. Women in advanced stages of disease (stages II -- IV), however, were found more likely to smoke than women in early stages (0 -- I). This may reflect a smoking-related decrease in the incidence of early-stage tumors as well as an increase in tumor invasiveness and metastases \[[@B27]\]. The risk of endometrial carcinomas may be reduced significantly by prolonged progestin therapy every month (for 10 days) alone or in combination with estrogen \[[@B28]\]. Since progestins are known to act as cofactors of cancerization in breast and cervical cancer such concepts are better interpreted cautiously \[[@B29]\]. Therefore hormonal preventive concepts need to undergo a general assessment of benefits and risks. To sum up, apart from excess body fat reduction and omission of unnecessary estrogen therapy, there appears to be no reasonable way of preventing endometrial carcinomas.
Early detection
===============
To date there is no procedure that seems appropriate as a screening method for early detection of endometrial carcinomas. Current guidelines of the American Cancer Society suggest informing patients of risks and symptoms involved with endometrial cancer and furthermore firmly emphasize the importance of reporting unexpected bleeding or spots to their physician \[[@B30]\]. The fact of most endometrial carcinomas (with the exception of the rare serous and clear cell types) showing these kinds of preliminary symptoms leads to the diagnosis of over 75% of cases while still in stage I \[[@B7]\]. A recent case-report on the usage of the Mirena^®^intrauterine system maintains that irregular menstrual bleeding should not be treated simply with this system without prior diagnostic \[[@B31]\].
The necessity of finding a screening method is discussed controversially in this context. So far, most epidemiological studies have failed to show significant effects of screening on mortality.
In some cases the Pap smear may lead to the diagnosis of endometrial cancer. However, in many cases cells from inside the uterus are not assessed by the sampling procedure. Positive cervical cytology was found to correlate with nodal spread in 91% of cases, whereas the risk of lymph node spread in patients with normal cervical cytology was estimated at around 2% \[[@B32]\]. It would seem too early to suggest that this could help to reach a decision on the necessity of lymphadenectomy. Maybe the ThinPrep Pap tests will be able to allow further conclusions in the future \[[@B33]\].
Transvaginal ultrasound has also been suggested as a potential means of early detection of endometrial carcinomas. A recent meta-analysis involving 9,031 patients and covering 57 separate studies on the diagnostic accuracy and positive predictive power of endometrial thickness measurement by pelvic ultrasound in patients with postmenopausal bleeding concluded that these measurements cannot solely be used to accurately rule out endometrial pathology. Measurement of both endometrial layers of ≤ 5 mm coincides with endometrial pathology in only 2.5% \[[@B34]\]. Other studies have used saline infusion sonography and color Doppler sonography to differentiate between endometrial cancer, endometrial hyperplasia, fibroids, endometriosis, myoma or tamoxifen induced endometrial thickness \[[@B35]-[@B42]\]. A recent comparison of saline infusion sonography and office hysteroscopy revealed similar ratings of patients\' pelvic pain during the procedures. Sensitivity and specificity coefficients as well as negative and positive predictive values were higher for the office hysteroscopy \[[@B43]\].
It seems that biopsy remains the only accurate way of diagnosing endometrial cancer \[[@B44]\]. Under optimal circumstances the gynecologist will remove a tissue sample from the uterine lining under hysteroscopic control \[[@B44]-[@B47]\]. If hysteroscopic control is neglected, the false negative rates for dilatation and curettage (D&C) will range between two and six percent, thereby emphasizing the limitations of D&C alone \[[@B45],[@B48]-[@B50]\].
Newer methods, like magnetic resonance imaging (MRI), positron emission tomography (PET), intraoperative ultrasound or three dimensional sonography are not likely to gain importance with respect to the diagnosis or early diagnosis of the disease \[[@B51]-[@B56]\]. It is, however, reasonable to believe that they are likely to deliver more information about the invasion depth of the myometrium or lymphatic metastases \[[@B52],[@B55],[@B57]\].
Classification of endometrial carcinoma
=======================================
The term endometrial carcinoma describes a variety of different tumor types originating from the inner ling of the uterus. Many authors differentiate between two basic types, which may be divided into estrogen-dependent and estrogen-independent types or tumors with favorable or unfavorable prognosis \[[@B58]\]. Although there are no cross-sectional studies comparing tumors from various ethnic groups and significant differences in tumor biology the frequency of tumors of a certain category in various geographical areas have been assumed. This may explain why mortality in the USA is higher in black women than in white women (5.8 deaths per 100,000 persons in black women vs. 3.1 deaths per 100,000 persons in white women) \[[@B59]\]. The different categories may be summarized as follows:
1\. It comprises of estrogen related tumors occurring in younger, perimenopausal women. These tumors are often said to be highly differentiated, mainly adenocarcinomas with positive steroid hormone receptor status (ER, PR), the known risk factors (estrogen etc.) originating from atypical endometrial hyperplasia. The patients in this group have a longer history, lower grade tumors, less myometrial invasion and low potential for lymphatic spread. They may be associated with concomitant carcinomas of the ovary, breast and colon and respond to progestin therapy. The overall prognosis is generally favorable.
2\. This category is comprised of tumors of patients with shorter history, higher grade tumors, deeper myometrial invasion and high risk of lymphatic spread. The tumors will not respond to progestin therapy and there are no associated tumors. Histologically these tumors are identified as serous carcinomas or clear cell adenocarcinomas. The prognosis of patients with this type of tumor is poor.
3\. Findings of carcinomas in atrophic endometrium being associated with an intermediate prognosis lead to the suggestion of a third category. In these cases the endometrioid carcinomas are not likely to be estrogen-related \[[@B21]\].
4\. It was furthermore suggested to incorporate endometrial neoplasia originating from an inherited predisposition into a fourth category. These types of tumors tend to develop about 15 years earlier and are associated with a favorable prognosis. Most patients in this category have a hereditary non-polyposis colorectal carcinoma syndrome (HNPCC).
Histological types according to the WHO classification
======================================================
The following section will briefly describe the most important characteristics of endometrial carcinomas. Detailed descriptions may be found in specialized pathological textbooks e.g. Anderson et al. \[[@B21]\].
• *Endometrioid adenocarcinoma*: The endometrioid adenocarcinoma, whose glands resemble those of the normal endometrium, is the most common type (60 -- 80%). This type is considered part of the 1^st^category
• *Endometrioid adenocarcinoma with squamous cell differentiation*(adenoacanthoma, adenosquamous carcinoma): Approximately 25% of endometrioid adenocarcinomas display partial squamous differentiation. The squamous elements are interpreted as *terminally differentiated*indicating that the tumor is incapable of independent growth. Prognosis depends on the glandular components of the lesion. Highly differentiated tumors (adenoacanthomata) have a favorable prognosis, whereas poorly differentiated tumors (adenosquamous carcinomas) have an unfavorable course.
• *Serous adenocarcinoma*: This tumor type is similar to the serous ovarian carcinoma. It is characterized by aggressive growth and poor prognosis. Lymphogenic and hematogenic metastases are usually already present at the time of diagnosis. Nearly all tumors are poorly differentiated. Serous adenocarcinomas belong to the 2^nd^category of endometrial cancer.
• *Clear cell adenocarcinoma*: This type comprises 3 to 6% of all endometrial carcinomas. Like the serous adenocarcinoma, clear cell adenocarcinomas tend to progress rapidly. They share the 2^nd^category.
• *Mucinous adenocarcinoma*: Diagnosis is based on the presence of mucus within the tumor cells. Purely mucinous carcinomas are rare, although a mucinous component within endometrioid carcinomas is more common. The tumors are usually highly differentiated and have a good prognosis. It is important to exclude a primary mucinous adenocarcinoma originating from the endocervix, spreading into the uterine body. Mucinous adenocarcinomas are part of the 1^st^category.
• *Squamous cell carcinoma*(2^nd^category): A very rare entity associated with very poor survival. Primary squamous cell carcinomas of the uterine cervix should be ruled out. Adjuvant platinum-based radiochemotherapy may result in improved survival.
• *Mixed carcinoma*: A carcinoma composed of two or more different non-squamocellular components with each component occupying at least 10% of the tumor. Prognosis varies, e.g. in case of a serous component, the prognosis is poor.
• *Undifferentiated carcinoma*: A rare carcinoma without glandular, squamous or sarcomatous differentiation. Prognosis is unfavorable.
• Rare types of endometrial carcinomas: small cell carcinomas, microglandular adenocarcinomas, signet-ring cell carcinomas, transitional cell carcinomas, glassy cell carcinomas, mucinous adenocarcinomas of the intestinal type, lymphoepithelioma-like carcinomas, and endometrial adenocarcinomas with trophoblastic differentiation have been reported.
Grading
=======
The tumor grading is a highly significant prognostic parameter although it is subjective with a considerable inter-observer and intra-observer variability. It is determined by the percentage of non-squamocellular, solid portions as follows: G1: 5% and less, G2: 5--50%, G3: more than 50%. A significant nuclear atypia increases the grade of differentiation by one grade.
Preoperative diagnostic procedures
==================================
After histological confirmation of an endometrial carcinoma clinical palpation and vaginal sonography should be performed. Several additional examinations have been suggested: Rectoscopy, cystoscopy, computed tomography and/or MRI. They may be omitted in clearly diagnosed early cases but are strongly recommended in advanced stages. Recent studies indicate some value of tumor marker diagnostics. At a cut-off level of 40 U/ml elevated CA125 serum levels indicate nodal metastases with a sensitivity of 77.8% and a specificity of 81.1% \[[@B60],[@B61]\].
Surgical therapy
================
In all stages of endometrial carcinomas, surgery is the primary treatment of choice. Preoperative intracavitary radiation treatment, often recommended in earlier times, is not considered advisable any more since the information about the depth of myometrial invasion and thus information on an important prognostic factors is lost \[[@B62]\]. A study indicates that the Maylard-type incision is superior to transverse (Pfannenstiel-type) or longitudinal incision \[[@B63]\]. If possible, an abdominal hysterectomy with removal of the adnexa and a peritoneal lavage should be performed. After removal of the uterus, the depth of tumor invasion into the myometrium has to be determined to estimate the probability of extrauterine spread. If the myometrium is infiltrated to more than 50%, a pelvic and paraaortic lymphadenectomy should be performed. For appropriate staging, more than 20 lymph nodes should be dissected \[[@B64]\]. But also in cases of additional adverse prognostic factors (poor grading, lymphangiosis, see below), pelvic and paraaortic lymphadenectomy are recommended by many \[[@B65]\]. A decision tree on primary therapy is given in figure [4](#F4){ref-type="fig"}. Unfortunately, studies found no correlation between depth of invasion, histological grade, cervical invasion, peritoneal cytology, menopausal status, preoperative serum CA125 level or primary tumor diameter. Only lymphvascular space involvement (P \< 0.0001) was significantly correlated to pelvic lymph node metastases, which lead the authors to the conclusion that all patients should undergo extended surgical staging, except when clinical or operative factors increase patients\' morbidity \[[@B66]\]. Lymphadenectomy may be omitted in cases of more favorable prognosis.
::: {#F4 .fig}
Figure 4
::: {.caption}
######
Decision tree concerning the primary treatment of endometrial carcinoma.Legend: RT = radiotherapy; HT = endocrine treatment; CHT = chemotherapy; D&C = dilatation and curettage
:::
:::
There is some controversy on the value of radical hysterectomy in stage II carcinomas. While several earlier studies advocated this procedure \[[@B67]-[@B70]\] a recent study reported no prognostic advantage \[[@B71]\]. Maybe only the patients with stage IIb tumors and clinically evident tumor infiltration profit from radical hysterectomy. This requires further investigation.
Complications after pelvic lymphadenectomy may be reduced by omentoplasty and omentopexy \[[@B72]\]. In advanced stages of the disease complete removal of all tumor sites is warranted. In case of serous histology and peritoneal spread, some authors also advocate omentectomy. A recent study indicates that optimal cytoreduction results in a significant survival benefit for stage IVB endometrial cancer patients with a reasonable surgical morbidity rate \[[@B73]\].
Vaginal hysterectomy as primary treatment of endometrial cancer has also been investigated especially in medically compromised women \[[@B72],[@B73]\]. Such approaches may be combined successfully with laparoscopically assisted radical vaginal hysterectomy \[[@B74]-[@B77]\].
The aforementioned basic goals of surgery (hysterectomy, removal of the adnexa and lymphadenectomy in stages Ic and higher) should be reached especially in medically fit patients, because treatment along the recognized guidelines has been found to be prognostically favorable. Some studies, however, have reported the standards mentioned in many cases as not having been realized \[[@B78]-[@B80]\]. As shown in Italy there seems to be certain reluctance towards bringing current topics discussed in literature into practice \[[@B81]\].
The problem mentioned may partly be due to the fact that the current guidelines are insufficiently supported by randomized surgical trials. Interestingly the COSA-NZ-UK Endometrial Cancer Study Group trial showed that lymphadenectomy showed no advantage for endometrial cancer if primary surgery was followed by adjuvant radiotherapy \[[@B82]\]. Therefore studies on all surgical aspects are warranted. This also includes newer surgical approaches which await further evaluation in prospective studies.
Staging
=======
Endometrial carcinomas are staged surgically. Procedures previously used for determination of stages, such as fractional dilatation and curettage to differentiate between stage I and II, are no longer applicable unless the patient is to be treated by primary radiation therapy. The old (1971) and new (1988) staging system of the International Federation of Gynecology and Obstetrics (FIGO) are shown in table [2](#T2){ref-type="table"}. The prognostic importance of adequate surgical staging was recently demonstrated \[[@B83]\].
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Tumor classification of the international Federation of Obstetrics and Gynecology (FIGO). The surgical staging system is obligatory unless patients are to undergo primary radiotherapy when the older clinical staging system may be used.
:::
Stage Stage -- Clinical Staging Stage -- Surgical Staging
--------- --------------------------- --------------------------------------------------------------------- ------ ---------------------------------------------------------------------------
**I** I Carcinoma confined to corpus Ia Tumor limited to endometrium
Ia Length of uterine cavity ≤ 8 cm Ib Invasion ≤ 1/2 myometrium
Ib Length of uterine cavity \> 8 cm Ic Invasion \> 1/2 myometrium
**II** II Carcinoma involves corpus and cervix IIa Endocervical glandular involvement only
IIb Cervical stromal invasion
**III** III Carcinoma extends outside uterus but not outside the true pelvis IIIa Tumor invades serosa or adnexa or positive peritoneal cytology
IIIb Vaginal metastasis
IIIc Metastases to pelvic or para-aortic lymph nodes
**VI** IV Carcinoma extents outside true pelvis or involves bladder or rectum IVa Tumor invades bladder, bowel mucosa, or both
IVb Distant metastases, including intra-abdominal and/or inguinal lymph nodes
:::
Prognostic and predictive factors
=================================
Tumor stage, patient age, histologic type and grade, hormone receptors and DNA ploidy represent the traditional prognostic factors. Respective of the response to progestin therapy steroid hormone receptors may also be regarded a predictive factors in recurrent and advanced disease \[[@B8]\]. The strong prognostic impact of tumor stage is underlined by the cumulative 5-year survival rates (surgical/pathological staging) which are 85% for stage I, 70% for stage II, 49% for stage III and 18% for stage IV disease. Obviously both major factors which make up the staging system of endometrial carcinomas, depth of invasion into the myometrium and lymph node status, are major prognostic factors \[[@B84],[@B85]\]. A recently published analysis draws the attention to lymphvascular space involvement and suggests that its presence should indicate lymphadenectomy or adjuvant therapy \[[@B86]\]. Lymphvascular space involvement was also closely linked with advanced stage (unpublished observation). Furthermore, several additional prognostic factors have been suggested: Nulliparity, high tumor cell proliferation (KI-67), high tumor vessel density (angiogenesis), oncogene amplification or overexpression (HER2/neu, K-ras) and alterations of tumor suppressor genes (PTEN, p53, p21, p16) are believed to be associated with adverse prognoses. Especially Ki-67 could be of greater importance seeing that this parameter proved to be independent in multifactorial analyses in a prospective study \[[@B87]\].
There is a divergence of opinion concerning the value of a positive peritoneal cytology as an independent prognostic factor \[[@B88]\]. In stage I, depth of myometrial invasion, vascular invasion, mitosis count and progesterone receptor negativity are statistically significant prognostic factors \[[@B89],[@B90]\]. Overexpression of p53 is observed in approximately 20% of all endometrial carcinomas and in up to 90% of serous carcinomas \[[@B91]\]. The plasminogen activator inhibitor type 2 has been discussed as a possible independent prognostic marker \[[@B92]\]. Overexpression of the oncogene Her-2/neu is significantly more common in advanced than in early stages. Finally, ploidy status is possibly an independent prognostic factor \[[@B93]\], with aneuploidy being mainly associated to prognostically unfavorable serous, clear cell and poorly differentiated carcinomas. There are, unfortunately, still too many controversies to draw final conclusions or even to make suggestions on factors to be determined routinely.
Radiotherapy
============
Primary radiotherapy
--------------------
In medically compromised women, primary irradiation may be suitable. Analyses from Germany show that approximately 20% of all patients undergo primary radiotherapy \[[@B7],[@B64]\]. There are three possible approaches: The after loading technique alone, a combination of after loading and percutaneous techniques or by percutaneous radiotherapy alone. The literature also reports on considerable variation in the number of fractions and the doses of each fraction \[[@B64]\]. In case of brachytherapy only 5 × 8 Gy or 8 × 5 Gy may be applied. Combined therapy usually delivers 50 Gy percutaneously with partial blocking of the bladder and intestines after 24 and 30 Gy and 2 to 5 fractions via brachytherapy. Brachytherapy may also be applied before commencement of percutaneous therapy. The selected dose may be applied by a variety of techniques e.g. by Heyman capsules, double-rod-shaped applicators, indwelling applicators etc. \[[@B64]\]. An appropriate applicator should ensure adequate irradiation of the entire uterus \[[@B94]\]. Nowadays, computer controlled treatment planning allows optimal treatment planning and an individual adaptation of the dose distribution to the uterine cavity. This may vary between patients of course.
Only few clinical trials on primary radiotherapy have been performed. The results suggest that dividing doses into smaller fractions allows better tumor control and has less side effects \[[@B95]\]. The old question on high-dose-rate or low-dose-rate after loading still remains unsolved. There is still limited data on the efficiency of primary radiotherapy in endometrial carcinomas. An analysis of 154 patients having undergone primary radiotherapy at our department showed that local recurrences are more common in this group compared to surgery and adjuvant radiotherapy. The rates of recurrences are stage dependent: 23.3% vs. 13.2% in stage I and 39.2% vs. 25.9% in stage II \[[@B7]\]. As shown in figure [5](#F5){ref-type="fig"} there is a significant difference in overall survival between both groups. Thus, primary radiotherapy represents an effective but suboptimal measure for this group of patients being in generally poorer physical condition.
::: {#F5 .fig}
Figure 5
::: {.caption}
######
Decision tree concerning the treatment of recurrent endometrial carcinoma.Legend: ENCA = endometrial carcinoma; QoL = Quality of Life; RT = radiotherapy; HT = endocrine treatment; CHT = chemotherapy;
:::
:::
Adjuvant postoperative irradiation
----------------------------------
Most studies indicate that advance age, grade 3 histology or deep myometrial invasion relate to a higher risk of disease recurrence. Therefore adjuvant radiotherapy seems important in this subset. External beam radiation should also be considered in cases of multiple infiltrated lymph nodes (\> 5). But many questions remain unanswered and the value of postoperative irradiation is still under debate. Survival rates of patients with early stage disease are excellent, no matter whether they underwent extended-surgical staging with more restricted use of the adjuvant therapy or simple hysterectomy bilateral salpingoophorectomy with more frequent use of adjuvant radiotherapy \[[@B96]\]. Prospective-randomized trials have so far only demonstrated improved local control yet no overall survival benefit, and have higher rates of treatment related complications \[[@B97]\]. This accords to larger retrospective analyses, most likely due to the fact that the majority of these recurrences can be salvaged through radiation therapy \[[@B98]\]. With respect to the importance of the problem, trials to evaluate the therapeutic benefit of adjuvant radiotherapy in the several subsets of patients at higher risk are warranted.
The American Brachytherapy Society has now issued recommendations for brachytherapy for carcinomas of the endometrium \[[@B94]\]. According to these, the applicator selection should be based on patient and target volume geometry, the dose prescription point should be clearly specified and the treatment plan should be optimized. For intravaginal brachytherapy selection of the largest diameter applicator is to ensure close mucosal apposition. Finally, doses should be reported both at the vaginal surface and at 0.5-cm depth irrespective of the dose prescription point.
Adjuvant medical treatment
==========================
The data on adjuvant medical treatment is not conclusive. Most studies have their limitations and therefore there is still no final answer to the question, who should receive what type of adjuvant treatment. Merely in cases of uterine papillary serous carcinomas, which affect 1% to 10% of patients, there is consensus that patients should receive chemotherapy (with or without adjuvant radiotherapy) with a platinum-based regimen, combined with doxorubicin and cyclophosphamide. Newer regimens consider paclitaxel, with or without platinum \[[@B99]\].
Adjuvant hormonal treatment
---------------------------
A multicenter, open, controlled, prospectively randomized trial on adjuvant endocrine treatment with medroxyprogesterone acetate (MPA) or tamoxifen in stage I and II endometrial carcinomas failed to detect differences in the disease-free and overall survival rates for a tamoxifen group compared with a control or a MPA group \[[@B100]\]. However, the total number of patients on trial (n = 388) seems too low in relation to the favorable prognosis of early stage disease and the low total response rate of tamoxifen which ranged around 10% in this situation \[[@B101]\]. In the aforementioned study tamoxifen demonstrated only modest activity which lead the authors to the conclusion that tamoxifen does not warrant further investigation as a single agent but perhaps a sequential use of tamoxifen and progestational agents \[[@B101]\].
Adjuvant chemotherapy
---------------------
The generally good prognosis of endometrial carcinomas does not justify a general recommendation of chemotherapy, especially in the early stages. Even so, patients at high risk (unfavorable histological type, deep myometrial invasion and lymph-vascular space involvement) seem to profit from adjuvant chemotherapy \[[@B102],[@B103]\]. Studies on the subject unfortunately often lack a control arm so the effects of chemotherapy remain unclear \[[@B104]\]. Well designed studies in the group of high risk patients are warranted.
Preservation of fertility
=========================
The development of endometrial cancer in young patients is usually related to unopposed estrogen stimulation. In patients with continuing a desire to have children, approaches have been made to preserve fertility. Primary hormonal therapy with progestins was suggested as an alternative treatment for surgery hereby offering the preservation of fertility. These must be interpreted with caution because of low case numbers and a publication bias. Treatment with megestrol acetate at 160 mg/day for 3 months or medroxyprogesterone acetate (MPA) at 200--800 mg/day for 2--14 months resulted in disease regression in 60 to 75%, however, the percentage of patients who actually delivered healthy children was much lower, ranging from 20 to 25% \[[@B105]-[@B107]\]. Furthermore, in several patients, persistent or recurrent disease was observed at the time of a later hysterectomy. In all these cases of unsatisfactory progestin therapy and delayed definitive surgical treatment may have adversely affected patient prognosis.
In essence, the problems regarding the optimal selection of patients for conservative progestin therapy are unsolved. Only cases with good prognostic factors are to be selected: Well differentiated tumors and tumors with known favorable prognoses (e. g. endometrioid type tumors) with no or minimal myometrial invasion and early stage disease. The greatest problem lies in the difficulty of appropriate staging, resulting in a potential underestimation of the problem. Maybe positron emission tomography, magnetic resonance imaging or a combination of both may be useful in this respect in due course.
Patients have to be carefully informed that this fertility preserving concept is still experimental. Moreover, these women must realize the low overall pregnancy rate which may partially be related to the origin of the disease (obesity, irregular menses, polycystic ovarian disease (PCOD) with chronic anovulation and infertility) \[[@B108]\]. Nowadays when treatment of infertility is frequently offered to elderly women, such conservative treatments should, however be investigated more thoroughly. Further questions concern follow-up of these patients. In the aforementioned studies, many patients were treated with maintenance therapy (oral contraceptives or cyclic progestins) to prevent recurrence, which was excluded by routine combination of sonography and D&C every 3--6 months.
Palliative treatment -- treatment of local recurrences
======================================================
Generally the prognosis for patients with recurrent disease is poor, therefore a thorough staging procedure should be performed. Analyses from other tumor entities, e.g. ovarian carcinomas, have demonstrated that a second surgical intervention may be useful in improving patients\' overall survival \[[@B109],[@B110]\]. Thus, even in absence of clinical studies on the subject, patients with single site recurrence should be evaluated for their suitability to surgery at relapse. In patients with isolated central recurrences, pelvic exenteration may be a potential option for cure \[[@B111]\]. The choice with respect to therapy of cancer recurrence strongly depends on prior treatment. In case of prior radiotherapy, a second intervention may often not be possible. If radiotherapy is possible, only nonbulky vaginal recurrences (\< 0.5-cm thick) should be treated by intracavitary brachytherapy. Patients with bulky (\> 0.5-cm thick) recurrences should receive interstitial techniques \[[@B94]\]. A general decision tree regarding the procedures in case of a recurrence is depicted in figure [6](#F6){ref-type="fig"}. Regarding the decision, whether or not to start with endocrine or cytotoxic treatment, the individual risk seems important. But equally important are aspects of patients\' quality of life. A risk assessment scale, which was originally introduced by Possinger for breast cancer, seems helpful \[[@B108]\]. Similar to the adjuvant situation, treatment with tamoxifen, medroxyprogesterone acetate (MPA) alone or in combination may be used. However, there are only few, relatively old studies on the topic, all of which do not allow a final conclusion on the value of such therapies. MPA seems to be the best substance producing remission rates up to 80% in receptor positive tumors \[[@B113]\]. Remission rates of tamoxifen range around 25--30%. After failure of MPA, tamoxifen may be added to MPA producing remission rates in this combination of 50--60% \[[@B114]\]. Also aminogluthemide seems to be active in endometrial cancer \[[@B115]\]. Responses to all kinds of treatments unfortunately do not tend to last for long.
::: {#F6 .fig}
Figure 6
::: {.caption}
######
Comparison of overall survival between patients undergoing surgery and radiotherapy or primary radiotherapy. Note that patients in the group undergoing primary radiotherapy were generally older
:::
:::
Some studies also suggest a potential benefit for luteinizing hormone-releasing hormone analogues (GnRH), although studies in endometrial carcinomas have not shown any convincing activity \[[@B116]-[@B118]\]. To sum, up on the background of the few and old studies with often low case numbers, newer large studies on endocrine strategies are warranted.
With respect to chemotherapy, several cyctostatic compounds have demonstrated efficiency. Among these are paclitaxel, carboplatinum, doxorubicin, cisplatin, etoposide, and 5 fluorouracil. Topotecan showed only limited activity \[[@B119]-[@B124]\]. All drugs may be used alone or in combination, perhaps even in combination with hormonal therapy \[[@B125],[@B126]\]. Again, realizing that combination chemotherapy produces greater side effects, every option, endocrine treatment, single drug or combination chemotherapy should be considered.
Follow-up
=========
Generally recommendation for clinical follow-up of patients advocate patients to be monitored at three-monthly intervals during the first 3 years, at 6-monthly intervals up to the 5th year and at yearly intervals thereafter. Apart from provision of general information on all aspects of the disease and its treatment including unconventional cancer therapies, the documentation of patients\' history, a clinical gynecological examination including a pap smear, a vaginal sonography and even the determination of tumor markers (SCC, CA125) have been recommended. Up to 95% of all recurrences may be detected early this way \[[@B127]\]. Again there are no prospective studies on the subject to enforce such recommendations. On the contrary, several retrospective studies indicate that an intensive follow-up does not result in a survival advantage for patients with recurrent disease but merely increases costs \[[@B7],[@B128]-[@B131]\]. Also in this area the most appropriate management of endometrial carcinomas remains to be determined. In any case an annual examination of the breast, including mammography, is recommended, due to the fact of the frequent coincidence of malignancies of the breast \[[@B7]\].
Estrogen replacement therapy in endometrial cancer patients
===========================================================
Hormone replacement therapy (HRT) with estrogen with or without progestins is frequently used to alleviate menopausal symptoms but also to reduce the risk of osteoporosis and cognitive dysfunctions. In endometrial carcinomas HRT may be believed to be critical due to fear of initiating growth of occult residual tumor cells, resulting in disease recurrence and shortened survival. As summarized by the American College of Obstetricians and Gynecologists (ACOG) there is not enough data to draw any final conclusion. Any decision on the subject should thus be individualized based on potential benefit and risk to the patient \[[@B132]\]. Some studies which have addressed this subject covered only a small number of patients in regard to the overall excellent prognosis of endometrial cancer patients. Of all these studies, however, none produced evidence that patients should not receive estrogen \[[@B133]-[@B136]\]. On the contrary, some studies reported an even prolonged survival for patients (with low-risk factors for recurrence, namely, low tumor grade (grades 1 and 2), less than 1/2 myometrial invasion, and no metastases to lymph nodes or other organs,) who received estrogen \[[@B134],[@B136]\]. Furthermore, the introduction of selective estrogen receptor modulators (SERM) has also offered new treatment options which will also have to be studied in the future.
Final remarks
=============
Endometrial carcinomas represent a very frequent tumor entity in industrialized countries. It is hard to believe how little evidence-based data exists on even the important aspects of the disease. This may be partially due to the overall good prognosis even if surgery is reduced to hysterectomy and adnectomy. However, the high incidence in the developed world and consequently many women suffering relapses, necessitates the research of new approaches for cancer recurrence. Recent research suggests that therapy with trastuzumab (Herceptin^®^) could perhaps improve the outcome in HER-2/neu overexpressing tumors \[[@B137]\]. Further research will focus on molecules and pathways responsible for the initiation and growth of endometrial carcinomas, including tumor suppressor genes, DNA mismatch repair genes, oncogenes, molecules involved in adhesion and invasion and angiogenesis \[[@B138]\]. This research will hopefully allow the development of specific and selective inhibitors.
Some advances may also be possible with -- conventional treatment, especially radiotherapy. Recent findings of a retrospective analysis suggest that tumor oxygenation may play an important role during adjuvant radiotherapy of endometrial carcinomas. Patients with normal hemoglobin levels during therapy (according to the definition of the EORTC = 12.0 g/dl) have a substantially better overall and recurrence free survival (Figure [7](#F7){ref-type="fig"}) \[[@B139]\]. Due to this strong impact on patients\' health, these aspects require further studies.
::: {#F7 .fig}
Figure 7
::: {.caption}
######
Comparison of recurrence-free survival between in patients undergoing adjuvant radiotherapy with respect to anemia. Log Rank = 9.1; df = 1; p \< 0.003.
:::
:::
| PubMed Central | 2024-06-05T03:55:46.629420 | 2004-7-21 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC506786/",
"journal": "World J Surg Oncol. 2004 Jul 21; 2:24",
"authors": [
{
"first": "Karsten",
"last": "Münstedt"
},
{
"first": "Phillip",
"last": "Grant"
},
{
"first": "Joachim",
"last": "Woenckhaus"
},
{
"first": "Gabriele",
"last": "Roth"
},
{
"first": "Hans-Rudolf",
"last": "Tinneberg"
}
]
} |
PMC506787 | Findings
========
Closed head injury (CHI) is the leading cause of mortality and persisting neurological impairment in young people in industrialized countries \[[@B1],[@B2]\]. The neuropathological sequelae of brain injury are mediated in large part by a profound host-mediated intracranial inflammatory response \[[@B3]-[@B5]\]. The pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-18 have been identified as crucial mediators of neuroinflammation after brain injury \[[@B6]-[@B9]\]. This notion has been supported by experimental studies in rodents which demonstrated neuroprotective effects by pharmacological inhibition of either TNF or IL-18 after CHI \[[@B9]-[@B11]\]. In recent years, the concept of a \"dual role\" evolved with regard to concomitant beneficial and adverse effects of pro-inflammatory mediators, depending on the kinetic of their expression and posttraumatic regulation in the injured brain \[[@B3],[@B12],[@B13]\]. However, the TNF-dependent regulation of IL-18 in the injured brain has not yet been investigated. We sought to determine the interrelationship between intracranial TNF and IL-18 levels in a clinical study on patients with severe CHI and in an experimental model in mice.
Patients with isolated severe closed head injury (*n*= 10, Glasgow Coma Scale score ≤ 8) and indication for intraventricular catheters for cerebrospinal fluid (CSF) drainage due to increased intracranial pressure (ICP \> 15 mm Hg) were included in this study. Drained CSF was collected daily for up to 14 days after trauma or until catheters were removed. The patient characteristics are shown in Table [1](#T1){ref-type="table"}. No patient was treated with steroids. The protocol for daily CSF collection is in compliance with the Helsinki Declaration and was approved by the University\'s Ethics Board Committee. Control CSF was collected from patients undergoing diagnostic spinal tap (*n*= 10) and revealed no inflammatory CNS disease, based on normal CSF protein and glucose levels and normal white cell counts. All samples were kept on ice at 4°C and immediately centrifuged after collection, aliquoted, and frozen at -70°C until analysis.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Clinical data and intrathecal cytokine levels in patients with severe closed head injury.
:::
**Patient** **Age (years) / Gender** **Type of brain injury (Marshall score)** **Outcome (GOS)** **TNF in CSF (pg/mL)** **IL-18 in CSF (pg/mL)** **Correlation *r***~S~
-------------- -------------------------- ------------------------------------------- ------------------- ------------------------ -------------------------- ------------------------ --------------- ---------------
**Mean** **Range** **Mean** **Range**
**1** 38 / M EML 4 6.4 1.0 -- 11.5 40.6 6.5 -- 155.2 \- 0.804 \*\*
**2** 30 / M DI II° 3 3.6 1.0 -- 7.7 114.3 29.7 -- 286.4 \- 0.580 \*
**3** 56 / M EML 4 6.3 1.0 -- 10.0 35.1 11.2 -- 100.3 \- 0.530
**4** 57 / F DI II° 5 6.0 1.0 -- 11.7 20.1 5.0 -- 168.8 \- 0.761 \*\*
**5** 44 / M EML 4 1.6 1.0 -- 3.4 39.8 22.6 -- 74.5 \- 0.751 \*
**6** 26 / M EML 4 3.2 1.0 -- 10.3 108.5 5.0 -- 328.6 \- 0.832 \*\*
**7** 47 / M EML 1 1.1 1.0 -- 1.4 268.5 78.3 -- 462.0 \- 0.372
**8** 25 / M EML 4 2.2 1.0 -- 4.0 91.6 10.3 -- 290.0 \- 0.195
**9** 37 / F DI III° 3 1.6 1.0 -- 2.7 183.7 21.5 -- 382.2 \- 0.844 \*\*
**10** 35 / M DI II° 4 2.0 1.0 -- 5.8 209.4 19.9 -- 391.8 \- 0.772 \*
**Controls** **(*n*= 10)** 1.0 1.0 -- 7.1 5.0 5.0 -- 8.4
Statistical analysis for assessment of the correlation between tumor necrosis factor (TNF) and interleukin (IL)-18 levels in serial cerebrospinal fluid samples for up to 14 days after trauma was performed by Spearman\'s rank correlation (\**P*\< 0.05, \*\**P*\< 0.01). The patients\' outcome was determined at 3 months after injury by the Glasgow Outcome Scale (GOS) score \[33\]: 5 = asymptomatic, 4 = moderate disability, 3 = severe disability, 2 = persisting vegetative state, 1 = death. The type of brain injury was classified by the CT-scan criteria established by Marshall et al. \[34\] into diffuse injury (DI) grade I-III and evacuated *vs.*non-evacuated mass lesions (EML, NEML).
:::
Quantification of IL-18 and TNF levels in human CSF samples and in murine brain homogenates was performed by species-specific commercially available ELISA (R&D Systems, Abingdon, UK). According to the information provided by the manufacturer, the IL-18 assay recognizes both the mature and the pro-form of IL-18. All concentrations below the detection limit of 5 pg/mL (IL-18) or 1 pg/ml (TNF) were assigned a value of 5 pg/mL, and 1 pg/ml respectively. All samples were run undiluted in duplicate wells. The concentrations were calculated from the mean OD of duplicate samples, determined by spectrophotometer (Dynatech Laboratories Inc., Chantilly, VA, U.S.A.) at an extinction wavelength of 405 nm.
The experimental part of the study was set-up on two different protocols with the aim to assess the TNF-dependent regulation of IL-18 in the murine brain:
*(1)*The first part of the experimental study was designed to investigate a potential role of TNF-dependent regulation of intracranial IL-18 expression in a standardized model of CHI, using mice double-deficient in genes for TNF and lymphotoxin-α (TNF/LT-α-/-) \[[@B14]\]. These knockout mice were selected in order to compensate for potential redundant functions of TNF by LT-α which binds to the identical common receptors (i.e. TNF receptors p55 and p75) \[[@B14]-[@B16]\]. The generation and development of the TNF/LT-α-/- mice on a mixed C57BL/6 × 129Sv/Ev (B6 × 129) genetic background has been previously described \[[@B14]\]. Knockout mice and wild-type (WT) littermates of the B6 × 129 strain were subjected to a CHI (*n*= 10 per group) using a standardized weight-drop model, as previously described \[[@B9],[@B17]\]. In brief, following ether anesthesia, a midline longitudinal scalp incision was performed. Trauma was applied to the left anterior frontal area of the exposed skull by a 330 g weight with a silicon tip dropped from a height of 2 cm, resulting in a focal closed injury to the left hemisphere. Mice received supporting oxygenation with 100% O~2~until they awakened and were then brought back to their cages. Control animals were subjected to anesthesia and sham operation only (*n*= 10 per group). In addition, mice with anesthesia alone (*n*= 8) were used as internal control and untreated control animals (*n*= 10) were analyzed for baseline evaluation of intracerebral cytokine profiles in these mice.
*(2)*In the 2^nd^part of the experimental study, mice of the C57BL/6 strain (*n*= 10 per group) were treated by stereotactic intracerebroventricular (i.c.v.) injection of either 200 ng mouse recombinant TNF in 10 μl PBS, or vehicle solution only (10 μl PBS), into the left hemisphere using a sterile 27-gauge syringe, under ether anesthesia. According to data from previously published studies \[[@B18]-[@B20]\], as well as based on titration studies from our own laboratory, the i.c.v. injection of 200 ng mouse-recombinant TNF (R&D Systems) elicited an evident induction of inflammatory changes in the murine CNS, such as intracranial recruitment of leukocytes and development of brain edema in the injected hemisphere within 24 hours (data not shown). Animals from all groups (CHI and i.c.v. injection) were sacrificed at 24 h after the respective procedure, which corresponds to the time-point of maximal extent of intracerebral inflammation in the model of CHI used in this study \[[@B17]\].
For assessment of intracerebral IL-18 levels, the murine brains were immediately removed after decapitation. Tissue homogenization was performed using a Polytron homogenizer (Kinematica, Kriens, Switzerland) with a dilution of 1:4 in ice cold extraction buffer containing 50 mmol/L Tris buffer (pH 7.2), NaCl 150 mmol/L, Triton-X-100 1%, and protease inhibitor cocktail (Roche, Mannheim, Germany). The homogenates were shaken on ice for 90 minutes, centrifuged for 15 minutes at 3,000 g and 4°C. Thereafter, the supernatants were aliquoted and stored at -70°C until analysis. The concentrations of total protein in the brain extracts were measured by Bradford assay (Bio Rad Laboratories, Munich, Germany). The intracerebral protein concentrations were in a similar range in all mice assessed (11.7 ± 2.4 mg/mL; mean ± SD). The quantification of IL-18 levels in murine brain homogenates was performed as described above for the human samples. All mice used in this study were males, in order to avoid a bias in gender, age 12 to 16 weeks with body weights of 25 to 32 g. The animal experiments were performed in compliance with the guidelines of the Federation of European Laboratory Animal Science Association (FELASA) and approval was granted by the Institutional Animal Care Committee of the University of Zurich and of the Hebrew University of Jerusalem.
In the clinical part of the study on CHI patients, the mean IL-18 concentrations in ventricular CSF collected up to 14 days after trauma were significantly higher than in control lumbar CSF from patients undergoing diagnostic spinal tap (*P*\< 0.05, repeated measures ANOVA; Table [1](#T1){ref-type="table"}). These findings are coherent with data from previously published studies which demonstrated significantly elevated intracranial IL-18 levels after brain injury, both in humans as well as in experimental model systems \[[@B8],[@B9]\]. With regard to TNF, the mean levels in individual serial cerebrospinal fluid samples were significantly elevated in 50% of all head-injured patients, compared to controls (patients \#1,2,3,4,6; Table [1](#T1){ref-type="table"}). Elevated intracranial TNF levels after traumatic brain injury have been previously reported in various clinical and experimental studies \[[@B21]-[@B26]\]. In the present study, the individual daily TNF levels in CSF were up to 10- to 100-fold lower than the corresponding IL-18 levels (Table [1](#T1){ref-type="table"}). Interestingly, despite the fairly low TNF levels in CSF we found an inverse correlation between the daily individual intrathecal TNF and IL-18 levels in all trauma patients, as demonstrated by a negative Spearman\'s rank correlation coefficient (*r*= -0.195 to -0.844). In 7 of 10 patients, this inverse correlation was statistically significant, with a *P*-value \< 0.05 in three patients (\# 2,5,10) and *P*\< 0.01 in four patients (\# 1,4,6,9; Table [1](#T1){ref-type="table"}). Since the quality of the blood-brain barrier has not been determined in this cohort of head-injured patients, due to the lack of matching serum samples for assessment of albumin levels, the source of elevated cytokines (intrathecal compartment *vs.*peripheral serum) remains unclear.
In the experimental study, IL-18 was found to be constitutively expressed in the brain of untreated mice (27.7 ± 5.4 ng/mL, mean ± SEM; \"baseline\", Fig. [1](#F1){ref-type="fig"}), which is in accordance with data from previous studies revealing constitutive IL-18 expression in the CNS of normal rats and mice \[[@B8],[@B9],[@B27],[@B28]\]. Microglia may represent the cellular source of constitutive intracranial IL-18 levels in these mice, since Prinz and colleagues have previously shown that microglial cells, but not astrocytes, produce IL-18 in the murine CNS \[[@B28]\]. The intracerebral IL-18 levels increased significantly in the head-injured group by 24 hours after experimental CHI (56.9 ± 4.7 ng/mL, *P*\< 0.01 *vs.*baseline, Fig. [1](#F1){ref-type="fig"}). Knockout mice lacking TNF and LT-α genes also showed significantly elevated IL-18 concentrations at 24 h after CHI (58.4 ± 7.8 ng/mL) which were in a similar range as in head-injured WT mice, as shown in Fig. [1](#F1){ref-type="fig"}. This lack of differences in mice deficient in the ligands for TNF receptors may be explained by alternatively expressed inflammatory mediators or modified pathways of IL-18 regulation in these genetically engineered mice which have been shown to have significantly altered immune responses \[[@B14]\]. Interestingly, the intracerebral injection of vehicle alone (10 μl PBS i.c.v.) induced significantly elevated IL-18 levels in brains of normal WT mice, compared to normal untreated mice, which were in a similar range as in the brain-injured groups (53.6 ± 9.6 ng/ml, Fig. [1](#F1){ref-type="fig"}). These data imply that a minor penetrating injury by i.c.v. injection of a small volume of buffer solution represents a procedure which is sensitive enough to induce significant IL-18 production in murine brains within 24 hours. In contrast, the intracerebral injection of murine recombinant TNF (in 10 μl PBS) reduced the elevated IL-18 levels in murine brains significantly to levels than were even lower than baseline concentrations (22.13 ± 7.1, *P*\< 0.01 *vs.*vehicle-injected mice), as shown in Fig. [1](#F1){ref-type="fig"}.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Intracerebral IL-18 concentrations in mice, as determined by ELISA in murine brain homogenates (*n*= 10 animals per group). Untreated normal mice were used for determination of baseline IL-18 levels in this study. The four treatment groups were sacrificed after 24 hours for assessment of intracerebral IL-18 levels, as described in the text. Mice deficient in genes for TNF and lymphotoxin-α (TNF/LT-α-/-) and wild-type (WT) littermates were subjected to focal closed head injury (CHI) and sacrificed after 24 hours. Two additional groups of WT mice were given an intracerebro-ventricular (i.c.v.) injection of 200 ng mouse-recombinant TNF in 10 μl PBS or by vehicle alone (10 μl PBS i.c.v.). The data are presented as means ± SEM. \**P*\< 0.01 vs. baseline / TNF-injection (unpaired Student\'s *t*-test).
:::
:::
The findings from these experimental investigations corroborate the data from the clinical study, where an inverse correlation of intrathecal TNF and IL-18 levels during the first 14 days after severe CHI was found, suggesting that the inhibition of IL-18 may represent a new potential anti-inflammatory mechanism after CHI. Such a \"dual role\" of TNF has been suggested previously in terms of concomitant pro- and anti-inflammatory effects and detrimental as well as beneficial neuroprotective properties after brain injury \[[@B12]\]. While the pro-inflammatory effects mediated by TNF in the CNS have been thoroughly investigated in the past two decades \[[@B15],[@B29],[@B30]\], the concept of anti-inflammatory effects mediated by cytokines which have been formerly designated as \"pro-\"inflammatory mediators, is still challenging and novel. Scherbel and colleagues were the first to provide evidence of beneficial effects of TNF in the later phase (i.e. 4 weeks) after traumatic brain injury, based on studies in TNF-/- mice subjected to controlled cortical impact brain injury \[[@B31]\]. In these experiments, the TNF-deficient mice showed a significantly attenuated neurobehavioral impairment than WT littermates in the first 48 hours post trauma, in terms of early detrimental effects mediated by TNF \[[@B31]\]. However, the expected neurobehavioral recovery was absent in TNF-/- mice after 4 weeks and cortical tissue loss was significantly increased at this time-point, compared to WT littermates, implying that at later time-points the lack of TNF leads to adverse outcome after brain injury \[[@B31]\]. Barger and colleagues have previously shown in an *in vitro*model of amyloid β-mediated neurotoxicity that both TNF and LT-α (formerly designated TNF-β) can significantly attenuate neuronal degeneration by induction of antioxidative pathways through activation of the transcription factor NFκb, thus strengthening the notion of neuroprotective effects mediated by these cytokines \[[@B32]\]. This assumption was further corroborated in the setting of experimental CHI, where mice lacking both TNF and LT-α genes were shown to have a significantly increased mortality within one week after trauma, compared to WT littermates \[[@B17]\]. Overall, these data support the notion that TNF exerts detrimental effects in the early phase and beneficial neuroprotective effects in the later phase after head injury \[[@B12]\]. However, the assumptive underlying regulatory mechanisms of TNF-mediated neuroprotection and of TNF-mediated suppression of IL-18 in the injured brain remain unclear and have to be investigated in future experimental studies.
List of abbreviations
=====================
Central nervous system (CNS), cerebrospinal fluid (CSF), closed head injury (CHI), intracerebroventricular (i.c.v.), interleukin (IL), lymphotoxin-α (LT-α / TNF-β), nuclear factor κB (NFκB), tumor necrosis factor (TNF), phosphate-buffered saline (PBS), intracranial pressure (ICP), wild-type (WT), enzyme-linked immunosorbent assay (ELISA).
Competing interests
===================
There are no financial interests by any of the authors with regard to the present project.
Authors\' contributions
=======================
OIS, MCMK, CEH, ES, and PFS were responsible for conception and planning of the experiments, as well as for performing the animal experiments, collection of the human cerebrospinal fluid samples and cytokine measurements in human and murine tissue samples, as well as for writing of the manuscript. DP and IY performed the experimental i.c.v. injection experiments. WE contributed to the interpretation of the results and writing of the manuscript.
Acknowledgments
===============
The authors thank Dr. Hans-Pietro Eugster (Division of Clinical Immunology, University of Zurich, Switzerland) for providing the TNF/lymphotoxin-α knockout mice. Dr. Volkmar Hans (Department of Neuropathology, Gilead Clinic, Bielefeld, Germany) is acknowledged for critical review of the manuscript.
| PubMed Central | 2024-06-05T03:55:46.633544 | 2004-7-28 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC506787/",
"journal": "J Neuroinflammation. 2004 Jul 28; 1:13",
"authors": [
{
"first": "Oliver I",
"last": "Schmidt"
},
{
"first": "Maria Cristina",
"last": "Morganti-Kossmann"
},
{
"first": "Christoph E",
"last": "Heyde"
},
{
"first": "Daniel",
"last": "Perez"
},
{
"first": "Ido",
"last": "Yatsiv"
},
{
"first": "Esther",
"last": "Shohami"
},
{
"first": "Wolfgang",
"last": "Ertel"
},
{
"first": "Philip F",
"last": "Stahel"
}
]
} |
PMC506788 | Viruses have long been a source of key scientific discoveries. Historically, they have contributed to our knowledge of transcription, cell cycle, and apoptosis. To date activated transcription in higher eukaryotic cells with or without chromatin is a great area of active research and many researchers use viral activators, including herpes virus VP16, adenovirus E1A, HIV-1 Tat and HTLV-I Tax to not only understand viral, but also basic mechanisms related to host control of vital cellular machineries, including transcription. Eukaryotic transcription has five distinct phases, pre-initiation, initiation, promoter clearance, elongation and termination, and is a tightly regulated and coupled process \[[@B1]\]. Viral transactivators, such as Tax, have long been shown to target the earliest steps of transcription by forming quaternary complexes with sequence specific transcription factors and histone-modifying enzymes in the LTR of HTLV-I. These Tax-containing complexes allow for increased recruitment of TBP (TFIID), GTFs, and RNAP II within the core promoter region, leading to the synthesis of viral RNA. However, determination of those cellular factors important for enhanced transcriptional activity, as well as the full scope of Tax transactivation, is still not fully elucidated.
In the report by Ching *et al.*\[[@B2]\] the authors directly compare which HTLV-I enhancer motif is preferred by Tax. Each enhancer element (21-bp, CRE, AP1, SP1, κB, or SRE) was placed in an identical TATAA-context to generate a minimal HTLV-I promoter. Previous studies had utilized various promoters (which contain additional DNA elements) to highlight a particular enhancer element necessary for Tax transactivation. Thus, this is the first study to directly compare these elements in an identical setting. In the presence of Tax, the 21-bp repeat (also known as the viral CRE elements or TxREs) was found to be most responsive (70-fold above basal levels). The 21-bp repeat was clearly preferred by Tax, since other enhancer elements were only stimulated 10-fold or less. Previously, several studies suggested that Tax activation of the 21-bp repeats may be mediated by ATF-4 \[[@B3]-[@B5]\]. It was shown that Tax was able to interact with ATF-4 bound to the 21-bp repeats, enhance the binding of ATF-4 to the enhancer, and recruit CREB binding protein (CBP) to the viral promoter \[[@B5]\]. Recently, CREB1 and ATF-4, in addition to ATF-1 and ATF-2, were found to be present *in vivo*on the 21-bp repeats (viral CRE elements) in HTLV-I infected cells through chromatin immunoprecipitation (ChIP) assays \[[@B6]\]. By using dominant negative mutants of CREB1, ATF-4 (CREB2/TAXREB67), Fos, and LZIP, Ching *et al.*demonstrated that among the various bZIP proteins, CREB1 was clearly favored for Tax transactivation of the 21-bp repeats. Additionally, CREB1 has also been found to primarily bind at the 5\' LTR (rather than the 3\' LTR) *in vivo*within HTLV-I infected cells, lending support to the idea that CREB1 is important for HTLV-I activated transcription \[[@B7]\].
If CREB1 is the dominant bZIP protein that is needed for Tax transactivation of the LTR, then what is the purpose of the additional bZIP proteins? Besides contributing to Tax transactivation, could these bZIP proteins help to exclude negative regulators from the LTR? A report by Basbous *et al.*\[[@B8]\] suggested that HBZ, which negatively down-regulated transcription from the HTLV-I LTR, heterodimerized with ATF-4 and subsequently this complex was no longer able to bind to the 21-bp repeats. Only over-expression of ATF-4 was found to reverse the negative effects of HBZ on Tax activity. However, additional studies are still needed to understand the respective contribution of CREB1 and other bZIP proteins, such as ATF-4, to Tax transactivation in the context of wildtype virus and stably integrated viral promoters (i.e. correctly assembled chromatinized DNA templates both *in vitro*and *in vivo*).
Lastly, Ching *et al.*presented the intriguing possibility of Tax enhancing transcription following transcription initiation. To determine whether Tax functioned solely to target TBP to the TATAA-element or if additional events subsequent to TBP (TFIID) recruitment were promoted by Tax, the authors constructed four independent reporters. Each promoter contained the minimal TATAA-element from HTLV-I, HIV-1, SV-40, or E1b promoters, two 21-bp repeats, and five copies of the Gal4-binding site. TBP was artificially targeted to the TATAA-element thru Gal4-TBP. The authors reasoned that if Tax functioned strictly to recruit TBP to the TATAA-element, then additional enhancement of transcription would not be observed when Tax and Gal4-TBP were present. Interestingly, only the Tax-responsive promoters, *i.e.*HTLV-I and HIV-1, were both synergistically stimulated by the addition of Tax and Gal4-TBP. These results suggest that Tax may control downstream transcription subsequent to the initiation phase.
Other viral transactivators have been shown to have a role at initiation and downstream events, such as elongation. The most notable of these has been Tat, the viral transactivator of HIV-1. Without cellular stimulation and Tat expression, RNAP II transcriptional elongation was shown to be inefficient, producing only short transcripts \[[@B9]\]. One major contributing factor of Tat-dependent transactivation is the elongation factor, pTEFb. pTEFb, composed of cyclin T1 and cdk9, associates with Tat leading to increased phosphorylation at specific sites on the heptad repeats of the CTD of RNAP II and promoting elongation. Elongation is highly dependent on the status of RNAP II CTD, since dissociation/association of factors have been shown to be dependent on CTD serine 5/serine 2 phosphorylation \[[@B1],[@B10]\]. Hyperphosphorylation of CTD at serine 5 is associated with promoter clearance/early elongation, whereby initiation factors are released and the 5\'capping machinery subsequently recruited. During processive elongation, there is a switch in CTD phosphorylation to serine 2 phosphorylation resulting in the loss of the capping machinery and the association of splicing, elongation and chromatin remodeling factors. In the case of HTLV-I, Tax has been shown not to associate with a CTD kinase \[[@B11]\] and a dominant negative mutant of cdk9 (the catalytic subunit of pTEFb) was found to increase Tax transactivation of the HTLV-I promoter \[[@B12]\]. Therefore, there is the possibility that other kinase complexes (small vs. large pTEFb complex or other cdk kinases) may aid in increased Tax transactivation. In this context, HTLV-I infected cells contain increased levels of cyclin E/cdk2 kinase activity, through sequestration of cdk inhibitor, p21/waf1, by cyclin D~2~/cdk4 complexes \[[@B13],[@B14]\]. This kinase complex was able to phosphorylate RNAP II CTD and antibodies against cyclin E co-immunoprecipitated only the phosphorylated form of RNAP II from HTLV-I infected cells. Thus, if only indirectly, Tax may increase kinase activity resulting in enhanced CTD phosphorylation for steps following initiation, such as promoter clearance and/or elongation.
Processive elongation is highly dependent on remodeling of chromatin structure \[[@B1],[@B10]\]. A study by Corey *et al.*\[[@B15]\] demonstrated that disruption of SWI/SNF recruitment by an activator resulted in lack of chromatin remodeling, transcription elongation, and production of full-length *hsp70*mRNA. Tax has been shown to associate with BRG1 components of the ATP-dependent chromatin remodeling complex, SWI/SNF, and increase Tax transactivation \[[@B16]\]. Disruption of BRG1 by siRNA led to a decrease in Tax transactivation. Therefore, Tax may target SWI/SNF complexes downstream of RNAP II in order to prevent stalling of RNAP II. This raises a number of questions such as does Tax bind to an elongating RNAP II complex? Does Tax help to recruit elongation factors, such as TFIIS or TFIIF? Finally, it should be emphasized that each stage of transcription is not an independent process; coupling of the transcriptional and RNA processing machinery is thought to increase the rate and specificity of these enzymatic reactions \[[@B1]\]. As shown in Figure [1A](#F1){ref-type="fig"}, acetylation of nucleosomes and other transcription factors/coactivators promote an open complex structure and RNAP II holoenzyme assembly. Initiation by Tax is dependent on the recruitment of CBP/p300 and p/CAF by transcription factor/Tax complex at the 21-bp repeats (viral CRE elements). Phosphorylation of RNAP II CTD is important for loading of the 5\' capping machinery to allow for rapid capping of nascent pre-mRNA, ensuring protection for the transcript from degradation. During promoter clearance (early elongation), site specific phosphorylation of the CTD is modified to allow for sequestration of splicing machinery and elongation factors, and release of the capping machinery. Assembly of SWI/SNF factors with Tax downstream of the elongation phase RNAP II complex remodels chromatin structure, promoting RNAP II processivity. Thus, the presence of Tax for initiation and possibly promoter clearance and/or elongation will help to increase viral transcription and mRNA processing overall (Figure [1B](#F1){ref-type="fig"}). While the results by Ching *et al.*are preliminary at this time, Tax transactivation post-initiation is indeed a novel concept. Further detailed analysis of Tax at both the LTR of HTLV-I and downstream of this region will help to resolve many of these questions and provide important insight into the transcription field.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Effect of Tax on transcription.**A) Schematic representation of proximal promoter of HTLV-I. Tax binding to CBP/p300 with either p/CAF or bZIP transcription factors (*e.g.*CREB1) leads to increased acetylation and interaction with the basal transcription machinery. Tax binding to SWI/SNF downstream of start site may help to remodel restrictive chromatin structure and aid in promoter clearance and elongation. B) The possible effect of Tax on gene expression network. The sequential steps of transcription (initiation, elongation, and termination) are intricately linked together and to mRNA processing and export (adapted from ref. 1). Thus, the effect of Tax on initiation and possibly elongation (both early promoter clearance and processive elongation events) would contribute, albeit indirectly, to RNA processing and export.
:::
:::
Abbreviations
=============
HTLV-I, human T cell leukemia virus, type I
CRE, cAMP response element
CREB, cAMP response element binding protein
ChIP, chromatin immunoprecipitation
RNAP II, RNA polymerase II
CTD, C-terminal domain
HIV-1, human immunodeficiency virus, type 1
LTR, long terminal repeat
TBP, TATA binding protein
TxREs, Tax-responsive elements
GTFs, general transcription factors
TAR, transactivation region
Competing Interests
===================
None declared.
Authors\' contributions
=======================
Both authors contributed equally to the structure and content of the manuscript.
| PubMed Central | 2024-06-05T03:55:46.635356 | 2004-7-30 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC506788/",
"journal": "Retrovirology. 2004 Jul 30; 1:19",
"authors": [
{
"first": "Cynthia",
"last": "de la Fuente"
},
{
"first": "Fatah",
"last": "Kashanchi"
}
]
} |
PMC509016 | Background
==========
Microarray experiments can provide a comprehensive picture of gene expression levels in biological samples. In a typical application they compare expression of several thousand genes under two different conditions (e.g. healthy vs. diseased tissue, wild type vs. mutant animals, drug-treated vs. control cells), using a small number of replicate experiments. Various techniques have been developed to rank genes according to their expression changes, e.g. based on the t-statistic \[[@B1]\] or the strong non-parametric RankProducts \[[@B2]\]. The resulting list of genes can then be restricted to those genes that fulfill a certain statistical criterion, usually an arbitrarily chosen maximum accepted false discovery rate.
The main challenge to the biologist is contained in the next step of the analysis. It consists in identifying the biologically relevant expression changes, the \"big picture\" of the experiment. As microarray experiments tend to generate unexpected observations in areas outside the specialized expertise of the experimentalist, this can be quite difficult and time-consuming. A principled mechanism to identify the significant higher-level features of the experimental results would therefore be very useful.
The biological interpretation process consists to a large extent of finding evidence connecting certain genes that are differentially expressed. This evidence can consist, e.g., of joint participation in some physiological process, physical interaction at the protein level, reported co-expression in earlier microarray experiments, a shared functional annotation, etc. This kind of evidence can intuitively be represented as a graph, and this feature is regularly used to visualize biological data, in the form of metabolic or signaling pathways or protein interaction maps. The task can then be described as the identification of subgraphs that as a whole show a statistically significant expression change. This would allow the biologist to focus her analysis on the most promising areas, without prior bias, while at the same time presenting the relevant evidence underlying each association for critical evaluation.
Results and Discussion
======================
The Algorithm
-------------
We have recently developed an approach, iterative Group Analysis (iGA) that identifies significantly changed functional classes of genes in a microarray experiment \[[@B3]\]. In contrast to similar approaches such as \[[@B4]-[@B8]\], the iGA method does not require a previous delimitation of a set of \"differentially expressed genes\", but uses an iterative calculation of p-values to determine the subset of class members that is most likely to be changed. Due to this feature, the iGA method is more sensitive in identifying functional classes that are slightly but consistently regulated, and works well on noisy data with small numbers of replicates, where the delimitation of gene lists can be overly restrictive.
Here we extend this approach to the analysis of \"evidence graphs\", which offers much larger flexibility of the annotations that can be used and allows substantially improved visualization. Evidence graphs can be represented as bigraphs with two types of nodes, one for genes and one for the associated \"evidence\" (Fig. [1A](#F1){ref-type="fig"}). For evaluation purposes we focused on two types of networks, one where the evidence consists of GeneOntology annotations (GO network) and one where the evidence comprises enzyme substrates (metabolic network). The construction of these networks from gene annotation files is fast and simple, but more unconventional networks are also straightforward (e.g. regulatory networks inferred from previous gene expression experiments, or \"literature networks\" based on co-occurrence of genes in publications). Before the calculation the bigraph is converted to a simple graph, eliminating the evidence nodes and introducing pairwise edges between all nodes that were connected to a common evidence node (Fig. [1B](#F1){ref-type="fig"}).
In addition to the graph, a complete list of genes sorted by differential expression is provided. All nodes without corresponding expression data are eliminated from the network. In the first step of the analysis, each gene node is assigned its rank in the list of genes, such that the node for the most strongly changed gene is labeled \"1\" (Fig. [1C](#F1){ref-type="fig"}), the second most changed gene is labeled \"2\", etc. Then, local minima are identified in the graph, i.e. those nodes that have a lower rank than all their direct neighbors in the graph (Fig. [1D](#F1){ref-type="fig"}). In the next step, subgraphs are iteratively extended from each of those local minima by including the neighboring node with the next highest rank (*m*) and, if present, all adjacent nodes of ranks equal or smaller than *m*. Hence, at each step of the extension process the newly extended subgraph is not adjacent to any outside node with a rank lower than *m* (Fig. [1E,1F,1G,1H](#F1){ref-type="fig"}). At each extension step we thus obtain a subgraph with *n* members with a maximum rank *m* and can calculate the *p*-value for observing all *n* of *n* genes at rank *m* or better in a list of *N* total genes in the graph, , which follows easily from inserting these values in the cumulative hypergeometric equation, which is also used for iGA \[[@B3]\].
The extension process is continued until all nodes reachable from the local minimum are included or the subgraph reaches an arbitrary maximum size. After extending the subgraphs, for each local minimum the subgraph yielding the smallest p-value is selected as its \"regulated neighborhood\" and all local minima are sorted by increasing p-value of these regulated neighborhoods. The subgraphs at the top positions of the resulting list should contain the most relevant regions of the total evidence network.
Comparison with Previous Approaches
-----------------------------------
The method devised by Ideker et al. \[[@B9]\] for the determination of signaling and regulation circuits from a combination of protein interaction information and expression data could easily be extended to cover the more general case of microarray interpretation addressed by GiGA. This would only require the extension of \"interaction\" to include, e.g., participation in a shared cellular process or shared functional annotation. Their approach uses aggregate z-scores to evaluate the quality of each subgraph. This requires a relatively complex parameter estimation procedure followed by simulated annealing. In contrast, the rank-transform of the data that is the basis of GiGA allows non-parametric p-value calculations and is thus much faster (and computationally less demanding) than the method described by Ideker et al. A disadvantage of both methods is that they both do not guarantee to find the optimally scoring subgraph.
The recently released commercial Pathway Analysis software from Ingenuity Systems <http://www.ingenuity.com> seems to be based on a similar concept as GiGA, i.e. the determination of regulated subgraphs in annotation networks. However, it just classifies genes as changed (\"focus genes\") or unchanged based on an arbitrary selected significance cut-off and thus discards most of the relative-change information (gene ranks) used by GiGA <http://www.ingenuity.com/products/Circadian_Application_Note0104.pdf>. Therefore, this method is difficult to apply to very noisy or unreplicated experiments where a reliable delimitation of the \"changed\" genes becomes impossible.
Experimental Case Study
-----------------------
To validate the GiGA approach, we used the yeast diauxic shift experiment by DeRisi et al. \[[@B10]\]. In this classical study the authors examined the response of yeast cells to glucose depletion in the growth medium. As the biology of this process is extremely well understood and the functional annotation of the yeast genome is very comprehensive, we could use this dataset to test the ability (and reliability) of GiGA to identify the relevant subgraphs of interest.
In their original publication, DeRisi et al. highlight the following changes during starvation: Rechanneling of metabolites into the tricarboxylic acid (TCA) and glyoxylate cycle, increase in aerobic respiration (cytochrome c oxidase and reductase), gluconeogenesis, and carbohydrate storage (glycogen and trehalose biosynthesis). In contrast, 95% of ribosomal proteins, as well as tRNA synthetases and translation elongation/initiation factors were strongly down-regulated. Twenty hours after the initial inoculation of the sample about 20% of all genes showed at least a two-fold change in expression.
Table [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"} show the result of an iterative Group Analysis of these expression data. Seven time points after inoculation were examined. Significant expression changes become apparent at 13.5 hours. It can be seen that all the processes identified manually by DeRisi et al. are already apparent at this level of analysis. In addition, iGA highlights the up-regulation of sugar transporters at the early stages of starvation, obviously a desperate attempt by the cells to take up the last remaining sugars from the medium. It also highlights the induction of heat shock proteins and the repression of ribosome biosynthesis processes in the nucleolus, which were not discussed in the original paper. What is missing in these lists, however, is the automatic identification of the interrelationships between the identified processes, which would be particularly informative in more realistic applications with a less well understood biology.
This connectivity between genes and functional classes is provided by GiGA. Table [3](#T3){ref-type="table"} summarizes the results for the 20.5 hour time point, using two different networks, one for GeneOntology classes, and one for enzyme substrates, extracted from the SwissProt catalytic activity descriptors of yeast proteins. For both up- and down-regulated genes, the most significant subgraph is widely separated from the next best one, contains the largest number of genes, and comprises almost all the processes detected by iGA and the original authors. (We here restricted the size of subgraphs to a maximum of 40 genes to keep navigation of the results simple.) Figures [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"} show the automatically generated visualization of the corresponding most significant subgraphs and the associated annotation. It is obvious how GiGA highlights the functional connections between different enzymes. In Fig. [2](#F2){ref-type="fig"}, the association between small and large ribosomal subunits, nucleolar rRNA processing and translational elongation is faithfully reconstructed. In Fig. [3](#F3){ref-type="fig"}, which uses the enzyme substrate network (which we considered to be more challenging for the algorithm), the interplay between the TCA cycle, the overlapping glyoxylate cycle, and all the relevant protein complexes of the respiratory chain are readily apparent. The agreement with the manual interpretation by DeRisi et al. extends down to the single gene level, while at the same time adding additional, obviously relevant connections, e.g. from the TCA cycle to the ATP synthase complex. Fig. [3](#F3){ref-type="fig"} also shows the second best subgraph, which contains the cytochrome c oxidase subunits together with two catalase genes that may be involved in the detoxification of the hydrogen peroxide generated by the respiratory burst induced by starvation.
The performance of GiGA (as well as iGA) is best appreciated when compared to the results of an extensive expert interpretation of the same data. Table [1](#T1){ref-type="table"} to [3](#T3){ref-type="table"}, and Fig. [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"} show how both techniques succeed in detecting and condensing exactly the genes and processes that were considered relevant by the expert biologists when first interpreting the same data \[[@B10]\]. GiGA effectively summarizes the original publication in three subgraph pictures (Fig. [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}). This is even more astonishing when considering that these results are achieved for each single time-point separately (see, e.g., Tab. [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}). This reveals the stability of the approach towards the measurement variance inherent in any unreplicated microarray experiment.
It is important to be aware that each of the highlighted subgraphs has to be carefully evaluated for its biological relevance. On the one hand, the sheer number of possible subgraphs in an evidence network creates a major multiple-testing problem, which means that some of the detected associations may be due to chance. Random permutations of the expression data -- which can be generated by the GiGA software -- can give an idea of the expected false-discovery rate. On the other hand, functional annotations are at present notoriously unreliable and spurious edges may affect the details of the results. Also, not all genes within a detected subgraph will necessarily show a strong expression change, because sometimes less strongly affected genes may connect those genes that do change. Such a relation is for example expected for many transcription factors and their targets \[[@B9]\]. Nonetheless, GiGA is able to direct the user to the most interesting areas in the evidence network.
The GiGA method is not restricted to use with exhaustively annotated genomes. It can work on a wide variety of \"evidence\" to build the necessary network, including hypothetical predicted functions or associations. It is even possible to apply GiGA to metabolomics results, which are characterized by the absence of any significant amount of annotation -- usually only exact molecular masses and their differential abundance are known. In that case, an evidence network can be built from the measured masses themselves, linking compounds *m*~1~ and *m*~2~ whenever their mass difference (Δ*m* = \|*m*~1~ - *m*~2~\|) can be explained by a common biochemical transformation (e.g. dehydrogenation: Δ*m* = 2\* *mass*(*hydrogen*)). The set of relevant transformations can easily be collected from any biochemistry textbook. In addition, one can introduce edges for condensation reactions between observed masses, i.e. if m~1~ + m~2~ = m~3~ + mass(H~2~O) then edges between m~1~ and m~3~, and m~2~ and m~3~ are added to the evidence network. We are currently developing the application of GiGA to this kind of data in the Sir Henry Wellcome Functional Genomics Facility at the University of Glasgow <http://www.gla.ac.uk/functionalgenomics/>; data not shown).
Conclusions
===========
The present analysis of a biologically well-understood test case demonstrates the reliable performance of GiGA. The method automatically identifies all relevant physiological processes, puts them into context, summarizes them in an intuitive format, and associates them with the underlying evidence (Fig. [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}). It can be applied to experiments with very small numbers of replicates (a single time point in the diauxic shift test case) and can be used with any available functional annotation, including protein interaction networks, co-expression data or literature mining results, as well as in areas beyond microarray analysis. For visualization, we have used the graph-layout software aiSee, but output files suitable for a variety of graphical tools can easily be generated by slight modifications in the implementation. GiGA can be used as a stand-alone tool, but we expect that it will be most useful when integrated into existing microarray analysis software, and for that reason the GiGA algorithm is freely available without restrictions.
Methods
=======
Yeast gene expression data for the diauxic shift experiment were obtained from the Stanford Microarray Database <http://genome-www5.stanford.edu/>. GeneOntology annotations <http://www.geneontology.org> were obtained from Affymetrix <http://www.affymetrix.com/analysis/download_center.affx>. The enzyme substrate networks were built based on information contained in the annotation of the yeast proteome in the SwissProt database <http://us.expasy.org/sprot/>. The GiGA algorithm has been implemented as a Perl script and compiled as a Windows command line executable. These files are available (together with a manual and example files) as [Additional files 1](#S1){ref-type="supplementary-material"} to [8](#S8){ref-type="supplementary-material"}.
Authors\' contributions
=======================
RB devised and implemented the GiGA technique and drafted the manuscript. AA and PH supervised the project. All authors read and approved the final manuscript.
Supplementary Material
======================
::: {.caption}
###### Additional File 1
GiGA program. For use from the Windows command line.
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###### Additional File 2
GiGA source code.
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###### Additional File 3
GiGA manual. Describes the use of GiGA applied to the example data (Additional files 4 to 6).
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###### Additional File 4
Gene expression data. Sorted list of genes, based on expression during the yeast diauxic shift.
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###### Additional File 5
Evidence network. List of gene pairs connecting genes whenever their gene products are enzymes that share a common substrate. Based on annotation derived from SwissProt.
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###### Additional File 6
Genenames file. Contains descriptive names of the yeast genes contained in Additional file 4.
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###### Additional File 7
Example output in text format. List of significantly affected subgraphs detected in the experimental data (Additional file 4) using GiGA with default settings.
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###### Additional File 8
Example output in graph-description language format. Contains the same results as Additional file 7, but in a format that can be visualized and explored using graph-layout software, e.g. aiSee, which is freely available for academics at <http://www.aisee.com>.
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Acknowledgements
================
We thank Dr. Mike Barrett for stimulating discussions on the application of GiGA to metabolomics data. We also thank the referees for their very constructive evaluation of the software. This work was supported by BBSRC grants 17/GG17989 and 17/P17237.
Figures and Tables
==================
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Principle of Graph-based Iterative Group Analysis. **A** Evidence network. Genes are associated with their annotation in the form of a bigraph (two types of nodes). **B** The same evidence represented as a simple network. Genes that share an annotation are connected. **C-H** Example of a GiGA analysis using fictitious microarray results. **C** Genes are assigned their ranks based on observed expression changes. **D** Local minima are found, i.e. genes that have no connection to genes with a better rank. **E-H** Iterative expansion of subgraphs from one of the local minima, gene 2 (rank 1). **E** The neighboring node with the smallest rank is included (gene 4, rank 4), which leads to the additional inclusion of genes 5 (rank 3) and 6 (rank 2). **F** Gene 3 (rank 5) is included). **G** Gene 7 (rank 7) is included, leading to the inclusion of gene 8 (rank 6). **H** The last gene reachable from this local minimum, gene 1 (rank 8), is included and the process terminates. For each of the subgraphs a p-value can be calculated (see text) and the subgraph with the smallest p-value is declared the \"regulated neighborhood\" of the local minimum. In the example, genes 2, 4, 5, and 6 form a regulated neighborhood (p = 0.014). The graph expansion process would then be repeated for the remaining two local minima.
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Visualization of the most significant \"down-regulated neighborhood\" identified by GiGA using a GeneOntology-based network. The expression data are taken from the 20.5 h timepoint of the yeast diauxic shift (DeRisi et al., 1997). The layout was generated from the output of GiGA by the free software aiSee <http://www.aisee.com> using a force-directed algorithm with default parameters. The same software can also be used for the versatile real-time navigation of the network. Colored boxes show the regulated genes (darker shading indicates stronger regulation), white boxes show the evidence linking the genes (in this case GeneOntology numbers and terms). Several important components of this regulatory neighborhood are indicated (small and large ribosomal subunit proteins, rRNA processing/snRNP, nucleolar proteins, translation elongation factors). These components were also identified in the original publication after manual analysis. GiGA finds them automatically, and also detects the -- biologically obvious -- connections between them. As all the evidence is included in the same picture, the biologist can then use her expertise to assess the relevance of each link, without having to make the connections *ad hoc* by tedious literature studies.
:::
:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
Visualization of the two most significant \"up-regulated neighborhoods\" identified by GiGA using a metabolic network derived from Swissprot annotations. The expression data are taken from the 20.5 h timepoint of the yeast diauxic shift (DeRisi et al., 1997). The layout was generated as in Fig 2. Colored boxes show the regulated genes (darker shading indicates stronger regulation), white boxes show the substrates that are in common between genes. Important components of this regulatory neighborhood are indicated (TCA cycle and glyoxylate cycle enzymes, and the various respiratory chain complexes). Here it can be seen that GiGA not only detects protein complexes (such as ribosomes or the respiratory chain complexes), but also \"linear\" metabolic pathways such as TCA cycle and glyoxylate cycle (and potentially signal transduction pathways or regulatory cascades etc.). Almost all the enzymes discussed by DeRisi et al. (1997) are included in these two subgraphs, plus the relevant enzymatic information necessary to assess the relevance of each link, without the danger of missing some genes (unless the annotation is incomplete).
:::
:::
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Iterative Group Analysis of gene expression during the yeast diauxic shift. Up-regulated groups.
:::
**0 h** **9.5 h** **11.5 h** **13.5 h** **15.5 h** **18.5 h** **20.5 h**
--------- ----------- -------------------------------------- ------------------------------------------------- ---------------------------------------------------------------- ---------------------------------------------------------------- ----------------------------------------------------------------
6144 -- purine base metabolism **6099 -- tricarboxylic acid cycle** **6099 -- tricarboxylic acid cycle** 3773 -- heat shock protein activity **6099 -- tricarboxylic acid cycle**
9277 -- cell wall (sensu Fungi) 3773 -- heat shock protein activity **5749 -- respiratory chain complex II (sensu Eukarya)** **6099 -- tricarboxylic acid cycle** 3773 -- heat shock protein activity
297 -- spermine transporter activity 6950 -- response to stress **6121 -- oxidative phosphorylation, succinate to ubiquinone** **5977 -- glycogen metabolism** **5749 -- respiratory chain complex II (sensu Eukarya)**
15846 -- polyamine transport 297 -- spermine transporter activity **8177 -- succinate dehydrogenase (ubiquinone) activity** 6950 -- response to stress **6121 -- oxidative phosphorylation, succinate to ubiquinone**
**4373 -- glycogen (starch) synthase activity** 3773 -- heat shock protein activity **4373 -- glycogen (starch) synthase activity** **8177 -- succinate dehydrogenase (ubiquinone) activity**
15846 -- polyamine transport **4373 -- glycogen (starch) synthase activity** **4129 -- cytochrome c oxidase activity** 6537 -- glutamate biosynthesis
5353 -- fructose transporter activity 7039 -- vacuolar protein catabolism **5751 -- respiratory chain complex IV (sensu Eukarya)** **6097 -- glyoxylate cycle**
15578 -- mannose transporter activity 6950 -- response to stress **5749 -- respiratory chain complex II (sensu Eukarya)** **5750 -- respiratory chain complex III (sensu Eukarya)**
7039 -- vacuolar protein catabolism **4129 -- cytochrome c oxidase activity** **6121 -- oxidative phosphorylation, succinate to ubiquinone** **9060 -- aerobic respiration**
8645 -- hexose transport **5751 -- respiratory chain complex IV (sensu Eukarya)** **8177 -- succinate dehydrogenase (ubiquinone) activity** **4129 -- cytochrome c oxidase activity**
4396 -- hexokinase activity 4396 -- hexokinase activity 30162 -- regulation of proteolysis and peptidolysis **5751 -- respiratory chain complex IV (sensu Eukarya)**
5215 -- transporter activity 297 -- spermine transporter activity 4364 -- glutathione transferase activity 16491 -- oxidoreductase activity
**5977 -- glycogen metabolism** **6101 -- citrate metabolism**
For this analysis, genes were assigned to groups based on GeneOntology annotations <http://www.geneontology.org> obtained from Affymetrix <http://www.affymetrix.com/analysis/download_center.affx>. All groups that are changed with a minimal p-value smaller than 1/ \[number of annotated genes\] (1/4087 = 2.4E-4) are shown, sorted by significance. Numbers and names are the standardized GeneOntology identifiers. Groups shown in bold were also reported as changed in the original publication (DeRisi et al., 1997) Up-regulated groups
:::
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Iterative Group Analysis of gene expression during the yeast diauxic shift.
:::
**0 h** **9.5 h** **11.5 h** **13.5 h** **15.5 h** **18.5 h** **20.5 h**
--------------------------------------------------- ----------- ------------ ----------------------------------------- ------------ --------------------------------------------------------------- ---------------------------------------------------------------
7152 -- spore wall assembly (sensu Saccharomyces) **5730 -- nucleolus** **30490 -- processing of 20S pre-rRNA** **5843 -- cytosolic small ribosomal subunit (sensu Eukarya)**
**30490 -- processing of 20S pre-rRNA** **5732 -- small nucleolar ribonucleoprotein complex** **5842 -- cytosolic large ribosomal subunit (sensu Eukarya)**
**7046 -- ribosome biogenesis** **5730 -- nucleolus** **30490 -- processing of 20S pre-rRNA**
**30515 -- snoRNA binding** **42273 -- ribosomal large subunit biogenesis** **5730 -- nucleolus**
**5842 -- cytosolic large ribosomal subunit (sensu Eukarya)** **6414 -- translational elongation**
**30515 -- snoRNA binding** **5732 -- small nucleolar ribonucleoprotein complex**
3938 -- IMP dehydrogenase activity **27 -- ribosomal large subunit assembly and maintenance**
6183 -- GTP biosynthesis **42273 -- ribosomal large subunit biogenesis**
**154 -- rRNA modification** **30515 -- snoRNA binding**
**3723 -- RNA binding**
**154 -- rRNA modification**
Down-regulated groups. See Table 1 for details. Down-regulated groups.
:::
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Graph-based iterative Group Analysis of gene expression during the yeast diauxic shift.
:::
**Down-regulated genes using GeneOntology-based network**
----------------------------------------------------------- ---------------------------------------------------------------- --------------------- ------------- ------- ---------------
**anchor locus** **group description** **minimal p-value** **E-value** **N** **max. rank**
YHL015W ***ribosomal proteins and rRNA processing*** 5.87E-86 \<0.01 39 48
YMR217W amino acid and nucleotide biosynthesis 3.38E-13 2.7 9 172
YDR144C cell wall biogenesis 4.06E-08 4.5 6 242
YNL065W membrane transporter 4.02E-05 9.3 3 141
YLR062C bud site selection 6.41E-05 9.9 4 367
YGL225W protein glycosylation in Golgi 1.12E-04 10.8 4 422
YPR074C pentose phosphate pathway 1.44E-04 11.2 4 449
**total genes measured in network: 4087.**
**Down-regulated genes using metabolic network**
**anchor locus** **group description** **minimal p-value** **E-value** **N** **max. rank**
YNL141W ***nucleotide and amino acid biosynthesis, tRNA synthetases*** 4.67E-59 \<0.01 39 45
YOR224C RNA polymerases 2.59E-13 1.1 23 219
**total genes measured in network: 744.**
**Up-regulated genes using GeneOntology-based network**
**anchor locus** **group description** **minimal p-value** **E-value** **N** **max. rank**
YER065C ***TCA and glyoxylate cycle, respiratory chain*** 8.57E-77 \<0.01 39 66
YKL217W membrane transporters (sugar, amino acids) 1.76E-15 2.3 8 62
YAL017W protein kinases 1.07E-07 4.8 6 284
YBL043W cell wall biogenesis 3.81E-07 5.4 4 103
YGR248W carbohydrate metabolism 5.66E-07 5.5 5 232
YEL011W ***glycogen metabolism*** 1.01E-06 5.8 3 42
YER037W protein phosphatases 1.07E-06 5.8 8 736
YJL137C ***glycogen biosynthesis*** 7.46E-06 7.3 4 215
YDL085W disulfide oxidoreductases 1.05E-05 7.6 4 234
YNL173C mating signal transduction 1.65E-05 8.2 4 262
YNL134C alcohol dehydrogenase 1.34E-04 11.1 3 210
YBL038W ***mitochondrial large ribosomal subunit*** 1.99E-04 11.9 4 487
**total genes measured in network: 4087.**
**Up-regulated genes using metabolic network**
**anchor locus** **group description** **minimal p-value** **E-value** **N** **max. rank**
YER065C ***TCA and glyoxylate cycle, respiratory chain*** 4.96E-53 0.11 39 54
YGR088W ***cytochrome c oxidase*** 3.09E-10 1.2 11 106
YFR015C ***glycogen synthases*** 2.08E-04 3.6 3 45
YJR073C methyltransferases 3.85E-04 4.0 5 156
YDR001C ***trehalases*** 5.01E-04 4.2 3 60
YCR014C DNA and RNA polymerases 5.44E-04 4.2 17 481
YIR038C glyoxalases 8.64E-04 4.5 5 183
**total genes measured in network: 744.**
The evidence network was constructed either from GeneOntology information (nodes are connected if they share a GeneOntology annotation) or from enzyme activity information obtained from Swissprot <http://www.expasy.ch>. In the latter case, genes are connected if their encoded proteins convert the same substrate (as product or educt, i.e. the direction of the reaction is not taken into account here). This type of network is much smaller (only 744 genes), as only genes coding for enzymes are included. All groups that are changed with a minimal p-value smaller than 1/\[number of annotated genes\] are shown, sorted by significance. The corresponding E-value as estimated by the analysis of 100 random permutations of the data is also shown. The employed threshold for inclusion in the table is very generous and does not guarantee that all subgraphs shown are statistically significant. The local minimum anchoring each regulated neighborhood is indicated by its genetic locus name (for overlapping neighborhoods, only the best-ranking minimum is shown). Descriptive group names were added manually. Groups that correspond to processes discussed in the original paper are highlighted in italics. It can be seen that the highest ranking group in each case is the largest and contains the central biological processes detected by DeRisi et al. (1997) and by iGA (see Table 1 and 2). N, number of genes in each subgraph.
:::
| PubMed Central | 2024-06-05T03:55:46.636364 | 2004-7-23 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509016/",
"journal": "BMC Bioinformatics. 2004 Jul 23; 5:100",
"authors": [
{
"first": "Rainer",
"last": "Breitling"
},
{
"first": "Anna",
"last": "Amtmann"
},
{
"first": "Pawel",
"last": "Herzyk"
}
]
} |
PMC509235 | Background
==========
Precise estimation of genetic relatedness between isolates of a microorganism is important for determination of phylogenetic relationships, which has important applications in studies of disease transmission \[[@B1],[@B2]\]. The definitive standard for assessing genetic relatedness among organisms is the complete genome sequence of nucleotide bases \[[@B3]\]. However, nucleotide sequencing is expensive and time-consuming, thus, generally it is impractical for use in most investigations, particularly when a large number of samples is analyzed.
Currently, one genotyping technique used frequently as an alternative to complete genome sequencing is restriction fragment analysis (RFA), in which restriction endonuclease enzymes cleave the genome at specific sites, producing DNA fragments that are then separated by size using electrophoresis \[[@B4]\]. The percentage of fragments matching in size has been commonly used as an index to represent the genetic similarity between samples \[[@B5],[@B6]\]. The accuracy of RFA in determining the true genetic relationships can be influenced by several factors, including the number of restriction enzymes used, the specific enzymes selected for DNA digestion, and laboratory conditions \[[@B7]-[@B9]\].
Another common alternative to complete genome sequencing is partial genome sequencing, i.e., the nucleotide sequencing of a particular gene or segment of the genome \[[@B8],[@B10]\]. The gene or genome segment is often targeted by polymerase chain reaction (PCR). Selection of an appropriate gene or region for analysis is critical for accurately representing phylogenetic relationships \[[@B11],[@B12]\].
In a comparison of RFA and partial genome sequencing with respect to their similarity in interpreting a disease outbreak caused by pseudorbabies virus in a swine producing region in Illinois, USA, both genotyping methods generated similar conclusions about patterns of spread of the virus \[[@B13]\]. However, the accuracy of each genotyping method in representing the complete genome was not evaluated.
Restriction fragment analysis detects genetic variation by surveying specific endonuclease restriction sites over the entire genome; in contrast, partial genome sequencing detects genetic variation by comparing nucleotide bases from a specific region of the genome. Each method detects a different dimension of genetic variation, and each can detect only a proportion of the genetic variation present in the entire genome. Therefore, it is important to determine which method, using partial information, provides a more accurate estimation of genetic relatedness.
The primary purpose of this study was to compare both restriction fragment analysis and partial genome sequencing to complete genome sequencing, with regard to their agreement in estimating genetic relationships and in reconstructing phylogenies under the ideal conditions of absence of laboratory error. Computer simulation of the genotyping analysis was conducted, using completely sequenced papillomavirus isolates obtained from Genbank.
Results
=======
Table [1](#T1){ref-type="table"} provides descriptive statistics on fragment size distributions for RFA (using the MaeI enzyme as an example) showing that a moderate number of fragments (mean \> 20) were produced by simulated digestion. Fragment sizes were large (median ≈ 280 bps for example enzyme), with only 4 samples having one fragment each ≤ 20 bps. Table [2](#T2){ref-type="table"} shows that with an increase in the number of restriction enzymes, the correlation between the RFA and the complete genome sequencing genetic distance matrices increased slightly and the partition metric measuring dendrogram topological dissimilarity decreased slightly. The highest agreement with complete genome sequencing obtained for RFA was for a 4-enzyme combination, which achieved a maximum Mantel\'s r = 0.60 and minimum partition metric = 18. Table [3](#T3){ref-type="table"} shows that the similarity with complete genome sequencing in estimating genetic relatedness was much higher for partial genome sequencing, particularly for the E1 and L1 genes, which had the relatively longer sequences (averaging 24.2% and 19.6% of genome, respectively), although all genes selected had Mantel\'s r ≥ 0.88. The minimum value of the partition metric was 10, and the maximum value was 14, compared to a minimum of 18 for RFA. Phylogenetic trees are presented for complete genome sequencing (Fig. [1](#F1){ref-type="fig"}), RFA (the 4-enzyme condition with the highest agreement with complete genome sequencing) (Fig. [2](#F2){ref-type="fig"}), and sequencing of the E1 gene (the longest gene) (Fig. [3](#F3){ref-type="fig"}). Tree stability, as indicated by bootstrap values, was higher for complete genome sequencing (Fig. [1](#F1){ref-type="fig"}: all bootstrap values \> 0.90) than for partial genome sequencing of the E1 gene (highest Mantel\'s r). However, the E1 gene tree structure for the most closely related samples was stable and nearly identical to complete genome sequencing. In contrast to the RFA example given (Fig. [2](#F2){ref-type="fig"}), which did not clearly differentiate the papillomavirus samples into subgroups, partial genome sequencing of the E1 gene identified 2 subgroups with the same composition (and BPV2 as an outlier) as did complete genome sequencing.
Discussion
==========
Sequencing entire genomes is impractical in most investigations of genetic relationships. The computer simulation conducted here determined that compared to restriction fragment analysis, partial genome sequencing had higher agreement with complete genome sequencing in estimating genetic relatedness and greater similarity in the topology of the dendrograms of phylogenetic relationships derived from these estimates. These results using papallomavirus sequences with a genome length averaging less than 8 kb, indicate that for microorganisms with small genomes, partial genome sequencing targeting genes comprising approximately 20--25% of the total genome length can provide a very good estimate of genetic relatedness. The topological structure of phylogenetic trees was also stable for partial genome sequencing, particularly for the most closely related samples. The degree to which these results generalize to larger genomes is unknown, in part because microorganisms with large genomes are rarely, if ever, sequenced in their entirety. There are also other considerations in selecting partial genome sequencing as a genotyping method, such as presence of the gene in all isolates, and sufficient variability to differentiate isolates \[[@B12]\]. In addition, whether genetic variation is random or due to natural selection needs to be taken into account \[[@B14]\], because in the latter case genetic dissimilarity may not reflect time since divergence, thus making it more difficult to infer evolutionary relationships, which are important for making inferences about pathogen transmission. These limitations should be considered as well for restriction fragment analysis.
One might expect that increasing genome size would diminish the advantage of partial genome sequencing compared to restriction fragment analysis. As total genome size increases, the number of restriction sites cut by restriction enzymes is expected to increase, providing more fragments and more genetic information for estimating genetic relatedness at no increased cost. This also needs to be taken into account in the selection of a genotyping method. However, it has been argued that if a gene is selectively neutral (i.e., variations are not subject to natural selection), it is only the length of the gene sequenced, not the ratio of sequenced gene length to genome size, that is important for determining the degree of divergence from a common ancestor \[[@B14]\]. To the extent that these conditions are satisfied, the results of this study indicate that specific gene sequencing is likely to provide a better estimate of genetic relationships than restriction fragment analysis of the complete genome under a wider variety of genome sizes.
The general conditions under which partial genome sequencing is more accurate than restriction fragment analysis in representing true genetic relatedness have not been addressed in the analysis conducted here. However, another study from our laboratory \[[@B15]\], using simulated genomes of various size with different nucleotide substitution rates, and varying degrees of genetic diversity among samples, found that only under conditions of both short partial genome sequence length and low rates of nucleotide substitution did RFA provide a more accurate topological reconstruction of phylogenetic relationships than did partial genome sequencing; the degree of genetic diversity among samples did not affect the advantage partial genome sequencing had in accurately depicting phylogenetic relationships. Thus, whether one is investigating the genetic relatedness among samples collected from a single disease outbreak or a diverse collection of samples from different times and geographic regions, under most conditions partial genome sequencing will represent genetic relationships more accurately than does RFA. Genotyping using partial genome sequencing and phylogenetic reconstruction (using the neighbor-joining algorithm) have become standard for several virus species, including not only papillomavirus \[[@B16],[@B17]\], but also human immunodeficiency virus \[[@B18]\], classical swine fever virus \[[@B19]\], porcine reproductive and respiratory syndrome virus \[[@B20]\], and foot-and-mouth disease virus \[[@B21]\].
The simulated genotyping conducted here assumed no error of measurement. The sources of error in restriction fragment analysis are well known \[[@B22]-[@B24]\]. Fragments of similar size in the same lane of a gel may be indistinguishable, thus appearing to form one fragment. Fragments of small size may be undetectable. The relationship between migration distances and fragment size may be affected by variation in gel density both between and within gels. There are also differences in measurement error between laboratories \[[@B25],[@B26]\]. These deficiencies are accounted for by use of marker DNA fragments of known nucleotide base pair length to assist in estimating cleaved DNA fragment sizes; however, acknowledgement of remaining error of measurement of the size of detectable fragments is inherent in the application of a tolerance range for considering fragments of similar but different sizes as a \"match\" \[[@B27]\]. Laboratory error is also inherent in partial genome sequencing \[[@B28]\]. With the commonly used polymerase chain reaction (PCR) methodology for detection and amplification of genes for sequencing, there can be error in primer development because primer sites may not be specific to the gene sequences or too specific to demarcate all occurrences of the gene. Heterogeneity of amplified DNA, due to replication error, recombination, low primer specificity, or impurity of the template can result in a failure to produce consistent sequencing results. In the comparison of the degree of similarity of DNA sequences between samples, alignment of sequences with unequal sequence lengths due to deletion or duplication, or the management of inverted sequences presents additional challenges for estimating genetic similarity and phylogenetic affinity \[[@B14]\]. The relative magnitude of sources of error in RFA versus partial genome sequencing is unknown and, thus, the conclusions presented here are those based upon the assumption of the absence or minimization of laboratory error.
In practical terms, laboratory error and cost need to be taken into account in the selection of a genotyping method. However, when the impact of these factors is minimized, the computer simulation analysis conducted here indicates that partial genome sequence becomes the preferred alternative for representing genetic relationships.
Conclusions
===========
For small genomes, partial genome sequencing of target genes comprising 20--25% of the total genome provides a more accurate estimate of genetic relatedness and more accurate representation of evolutionary and transmission histories than does restriction fragment analysis and thus is indicated to be the preferred genotyping method for phylogenetic reconstruction under these conditions. The degree to which these results are generalizable to larger genomes and conditions of laboratory error remains to be determined.
Methods
=======
Sample DNA sequences
--------------------
The source of information on nucleotide sequences was the Genbank database \[[@B29]\]. The organism selected for analysis was papallomavirus, for which a moderately large number of isolates with complete genome sequences was available. Human, bovine, canine, and chimpanzee papillomaviruses were considered. Among human papillomavirus (HPV) with complete genome sequencing available, 12 samples were selected at random: HPV 4, 6a, 6b, 20, 24, 49, 63, 13, 29, 32, 54, and 26. For bovine papillomavirus (BPV), complete genome sequences were available for BPV1, BPV2, and BPV4. Because the E1 gene of BPV1 (of interest for partial genome sequencing) could not be located, only BPV2 and BPV4 were chosen and included in the study. One type of canine oral papillomavirus (caninePV) and one type of common chimpanzee papillomavirus (chimPV) were available in the database, and these were chosen. Thus, a total of 16 types or subtypes of papillomaviruses that have been completely sequenced and stored in Genbank were used (Table [1](#T1){ref-type="table"}).
The complete DNA sequences of the 16 papillomavirus samples were aligned using ClustalW software \[[@B30]\]. The genetic distances among these sequences were then calculated using the Kimura correction \[[@B31],[@B32]\].
Computer simulated restriction fragment analysis
------------------------------------------------
### Restriction endonuclease enzymes
Commonly used restriction endonuclease enzymes were selected \[[@B33]\], based on the following criteria: (1) Only enzymes with 4-base pair recognition sites were selected, in order to produce a sufficient number of fragments for analysis. (2) Among enzymes having the same recognition site, only one was selected. (3) For simplicity, enzymes with multiple recognition sites were excluded. Using these criteria, 15 restriction enzymes were included (AccII, AciI, AluI, BsuRI, CviRI, HapII, HhaI, MaeI, MaeI, MboI, MseI, NlaIII, RsaI, TaqI, TspEI).
### Digestion
Simulated digestion of each papillomavirus DNA sample by each restriction enzyme was conducted using the DIGEST program \[[@B34]\]. The resulting restriction fragments for each sample were sorted by size (number of nucleotide base pairs).
### Calculation of genetic distances
Based on the distribution of restriction fragment sizes, the genetic similarity between any two papillomavirus samples was calculated for each restriction enzyme using the Dice coefficient \[[@B5],[@B6]\]: S~xy~= 2n~xy~/(n~x~+n~y~), where n~xy~is the number of fragments matching in size for samples x and y, and n~x~and n~y~are the number of fragments in samples x and y, respectively. Then, D~xy~= 1-S~xy~was calculated as a distance measure. Pairwise distances between samples were computed for each individual enzyme. Also, pairwise distances were obtained for up to 4 enzymes, by using for each condition (2, 3, and 4 enzymes) the fragment size distributions for 30 randomly selected combinations of enzymes, and calculating the composite distance \[[@B35]\].
Partial genome sequence analysis
--------------------------------
The E1, E6, E7, and L1 genes, which have been of interest in studies of papillomavirus, were used for estimating genetic relatedness. The ClustalW program \[[@B30]\] was used for sequence alignment, and the genetic distances (with the Kimura correction) were calculated for each gene.
Agreement between genotyping methods
------------------------------------
### Correlation between distance matrices
The matrix of genetic distances based on complete DNA sequences was considered the definitive standard. The genetic distance matrices based on RFA and partial genome sequencing were compared to complete genome sequencing by calculating Mantel\'s coefficient of correlation between matrices (Mantel\'s r) \[[@B36]\].
### Comparison of phylogenetic trees
The genetic distance matrices for RFA, partial genome sequencing, and complete genome sequencing were used to construct phylogenetic trees, using the Neighboring-joining algorithm \[[@B37]\], as implemented by MEGA software \[[@B38]\]. Trees were rooted at the midpoint between the most distantly related samples \[[@B39]\]. Bootstrap values indicating stability of tree topology were added to trees based on partial and complete genome sequencing \[[@B14]\]. The trees based on RFA and specific gene sequences were compared to the tree for complete genome sequencing, by using the COMPONENT software \[[@B40]\] to calculate the partition metric, which measures the difference in tree topology \[[@B41],[@B42]\]. A lower value of partition metric indicates greater topological similarity.
List of abbreviations
=====================
bps: base pairs
BPV: bovine papillomavirus
caninePV: canine papillomavirus
chimpPV: chimpanzee papillomavirus
HPV: human papillomavirus
kb: kilobase
Mantel\'s r: Mantel\'s coefficient of correlation between matrices
PCR: polymerase chain reaction
RFA: restriction fragment analysis
Authors\'s contributions
========================
BQ designed the investigation, collected the data, conducted the data analysis, and wrote the manuscript. RW identified the problem to be investigated, provided statistical guidance, assisted in interpretation of results, and edited the final drafts of the manuscript. Both authors read and approved the final manuscript.
Figures and Tables
==================
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Tree of phylogenetic relationships among Papillomavirus samples, based on complete genome sequences. Classification achieved using the Neighbor-joining algorithm. The tree was rooted at the midpoint between the most disparate samples. Numbers on branches indicate bootstrap values.
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Tree of phylogenetic relationships among Papillomavirus samples, based on restriction fragment analysis with four restriction enzymes. Classification achieved using the Neighbor-joining method. The tree was rooted at the midpoint between the most disparate samples.
:::
:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
Tree of phylogenetic relationships among Papillomavirus samples, based on DNA sequencing of the E1 gene. Classification achieved using the Neighbor-joining method. The tree was rooted at the midpoint between the most disparate samples. Numbers on branches indicate bootstrap values.
:::
:::
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Sequence lengths and fragment size distribution for papillomavirus samples obtained from Genbank
:::
Type of Papillomavirus Genbank Accession Number Length of Complete Genome (bps) Length of Genes (bps) Fragment Size Distribution (digested by MaeI enzyme^1^)
------------------------ -------------------------- --------------------------------- ----------------------- --------------------------------------------------------- ----------- ------------ ---------- ----------- ---------- -----------
HPV4 X70827 7353 1800 422 303 1550 24 215 54 817
HPV6a L41216 8010 1886 452 297 1502 18 249 43 1151
HPV6b X00203 7902 1930 452 297 1502 17 207 47 1241
HPV20 U31778 7757 1818 497 309 1550 24 294 9 781
HPV24 U31782 7452 1824 422 291 1538 13 501 55 1354
HPV49 X74480 7560 1830 416 312 1529 18 232 24 1210
HPV63 X70828 7348 1857 425 267 1523 23 221 29 968
HPV13 X62843 7880 1941 452 306 1499 21 352 9 765
HPV29 U31784 7916 1983 446 273 1511 16 435 33 1003
HPV32 X74475 7961 1929 428 315 1511 21 276 36 881
HPV54 U37488 7759 1902 434 288 1493 28 158 20 815
HPV26 X74472 7855 1917 452 315 1511 20 341 47 877
BPV2 M20219 7937 1815 413 384 1493 27 204 17 809
BPV4 X05817 7265 1932 300 363 1562 19 323 31 750
CaninePV D55633 8607 1794 434 294 1511 24 303 24 715
ChimPV AF020905 7889 1947 458 300 1505 18 236 35 1504
**Mean** **7778.1** **1881.6** **431.4** **307.1** **1518.1** **20.7** **284.1** **32.2** **977.5**
1: Data reported for MaeI as an example.
:::
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Similarity of restriction fragment analysis to complete genome sequencing in estimating genetic relatedness between papillomavirus samples
:::
Number of Enzymes Mantel\'s r Partition Metric
------------------- ------------- ------------------ ------ ------- ------ ----
1 0.37 0.13 0.54 23.33 1.23 20
2 0.42 0.09 0.55 22.60 1.19 20
3 0.46 0.08 0.58 22.07 1.44 20
4 0.49 0.08 0.60 21.40 1.44 18
Mantel\'s r is the correlation between matrices of genetic similarity. The partition metric indicates topological similarity of dendrograms, with lower values indicating greater similarity.
:::
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Similarity of partial genome sequencing to complete genome sequencing in estimating genetic relatedness between papillomavirus samples
:::
Gene Mantel\'s Correlation Coefficient Partition Metric
------ ----------------------------------- ------------------
E1 0.97 12
E6 0.92 10
E7 0.88 14
L1 0.96 12
Mantel\'s r is the correlation between matrices of genetic similarity. The partition metric indicates topological similarity of dendrograms, with lower values indicating greater similarity.
:::
| PubMed Central | 2024-06-05T03:55:46.640133 | 2004-7-27 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509235/",
"journal": "BMC Bioinformatics. 2004 Jul 27; 5:102",
"authors": [
{
"first": "Baozhen",
"last": "Qiao"
},
{
"first": "Ronald M",
"last": "Weigel"
}
]
} |
PMC509236 | Background
==========
The high-density oligonucleotide microarray, also known as GeneChip^®^, made by Affymetrix Inc (Santa Clara, CA), has been widely used in both academic institutions and industrial companies, and is considered as the \"standard\" of gene expression microarrays among several platforms. A single GeneChip^®^can hold more than 50,000 probe sets for every gene in human genome. A probe set is a collection of probe pairs that interrogates the same sequence, or set of sequences, and typically contains 11 probe pairs of 25-mer oligonucleotides \[[@B1]-[@B3]\]. Each pair contains the complementary sequence to the gene of interest, the so-called perfect match (PM), and a specificity control, called the Mismatch (MM) \[[@B3]\]. Gene expression level is obtained from the calculation of hybridization intensity to the probe pairs and is referred to as the \"signal\" \[[@B4]-[@B10]\]. The normalization method used in GeneChip software is called scaling and is defined as an adjustment of the average signal value of all arrays to a common value, the target signal value in order to make the data from multiple arrays comparable \[[@B4],[@B11]\].
The purpose of data normalization is to minimize the effects of experimental and/or technical variations so that meaningful biological comparisons can be made and true biological changes can be found among multiple experiments. Several approaches have been proposed and shown to be effective and beneficial. They were mostly from studies on two-color spotted microarrays \[[@B12]-[@B19]\]. Some authors proposed normalization of the hybridization intensities, while others preferred to normalize the intensity ratios. Some used global, linear methods, while others used local, non-linear methods. Some suggested using the spike-in controls, or house-keeping genes, or invariant genes, while others preferred all the genes on the array. For GeneChip data, some have proposed different models to normalize signal values or normalize probe pair values \[[@B10],[@B20]-[@B24]\]. Despite the presence of other alternatives, many biologists still use the default scaling method and consider that such method is satisfactory and is useful to identify biological alterations \[[@B23],[@B25],[@B26]\]. With the increasing awareness and usage of GeneChip technology and willingness to continue to use GeneChip software among many biologists, it is worth improving the performance or correcting the problems of the software. In this report, the author has demonstrated that in the scaling algorithm excluding 2% of the probe sets with the highest and the lowest values did not have much benefit. However, the logarithmic transformation of signal values prior to scaling proved to be the optimum normalization strategy and is strongly recommended.
Results
=======
The statistical algorithm in current GeneChip software (MAS 5 and GCOS 1) for gene expression microarray data has eliminated the negative gene expression values, a problem present in earlier versions of the software \[[@B5],[@B7]\]. It uses a robust averaging method based on the Tukey biweight function to calculate the gene expression level from the logarithm transformed hybridization data \[[@B3]-[@B5],[@B11]\]. The reported data of a probe set is the antilog of the Tukey biweight mean multiplied by a *SF*and/or a normalization factor (*NF*~*affy*~). When both the *SF*and *NF*~*affy*~are equal to 1, there is no normalization or manipulation of original data. Both *NF*~*affy*~and *SF*are computed in virtually the same way. *NF*~*affy*~is calculated in comparison analysis to compare the array average of one experiment with that of a baseline experiment, while *SF*is obtained from the signal average of one experiment comparing with a common value, the target signal in absolute analysis \[[@B3]-[@B5],[@B11],[@B22]\]. The average value used in GeneChip is a trimmed average. It is not calculated from all probe sets, but from 96% of the probe sets after the 2% of the probe sets with the highest and the 2% of the lowest signals were removed.
In this report, a total of 76 experiments with rat U34A GeneChip were analyzed. As shown in Table [1](#T1){ref-type="table"}, the total hybridization signals varied although all arrays were hybridized with the same amount of biotin-labeled cRNA and scanned with the same scanner of identical settings. The array of the highest hybridization intensities had 2.8 times more signals than that of the lowest. The average array signals had 25.8% variation in terms of coefficient of variation. The mean signals were significantly greater than the median signals on each array, indicating a non-normal distribution. The density plot showed a long-tailed and skewed distribution (not shown) and the average of such data is known to be sensitive to the larger values in the data set.
The rat U34A GeneChip contained 8799 probe sets; hence 2% was about 176 probe sets. The sum of the 2% of the probe sets with the lowest signals accounts for less than 0.1% of the total signals (0.05% ± 0.01%, mean ± SD, n = 76) and its impact on *SF*calculation can be ignored. However, the sum of the 2% of the probe sets with the highest signals, the *TrimTotal*as used in this report, was responsible for about 40% of the total signals (from 34% to 54%, Table [1](#T1){ref-type="table"}). The remaining 96% of the probe sets used for *SF*calculation, produced only about 60% of the signals. Excluding 4% of the probe sets did not reduce the variation, but rather slightly increased the variation, which in turn resulted in a wider range of *SF*s (Table [1](#T1){ref-type="table"}). It was also found that the *TrimTotal*was highly correlated with total signal (R = 0.928), but less with medians (R = 0.536) and the mean of log signals (R = 0.643). The trimmed percentage (*Tp*) was found to be negatively associated with the median (R = 0.558, b = -1.116) and the mean of log signals (R = 0.495, b = -0.968), but not with the total signal of all probe sets.
Among other approaches to global linear normalization, one can also use the median signal or the mean of logarithm transformed signals to calculate the NF. *NFLogMean*showed a higher correlation with *NFMedian*than with *SF*. There were larger differences between *NFLogMean*and *SF*than those between *NFLogMean*and *NFMedian*(Fig. [1](#F1){ref-type="fig"}). To test if the larger difference was a result of removing 4% of the probe sets from the calculation, another NF, the *NFTrimLogMean*was obtained using the same data as for *SF*, but with a log transformation. There is a very significant correlation between *NFTrimLogMean*and *NFLogMean*(R = 0.9998). The 4% of the probe sets that was removed from *NFTrimLogMean*calculation reduced the total data by only 4% after log transformation.
Since it is impossible to obtain the true normalization factor, an average of the four global linear *NF*s mentioned above was used instead to estimate the \'true\' NF. To compare them with the true NF, a score (*NFscore*) is introduced. Each NF is calculated against the respective \'true\' NF to obtain its *NFscore*. The average *NFscore*(± SD) is 7.01% (± 6.24%), 4.51% (± 3.48%), 2.25%(± 2.33%) and 1.95% (± 1.61%), and the sum of *NFscore*is 5.33, 3.43, 1.71 and 1.48 for *SF*, *NFMedian*, *NFTrimLogMean*and *NFLogMean*, respectively (Fig. [1](#F1){ref-type="fig"}). The sum of *NFscore*indicated an accumulated variation from the true NF, and the larger the number, the larger the accumulated variation. An attempt to add a 5th NF obtained from the arithmetic mean of all probe sets of the array was also made to calculate and compare *NFscore*with each NFs, and the results showed the same conclusion (data not shown). It is fair to conclude that *NFLogMean*produced the least variation.
Discussion
==========
Logarithmic transformation is a well-accepted approach for stabilizing variance and has become a common choice for data transformation and normalization for spotted microarrays \[[@B12],[@B16]\]. Much improvement has been made in GeneChip microarray technology and accompanying software during the past few years. The current version of GeneChip software has improved its performance and is better than the earlier versions that used the Average Difference to express levels of gene expression \[[@B3],[@B4]\]. However, the normalization algorithm was inherited and remains the only and default option for gene expression data processing in both MAS 5 and the newly released GeneChip Operating Software (GCOS) software. They continue to use the arithmetic mean of signals to obtain the *SF*in absolute analysis (single array) and the *NF*in comparison analysis (two arrays) \[[@B3]-[@B5],[@B7],[@B11],[@B22]\]. It is clearly shown here that the trimmed average and the resulting *SF*had a larger variance than the median-based NF, or the NF based on the mean of log transformed signals. Similar results were observed in other GeneChip expression arrays, such as mouse U74A and human U133A (data not shown). Elimination of the highest and the lowest 2% of the probe set signals did not stabilize the trimmed means. When intra-array variance was reduced by 40%, this approach cannot be considered to be optimal. The logarithmic transformation of signals stabilized the variation well and made the normalization process much less dependent upon the mean and less affected by the outliers.
Although simple and popular, the global linear normalization has its drawbacks, especially when the relationship among multiple experiments or genes is not linear. To address such problems, several methods have been proposed to conduct local and non-linear normalization, \[[@B12],[@B14]-[@B17],[@B20],[@B22],[@B27]\]. Data normalization is a very critical and important step for microarray data mining process. The use of different approaches to normalization may have a profound impact on the selection of differentially expressed genes and conclusions about the underlying biological processes especially when subtle biological changes are investigated \[[@B12],[@B16],[@B28]\].
Conclusions
===========
Normalization of microarray data allows direct comparison of gene expression levels among experiments. A global linear normalization, called scaling has been widely used in GeneChip microarray technology for gene expression analysis. The scaling factor (*SF*) is calculated from a trimmed average of gene expression level after excluding the 2% of the data points of the highest values and the lowest values. It is shown here that the 2% of the probe sets of the highest signals contained from 34% to 54% of the total signals. Elimination of the outliers did not reduce, but increased the variation among multiple arrays. Instead, normalization factors obtained from the mean of the log transformed signals had the best performance. Thus, the current scaling method, although widely used, is not optimal and needs further improvement. The mean of logarithm transformed signals is highly recommended to use for normalization factor calculation.
Methods
=======
GeneChip experiments and data
-----------------------------
Total RNA was isolated from rat tissues or cells in Trizol reagent and purified with Qiagen Rneasy kit. cDNA was synthesized in presence of oligo(dT)24-T4 (Genset Corp, La Jolla, CA) and biotinlated UTP and CTP were used to generate biotin labeled cRNA according to the recommended protocols \[[@B29]\]. Rat genome microarray, U34A GeneChip (Affymetrix Inc., Santa Clara, CA) was used and hybridized with 15 μg of gel-verified fragmented cRNA. Hybridization intensity was scanned in GeneArray 2500 scanner (Agilent, Palo Alto, CA) with Microarray Suite (MAS) 5.0 software \[[@B4]\]. Data from a total of 76 independent GeneChip experiments were used in this study.
Normalization factor (NF)
-------------------------
Gene expression data exported from MAS 5.0 were submitted to a Perl script to calculate different normalization factors. In the scaling approach, a trimmed average signal is calculated after excluding 2% probe sets with the highest signals and 2% with the lowest signal values. The scaling factor (*SF*) is obtained using equation (1) in comparison with a chosen fixed number, called the target signal (*TS*) and is verified with the results from MAS 5.0 of the same settings \[[@B3],[@B4],[@B11]\].
*SF*~*j*~= *TS*/ *S*~*TrimMeanj*~ (1)
Other normalization factors for comparison were obtained by the following:
*NFMedian*~*j*~= *TS*/ *S*~med*j*~ (2)
*NFLogMean*~*j*~= 2 ^*nf*^~*j*~
where *i*= 1\..., n represents the probe sets, *j*= 1\..., J represented the array experiments, *Si*is the signal of the anti-log of a robust average (Tukey biweight) of log(PM-MM) reported from MAS 5.0 \[[@B5]\], *S*~med*j*~is the median signal on the array *j*, *S*~*TrimMeanj*~is the trimmed average on array *j*after excluding 2% of the probe sets with the highest and the lowest signals \[[@B3],[@B4],[@B11],[@B22]\]. *NFMedian*~*j*~is obtained by using the median signal on array *j*, and *NFLogMean*~*j*~is obtained by using the mean of log transformed signals. *TS*was set to 150, 38 and 38 for *SF*, *NFMedian*and *NFLogMean*, respectively in order to have similar NFs.
In comparison with different NFs, a score, *NFscore*is introduced. *NFscore*~*j*~= (*NF*~*j*~- *TrueNF*~*j*~)/*TrueNF*~*j*~, and *TrueNF*~*j*~= (*SF*~*j*~+ *NFMedian*~*j*~+ *NFLogMean*~*j*~+ *NFTrimLogMean*~*j*~)/4, where *NFTrimLogMean*~*j*~, was calculated from equation (3) excluding the 2% of the probe sets with the highest and lowest signals, *TrueNF*~*j*~was used as a \'true\' NF. Sum of .
Other analysis
--------------
Unless otherwise specified, logarithm transformation is carried out with the logarithm base 2. Trimmed total signal *TrimTotal*is the sum of the signals from the 2% of the probe sets with the highest signal values. Total signal *Total*is the sum of the signals of all probe sets in the array, and trimmed percentage *Tp*~*j*~= (*TrimTotal*~*j*~/ *Total*~*j*~) × 100%.
Abbreviations
=============
GeneChip^®^is the registered trademark owned by Affymetrix Inc.
PM: perfect Match; MM: mismatch; SF: scaling factor; NF: normalization factor; TS: target signal Short phrase: Normalization of GeneChip microarray data
Acknowledgements
================
I would like to acknowledge the support from Dr. H. D. Lipshitz, Dr. S. Scherer, The Centre for Applied Genomics, and The Hospital for Sick Children. The excellent technical work by Lan He is highly appreciated. I would also like to thank Drs. P. Liu, M. Post, K. Tanswell, G. Fantus and S. Keshavjee for sharing their U34A data. Review and comments from Drs. C. Greenwood, J. Beyene, C.E. M\'lan and P. McLoughlin are highly appreciated. Finally, suggestions to improve this paper from the editor and referees are deeply appreciated.
Figures and Tables
==================
::: {#F1 .fig}
Figure 1
::: {.caption}
######
\(A) Comparison among different normalization factors. *NFLogMean*(x-axis) is plotted against *SF*(red open triangle) and *NFMedian*(black closed circle). The correlation between *NFLogMean*and *NFMedian*is higher (R = 0.971) than that between *NFLogMean*and *SF*(R = 0.918). (B) The NF score, *NFscore*, for *SF*(red open triangle), *NFMedian*(blue open diamond) and *NFLogMean*(black closed circle) is expressed as a function of respective \'*true NF*\'. *NFTrimLogMean*is not shown here to simplify the graph since it is similar to *NFLogMean*. See also in*Methods*.
:::
:::
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Summary of signal data in 76 rat genome U34A GeneChip microarrays.
:::
Lowest Highest Mean SD CV (%)
---------------------------- ----------- ------------- ------------- ----------- --------
Total signal 832,561.4 3,161,392.7 2,039,655.7 526,295.0 25.80%
Sum of signals used for SF 524,513.7 1,986,236.9 1,212,296.5 336,138.0 27.73%
Trimmed total 308,047.7 1,240,257.3 827,359.1 215,325.1 26.03%
Mean signal 94.6 359.3 231.0 59.8 25.80%
Median of signals 17.8 54.8 35.7 8.7 24.41%
Mean of log signals 4.3 5.8 5.1 0.4 7.17%
Trimmed percentage 34.4 54.1 40.7 4.4 10.70%
\"Total signal\" is the sum of all the signals on each array. \"Sum of signals used for *SF*\" is the sum of signals excluding the trimmed data and used to calculate *SF*. \"Trimmed total\" is the sum of the 2% probe sets with the highest signals on the array. \"Mean of log signals\" is the mean of log~2~transformed signals. \"Trimmed percentage\" = (Trimmed total/Total signal) × 100%. See also in *Methods*. The \"lowest\" and \"highest\" showed the lowest and highest number in the category among the 76 chips, respectively. The mean, standard deviation (SD) and coefficient of variation (CV) were also calculated.
:::
| PubMed Central | 2024-06-05T03:55:46.644088 | 2004-7-29 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509236/",
"journal": "BMC Bioinformatics. 2004 Jul 29; 5:103",
"authors": [
{
"first": "Chao",
"last": "Lu"
}
]
} |
PMC509237 | Background
==========
Spotted cDNA microarrays are used in high-throughput experiments that interrogate the relative expression of thousands of genes simultaneously for many biological processes with wide applications in biological and medical research. Typically in a two-dye spotted cDNA microarray experiment, two mRNA samples are transcribed into cDNAs, labeled with two different fluorescent dyes, commonly Cy3 and Cy5, and hybridized on the same slide. The relative gene expression level is then measured as a ratio of the intensities of the fluorescent dyes. However, the signal intensity of the dye, which indirectly represents the gene expression level, can be affected by many other sources of error such as dye efficiency, sample preparation, and the variability of the biological samples \[[@B1],[@B2]\].
An important question is how to identify differentially expressed genes, some of which change only minimally (\<2-fold), given many known and potentially unknown sources of variance in the microarray experiment. In order to reduce false positive rates, many published experiments use a cut-off of 2- to 3-fold \[[@B3]-[@B5]\]. This limits the ability of the microarray experiment to detect small, but biologically important changes. In fact, recent reports have shown that microarrays can significantly underestimate gene expression changes and therefore a high cut-off will miss important changes \[[@B6]\]. Although more sophisticated statistical methods have been proposed for single slide analysis \[[@B7]-[@B13]\], it is becoming clear that in order to reduce random variance, replication becomes more and more important in microarray experimental design by greatly increasing the power of the experiment to measure small gene expression changes \[[@B2],[@B13]-[@B17]\]. As a relatively new technique, many new theories have been developed for data analysis and experimental design, but few of these theories have been rigorously tested against a well-established standard method such as the Northern blot.
In this paper we compared two experimental design and analysis methods performed on quadruplicate arrays that include a dye-swap design \[[@B18],[@B19]\] and a modified reference design method that uses a control-control hybridization to correct for systematic experimental errors, that we refer to as the \"control correction method\" (CCM). We demonstrate that both experimental designs accurately identified small (\<2-fold) gene expression changes after a 24-hour treatment of MCF10AT breast epithelial cells with the growth and differentiation factor neuregulin. These changes correlate well with the anti-proliferative effects of neuregulin resulting in a relative decrease in proliferative genes and increase in anti-proliferative genes that will be important for future investigations.
Results
=======
The results presented in this paper demonstrate two, complementary cDNA microarray methods capable of reliably revealing small changes in gene expression in transformed human breast epithelial MCF10AT cells after treatment with neuregulin. Since, as shown in Fig. [1](#F1){ref-type="fig"}, treatment of these cells with neuregulin significantly slows their growth rate, identifying early gene expression changes in this process will be important in understanding how neuregulin regulates cell growth in both normal and malignant breast epithelium, and will also provide both biological markers and potential targets in breast cancer. Large quantities of highly purified total RNA were isolated from MCF10AT cells treated with or without neuregulin for 24 hours and used both for microarray experiments and northern blot confirmation studies.
Experimental designs to address systematic errors
-------------------------------------------------
As with most experimental methods, replicate measurements can reduce random errors. Equally important are systematic errors. Systematic errors result from a constant tendency to over- and under-estimate true values and cannot be eliminated by replicate analysis, since they are often highly reproducible. An example of such a systematic error is a gene-specific dye effect, also called \"dye--gene\" interaction \[[@B18]\], and is shown in Fig. [2A](#F2){ref-type="fig"}. For a given gene spotted in duplicate (arrows), the red signal labeling the treated sample (T) is much brighter than the green signal for the control sample (C). This was highly reproducible for both spots on the same array and between multiple arrays. One way to determine whether the apparent up-regulation of this gene is true, is to use the same control sample labeled with both red and green dyes and perform a control/control (C/C) hybridization. Fig. [2A](#F2){ref-type="fig"} shows that the same intense red signal is seen in the C/C hybridization as was seen in the treated/control (T/C), demonstrating that this signal is a systematic error producing a false positive gene expression change.
Given the unavoidable presence of these systematic errors, methods to correct these errors are needed. One way to correct for systematic errors in microarray experiments is to take advantage of C/C hybridizations to correct the T/C hybridizations. This requires a modified reference design, which we refer to as a \"control correction\" design. This is different from a common reference design used previously \[[@B19],[@B20]\]. Here, each spot of the T/C hybridization is \"corrected\" by the same spot from the C/C hybridization for systematic errors. A second method that will also correct for systematic errors is a \"dye-swap\" design \[[@B16],[@B17],[@B19]\]. The dye-swap design uses an ANOVA to calculate gene expression changes from replicate cDNA microarrays probed with T/C hybridizations performed where the dye color is swapped. Included in the ANOVA are factors to correct for systematic errors such as dye and dye-gene interactions. The \"control correction\" and the dye-swap designs are compared in Fig. [2B](#F2){ref-type="fig"}. Each of these experimental designs was performed on quadruplicate arrays. Each of these two designs required its own analysis method. While we used an analysis method that utilizes individual t-tests for each spot for the CCM, we compared both ANOVA and Bayesian analysis methods for the dye-swap design.
Control correction method experimental design and results
---------------------------------------------------------
A flow chart for the control correction method is shown in Fig. [2C](#F2){ref-type="fig"}. All microarrays used in this study were from the same lot of 3333 gene spotted cDNA slides (similar to the commercially available NEN MicroMax 2400 slides with 933 additional genes (Alphagene Inc., Woburn, MA) where each gene was spotted in duplicate, and hybridized using an optimized, two-step hybridization protocol with either Cy3 or Cy5-labeled dendrimer complexes (Genisphere, Hatfield, PA). A key advantage of the Genisphere Dendrimer system is the need for only 3 μg of total RNA per array without the need for a potentially non-linear amplification step to boost the signal. After scanning and spot-wise local background correction (Imagene Software, Biodiscovery, CA), a log Cy5/Cy3 ratio versus log signal intensity MA plot was prepared and shown in Fig [3A](#F3){ref-type="fig"}\[[@B20]\]. Without any correction, the ratio vs. intensity plot shows a banana shape as ratios trend downward in the low intensity range. This suggests an intensity-dependent dye effect. In order to correct this and to normalize data sets between different slides, an intensity-dependent normalization procedure was performed that fits the data to a lowess curve as a function of signal intensity \[[@B21]\]. After normalization, the log ratios became more evenly distributed around zero (Fig. [3B](#F3){ref-type="fig"}).
However, despite this relatively even distribution, histograms of normalized log ratios for T/C and C/C display long tails to the left as shown in the histograms in Fig. [4A](#F4){ref-type="fig"} and the quantile-qauntile plots in Fig. [4B](#F4){ref-type="fig"}. Since there should be no treatment effects on the C/C slides, a symmetric, normal distribution would have been expected. The skewed appearances of the normalized distributions indicate additional, uncorrected systematic errors in both T/C and C/C hybridizations. \"Correction\" of each spot by subtracting the log (C/C) ratios from the log (T/C) ratios produces an approximately normal distribution of the log (T/C) ratios (shown on the bottom of Figs. [4A](#F4){ref-type="fig"} and [4B](#F4){ref-type="fig"}). In addition to the systematic errors that occur on a spot-by-spot basis shown in Fig. [2A](#F2){ref-type="fig"}, systematic errors were found as a function of slide location, particularly at the edge of the arrays. These errors were also corrected by this method (data not shown). Yang and Dudoit proposed a within slide normalization for this type of spatial effect \[[@B21]\], however, one concern for within slide normalization is that if the number of genes is small in each spatial group, the assumption that there will be an equal proportion of up- and down-regulated genes may be untrue.
As a final step, a t-test was performed to compare the normalized log ratios of T/C and C/C for each gene. This yields p values for each control-corrected fold change calculated as log (T/C)-log (C/C). In Fig. [5](#F5){ref-type="fig"}, the average and standard deviation of gene expression ratios for the log (T/C) and log (C/C) are plotted for the genes using 1.7-fold and p \< 0.05 cut-offs. This clearly demonstrates the importance of correcting each log (T/C) value with the corresponding log (C/C) control value. For example, while some log (T/C) ratios are close to zero, by using the log (C/C) as baseline, true gene expression changes above or below this were identified that would otherwise have been missed. The 1.7-fold cutoff was chosen to be within the detection range of northern blot analyses, which we felt would be the most sensitive method to confirm these small changes. A volcano plot, shown in Fig. [6A](#F6){ref-type="fig"}, summaries 46 differentially-regulated genes that met these criteria for the CCM.
Comparison of the control correction to the dye-swap design
-----------------------------------------------------------
Many have proposed that a dye-swap experimental design combined with an ANOVA will correct for systematic errors \[[@B17]-[@B19]\]. To verify this and compare the dye-swap design to the control correction design, a dye-swap experiment was performed on quadruplicate arrays using the same RNA samples and the two interconnect ANOVA model of Wolfinger et al \[[@B22],[@B23]\]. Using this experimental design with the same cut-off values, 14 differentially expressed genes were identified and are presented as a volcano plot alongside that of the CCM (Fig. [6B](#F6){ref-type="fig"}).
Table [1](#T1){ref-type="table"} lists those genes that met our selection criteria, together with their fold-change, p values, and functional classifications. Only 5 genes were found in common for both methods. The genes have been broadly grouped into proliferation, differentiation, and unclassified genes in order to observe trends in the neuregulin-induced gene expression changes that could be important in regulating cell growth. A general trend showing a down-regulation of proliferation genes and up-regulation of differentiation genes was observed. This includes several oncogenes, cell cycle control and cell proliferation genes that were all down-regulated; and tumor suppressor genes, growth inhibition and differentiation genes were up-regulated. This pattern is consistent with the anti-proliferative/differentiation effects of neuregulin on MCF10AT human breast epithelial cells.
Verification of microarray accuracy by northern blot analysis
-------------------------------------------------------------
To confirm these gene expression changes and to determine the accuracy of each experimental method, we selected 23 genes for verification by northern blot. We chose all 5 genes detected by both methods, 6 up-regulated and 5 down-regulated genes from the control correction design, and 7 genes from the dye-swap experiment. The selection of genes was not random, as we selected a balanced complement of genes of variable intensity that were both up- and down-regulated. The probes used for northern blots were generated by PCR from clones used to spot the arrays. Each blot contained triplicate control and treated samples and was re-probed multiple times. Fig. [7](#F7){ref-type="fig"} summarizes the northern blot results for these 23 genes. The band intensities were quantified, normalized to total ribosomal RNA for each gel, and averaged to produce a fold change that was compared directly to the fold change from the microarrays. In general, differential gene expression was confirmed by the northern blots for both array design methods. For the dye-swap method only 1 of 12 genes was a false positive, while 4 out of 16 genes were false positives in the control correction method. Down-regulated genes were verified more reliably in the control correction method (10/10) than up-regulated genes (2/6). All differentially expressed genes common to both methods were confirmed..
Since the ANOVA method we used can sometimes underestimate the variance, we re-analyzed our dye-swap data with a Bayesian method using a regularized t-test as implemented in Cyber-T \[[@B24]\]. This analysis revealed 16 differentially expressed genes using the same cut-offs, 10 of which were in common with the ANOVA method (Table [1](#T1){ref-type="table"}). A greater number of genes were identified using the regularized t-test, and the corresponding p values for these genes were lower. Based on the previous northern blot data, 8/9 (89%) of these were confirmed.
Discussion
==========
Gene expression changes in MCF10AT cells suggest a rapid anti-proliferative effect of neuregulin
------------------------------------------------------------------------------------------------
MCF10AT cells are a human breast epithelial cell line stably transfected with a mutant ras oncogene. These cells are pre-malignant, but can progress to invasive carcinoma \[[@B25],[@B26]\]. Given that neuregulin can differentially affect the growth properties of different cell lines, we used the MCF10AT cell line as model system to identify genes that may be down-stream from neuregulin activation and could thus be studied further for their roles in breast cancer cells that respond differentially to neuregulin. Combining two cDNA microarray experimental design methods, we have identified genes differentially expressed by neuregulin treatment that correlated with a significant decrease in their growth rate. The pattern of expression clearly shows an anti-proliferative effect of neuregulin on the MCF10AT cells with a reduction in genes associated with proliferation such as heat shock proteins, oncogenes, cell cycle control genes, genes involved in fatty acid and sugar synthesis, transcription and translation together with an increase in differentiation genes including tumor suppressor genes, DNA damage repair genes, growth inhibition genes and differentiation genes. We further showed that these effects are biologically consistent with the rapid, anti-proliferative effects of neuregulin on cell number. Additional experiments have shown that these genes are important biological markers for the degree of malignancy in other breast epithelial cell lines that have differential proliferation responses to neuregulin (Li Q, Ahmed S, and Loeb JA, unpublished results).
Both experimental designs demonstrate a high confirmation rate for small changes in gene expression
---------------------------------------------------------------------------------------------------
One of the important tasks in microarray technology is to design experiments and develop statistical tools to obtain data efficiently and accurately to answer fundamental questions in biology. In many experiments, this requires the ability to detect small changes in gene expression with high fidelity. In this study we compared two common experimental design paradigms for cDNA microarrays and determined their accuracy by northern blot. Both methods identified small expression changes with considerable accuracy. In the control correction design, we used control hybridizations to correct for systematic errors on a spot-by-spot basis. The method is based on an assumption that systematic errors from slides made from the same lot and processed identically do not vary significantly. To minimize the possible variance of systematic errors in T/C slides and C/C slides we maintained strict experimental conditions, such as same-day sample preparation and same-day hybridization. We also used the same control samples for both the T/C and C/C hybridizations instead of using an arbitrary control sample that might be quite different in mRNA composition \[[@B19]\]. This results in similar spot intensities for each gene both in the treatment and the control and will minimize any differences that could be caused by the different mRNA compositions from different samples. This spot-by-spot control correction can eliminate systematic errors that cannot be corrected with slide-wise normalization. Similarly, in the dye-swap design, two different dyes are used to label the same sample, which enables the correction of dye-gene interactions in the ANOVA model.
A summary of the results from this study are shown in the Venn diagrams in Fig. [8](#F8){ref-type="fig"}. Using the 1.7-fold and p \< 0.05 cut-offs, the overall verification rate was 75% for the CCM and 92% for the dye-swap method using ANOVA. Among the 18 confirmed expression changes, all were below 3-fold and only six were above 2-fold. Many of the expression changes below 2-fold on the microarrays underestimated the fold-change measured by northern blotting. The accuracy was not dependent on microarray spot intensity as genes with both low and high signal intensities had similar verification rates (data not shown). The confirmation rates for both methods are comparable to methods reported by Mutch (87.5%) \[[@B27]\] and Tusher (92%) \[[@B28]\]. Of particular importance in this study is our high confirmation rates for genes differentially expressed by 2-fold or less.
The t-test used for the CCM and ANOVA for the dye-swap method depend on assumptions of Gaussian distributions that may or may not be present in a microarray experiment with a small number of replicates. Some efforts have been made to develop Bayesian frameworks that incorporate prior distributions in order to estimate the noise \[[@B24],[@B29],[@B30]\]. We therefore re-analyzed our dye-swap data using a \"regularized\" t-test \[[@B24]\]. Using this, we identified 16 genes that met our cut-off criteria, 10 of which were in common with the ANOVA analysis. Of those genes that we measured by northern blot analysis, 8/9 or 89% were verified. In summary, the regularized t-test revealed more genes than the ANOVA method with generally lower p values. If we eliminate the 1.7-fold cut-off, but maintain the p value \<0.05, the CCM identified 493 genes, the ANOVA identified 499 genes, and the regularized t-test identified 729 differentially expressed genes (Fig. [8B](#F8){ref-type="fig"}). Among these, 399 were in common between the regularized t-test and ANOVA, 248 in common between the CCM and the regularized t-test, and 188 in common between the CCM and the ANOVA. These results demonstrate that if the false-positive rate remains the same, the regularized t-test is more sensitive than the traditional ANOVA and has extensive overlap, while the CCM has the least overlap between the other methods, but identifies different genes with slightly less specificity.
In our analysis, we selected genes based on their p values obtained from replicates of individual spots and did not adjust these p-values for multiple comparisons. This may be a major cause for the higher false positive rates for both of our experimental designs. For the CCM, if we apply Bonferroni correction, while we can eliminate all false positives, we would also miss a majority of the differentially expressed genes verified by Northern blotting. Therefore, if accuracy is the main purpose of a study, multiple comparison corrections should be used, while if sensitivity is the main purpose, then it should not be used with the understanding that the accuracy will be lower.
Comparison of a dye-swap versus a control correction method experimental design
-------------------------------------------------------------------------------
For our experimental design, the dye-swap method had a higher confirmation rate than the control correction method. This is, in part, due to the smaller variance that results from an effective doubling of the number of treated samples in the dye-swap method compared to the control correction method. Despite the higher degree of accuracy, the dye-swap design identified fewer genes and only detected down-regulated genes, whereas the control correction identified 3-times the number of genes that were both up- and down-regulated. However the control correction method was less specific for up-regulated genes. These differences may not solely reflect methodological differences, but likely result from experimental variability produced by performing the experiments independently on different days. Nonetheless, the results presented here suggest that both methods have clear merit in their abilities to show true gene expression changes, particularly for expression changes of 2-fold or less, and for genes with low signal intensities and/or low abundance.
The final decision as to which method is preferred depends on the experimental design. For example, the amount of sample and number of replicates required are important considerations both in terms of how difficult the RNA is to obtain and the number of samples that need to be compared. This also translates into the cost to perform the experiment. For instance, the dye-swap method generates a larger sample size for the same number of slides, thus producing greater significance when comparing gene expression between two samples. However this method requires a minimum of two slides and two different labeling reactions per sample. If the amount of sample is limited or population level replication is more desirable than individual sample replication, the control correction is more efficient since individual replicates for reverse dye labeling are not required and each sample can be run with only one slide. For example, to compare 6 treatment samples with a single control sample would require a minimum of 12 microarrays using the dye-swap method, whereas the minimum number of 8 arrays is possible using the control correction method; 6 for treatment samples and 2 for controls.
Another common experimental design used for time course or dose response studies is the reference design. In fact, the control correction method described here is essentially a modified reference design method where the zero time or dose point is the control-control comparison. As discussed above, using a very similar control sample to correct the series will give less false positives and negatives and a more accurate absolute value of the observed change than a dissimilar, pooled reference sample.
Under-estimation of fold changes by cDNA microarrrays
-----------------------------------------------------
Although our cDNA microarray results were accurate, the measured changes generally underestimated the actual changes measured by northern blots. Yuen et al. \[[@B6]\] similarly found that both oligonucleotide arrays (GeneChips by Affymetrix) and cDNA arrays underestimate fold changes compared to quantitative RT-PCR. The cause for this underestimation is not clear, however, it may be due to the limited dynamic range of dye signal or non-specific binding of the dye. Nonetheless, the limitations in accuracy and fold change estimation are far outweighed by the ability of microarrays to identify biologically important gene expression changes.
Conclusions
===========
This study demonstrated that dye-swap and control correction experimental design paradigms for cDNA microarray experiments are capable of detecting small, biologically important changes in gene expression with excellent fidelity while revealing important down-stream anti-proliferative effects of neuregulin on breast epithelial cells for future studies.
Methods
=======
cDNA microarrays
----------------
Human cDNA glass microarrays, called the Alphamax Genechip, were obtained from Alphagene Inc. (Woburn, MA) containing 3333 cDNAs spotted in duplicate. The cDNAs used are identical to commercially available Micromax 2400 slides from Perkin Elmer Life Sciences (Boston, MA), most of which were derived from a human fetal brain cDNA library, with an additional 933 genes (gene list available upon request).
MCF10AT cell culture -- MCF10AT cells were from Dr. Robert Pauley at the Karmanos Cancer Institute (Detroit, MI). The cells were cultured in DMEM/F12 media (Invitrogen) supplemented with 5% horse serum (Invitrogen), 10 mM HEPES buffer (Invitrogen), 10 μg/ml insulin (Sigma), 20 ng/ml EGF (Upstate Biotechnology), 100 ng/ml cholera enterotoxin (CalBiochem) and 0.5 μg/ml hydrocortisone (Sigma) at 37°C in 5% CO~2~incubator.
Neuregulin treatment and RNA extraction -- A recombinant human NRG β1 polypeptide (amino acids 14--246) was generously provided by AMGEN (Thousand Oaks, CA). After 3 days of culture, MCF10AT cells were treated with human recombinant neuregulin β1 form for 24 hours. MCF10AT cells grown under similar conditions without neuregulin treatment were used as a control. The cells were then harvested and total RNA was extracted using Ultraspec (Biotecx laboratories). The total RNA was cleaned up by Rneasy kit (Qiagen) and quantified using a fluorescent dye binding assay, Ribogreen (Molecular Probes). RNA purity was assessed by agarose gel electrophoresis. Proliferation assays were performed by counting quadruplicate cultures plated at 5000 cells/well using a hemocytometer.
Microarray hybridization -- cDNA microarrays were used in a 2-step hybridization protocol that was optimized for the Genisphere dendrimer labeling method. Total RNA was reverse transcribed into cDNA containing a unique 5\' primer tag, using the Genisphere 3DNA expression array detection kit. In brief, for each reaction, 3 μg of total RNA was reverse transcribed using 0.2 μM oligo-dT-Genisphere capture primer, 0.5 mM dNTP, 200 U Superscript II (Invitrogen) in 1X first strand Superscript II buffer at 42°C for 2 h. The RNA from the DNA/RNA hybrids was denatured with 0.5 M NaOH / 50 mM EDTA at 65°C for 10 min. The reaction was neutralized using 1 M Tris-HCl ph 7.5. The contents of the tube containing the NRG-treated and control cDNA were then mixed together and 3 μl linear acrylamide (Ambion) and 250 μl of 3 M ammonium acetate were added to them. cDNA was precipitated by adding 100% Ethanol and incubating at -20°C for 30 min. The cDNA was collected in a pellet by centrifugation at 13000 rpm for 15 min in a microcentrifuge. and resuspended in Alternate (formamide-containing) Hybridization buffer (Genisphere) at 65°C for 10 min and modified LNA blocker (Genisphere) with denatured Cot 1 DNA.
The entire mixture was added to the pre-hybridized array (Alphamax) for hybridization at 55°C for at 36 hr. A clear increase in signal was obtained with a 36 h hybridization compared to 16 h. After hybridization, the arrays were washed with 2X SSC and 0.2% SDS at 60°C for 15 min, followed by a wash with 2X SSC and another with 0.2X SSC at room temperature. For fluorescence detection, a second hybridization with the dendrimer was optimal. 2.5 μl each of the Cy3 and Cy5 dendrimer in Hybridization Buffer (Vial 6, Genisphere kit) were mixed with denatured Cot1 DNA and differential expander and the mixture was added to the pre-hybridized slides for hybridization at 60°C for 2 hrs. The slides were washed again as described above.
Microarray data analysis method
-------------------------------
### Analysis of CCM experiment
Arrays were scanned with a GenePix 4000 A scanner (Axon Instruments, Inc., Union City, CA). Images were quantified using ImaGene Software (Biodiscovery, Inc. Marina del Rey, CA) that uses a local background subtracted from the signal. Signals not consistently detectable (background corrected signal lower than 2 times of background standard deviation) were eliminated.
We fitted loess curve to the log transformed data using the \"loess\" function in SAS software (SAS Institute Inc., NC) for intensity dependent normalization followed by a t-test to compare T/C with C/C ratio, gene by gene. The t-test was performed on the normalized log ratio with Welch correction for unequal variance. The control corrected fold change was calculated as:
log (fold) = log(T/C)-log(C/C)
### Analysis of dye-swap experiment
For the dye-swap method we performed the same background correction and data filtering for absent genes and log transformations. We then used a two interconnect ANOVA model \[[@B22],[@B23]\] and Mixed Model Analysis of Microarray Data (MANMADA) <http://statgen.ncsu.edu/ggibson/Manual.htm> to identify differentially expressed genes. First we use a normalization model for log-transformed intensity measurements:
*y*~*ij*~= μ + *A*~*i*~+ *D*~*j*~+ *AD*~*ij*~+ ε~*ij*~
Where μ is the sample mean, *A*~*i*~is the effect of *i*th array, *D*~*j*~is the effect of dye cy3 or cy5, *AD*~*ij*~is array dye interaction and ε~*ij*~is random error. The residue from normalization model is then used in following gene model to find treatment effects on each gene:
*r*~*ijkg*~= *A*~*ig*~+ *D*~*jg*~+ *T*~*kg*~
Where *r*~*ijkg*~is the residual of each gene from the normalization model, *T*~*kg*~is the treatment effect (control or treated), and *A*~*ig*~and *D*~*jg*~are the array and dye effects, respectively. The expression change for each gene is thus:
log (fold) = *T*~treated~-*T*~control~
Northern blots
--------------
**5**μg total RNA isolated from MCF10AT cells was run on a 1.3% Agarose/2.2M Formaldehyde gel as described previously \[[@B31]\]. Probes were prepared by PCR from the same clones used to spot the slides provided by Alphagene Inc except for AJ224442, X86779 and U62739, where clones BC011696, BI754516 and BG763631, with of over 99% identity, were used as substitutes. Probes were generated by random priming using PrimiT II kit (Stratagene) radiolabeled probes. The auto-radiographs within the linear range of the film were scanned with a flatbed scanner with transparency adapter and quantified using MetaMorph (Universal Imaging) analysis software as described previously \[[@B32]\]. For time course measurements, the amount of signal normalized for loading with either 18S RNA or GAPDH were plotted together after first setting 100% to the intensity of the control measurement at 48 hours and setting the lowest intensity value to 0%.
Authors\' contributions
=======================
BY analyzed microarray data. SR carried out microarray experiment. QL conducted MCF10AT cell culture and mRNA extraction. SA carried out northern blots experiments. RK provide input on microarray experiments. SD contributed ideas to data analysis. JAL conceived and design the experiment.
Acknowledgements
================
This work was supported by the Ralph C. Wilson, Sr. and Ralph C. Wilson, Jr. Medical Research Foundation (JAL), NINDS (NIH) R01 NS45207 (JAL), and the American Cancer Society 85-003-14 (JAL). SNR was supported by a pre-doctoral fellowship from the Epilepsy Foundation of America. We thank Robert Getts from Genisphere for helpful discussions on optimizing the dendrimer labeling system and Thomas Beaumont for helpful comments on the manuscript.
Figures and Tables
==================
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Anti-proliferative effects of neuregulin on MCF10AT cells. Quadruplicate cultures of MCF10AT cells were treated with and without 1 nM neuregulin 3 days after plating and cell counts were performed demonstrating a significant decrease in their growth 24 and 48 hours after treatment. The p value for 24 hr was 0.0011, and for 48 hr was 1.46E-05.
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
(**A**) Highly reproducible systematic errors from gene-dye interactions. The arrows demonstrate intense red dye labeling for a given gene spotted in duplicate both for the T/C slide where the treated sample is labeled with red dye (Cy5) and control sample is labeled with green dye (Cy3), as well as in the C/C slide where the same sample is labeled both red and green. (**B**) Experimental designs. Two different experimental methods were compared: A dye-swap approach, where the dye color is reversed for T/C hybridizations, and a \"control correction\" design, where T/C and C/C hybridizations are performed without reversing the dyes. T denotes the neuregulin treated cells, while C denotes the untreated, control cells. Each arrows represent a replicate and the tails of the arrows indicate cy5 labeling and the heads indicate cy3 labeling. (**C**) Data processing flow chart for the control correction method.
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:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
Array and intensity-dependent variation can be corrected by normalization based on intensity. (**A**) This is an MA-plot before normalization for one of T/C slides that plots the log intensity ratios against the averaged intensities at both wavelengths: M = log (T/C) and A = 1/2log(T\*C). The majority of the data is less than zero in a \"banana\" or \"comma\" shaped distribution. This demonstrates a systematic, intensity-dependent dye effect, prominent at lower intensities. (**B**) After normalization using the lowess function, the MA-plot shows a more even distribution at all intensities.
:::
:::
::: {#F4 .fig}
Figure 4
::: {.caption}
######
Control correction of each spot markedly improves the distribution of log ratios. (**A**) Histograms show that T/C and C/C log ratio distributions after lowess normalization still have a marked asymmetry with a larger tail towards the left (increased down-regulated genes). The distribution becomes symmetric after subtracting the log (C/C) from the log (T/C). (**B**) Quantile-quantile plots similarly show that the log ratio distribution becomes more normal after correction of each spot with the control ratio.
:::
:::
::: {#F5 .fig}
Figure 5
::: {.caption}
######
The control correction method identifies gene expression changes from spots with variable C/C ratios. The log ratios for each gene are plotted both for the T/C (□) and the corresponding C/C (▲) hybridizations for the 46 genes selected from control correction method. The error bars represent one standard deviation in each direction.
:::
:::
::: {#F6 .fig}
Figure 6
::: {.caption}
######
Both control correction and dye-swap methods reveal statistically significant changes in gene expression. Volcano plots of the control correction method (**A**) and the dye-swap method (**B**) reveal a small proportion of genes that met our arbitrary criteria of having \>1.7 fold changes with p values \<0.05, determined individually for each gene. The horizontal lines on each graph represent p = 0.05. The vertical lines represent 1.7 fold changes, both up- and down-regulated. Genes shown in blue in upper left and right areas were selected for northern blot confirmation.
:::
:::
::: {#F7 .fig}
Figure 7
::: {.caption}
######
Northern blots confirm a majority of gene expression changes for both methods. The far right-hand column shows northern blot results performed in triplicate for genes identified by each microarray design method. For each gene, the fold-change from the microarray together with the average fold-change quantified from the northern blots is shown. To correct for loading differences, in the northern blots each measurement was normalized to the corresponding amount of 18S rRNA measured on each gel. A representative example of the 18S rRNA is shown on the bottom of the figure. Two of the Z74615 northern blot bands were discarded due to contamination. Down-regulated, up-regulated, and false positive genes that were not confirmed by northern blots are indicated.
:::
:::
::: {#F8 .fig}
Figure 8
::: {.caption}
######
Summary of confirmation rates for the two methods. (**A**) A Venn diagram summarizes the number of genes identified by each experimental method using 1.7-fold and p \< 0.05 cut-offs and the verification rate by northern blot. While all 5 genes common to both methods were confirmed, 7 out of 11 genes from control correction method were confirmed, and 6 out of 7 genes from the dye-swap method using the ANOVA were confirmed. 8 out of 9 genes identified with the regularized t-test were confirmed. (B) A Venn diagram summarizes the number of genes identified by each experimental method of p \< 0.05 without a fold change restriction.
:::
:::
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
List of identified genes. Gene accession numbers, gene descriptions, fold-changes, and p-values for genes identified by the dye-swap method with ANOVA and regularized t-test analysis and the control correction method (CCM). Genes are broadly classified into three groups: proliferation-related, differentiation-related and unclassified.
:::
**Proliferation-Related Genes**
------------------------------------- ------------ ----------------------------------------------------------------------------------------------------------------- -------------- ----------------- ------------------- ---------------------- ---------------------- -------------------------
**Classification** **Acc\#** **Gene Description** **CCM fold** **CCM p-value** **Dye-Swap fold** **Dye-Swap p-value** **Regularized fold** **Regularized p-value**
**Heat shock proteins** NM\_006597 heat shock 70 kDa protein 8 -2.8 9.5E-6 -2.9 4.2E-7 -2.9 8.4E-13
M34664 heat shock 60 kDa protein 1 (chaperonin) -1.9 3.9E-6 -1.9 4.8E-10
L15189 heat shock 70 kDa protein 9B (mortalin-2) -2.2 1.4E-3 -1.8 1.8E-6 -1.8 6.5E-6
M22382 Human mitochondrial matrix protein P1 -1.8 1.5E-5 -1.7 2.9E-9
M94859 Human calnexin mRNA, complete cds -1.8 4.1E-3
L27706 Human chaperonin protein (Tcp20) gene complete cds -2.1 3.8E-4
**Transcription and translation** D29677 helicase with zinc finger domain -1.9 3.1E-5 -1.9 2.8E-10
X91257 seryl-tRNA synthetase -1.8 1.2E-3 -1.8 4.5E-8 -1.9 2.0E-11
D30655 eukaryotic translation initiation factor 4A, isoform 2 -1.7 5.5E-5
U76111 Human translation repressor NAT1 mRNA, complete cds -1.7 8.9E-3
M74719 transcription factor 4 -1.9 2.0E-3
X13293 Human mRNA for B-myb gene 1.7 4.2E-9
U00968 Human SREBP-1 mRNA -1.7 8.4E-9
L41490 eukaryotic translation elongation factor 1 alpha 1-like 14 -1.8 1.4E-5 -1.7 5.3E-5 -1.8 5.0E-12
**Fatty acid and sugar metabolism** D16481 Homo sapiens mRNA for mitochondrial 3-ketoacyl-CoA thiolase beta-subunit of trifunctional protein, complete cds -1.8 5.6E-5
Y00711 Human mRNA for lactate dehydrogenase B (LDH-B) -1.8 6.9E-5 -1.7 2.4E-8
D78130 Homo sapiens mRNA for squalene epoxidase, complete cds -1.9 1.2E-5 -1.8 9.5E-7
M37154 Human glutamate dehydrogenase (GDH) mRNA, complete cds -1.8 4.0E-3 -1.7 8.0E-4
U62961 3-oxoacid CoA transferase -2.0 1.2E-4
Y13647 Homo sapiens mRNA for stearoyl-CoA desaturase -1.9 1.5E-6
**GTPase** Y13286 Homo sapiens mRNA for GDP dissociation inhibitor beta -1.8 1.8E-5 -1.7 1.3E-6
X51408 Human mRNA for n-chimaerin 2.1 1.8E-2
**Cell cycle** U47413 Human cyclin G1 mRNA, complete cds -1.7 9.6E-7
AF139897 Homo sapiens BASS1 (BASS1) mRNA, partial cds -1.8 1.8E-3
D00265 Homo sapiens mRNA for cytochrome c, partial cds -2.0 2.0E-2
X68836 H. sapiens mRNA for S-adenosylmethionine synthetase -2.2 8.3E-6
U19251 Homo sapiens neuronal apoptosis inhibitory protein mRNA, complete cds 1.8 1.1E-2
S45630 alpha B-crystallin=Rosenthal fiber component \[human, glioma cell line, mRNA, 691 nt\] 2.1 2.7E-3
X86779 H. sapiens mRNA for FAST kinase 1.7 1.9E-2
**Oncogenes** X03541 Human mRNA of trk oncogene -1.8 3.1E-3
M19722 Human fgr proto-oncogene encoded p55-c-fgr protein, complete cds -1.9 5.0E-3
X77548 H. sapiens cDNA for RFG -2.1 1.5E-3
**Proliferation** D63390 platelet-activating factor acetylhydrolase, isoform Ib, beta subunit 30 kDa -1.8 4.4E-4
U62739 Human branched-chain amino acid aminotransferase (ECA40) mRNA, complete cds 1.7 1.2E-2
L12350 Human thrombospondin 2 (THBS2) mRNA, complete cds 1.9 2.2E-3
X06614 Human mRNA for receptor of retinoic acid 1.7 2.8E-2
Y07921 Human mRNA for serine protease -1.7 1.1E-6
**Differentiation-Related Genes**
**Tumor suppressor** AF042857 Homo sapiens lung cancer antigen NY-LU-12 variant A mRNA, complete cds 2.1 4.4E-3
**Differentiation** M21300 Human small proline rich protein (sprI) mRNA, clone 15B 1.7 2.0E-4
**ECM and vesicle trafficking** M15395 Human leukocyte adhesion protein (LFA-1/Mac-1/p150,95 family) beta subunit mRNA 2.0 2.8E-2
S72869 H4(D10S170) = putative cytoskeletal protein \[human, thyroid, mRNA, 3011 nt\] -1.9 3.2E-2
D21267 SNAP25 synaptosomal-associated protein, 25 kDa 1.9 1.7E-2
U95735 Human thrombospondin 2 (THBS2) mRNA, complete cds -1.8 1.5E-5
X68194 H. sapiens h-Sp1 mRNA -1.7 4.2E-2
Z74615 collagen, type I, alpha 1 2.5 5.3E-3
**Immune-response** U68030 chemokine (C-C motif) receptor 6 1.8 5.8E-3
X04701 Human mRNA for complement component C1r -2.4 3.3E-2
**DNA repair** D79983 ring finger protein 144 1.7 2.3E-2
D29013 Homo sapiens mRNA for DNA polymerase beta, complete cds 1.8 7.3E-3
**Unclassified Genes**
J02854 myosin, light polypeptide 9, regulatory 2.6 4.5E-2
X58141 Human mRNA for erythrocyte adducin alpha subunit 2.1 3.2E-2
AJ224442 Homo sapiens mRNA for putative methyltransferase 1.8 3.4E-3
X06661 Human mRNA for 27-kDa calbindin 2.1 9.0E-3
U24266 aldehyde dehydrogenase 4 family, member A1 1.7 2.5E-3
U62432 Human nicotinic acetylcholine receptor alpha3 subunit precursor, mRNA, complete cds 1.8 7.0E-3
U79259 Human clone 23945 mRNA, complete cds -1.7 4.2E-3
M18533 Homo sapiens dystrophin (DMD) mRNA, complete cds -2.5 1.1E-2
J05401 Human sarcomeric mitochondrial creatine kinase(MtCK) gene, complete cds 2.2 1.5E-2
D13315 Human mRNA for lactoyl glutathione lyase -1.7 4.6E-7
AB001740 Homo sapiens mRNA for p27 1.8 8.6E-4
M15661 Human ribosomal protein mRNA -1.7 1.3E-4
:::
| PubMed Central | 2024-06-05T03:55:46.645953 | 2004-7-23 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509237/",
"journal": "BMC Bioinformatics. 2004 Jul 23; 5:99",
"authors": [
{
"first": "Bin",
"last": "Yao"
},
{
"first": "Sanjay N",
"last": "Rakhade"
},
{
"first": "Qunfang",
"last": "Li"
},
{
"first": "Sharlin",
"last": "Ahmed"
},
{
"first": "Raul",
"last": "Krauss"
},
{
"first": "Sorin",
"last": "Draghici"
},
{
"first": "Jeffrey A",
"last": "Loeb"
}
]
} |
PMC509239 | Background
==========
The construction of genetic linkage maps based on molecular markers has become a routine tool for comparative studies of genome structure and organization and the identification of loci affecting complex traits in different organisms \[[@B1]\]. Statistical methods for linkage analysis and map construction have been well developed in inbred line crosses \[[@B2]\] and implemented in the computer packages MAPMAKER \[[@B3]\], CRI-MAP \[[@B4]\], JOINMAP \[[@B5]\] and MULTIMAP \[[@B6]\]. Increasing efforts have been made to develop robust tools for analyzing marker data in outcrossing organisms \[[@B7]-[@B12]\], in which inbred lines are not available due to the heterozygous nature of these organisms and/or long-generation intervals.
Genetic analyses and statistical methods in outcrossing species are far more complicated than in species that can be selfed to produce inbred lines. There are two reasons for this. First, the number of marker alleles and the segregation pattern of marker genotypes may vary from locus to locus in outcrossing species, whereas an inbred line-initiated segregating population, such as an F~2~or backcross, always has two alleles and a consistent segregation ratio across different markers. Second, linkage phases among different markers are not known *a priori*for outbred parents and, therefore, an algorithm should be developed to characterize a most likely linkage phase for linkage analysis.
To overcome these problems of linkage analysis in outcrossoing species, Grattapaglia and Sederoff \[[@B13]\] proposed a two-way pseudo-testcross mapping stratety in which one parent is heterozygous whereas the other is null for all markers. Using this strategy, two parent-specific linkage maps will be constructed. The limitation of the pseudo-testcross strategy is that it can only make use of a portion of molecular markers. Ritter et al. \[[@B7]\] and Ritter and Salamini \[[@B9]\] proposed statistical methods for estimating the recombination fractions between different segregation types of markers. Using both analytical and simulation approaches, Maliepaard et al. \[[@B10]\] discussed the power and precision of the estimation of the pairwise recombination fractions between markers. Wu et al. \[[@B11]\] formulated a multilocus likelihood approach to simultaneously estimate the linkage and linkage phases of the crossed parents over multiple markers. Ling \[[@B14]\] proposed a three-step analytical procedure for linkage analysis in out-crossing populations, which includes (1) determining the parental haplotypes for all of the markers in a linkage group, (2) estimating the recombination fractions, and (3) choosing a most likely marker order based on optimization analysis. This procedure was used to analyze segregating data in an outcrossing forest tree \[[@B15]\]. Currently, none of these models for linkage analysis in outcrossing species can provide a one-step analysis for the linkage, parental linkage phase and marker order from segregating marker data.
In this article, we construct a unifying likelihood analysis to simultaneously estimate linkage, linkage phases and gene order for a group of markers that display all possible segregation patterns in a full-sib family derived from two outbred parents (see Table [1](#T1){ref-type="table"} of Wu et al. \[[@B11]\]). Our idea here is to integrate all possible linkage phases between a pair of markers in the two parents, each specified by a phase probability, into the framework of a mixture statistical model. In characterizing a most likely linkage phase (or parental diplotype) based on the phase probabilities, the recombination fractions are also estimated using a likelihood approach. This integrative idea is extended to consider gene orders in a multilocus analysis, in which the probabilities of all possible gene orders are estimated and a most likely order is chosen, along with the estimation of the linkage and parental diplotype. We perform extensive simulation studies to investigate the robustness, power and precision of our statistical mapping method incorporating linkage, parental diplotype and gene orders. An example from the published literature is used to validate the application of our method to linkage analysis in outcrossing species.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Estimation from two-point analysis of the recombination fraction ( ± SD) and the parental diplotype probability of parent P () and Q () for five markers in a full-sib family of *n*= 100
:::
Parental diplotype *r*= 0.05 *r*= 0.20
-- -------------------- ----------- ----------- ----- ----- ----------------- ------------ ----------- ----------------- ------------ -----------
\| \| \| \|
*a* *b* *c* *d*
\| \| \| \| 0.530 ± 0.0183 0.2097 ± 0.0328
*a* *b* *a* *b* 0.9960 0.9972 0.9882 0.9878
\| \| \| \| 0.0464 ± 0.0303 0.2103 ± 0.0848
*a* *o* × *o* *a* 1 (0^*b*^) 0(1^*b*^) 1 (0^*b*^) 0(1^*b*^)
\| \| \| \| 0.0463 ± 0.0371 0.1952 ± 0.0777
*a* *b* *b* *b* 1 1/0^*c*^ 1 1/0^*c*^
\| \| \| \| 0.0503 ± 0.0231 0.2002 ± 0.0414
*a* *b* *c* *d* 1 1/0^*c*^ 1 1/0^*c*^
^*a*^Shown is the parental diplotype of each parent for the five markers hypothesized, where the vertical lines denote the two homologous chromosomes. ^*b*^The values in the parentheses present a second possible solution. For any two symmetrical markers (2 and 3), = 1, = 0 and = 0, = 1 give an identical likelihood ratio test statistic (Wu et al. 2002a). Thus, when the two parents have different diplotypes for symmetrical markers, their parental diplotypes cannot be correctly determined from two-point analysis. ^*c*^The parental diplotype of parent *P*~2~cannot be estimated in these two cases because marker 4 is homozygous in this parent. The MLE of *r*is given between two markers under comparison, whereas the MLEs of *p*and *q*given at the second marker.
:::
Two-locus analysis
------------------
### A general framework
Suppose there is a full-sib family of size *n*derived from two outcrossed parents P and Q. Two sets of chromosomes are coded as **1**and **2**for parent P and **3**and **4**for parent Q. Consider two marker loci  and , whose genotypes are denoted as 12/12 and 34/34 for parent P and Q, respectively, where we use / to separate the two markers. When the two parents are crossed, we have four different progeny genotypes at each marker, i.e., 13, 14, 23 and 24, in the full-sib family. Let *r*be the recombination fraction between the two markers.
In general, the genotypes of the two markers for the two parents can be observed in a molecular experiment, but the allelic arrangement of the two markers in the two homologous chromosomes of each parent (i.e., linkage phase) is not known. In the current genetic literatuire, a linear arrangement of nonalleles from different markers on the same chromosomal region is called the *haplotype*. The observable two-marker genotype of parent P is 12/12, but it may be derived from one of two possible combinations of maternally- and paternally-derived haplotypes, i.e., \[11\] \[22\] or \[12\] \[21\], where we use \[\] to define a haplotype. The combination of two haplotypes is called the *diplotype*. Diplotype \[11\] \[22\] (denoted by 1) is generated due to the combination of two-marker haplotypes \[11\] and \[22\], whereas diplotype \[12\] \[21\] (denoted by ) is generated due to the combination of two-marker haplotypes \[12\] and \[21\]. If the probability of forming diplotype \[11\] \[22\] is *p*, then the probability of forming diplotype \[12\] \[21\] is 1 - *p*. The genotype of parent Q and its possible diplotypes \[33\] \[44\] and \[34\] \[43\] can be defined analogously; the formation probabilities of the two diplotypes are *q*and 1 - *q*, respectively.
The cross of the two parents should be one and only one of four possible parental diplotype combinations, i.e., \[11\] \[22\] × \[33\] \[44\]), \[11\] \[22\] × \[34\] \[43\], \[12\] \[21\] × \[33\] \[44\] and \[12\] \[21\] × \[34\] \[43\], expressed as 11, 1, 1 and , with a probability of *pq*, *p*(1 - *q*), (1 - *p*)*q*and (1 - *p*) (1 - *q*), respectively. The estimation of the recombination fraction in the full-sib family should be based on a correct diplotype combination \[[@B10]\]. The four combinations each will generate 16 two-marker progeny genotypes, whose frequencies are expressed, in a 4 × 4 matrix, as
for \[11\] \[22\] × \[33\] \[44\],
for \[11\] \[22\] × \[34\] \[43\],
for \[12\] \[21\] × \[33\] \[44\] and
for \[12\] \[21\] × \[34\] \[43\]. Note that these matrices are expressed in terms of the combinations of the progeny genotypes for two markers  and , respectively.
Let **n**= (*n*~*j*1*j*2~)~4\ ×\ 4~denote the matrix for the observations of progeny where *j*~1~,*j*~2~= 1 for 13, 2 for 14, 3 for 23, or 4 for 34 for the progeny genotypes at these two markers. Under each parental diplotype combination, *n*~*j*1*j*2~follows a multinomial distribution. The likelihoods for the four diplotype combinations are expressed as
where *N*~1~= *n*~11~+ *n*~22~+ *n*~33~+ *n*~44~, *N*~2~= *n*~14~+ *n*~23~+ *n*~32~+ *n*~41~, *N*~3~= *n*~12~+ *n*~21~+ *n*~34~+ *n*~43~, and *N*~4~= *n*~13~+ *n*~31~+ *n*~24~+ *n*~42~. It can be seen that the maximum likeihood estimate (MLE) of r () under the first diplotype combination is equal to one minus  under the fourth combination, and the same relation holds between the second and third diplotype combinations. Although there are identical plug-in likelihood values between the first and fourth combinatins as well as between the second and third combinations, one can still choose an appropriate  from these two pairs because one of them leads to  greater than 0.5. Traditional approaches for estimating the linkage and parental diplotypes are to estimate the recombination fractions and likelihood values under each of the four combinations and choose one legitimate estimate of *r*with a higher likelihood.
In this study, we incorporate the four parental diplotype combinations into the observed data likelihood, expressed as
where **Θ**= (*r*, *p*, *q*) is an unknown parameter vector, which can be estimated by differentiating the likelihood with respect to each unknown parameter, setting the derivatives equal to zero and solving the likelihood equations. This estimation procedure can be implemented with the EM algorithm \[[@B2],[@B11],[@B16]\]. Let **H**be a mixture matrix of the genotype frequencies under the four parental diplotype combinations weighted by the occurring probabilities of the diplotype combinations, expressed as
where
Similar to the expression of the genotype frequencies as a mixture of the four diplotype combinations, the expected number of recombination events contained within each two-marker progeny genotype is the mixture of the four different diplotype combinations, i.e.,
where the expected number of recombination events for each combination are expressed as
Define
The general procedure underlying the {*τ*+ 1}th EM step is given as follows:
**E Step:**At step *τ*, using the matrix **H**based on the current estimate *r*^{*τ*}^, calculate the expected number of recombination events between two markers for each progeny genotype and ,
where *d*~*j*1*j*2~, *h*~*j*1*j*2~, *p*~*j*1*j*2~and *q*~*j*1*j*2~are the (j~1~*j*~2~)th element of matrix **D**, **H**, **P**and **Q**, respectively.
**M Step:**Calculate *r*^{*τ*+1}^using the equation,
The E step and M step among Eqs. (4) -- (7) are repeated until *r*converges to a value with satisfied precision. The converged values are regarded as the MLEs of **Θ**.
### Model for partially informative markers
Unlike an inbred line cross, a full-sib family may have many different marker segregation types. We symbolize observed marker alleles in a full-sib family by *A*~1~, *A*~2~, *A*~3~and *A*~4~, which are codominant to each other but dominant to the null allele, symbolized by *O*. Wu et al. \[[@B11]\] listed a total of 28 segregation types, which are classified into 7 groups based on the amount of information for linkage analysis:
A. Loci that are heterozygous in both parents and segregate in a 1:1:1:1 ratio, involving either four alleles *A*~1~*A*~2~× *A*~3~*A*~4~, three non-null alleles *A*~1~*A*~2~× *A*~1~*A*~3~, three non-null alleles and a null allele *A*~1~*A*~2~× *A*~3~*O*, or two null alleles and two non-null alleles *A*~1~*O*× *A*~2~*O*;
B. Loci that are heterozygous in both parents and segregate in a 1:2:1 ratio, which include three groups:
B~1~. One parent has two different dominant alleles and the other has one dominant allele and one null allele, e.g., *A*~1~*A*~2~× *A*~1~*O*;
B~2~. The reciprocal of B~1~;
B~3~. Both parents have the same genotype of two codominant alleles, i.e., *A*~1~*A*~2~× *A*~1~*A*~2~;
C. Loci that are heterozygous in both parents and segregate in a 3:1 ratio, i.e., *A*~1~*O*× *A*~1~*O*;
D. Loci that are in the testcross configuration between the parents and segregate in a 1:1 ratio, which include two groups:
D~1~. Heterozygous in one parent and homozygous in the other, including three alleles *A*~1~*A*~2~× *A*~3~*A*~3~, two alleles *A*~1~*A*~2~× *A*~1~*A*~1~, *A*~1~*A*~2~× *OO*and *A*~2~*O*× *A*~1~*A*~1~, and one allele (with three null alleles) *A*~1~*O*× *OO*;
D~2~. The reciprocals of D~1~.
The marker group A is regarded as containing *fully informative*markers because of the complete distinction of the four progeny genotypes. The other six groups all contain the *partially informative*markers since some progeny genotype cannot be phenotypically separated from other genotypes. This incomplete distinction leads to the segregation ratios 1:2:1 (B), 3:1 (C) and 1:1 (D). Note that marker group D can be viewed as fully informative if we are only interested in the heterozygous parent.
In the preceding section, we defined a (4 × 4)-matrix **H**for joint *genotype*frequencies between two fully informative markers. But for partially informative markers, only the joint *phenotypes*can be observed and, thus, the joint genotype frequencies, as shown in **H**, will be collapsed according to the same phenotype. Wu et al. \[[@B11]\] designed specific incidence matrices (**I**) relating the genotype frequencies to the phenotype frequencies for different types of markers. Here, we use the notation  for a (*b*~1~× *b*~2~) matrix of the phenotype frequencies between two partially informative markers, where *b*~1~and *b*~2~are the numbers of distinguishable phenotypes for markers  and , respectively. Correspondingly, we have . The EM algorithm can then be developed to estimate the recombination fraction between any two partial informative markers.
**E Step:**At step *τ*, based on the matrix (**DH**)\' derived from the current estimate *r*^{*τ*}^, calculate the expected number of recombination events between the two markers for a given progeny genotype and :
where , ,  and  is the (*j*~1~*j*~2~)th element of matrices (**DH**)\', **H**\', **P**\' and **Q**\', respectively.
**M Step**: Calculate *r*^{*τ*+1}^using the equation,
The E and M steps between Eqs. (8) -- (11) are repeated until the estimate converges to a stable value.
Three-locus analysis
--------------------
### A general framework
Consider three markers in a linkage group that have three possible orders ,  and . Let *o*~1~, *o*~2~and *o*~3~be the corresponding probabilities of occurrence of these orders in the parental genome. Without loss of generality, for a given order, the allelic arrangement of the first marker between the two homologous chromosomes can be fixed for a parent. Thus, the change of the allelic arrangements at the other two markers will lead to 2 × 2 = 4 parental diplotypes. The three-marker genotype of parent P (12/12/12) may have four possible diplotypes, \[111\] \[222\], \[112\] \[221\], \[121\] \[212\] and \[122\] \[211\]. Relative to the fixed allelic arrangement 1\|2\| of the first marker on the two homologous chromosomes **1**and **2**, the probabilities of allelic arragments 1\|2\| and 2\|1\| are denoted as *p*~1~and 1 - *p*~1~for the second marker and as *p*~2~and 1 - *p*~2~for the third marker, respectively. Assuming that allelic arrangements are independent between the second and third marker, the probabilities of these four three-marker diplotypes can be described by *p*~1~*p*~2~, *p*~1~(1 - *p*~2~), (1 - *p*~1~)*p*~2~and (1 - *p*~1~) (1 - *p*~2~), respectively. The four diplotypes of parent Q can also be constructed, whose probabilities are defined as *q*~1~*q*~2~, *q*~1~(1 - *q*~2~), (1 - *q*~1~)*q*~2~and (1 - *q*~1~) (1 - *q*~2~) respectively. Thus, there are 4 × 4 = 16 possible diplotype combinations (whose probabilities are the product of the corresponding diplotype probabilities) when parents P and Q are crossed.
Let *r*~12~denote the recombination fraction between markers  and , with *r*~23~and *r*~13~defined similarly. These recombination fractions are associated with the probabilities with which a crossover occurs between markers  and  and between markers  and . The event that a crossover or no crossover occurs in each interval is denoted by *D*~11~and *D*~00~, respectively, whereas the events that a crossover occurs only in the first interval or in the second interval is denoted by *D*~10~and *D*~01~, respectively. The probabilities of these events are denoted by *d*~00~, *d*~01~, *d*~10~and *d*~11~, respectively, whose sum equals 1. According to the definition of recombination fraction as the probability of a crossover between a pair of loci, we have *r*~12~= *d*~10~+ *d*~11~, *r*~23~= *d*~01~+ *d*~11~and *r*~13~= *d*~01~+ *d*~10~. These relationships have been used by Haldane \[[@B17]\] to derive the map function that converts the recombination fraction to the corresponsding genetic distance.
For a three-point analysis, there are a total of 16 (16 × 4)-matrices for genotype frequencies under a given marker order (), each corresponding to a diplotype combination, denoted by , where  for 1\|2\| or 2 for 2\|1\| denote the two alternative allelic arrangements of the second and third marker, respectively, for parent P, and  for 1\|2\| or 2 for 2\|1\| denote the two alternative allelic arrangements of the second and third marker, respectively, for parent Q. According to Ridout et al. \[[@B18]\] and Wu et al. \[[@B11]\], elements in  are expressed in terms of *d*~00~, *d*~01~, *d*~10~and *d*~11~.
Similarly, there are 16 (16 × 4)-matrices for the expected numbers of crossover that have occurred for *D*~00~, *D*~01~, *D*~10~and *D*~11~for a given marker order, denoted by , ,  and  respectively. In their Table [2](#T2){ref-type="table"}, Wu et al. \[[@B11]\] gave the three-locus genotype frequencies and the number of crossovers on different marker intervals under marker order .
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Estimation from three-point analysis of the recombination fraction ( ± SD) and the parental diplotype probabilities of parent P () and Q () for five markers in a full-sib family of *n*= 100
:::
Parental diplotype
------------------------------- -------------------- ----- --- ----- ----- ----------------- ----------------- -------- -------- ----------------- ----------------- --- -----
Recombination fraction = 0.05
\| \| \| \|
*a* *b* *c* *d*
\| \| \| \| 0.0511 ± 0.0175
*a* *b* *a* *b* 0.1008 ± 0.0298 0.9978 0.9986
\| \| \| \| 0.0578 ± 0.0269 0.0557 ± 0.0312
*a* *o* × *o* *a* 0.9977 0 0.0988 ± 0.0277 1 0
\| \| \| \| 0.0512 ± 0.0307 0.0476 ± 0.0280 1 1/0
*a* *b* *b* *b* 0.0932 ± 0.0301 1 1/0 1 1/0
\| \| \| \| 0.0514 ± 0.0229
*a* *b* *c* *d* 1 1
\| \| \| \|
Recombination fraction = 0.20
\| \| \| \|
*a* *b* *c* *d*
\| \| \| \| 0.2026 ± 0.0348
*a* *b* *a* *b* 0.3282 ± 0.0482 0.9918 0.9916
\| \| \| \| 0.2240 ± 0.0758 0.2408 ± 0.0939
*a* *o* × *o* *a* 0.9944 0 0.3241 ± 0.0488 1 0
\| \| \| \| 0.1927 ± 0.0613 0.1824 ± 0.0614
*a* *b* *b* *b* 0.3161 ± 0.0502 1 1/0 1 1/0
\| \| \| \| 0.2017 ± 0.0393
*a* *b* *c* *d* 1 1
\| \| \| \|
Case 1 denotes the recombination fraction between two adjacent markers, whereas case 2 denotes the recombination fraction between the two markers separated by a third marker. See Table 1 for other explanations.
:::
The joint genotype frequencies of the three markers can be viewed as a mixture of 16 diplotype combinations and three orders, weighted by their occurring probabilities, and is expressed as
Similarly, the expected number of recombination events contained within a progeny genotype is the mixture of the different diplotype and order combinations, expressed as:
Also define
The occurring probabilities of the three marker orders are the mixture of all diplotype combinations, expressed, in matrix notation, as
We implement the EM algorithm to estimate the MLEs of the recombination fractions between the three markers. The general equations formulating the iteration of the {*τ*+ 1}th EM step are given as follows:
**E Step:**As step *τ*, calculate the expected number of recombination events associated with *D*~00~(*α*), *D*~01~(*β*), *D*~10~(*γ*), *D*~11~(*δ*) for the (*j*~1~*j*~2~*j*~3~)th progeny genotype (where *j*~1~, *j*~2~and *j*~3~denote the progeny genotypes of the three individual markers, respectively):
Calculate , , ,  and , (*k*= 1,2,3) using
where *n*~*j*1*j*2*j*3~denote the number of progeny with a particular three-marker genotype, h~*j*1*j*2*j*3~, , , , , *p*~1(*j*1*j*2*j*3)~, *p*~2(*j*1*j*2*j*3)~, *q*~1(*j*1*j*2*j*3)~and *q*~2(*j*1*j*2*j*3)~are the (*j*~1~*j*~2~*j*~3~)th element of matrices **H**, **D**~00~, **D**~01~, **D**~10~, **D**~11~, **P**~1~, **P**~2~, **Q**~1~and **Q**~2~, respectively.
**M Step:**Calculate , ,  and  using the equations,
The E and M steps are repeated among Eqs. (19) -- (32) until *d*~00~, *d*~01~, *d*~10~and *d*~11~converge to values with satisfied precision. From the MLEs of the *g\'s*, the MLEs of recombination fractions *r*~12~, *r*~13~and *r*~23~can be obtained according to the invariance property of the MLEs.
### Model for partial informative markers
Consider three partially informative markers with the numbers of distinguishable pheno-types denoted by *b*~1~, *b*~2~and *b*~3~, respectively. Define  is a (*b*~1~*b*~2~× *b*~3~) matrix of genotype frequencies for three partially informative markers. Similarly, we define , and .
Using the procedure described in Section (2.2), we implement the EM algorithm to estimate the MLEs of the recombination fractions among the three partially informative markers.
### m-point analysis
Three-point analysis considering the dependence of recombination events among different marker intervals can be extended to perform the linkage analysis of an arbitrary number of markers. Suppose there are *m*ordered markers on a linkage group. The joint genotype probabilities of the *m*markers form a (4^*m*-1^× 4)-dimensional matrix. There are 2^*m*-1^× 2^*m*-1^such probability matrices each corresponding to a different parental diplotype combination. The reasonable estimates of the recombination fractions rely upon the characterization of a most likely parental diplotype combination based on the multilocus likelihood values calculated.
The *m*-marker joint genotype probabilities can be expressed as a function of the probability of whether or not there is a crossover occurring between two adjacent markers,  where *l*~1~, *l*~2~, \..., *l*~*m*-1~are the indicator variables denoting the crossover event between markers  and , markers  and , \..., and markers  and , respectively. An indicator is defined as 1 if there is a crossover and 0 otherwise. Because each indicator can be taken as one or zero, there are a total of 2^*m*-1^D\'s.
The occurring probability of interval-specific crossover  can be estimated using the EM algorithm. In the E step, the expected number of interval specific crossovers is calculated (see Eqs. (19) -- (22) for three-point analysis). In the M step, an explicit equation is used to estimate the probability . The MLEs of  are further used to estimate *m*(*m*- 1)/2 recombination fractions between all possible marker pairs. In *m*-point analysis, parental diplotypes and gene orders can be incorporated in the model.
Monte Carlo simulation
----------------------
Simulation studies are performed to investigate the statistical properties of our model for simultaneously estimating linkage, parental diplotype and gene order in a full-sib family derived from two outbred parents. Suppose there are five markers of a known order on a chromosome. These five markers are segregating differently in order, 1:1:1:1, 1:2:1, 3:1, 1:1 and 1:1:1:1. The diplotypes of the two parents for the five markers are given in Table [1](#T1){ref-type="table"} and using these two parents a segregating full-sib family is generated. In order to examine the effects of parameter space on the estimation of linkage, parental diplotype and gene order, the full-sib family is simulated with different degrees of linkage (*r*= 0.05 vs. 0.20) and different sample sizes (*n*= 100 vs. 200).
As expected, the estimation precision of the recombination fraction depends on the marker type, the degree of linkage and sample size. More informative markers, more tightly linked markers and larger sample sizes display greater estimation precision of linkage than less informative markers, less tightly linked markers and smaller sample sizes (Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}). To save space, we do not give the results about the effects of sample size in the tables. Our model can provide an excellent estimation of parental linkage phases, i.e., parental diplotype, in two-point analysis. For example, the MLE of the probability (*p*or *q*) of parental diplotype is close to 1 or 0 (Table [1](#T1){ref-type="table"}), suggesting that we can always accurately estimate parental diplotypes. But for two symmetrical markers (e.g., markers  and  in this example), two sets of MLEs,  = 1,  = 0 and  = 0,  = 1, give an identical likelihood ratio test statistic. Thus, two-point analysis cannot specify parental diplotypes for symmetrical markers even when the two parents have different diplotypes.
The estimation precision of linkage can be increased when a three-point analysis is performed (Table [2](#T2){ref-type="table"}), but this depends on different marker types and different degrees of linkage. Advantage of three-point analysis over two-point analysis is more pronounced for partially than fully informative markers, and for less tightly than more tightly linked markers. For example, the sampling error of the MLE of the recombination fraction (assuming *r*= 0.20) between markers  and  from two-point analysis is 0.0848, whereas this value from a three-point analysis decreases to 0.0758 when combining fully informative marker  but increases to 0.0939 when combining partially informative marker . The three-point analysis can clearly determine the diplotypes of different parents as long as one of the three markers is asymmetrical. In our example, using either asymmetrical marker  or , the diplotypes of the two parents for two symmetrical markers ( and ) can be determined. Our model for three-point analysis can determine a most likely gene order. In the three-point analyses combining markers , markers  and marker , the MLEs of the probabilities of gene order are all almost equal to 1, suggesting that the estimated gene order is consistent with the order hypothesized.
To demonstrate how our linkage analysis model is more advantageous over the existing models for a full-sib family population, we carry out a simulation study for linked dominant markers. In two-point analysis, two different parental diplotype combinations are assumed: (1) \[*aa*\] \[*oo*\] × \[*aa*\] \[*oo*\] (*cis*× *cis*) and (2) \[*ao*\] \[*oa*\] × \[*ao*\] \[*oa*\] (*trans*× *trans*). The MLE of the linkage under combination (2), in which two dominant alleles are in a repulsion phase, is not as precise as that under combination (1), in which two dominant non-alleles are in a coupling phase \[[@B12]\]. For a given data set with unknown linkage phase, the traditional procedure for estimating the recombination fraction is to calculate the likelihood values under all possible linkage phase combinations (i.e., *cis*× *cis*, *cis*× *trans*, *trans*× *cis*and *trans*× *trans*). The combinations, *cis*× *cis*and *trans*× *trans*, have the same likelihood value, with the MLE of one combination being equal to the subtraction of the MLE of the second combination from 1. The same relationship is true for *cis*× *trans*and *trans*× *cis*. A most likely phase combination is chosen corresponding to the largest likelihood and a legitimate MLE of the recombination fraction (*r*≤ 0.5) \[[@B10]\].
For our data set simulated from \[*aa*\] \[*oo*\] × \[*aa*\] \[*oo*\], one can easily select *cis*× *cis*as the best estimation of phase combination because it corresponds to a larger likelihood and a smaller  (Table [3](#T3){ref-type="table"}). Our model incorporating the parental diplotypes can provide comparable estimation precision of the linkage for the data from \[*aa*\] \[*oo*\] × \[*aa*\] \[*oo*\] and precisely determine the parental diplotypes (see the MLEs of *p*and *q*; Table [3](#T3){ref-type="table"}). Our model has great advantage over the traditional model for the data derived from \[*ao*\] \[*oa*\] × \[*ao*\] \[*oa*\]. For this data set, the same likelihood was obtained under all possible four diplotype combinations (Table [3](#T3){ref-type="table"}). In this case, one would select *cis*× *trans*or *trans*× *cis*because these two phase combinations are associated with a lower estimate of *r*. But this estimate of *r*(0.0393) is biased since it is far less than the value of 0.20 hypothesized. Our model gives the same estimation precision of the linkage for the data derived from \[*ao*\] \[*oa*\] × \[*ao*\] \[*oa*\] as obtained when the analysis is based on a correct diplotype combination (Table [3](#T3){ref-type="table"}). Also, our model can precisely determine the parental diplotypes ( =  = 0 ).
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Comparison of the estimation of the linkage and parental diplotype between two dominant markers in a full-sib family of *n*= 100 from the traditional and our model
:::
Traditional model Our model
--------------------------------------- ------------------- ----------------- ----------------- ----------------- -----------------
Data simulated from *cis*× *cis*
Correct diplotype combination Correct Incorrect Incorrect Incorrect
Log-likelihood^*a*^ -46.2 -92.3 -92.3 -46.2
under each diplotype combination 0.1981 ± 0.0446 0.5000 ± 0.0000 0.5000 ± 0.0000 0.8018 ± 0.0446
Estimated diplotype combination Selected
under correct diplotype combination 0.1981 ± 0.0446 0.1982 ± 0.0446
Diplotype probability for parent P () 1.0000 ± 0.0000
Diplotype probability for parent Q () 1.0000 ± 0.0000
Data simulated from *trans*× *trans*
Correct diplotype combination Incorrect Incorrect Incorrect Correct
Log-likelihood^*a*^ -89.6 -89.6 -89.6 -89.6
under each diplotype combination 0.8573 ± 0.1253 0.0393 ± 0.0419 0.0393 ± 0.0419 0.1426 ± 0.1253
Estimated diplotype combination Selected Selected
under correct diplotype combination 0.1426 ± 0.1253 0.1428 ± 0.1253
Diplotype probability for parent P () 0.0000 ± 0.0000
Diplotype probability for parent Q () 0.0000 ± 0.0000
^*a*^The log-likelihood values given here are those from one random simulation for each diplotype combination by the traditional model.
:::
In three-point analysis, we examine the advantage of implementing linkage analysis with gene orders. Three dominant markers are assumed to have two different parental diplotypes combinations: (1) \[*aaa*\] \[*ooo*\] × \[*aaa*\] \[*ooo*\] and (2) \[*aao*\] \[*ooa*\] × \[*aao*\] \[*ooa*\]. The traditional approach is to calculate the likelihood values under three possible gene orders and choose one of a maximum likelihood to estimate the linkage. Under combination (1), a most likely gene order can be well determined and, therefore, the recombination fractions between the three markers well estimated, because the likelihood value of the correct order is always larger than those of incorrect orders (Table [4](#T4){ref-type="table"}). However, under combination (2), the estimates of linkage are not always precise because with a frequency of 20% gene orders are incorrectly determined. The estimates of *r*\'s will largely deviate from their actual values based on a wrong gene order (Table [4](#T4){ref-type="table"}). Our model incorporating gene order can provide the better estimation of linkage than the traditional approach, especially between those markers with dominant alleles being in a repulsion phase. Furthermore, a most likely gene order can be determined from our model at the same time when the linkage is estimated.
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
Comparison of the estimation of the linkage and gene order between three dominant markers in a full-sib family of *n*= 100 from the traditional and our model
:::
MLE Traditional model Our model
---------------------------------------------------------------- ------------------- ----------------- ----------------- -----------------
Data stimulated from \[*aaa*\] \[*ooo*\] × \[*aaa*\] \[*ooo*\]
Correct gene order Correct Incorrect Incorrect
Estimated best gene order (%^*a*^) 100 0 0
0.2047 ± 0.0422 0.2048 ± 0.0422
0.1980 ± 0.0436 0.1985 ± 0.0434
0.3245 ± 0.0619 0.3235 ± 0.0618
0.9860 ± 0.0105
0.0060 ± 0.0071
0.0080 ± 0.0079
Data simulated from \[*aao*\] \[*ooa*\] × \[*aao*\] \[*ooa*\]
Correct gene order Correct Incorrect Incorrect
Estimated best gene order (%^*a*^) 80 11 9
0.1991 ± 0.0456 0.8165 ± 0.1003 0.9284 ± 0.0724 0.2104 ± 0.0447
0.1697 ± 0.0907 0.8220 ± 0.0338 0.1636 ± 0.0608 0.2073 ± 0.0754
0.3218 ± 0.0755 0.2703 ± 0.0586 0.7821 ± 0.0459 0.2944 ± 0.0929
0.9952 ± 0.0058
0.0045 ± 0.0058
0.0003 ± 0.0015
^*a*^The percents of a total of 200 simulations that have a largest likelihood for a given gene order estimated from the traditional approach. In this example used to examine the advantage of implementing gene orders, known linkage phases are assumed.
:::
Our model is further used to perform joint analyses including more than three markers. When the number of markers increases, the number of parameters to be estimated will be exponentially increased. For four-point analysis, the speed of convergence was slow and the accuracy and precision of parameter estimation have been affected for a sample size of 200 (data not shown). According to our simulation experience, the improvement of more-than-three-point analysis can be made possible by increasing sample size or by using the estimates from two- or three-point analysis as initial values.
A worked example
----------------
We use an example from published literature \[[@B18]\] to demonstrate our unifying model for simultaneous estimation of linkage, parental diplotype and gene order. A cross was made between two triple heterozygotes with genotype *AaVvXx*for markers ,  and . Because these three markers are dominant, the cross generates 8 distinguishable genotypes, with observations of 28 for *A*~-~/*V*~-~/*X*~-~, 4 for *A*~-~/*V*~-~/*xx*, 12 for *A*~-~/*vv*/*X*~-~, 3 for *A*~-~/*vv*/*xx*, 1 for *aa*/*V*~-~/*X*~-~, 8 for *aa*/*V*~-~/*xx*, 2 for *aa*/*vv*/*X*~-~and 2 for *aa*/*vv*/*xx*. We first use two-point analysis to estimate the recombination fractions and parental diplotypes between all possible pairs of the three markers. The recombination fraction between markers  and  is , whose the estimated parental diplotypes are \[*Av*\] \[*aV*\] × \[*AV*\] \[*av*\] or \[*AV*\] \[*av*\] × \[*Av*\] \[*aV*\]. The other two recombination fractions and the corresponding parental displotypes are estimated as , \[*Vx*\] \[*vX*\] × \[*VX*\] \[*vx*\] or \[*VX*\] \[*vx*\] × \[*Vx*\] \[*vX*\] and , \[*AX*\] \[*ax*\] × \[*AX*\] \[*ax*\], respectively. From the two-point analysis, one of the two parents have dominant alleles from markers  and  are repulsed with the dominant alleles from marker .
Our subsequent three-point analysis combines parental diplotypes and gene orders to estimate the linkage between these three dominant markers. The estimated gene order is . The MLEs of the recombination fractions are ,  and . The parental diplotype combination is \[*XAV*\] \[*xav*\] × \[*XAv*\] \[*xaV*\] or \[*XAv*\] \[*xaV*\] × \[*XAV*\] \[*xav*\]. The three-point analysis for these three markers by Ridout et al. \[[@B18]\] led to the estimates of the three recombination fractions all equal to 0.20. But their estimates may not be optimal because the effect of gene order on  was not considered.
Discussion
==========
Several statistical methods and software packages have been developed for linkage analysis and map construction in experimental crosses and well-structured pedigrees \[[@B2]-[@B6]\], but these methods need unambiguous linkage phases over a set of markers in a linkage group. For outcrossing species, such as forest trees, it is not possible to know exact linkage phases for any of two parents that are crossed to generate a full-sib family prior to linkage analysis. This uncertainty about linkage phases makes linkage mapping in outcrossing populations much more difficult than that in phase-known pedigrees \[[@B7],[@B9]\].
In this article we present a unifying model for simultaneously estimating the linkage, parental diplotype and gene order in a full-sib family derived from two outbred parents. As demonstrated by simulation studies, our model is robust to different parameter space. Compared to the traditional approaches that calculate the likelihood values separately under all possible linkage phases or orders \[[@B9],[@B10],[@B18]\], our approach is more advantageous in three aspects. First, it provides a one-step analysis of estimating the linkage, parental diplotype and gene order, thus facilitating the implementation of a general method for analyzing any segregating type of markers for outcrossing populations in a package of computer program. For some short-generation-interval outcrossing species, we can obtain marker information from grandparents, parents and progeny. The model presented here allow for the use of marker genotypes of the grandparents to derive the diplotype of the parents. Second, our model for the first time incorporates gene ordering into a unified linkage analysis framework, whereas most earlier studies only emphasized on the characterization of linkage phases through a multilocus likelihood analysis \[[@B11],[@B14],[@B15]\]. Instead of a comparative analysis of different orders, we proposed to determine a most likely gene order by estimating the order probabilities.
Third, and most importantly, our unifying approach can significantly improve the estimation precision of the linkage for dominant markers whose alleles are in repulsion phase. Previous analyses have indicated that the estimate of the linkage between dominant markers in a repulsion phase is biased and imprecise, especially when the linkage is not strong and when sample size is small \[[@B12]\]. There are two reasons for this: (1) the linkage phase cannot be correctly determined, and/or (2) there is a fairly high possibility (20%) of detecting a wrong gene order. Our approach provides more precise estimates of the recombination fraction because correct parental diplotypes and a correct gene order can be determined.
Our approach will be broadly useful in genetic mapping of outcrossing species. In practice, a two-point analysis can first be performed to obtain the pairwise estimates of the recombination fractions and using this pairwise information markers are grouped based on the criteria of a maximum recombination fraction and minimum likelihood ratio test statistic \[[@B2]\]. The parental diplotypes of markers in individual groups are constructed using a three-point analysis. With a limited sample size available in practice, we do not recommend more-than-three-point analysis because this would bring too many more unknown parameters to be precisely estimated. If such an analysis is desirable, however, one may use the results from these lower-point analyses as initial values to improve the convergence rate and possibly the precision of parameter estimation.
In any case, our two- and three-point analysis has built a key stepping stone for map construction through two approaches. One is the least-squares method, as originally developed by Stam \[[@B5]\], that can integrate the pairwise recombination fractions into reconstruction of multilocus linkage map. The second is to use the hidden Markov chain (HMC) model, first proposed by Lander and Green \[[@B2]\], to construct genetic linkage maps by treating map construction as a combinatorial optimization problem. The simulated annealing algorithm \[[@B19]\] for searching for optima of the multilocus likelihood function need to be implemented for the HMC model. A user-friendly package of software that is being written by the senior author will implement two- and three-point analyses as well as the algorithm for map construction based on the estimates of pairwise recombination fractions. This software will be online available to the public.
Our maximum likelihood-based approach is implemented with the EM algorithm. We also incorporate the Gibbs sampler \[[@B20]\] into the estimation procedure of the mixture model for the linkage characterizing different parental diplotypes and gene orders of different markers. The results from the Gibbs sampler are broadly consistent with those from the EM algorithm, but the Gibbs sampler is computationally more efficient for a complicated problem than the EM algorithm. Therefore, the Gibbs sampler may be particularly useful when our model is extended to consider multiple full-sib families in which the parents may be selected from a natural population. For such a multi-family design, some population genetic parameters describing the genetic structure of the original population, such as allele frequencies and linkage disequilibrium, should be incorporated and estimated in the model for linkage analysis. It can be anticipated that the Gibbs sampler will play an important role in estimating these parameters simultaneously along with the linkage, linkage phases, and gene order.
Authors\' contributions
=======================
QL derived the genetic and statistical models and wrote computer programs. YHC participated in the derivations of models and statistical analyses. RLW conceived of ideas and algorithms, and wrote the draft. All authors read and approved the final manuscript.
Acknowledgements
================
We thank two anonymous referees for their constructive comments on the manuscript. This work is partially supported by a University of Florida Research Opportunity Fund (02050259) and a University of South Florida Biodefense Grant (7222061-12) to R. W. The publication of this manuscript is approved as Journal Series No. R-10073 by the Florida Agricultural Experiment Station.
| PubMed Central | 2024-06-05T03:55:46.651283 | 2004-7-26 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509239/",
"journal": "BMC Genet. 2004 Jul 26; 5:20",
"authors": [
{
"first": "Qing",
"last": "Lu"
},
{
"first": "Yuehua",
"last": "Cui"
},
{
"first": "Rongling",
"last": "Wu"
}
]
} |
PMC509241 | Background
==========
In breast cancer, cell based experiments are largely conducted with a few spontaneously arising cell lines from late stages of disease that demonstrate unlimited proliferation (immortalization). However, the breadth of tumor heterogeneity and early stages of breast tumorigenesis remain under represented in such assays. We, and others have adapted tissue processing and culture conditions to enable the selective isolation and expansion of tumor cell populations from primary breast cancer \[[@B1]-[@B9]\]. Closely reflecting the biological heterogeneity and relatively slower growth rate of malignant cells in primary tumor tissue \[[@B10]\], *in vitro*tumor cell populations are highly variable at the microscopic level and generally require long intervals between passages. The vast majority of such cultures (\>90%) display a finite mitotic lifespan. While molecular changes underlying growth cessation in non-malignant breast epithelial cultures are well studied \[reviewed in \[[@B11]\]\], barriers to the continued proliferation of tumor-derived cultures remain undetermined.
In contradistinction to previous reports on the characteristics of rare immortal cell lines developed from primary tumors \[[@B4],[@B5],[@B7],[@B8],[@B12]\], we have focused on early passage tumor cultures (that may not necessarily culminate as immortal cell lines). This approach encompasses a broader range of disease since \<10% of primary breast tumors spontaneously develop into immortal cell lines. Although limited direct comparison of primary breast tumor tissue and corresponding short-term cultures has assisted in authenticating the malignant origin of tumor-derived cultures \[[@B1]-[@B3],[@B9]\], relatively little is known regarding the degree of phenotypic and functional concordance between malignant epithelial populations in human breast tissue and their counterparts *in vitro*. In depth comparative analysis of such isogenic malignant cell populations could provide important insights regarding cellular pathways that function independently of environmental constraints, as well as the genome wide consequences of constitutive growth arrest of breast tumor cells widely encountered in laboratory dishes. Here we describe a comprehensive comparison of matched samples of primary breast carcinoma tissue, and early passage tumor cultures. We have used cDNA microarray-based global gene expression profiling to determine common molecular phenotypes of tumor tissue and tumor-derived epithelial cultures, which distinguish them from cancerous and non-cancerous immortal cell lines.
Results
=======
In order to ensure the propagation of tumor epithelium without apparent contamination with fibroblasts and other cellular components, previously described methods including the selective release of nests of malignant cells from the connective tissue, were used \[[@B2],[@B3]\]. As illustrated in Figure [1A](#F1){ref-type="fig"}, the characteristic epithelial morphology, and microscopic heterogeneity between tumor cultures from independent cases was observed. To confirm that these cultures represented pure populations of epithelial cells, indirect cytokeratin immunostaining was performed (Figure [1A](#F1){ref-type="fig"}). Clinical information and additional cell culture details are listed in Table [1](#T1){ref-type="table"}.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**A.**Microscopic phenotype of primary tumor tissue and corresponding tumor-derived epithelial and stromal cell cultures. (**1--3**) -- H & E-stained frozen sections showing histology of representative cases processed for RNA isolation and cell culture. *Case number 061T*-- 1; *066T*-- 2; *068T*-- 3. Note abundant tumor cells in the samples. (**4--6**) -- Early passage epithelial cultures; brightfield, 100× magnification. Note morphological variation between cultures in the context of an epithelial phenotype. (**7--9**) -- abundant cytokeratin expression in the cytoplasm of cultured primary tumor cells analyzed by indirect immunofluorescence. Magnification -- 400×. (**10--12**) -- tumor-derived fibroblast cultures; brightfield, 100× magnification. Note morphological distinction between epithelial and stromal cells isolated from the same tissue sample. **B -- E.**cDNA-based gene expression profiles of tumor tissue and tumor derived epithelial cultures. Rows represent genes, and columns represent samples. **B, C**-- unsupervised two-dimensional hierarchical clustering of 17 samples. **B**-- cluster shows genes under expressed in primary tumor tissue and tumor cultures compared to immortal cell lines. **C**-- cluster shows genes over expressed in primary tumor cultures and tumor tissue compared to immortal cell lines. **D, E**-- Gene expression patterns, which distinguish between group 1, consisting of immortal cell lines, and group 2, consisting of primary tumor tissue and tumor cultures. **D**-- Results of SAM analysis showing a thumbnail of 681 differentially expressed genes. **E**-- SAM-identified genes which are in common with the cluster in panel B are shown by red vertical bar, and with the cluster in panel C are shown by green vertical bars. Color scale indicates expression level.
:::
:::
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Clinical characteristics of Primary Tumors used for Gene Expression Analysis
:::
**Primary Tumor Cultures**
---------------------------- --------- ---- ----- ---- --------- ----- -------
1 022T NA NA 1 IDC NA 3
2 044T 50 NA 3 IDC Neg 2
3 047T 79 II 2 IDC Pos 5
4 054T 59 IV 3 IDC Pos 8, 12
5 061T\* 62 III 2 ILC Pos 5, 6
6 066T\* 47 II 2 IDC NA 6, 7
7 068T\* 52 II 1 IDC Pos 5, 6
8 071T NA II 3 IDC NA 3
9 076T 51 III 2 IDC Pos 2
10 257T 41 IIB 3 IDC Neg 8
11 672T 60 II 3 IDC Neg 5
12 701T 59 I 1 IDC Pos 7
13 713T 77 II 3 IDC+ILC Pos 5, 7
14 1555T 38 NA 2 ILC NA 3, 4
15 1569T NA NA NA IDC NA 3, 4
16 1570T 66 IIA 2 IDC+ILC Pos 2
17 1599T\* 64 I 2 IDC NA 3
18 1607T 58 I 1 IDC NA 5
19 1617T 64 III 2 IDC Pos 2
20 1620T 70 III 3 IDC Pos 2
21 1625T 80 IIB 3 IDC Pos 2
\* Matched tissue samples analyzed by cDNA microarrays NA -- not available; IDC -- invasive ductal carcinoma; ILC -- invasive lobular carcinoma ER -- estrogen receptor; Neg -- negative; Pos -- positive
:::
Concordant aspects of global gene expression in breast cancer tissue and tumor-derived cultures
-----------------------------------------------------------------------------------------------
Seventeen RNA samples were analyzed by cDNA microarrays. These were comprised of 4 cases of primary breast tumor tissue and 1--2 matched tumor cultures, 2 additional unmatched tumor cultures, and 4 immortal breast cell lines. Cell lines were selected to represent estrogen receptor (ER) positive (T47D) and ER negative (SKBR3, BT20) tumors, and non-cancerous breast epithelium (ENUt7, ref. \[[@B13]\]).
Unsupervised clustering analysis of 7362 clones (4743 unique genes/ESTs) grouped the samples into three separate clusters representing immortal cell lines, tumor tissue, and tumor cultures. Figure [1B](#F1){ref-type="fig"} displays a cluster of genes, which were over expressed in immortal cell lines but under expressed in tumor tissue and tumor cultures, while Figure [1C](#F1){ref-type="fig"} displays a \'mirror image\' gene expression pattern (under expressed genes in immortal cell lines, which were over expressed in tumor tissue and tumor cultures). The clusters selected for illustration represent \~90% gene correlation. The entire data set displayed additional clusters \[Figure S1 -- see http://genome-www.stanford.edu/breast\_cancer/PTCC/\].
Comparisons of large data sets as described above frequently result in \"significant\" patterns of gene expression by chance alone. We employed the Significance Analysis of Microarrays (SAM) for independently verifying genes that are differentially expressed between classes or groups of samples. SAM analysis identified 930 clones, representing 681 unique genes/ESTs, whose expression was significantly (\>2-fold) different between group 1 comprised of immortal cell lines, and group 2 comprised of tumor tissue and tumor cultures (0.05% false discovery rate). A full list of the differentially expressed genes is provided in \[Table S2 -- see http://genome-www.stanford.edu/breast\_cancer/PTCC/\]. As expected on the basis of the similarity in gene expression observed between tumor tissue and tumor cultures in the unsupervised array data (Figure [1B,1C](#F1){ref-type="fig"}), these clusters were also present in the SAM profile (Figure [1D](#F1){ref-type="fig"}). As shown in Figure [1E](#F1){ref-type="fig"}, genes upregulated in immortal cell lines (indicated by red vertical bar) reflecting significantly shorter doubling times (for example, *RFC4*, *CENPA*, *TOP2A*, *CCNA*, *MCM7*, *PCNA*, *CDC2*) were primarily those associated with the \'proliferation\' cluster described by Ross et al \[[@B14]\]. In contrast, upregulated transcripts in tumor tissue and tumor cultures (Figure [1E](#F1){ref-type="fig"}, indicated by green vertical bar) included genes involved in epithelial differentiation (*MAL, MAFB, RUNX1, KRT5*), in the induction of apoptosis (*DAPK1*), and in tumor angiogenesis, and extravasation (*SPARC*) (Gene Ontology -- GO annotations, ref \[[@B15]\]).
Primary epithelial cell cultures, in contrast to fibroblasts and rapidly growing cell lines, undergo rapid growth arrest in 10% fetal calf serum (FCS). This is why we, and others have propagated cultures of primary tumor epithelium in 0--5% FCS (1--9). Immortal cell lines, however, grow optimally in 10% FCS; lower concentrations retard growth. Therefore, to optimize growth conditions for both, 2% FCS was chosen for primary tumor cultures and 10% FCS for cell lines. It is conceivable that an increased concentration of FCS may account for differences in gene expression between primary tumor cultures and immortalized cell lines. In this case, one would expect increased proliferation in the primary tumor cultures at 10% FCS, when in fact, growth is severely inhibited by this approach.
As summarized in Table [2](#T2){ref-type="table"}, based on microarray expression data of 38,999 cDNA clones, the average correlation between matched tumor tissue and short-term tumor cultures (7 sets) was 0.41 (sd = 0.03) in contrast to 0.10 (sd = 0.09) between tumor tissue and immortal cell lines (16 pairs). These correlation coefficients differed significantly in a Mann-Whitney rank sum test (p = 0.007) demonstrating that the gene expression profile of tumor tissue was more consistent with that of the corresponding tumor culture than it was with any of the immortal cell lines tested.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Pair wise correlations between primary tumors, matched epithelial cultures, and immortal breast epithelial cell lines
:::
**Matched Primary Tumor Pair** **Immortal Breast Epithelial Cell Lines**
-------------------------------- ------------------------------------------- ------ ------ ------ ------ ------
***R correlation***
1599T 1599TC 0.31 0.05 0.07 0.01 0.23
061T 061TC1 0.44 0.10 0.03 0.03 0.24
061TC2 0.43
066T 066TC1 0.44 0.16 0.02 0.03 0.20
066TC2 0.39
068T 068TC1 0.50 0.12 0.05 0.00 0.27
068TC2 0.54
:::
Determinants of replicative arrest in primary breast tumor-derived cultures
---------------------------------------------------------------------------
Towards the determination of specific molecular changes underlying the finite proliferative lifespan in tumor cultures, gene expression analysis, by QRT-PCR, was conducted on an expanded set of 39 samples comprised of multiple early passage epithelial cultures obtained from 16 primary breast cancers, 8 cases of normal breast epithelial organoids and, 12 immortal breast epithelial cell lines. As noted above, since genes in the proliferation cluster displayed minimal expression in primary tumor cultures, first we considered the possibility of growth arrest due to a lack of telomerase activity and subsequent telomeric attrition. The relative expression of *hTERC*and *hTERT*subunits of telomerase, encoding the structural RNA component, and the component with reverse transcriptase activity, respectively, was measured. While commonly used immortal cell lines (T47D, MDA231) displayed several-fold higher transcript levels for *hTERT*, undetectable to minimal levels were observed in 6/8 independent primary tumor cultures (1599T, 713T, 1569T, 1570T, 1617T, 1620T). The primary tumor culture with the highest relative expression of *hTERT*(257T) has developed into an immortal cell line (Figure [2A](#F2){ref-type="fig"}).
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**A.**QRT-PCR analysis of *hTERT*and *hTERC*in primary tumor cultures compared to levels in normal breast organoids, matched fibroblasts, and immortal breast epithelial cell lines (*T47D*, *MDA231*, and *ENUt7*). The Y-axis is minimized to display the range of relative gene expression. The highest *hTERT*expression level, denoted by the asterisk, was 22-fold (sample 257T). **B**. TRAP assay measurement of telomerase activity shown with, and without heat inactivation of cell lysates. Extracts equivalent to 1000 cells are displayed. IC -- internal PCR control. For sample 054T, 2 independent fractions of the tumor are shown; SP -- mechanically dissociated spillage, DIG -- enzymatically digested tissue.
:::
:::
To confirm the functional impact of *hTERC*and *hTERT*down regulation, telomerase activity was measured directly by the TRAP assay. As expected, primary cultures with detectable transcript levels, and immortal cell lines of cancerous and non-cancerous origin displayed significant telomerase activity, while those tumor cultures that did not display gene expression showed no activity. In the primary tumor sample, 257T, robust telomerase activity was found as early as passage 8. Similarly, telomerase activity was detectable in early passage epithelial cultures propagated from cells isolated by mechanical dissociation (SP -- spillage), or enzymatic digestion (DIG) of the tumor sample 054T (Figure [2B](#F2){ref-type="fig"}).
In the next step towards identifying the determinants of replicative arrest, primary tumor cultures were compared with immortal cell lines for relative expression of genes associated with the negative regulation of the cell cycle in general, and with epithelial cell proliferation in particular. This analysis included 9 candidate genes in 3 signaling pathways: (1) members of the CIP/KIP family of cyclin-dependent kinase inhibitors (CDKIs), *p21*^*CIP1/WAF1*^, *p27*^*KIP1*^, and *p57*^*KIP2*^(2) members of the INK family of CDKIs, *p15*^*INK4B*^, and *p16*^*INK4A*^(3) members of the TGF-β family, *TGFβI*, *TGFβII*and the signaling receptors, *TβRI*, and *TβRII*. As illustrated in Figure [3A](#F3){ref-type="fig"}, we observed that in 10/12 breast cancer cell lines, *p21*^*CIP1/WAF1*^levels were 2 to138 fold lower than steady state levels in normal breast epithelium. In contrast, 4/20 primary tumor culture samples showed this range of *p21*^*CIP1/WAF1*^expression (p = 0.0003). For the CDKIs, *p27*^*KIP1*^, and *p57*^*KIP2*^, several fold decrease in gene expression was observed in all primary tumor cultures and most immortal cell lines as well (p = 0.04 and 0.69, respectively). Similarly, no significant differences were apparent for *p15*^*INK4B*^, and *p16*^*INK4A*^gene expression in the two groups (p = 0.07 and 0.39, respectively). For members of the TGF-β family, while significant differences were not observed in the expression of *TGFβI andTβRI*, a median [increase]{.underline} of 2 fold and 4 fold were found in the expression of *TGFβII*and *TβRII* respectvely. In contrast, immortal cell lines, showed a median [decrease]{.underline} of 7-fold for *TGFβII*and 4-fold for *TβRII*(p = 0.0035 and 0.0011, respectively). All p values were derived by the Mann-Whitney test.
::: {#F3 .fig}
Figure 3
::: {.caption}
######
Comparative QRT-PCR analysis of genes encoding negative regulators of cell proliferation in primary tumor cultures and immortal cell lines. **A**-- Expression levels of individual genes represented as fold increase or decrease over gene expression in normal breast organoids (average of 8 independent reduction mammoplasty cases). Data shown are averages of triplicate measurements. Dots represent immortal cell lines (red) and primary tumor cultures (black) shown in panels ***B***and ***C***. **B**-- multivariate analysis of data in ***A***displayed as a hierarchical clustering dendrogram. The scale shows the distance between groups comprised of normal breast, primary tumor cultures, and immortal cell lines. For samples 1569T, 1555T, 713T, and 054T, gene expression was analyzed in epithelial cells at 2 different passages in culture (passage number indicated as 3\', 4\', 5\' etc.) **C**-- a plot of 3 principal components (U1, U2, and U3) of 9-dimensional space, representing the 9 genes evaluated in samples in panels ***A***and ***B***. Based on its level of gene expression, a place is assigned to each cell sample in this space. Data points representing epithelial organoids from the normal breast (pink) appear to be tightly clustered, whereas those representing primary breast tumor cultures (green) and immortal cancer cell lines (blue) show considerable scatter.
:::
:::
We used MANOVA to compare expression of the 9 above-mentioned genes in immortal cell lines, primary tumor cultures, and normal breast epithelium. It was apparent that the multivariate means for the 9 genes differed for each of the 3 groups above and that both immortal lines and primary tumor cultures differed from normal breast epithelium (p = 0.0001) as also depicted in the hierarchical clustering dendrogram of these samples (Figure [3B](#F3){ref-type="fig"}). A 3-D plot of the first three principal components, which together account for 83% of the total variation in expression in the 9-dimensional gene space, is shown in Figure [3C](#F3){ref-type="fig"}. This display format demonstrates that the three types of cell samples cluster in different parts of the three dimensional space and that primary tumor cultures and immortal cell lines are significantly different from normal breast and from each other in the expression of the negative growth regulators evaluated here (p = 0.0002). The relatively large Wilks\' lamda (0.271) for immortal cell lines vs. primary tumor cultures is most likely due to the greater spread of these groups in the 9-dimensional gene space, while normal samples are tightly grouped together. This finding may be related to the heterogeneity between individual tumors.
Overall, the genes in the array-based clusters, shown in Figure [1B](#F1){ref-type="fig"} and [1C](#F1){ref-type="fig"}, could be categorized as positive or negative regulators of proliferation respectively according to GO annotations. Genes such as, *PCNA*, *CKS1B*, *TPX2*, *UBE2C*, *CDC6*, confirmed by the statistically significant analysis of microarray data, SAM, were among the positive proliferation genes differentially expressed by immortal cell lines, and matched tumor tissue/cell culture samples (Figure [1E](#F1){ref-type="fig"}, also see Table S2 -- http://genome-www.stanford.edu/breast\_cancer/PTCC/, for full list). Expression levels of negative proliferation genes identified in the SAM data, such as, *TβRII*, and *CDKN1A*(*p21*^*CIP1/WAF1*^) were confirmed by QRT-PCR (Figure [3A](#F3){ref-type="fig"})
Discussion
==========
The cDNA microarray analysis of primary breast tumors and their epithelial counterparts propagated in cell culture has revealed close similarities in the expression of several hundred genes. Notably, increased expression of genes deemed to be \'growth limiting\' by virtue of their consistent down regulation in immortal cell lines, was observed. The patterns of gene up regulation or down regulation were remarkably consistent in independent immortal lines (including those derived from non cancerous breast epithelial cells) as was the contrasting gene \'signature\' in cultures derived from independent cases of breast carcinoma. Together, these observations have led us to conclude that the continuous selection of rapidly proliferating cells culminating in the immortalized phenotype favors the loss of several aspects of gene expression retained by early passage primary tumor cultures.
Our data suggests that the limited growth potential of primary tumor cultures results from two major proliferative barriers, (a) telomerase inactivation, potentially leading to telomere attrition, and (b) negative growth signaling by upregulated *TGFβ*resulting in a significant increase in transcription of the CDKI, *p21*^*CIP1/WAF1*^, consistent with the findings of induction or stabilization of this gene in a variety of immortalized cells exposed to [exogenous]{.underline} TGFβ \[[@B16],[@B17]\]. Elevated levels of *p21*^*CIP1/WAF1*^in turn appear to have a direct impact on telomerase regulation \[[@B18]\]. As expected of slow growing primary breast tumors, *in vivo*, p21^CIP1/WAF1^positive cells are detected in the majority of cases \[[@B19]\]. Also pertinent in this regard is the fact that substrate induced changes in cell shape upregulate *TGFβ*transcription \[[@B20]\]. Since the promoter region of the *TGFβ*gene contains a shear stress response element \[[@B21],[@B22]\], it seems likely that TGFβ induction occurs in response to unknown stresses in the *in vitro*environment and initiates a cascade of growth inhibitory events, including telomerase inactivation. While epithelial cells derived from most primary breast tumors rapidly revert to regulated growth dictated by environmental signals, immortal breast epithelial cells are insensitive to such cues, which may be the underlying basis for their continued selection *in vitro*. Notably, loss or functional inactivation of the cognate receptor often enables tumor cells *in vivo*to overcome the growth inhibitory effect of TGFβ early in tumorigenesis, however, during metastatic progression, autocrine TGFβ appears to play a tumor-promoting role, possibly enabling tumor cells to survive significant changes in microenvironment \[[@B23]\]. In this light, induction of *TGFβ*in response to the *in vitro*environment in primary breast tumor cultures portrays a critical phase of tumor progression. Additionally, towards the full manifestation of differentiated phenotypes in primary tumor cultures, cell propagation in a three-dimensional growth matrix, pioneered by Bissell and colleagues \[[@B24]\], is imperative. Such studies are currently ongoing in our laboratory.
Tumor-derived immortal cell lines generally display robust proliferation and have thus filled an important need for functional cancer cell model systems. While immortal cell lines continue to provide molecular and biological insights regarding proliferation-related hallmarks of malignancy, their functional application as indicators of efficacy in cancer drug development is relevant mostly to rapidly proliferating high-grade tumors. Many breast tumors do not fall into this category, and are not necessarily indolent. Thus, additional targets in diverse gene clusters must be identified for novel drug designing. In fact, such targets could be applicable to tumors at early or late stages as recent studies suggest that genes conferring invasive and/or metastatic characteristics late in progression often become dysfunctional at earlier stages of tumorigenesis \[[@B25]\]. Moreover, since our data demonstrate that primary tumor cultures routinely derived from surgical discard tissue display phenotypic, and most likely functional aspects of breast cancer, they provide a strong rationale for the experimental manipulation of such cells towards revealing the causative role of genetic polymorphisms in cancer susceptibility, an important goal which is unlikely to be fulfilled with currently used model systems.
Conclusions
===========
This microarray-based analysis demonstrates that epithelial cultures isolated from primary breast tumors retain phenotypes of the malignant tissue, which are eliminated during the selection of rapidly proliferating cell populations that comprise commonly used *in vitro*model systems. Thus the opportunity for basic and clinical application of [functional]{.underline} cells derived from the full range of pathological breast tissue, instead of a few immortal cell lines should not be missed.
Methods
=======
Clinical specimens and cell culture
-----------------------------------
Pathologically confirmed tumor tissue and non malignant reduction mammoplasty samples were collected as fresh specimens under IRB approved guidelines at the California Pacific Medical Center, San Francisco, Stanford University, and the University of California, San Francisco between 1997 -- 2000. Additional tumor samples were obtained from the NCI Cooperative Human Tissue Network. A portion of the tissue was snap frozen and cryopreserved for histological confirmation and nucleic acid isolation prior to cell culture. Tumor samples were processed as previously described \[[@B2],[@B3]\]. A total of 21 independent cultured specimens were used for gene expression analysis. Primary tumor cultures and the non-tumorigenic, *ENUt7*cell line were propagated in low calcium MCDB170 medium supplemented with 2% FCS. Breast cancer cell lines were cultured in the recommended growth media supplemented with 10%FCS; *MCF7*, *BT20*, *MDA231*, *MDA157*in DME-H21; *BT474, CAMA1*, *T47D*, *ZR75*in RPMI 1740; *MDA435*, *MDA134*, *MDA361*in L15.
To confirm the epithelial phenotype of primary tumor derived cells, cultures fixed with 50% ethanol: acetone were immunostained with anti pan-cytokeratin, as previously described \[[@B1]\].
RNA isolation, microarray methods and data analysis
---------------------------------------------------
Frozen tissue was trimmed to yield \>90% tumor cell enrichment. Total RNA was extracted with the RNAeasy Mini kit (Qiagen), amplified using an optimized T7 based protocol \[[@B26]\], labeled with Cy5, and hybridized to \~47,000 feature cDNA microarrays as previously described \[[@B26],[@B27]\]. Hybridized arrays were scanned (Axon) and images were analyzed using GenePix^®^Pro 4.0 software (Axon Instruments). Spots were selected for analysis if signal intensities in both Cy3 and Cy5 channels were 2.5 times higher than background. The arrays were clustered using a hierarchical clustering algorithm \[[@B28]\], which groups genes and samples on the basis of their expression similarities. The results were visualized using TreeView software \[[@B29]\]. The clustering was performed on clones whose expression varied at least 3-fold from the mean in one or more samples and was measurable in over 80% of the samples.
Significance Analysis of Microarrays \[[@B30],[@B31]\], used for identifying genes whose expression differed significantly between groups, was performed on data from 38,999 cDNA clones. After the selection of spots 2.5 times over background, and the elimination of clone redundancy, data was retrieved on 930 clones, representing 681 unique genes.
Quantitative Real Time PCR (QRT-PCR)
------------------------------------
RNA samples were treated with RNAse-free DNAse (Roche) to remove genomic DNA, and reverse transcribed. Fifty ng of cDNA was used as template for PCR amplification with specific oligonucleotide primers, (designed using Primer Express 1.5 software). Following denaturation and cycling reactions (40 cycles) the products were analyzed (Applied Biosystems 5700 Sequence Detection System). The cycle number at which the amount of amplified target reached a fixed threshold was designated as the threshold cycle (C~T~). The higher the initial amount of transcript template, the lower the C~T~value. For quantitation of gene expression, the target gene value normalized to the expression of an endogenous reference (*β actin*) was designated as ΔC~T~. Relative gene expression was calculated by the formula 2^-ΔΔCT^. ΔΔC~T~was obtained by subtracting the ΔC~T~of test sample from the ΔC~T~of normal breast epithelial fractions, called \'organoids\', isolated from mechanically and enzymatically dissociated reduction mammoplasty tissue (average of 8 independent specimens).
Telomerase activity
-------------------
Cell lysates prepared from primary tumor cultures, immortal cell lines, and normal breast organoids were analyzed by the telomerase repeat amplification protocol (TRAP) as per manufacturer recommendation (Intergen). Briefly, trypsinized cell pellets were resuspended in lysis buffer at 500 cells/microliter, incubated on ice for 30 minutes, centrifuged at 12,000 × g for 20 mins, and the telomerase containing supernatant used for PCR amplification. Reaction products were resolved in 10% PAGE gels and visualized with SYBR Green-1 (Molecular Probes). Heat inactivated controls were included for each sample. Linearity of the assay was determined with cell dilutions ranging from 100 to 10, 000 cells.
Statistical methods
-------------------
We calculated Pearson correlations from the microarray data generated from 38,999 clones to measure similarities among 7 matched tumor tissue: cell culture sample pairs and among 16 tumor tissue: immortal cell line pairs. For QRT-PCR data, multivariate analysis of variance (MANOVA) was used to compare the expression levels of genes involved in negative growth regulation between immortal cell lines, primary tumor cultures, and non malignant breast epithelial cells. Tests for statistical significance were based on Wilks lamda criterion, a multivariate analog of the F-test for univariate analysis of variance (ANOVA), which tests the equality of means. Calculations were made in Data Desk, version 6.2. Principal components for all genes over all the data were computed and S-Plus was used to plot the data for the first 3 principal components.
Authors\' contributions
=======================
SHD conceived the study, provided overall direction and coordination to the research, and drafted the manuscript. YJ carried out the microarray analysis. YB carried out QRT-PCR, and other molecular analyses. DHM performed the statistical analysis of data. ZM performed the pathology review and tissue dissection. SSJ participated in the design and analysis of microarray-based experiments and in editing portions of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
================
This work was supported by the California Breast Cancer Research Program (8WB-0032). We thank Ms. Stacey Champion for technical assistance, and Ms Susan Overholser for creating web supplement.
| PubMed Central | 2024-06-05T03:55:46.655637 | 2004-7-19 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509241/",
"journal": "BMC Genomics. 2004 Jul 19; 5:47",
"authors": [
{
"first": "Shanaz H",
"last": "Dairkee"
},
{
"first": "Youngran",
"last": "Ji"
},
{
"first": "Yong",
"last": "Ben"
},
{
"first": "Dan H",
"last": "Moore"
},
{
"first": "Zhenhang",
"last": "Meng"
},
{
"first": "Stefanie S",
"last": "Jeffrey"
}
]
} |
PMC509242 | Background
==========
Chemokines are a superfamily of small chemotactic proteins that have been classified into four major subfamilies, namely CXC, CC, C, and CX3C, based on the presence or absence and positional arrangement of N-terminal cysteine (C) residues \[[@B1]-[@B3]\]. One of the hallmarks of chemokine function is to facilitate trafficking and recirculation of immune cells from the circulation and tissues into secondary lymphatic organs and various peripheral tissues to maintain immune homeostasis *in vivo*\[[@B4]\]. These ligands also control the selective recruitment of specific leukocyte subsets to sites of inflammation and immune reactions. Besides migration, chemokines also induce the rapid activation of integrin molecules. The two CXC chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC), are members of the CXCL1 subfamily containing a glutamate-leucine-arginine (ELR) motif that are well known for their ability to induce the activation and recruitment of neutrophils *in vitro*and *in vivo*\[[@B5]-[@B7]\]. These chemokines are also believed to be the murine structural and functional homologues of human CXCL8, IL-8 and chemokine growth-related oncogene (GRO) \[[@B8],[@B9]\]. MIP-2 and KC display high affinity binding and signaling through the murine CXCR2, a 7-transmembrane G protein-coupled receptor \[[@B10]\].
It has been well established that endotoxin and various proinflammatory cytokines (e.g., TNF-α and IL-1) stimulate the expression of MIP-2 by macrophages, neutrophils and epithelial cells \[[@B11]-[@B13]\]. Numerous studies have also demonstrated a pathophysiological role for MIP-2 and KC in several inflammatory disease states including endotoxemia-induced lung injury \[[@B14]\], 1996), glomerulonephritis \[[@B15]\], bacterial meningitis \[[@B16]\] and herpes simplex virus type 1 (HSV-1) infection \[[@B17]\]. In mice, the cellular sources of MIP-2 have been confirmed to be macrophages \[[@B6]\], epithelial cells \[[@B12]\], bone marrow endothelial cells \[[@B18]\], astrocytes \[[@B19]\] and mast cells \[[@B20]\]. In humans, in addition to macrophages, monocytes, T, NK and B cells have also been shown to produce and respond to CXCL8 \[[@B21]-[@B24]\]. Despite all of these reports, few studies have focused on chemokine production by B cells and the relevance of such production in cell-mediated immune responses.
Age-related dysfunction of the immune system has often been attributed to a variety of measurable changes in the functional activity of many immunomodulatory factors. This immune deterioration with age is believed to contribute to the morbidity and mortality in humans, possibly due to the greater incidence of infection, autoimmunity and cancer in the elderly. Dysregulation of lymphocyte function is thought to play a critical part in these processes. Many factors are believed to contribute to age-associated immunodeficiencies including defects in cellular signaling, stem cell and bone marrow defects, thymus involution, alterations in hormone and growth factor production, and replicative senescence. Chemokines are believed to play a pivotal role in the complex communication network between different cell types that enable the selective trafficking of many immune effector cells to the necessary sites at the appropriate times. Several reports have also demonstrated increased expression of inflammatory cytokines and chemokines in the circulation and by peripheral blood cells with age suggesting that uncontrolled inflammation may contribute to the increased susceptibility to infection and injury in certain aged cohorts. For example, IL-8 chemokine production by human T, NK and monocytes is altered with age in response to bacteria products \[[@B25]-[@B27]\]. Alterations in chemokine production and responsiveness may have a significant impact on the capacity of aged subjects to control or even mount an immune response.
The objective of the current study is to explore both young and aged murine splenic B cells and B cell subsets for their ability to express the CXC chemokines, MIP-2 and KC, in response to LPS and other stimuli. Three B cell subpopulations were studied here including: (1) Marginal zone (MZ) B cells, which are uniquely positioned in the marginal sinus where they interact with efficient Ag trapping circulating cells and are thought to be involved in the early response against thymus-independent (TI) blood-borne Ags \[[@B28],[@B29]\]; (2) Follicular (FO) B cells, which are long-lived recruiting B cells located at the border of the B cells follicle and the T cell containing PALS zone that facilitate responses to thymus-dependent (TD) Ags and give rise to both germinal center (GC) B cells and plasma cells \[[@B30]\]; and (3) Newly formed (NF) B cells, which correspond to the recently immigrated B cells from the bone marrow and have been described as immature/transitional B cells and the precursors of FO and MZ B cells \[[@B31]\]. We also investigated the effect of these B cell-derived chemokines on splenocyte migration and the influence of aging on this event. The relevance of these findings to the generation of humoral and cellular immune responses shall be discussed.
Results and Discussion
======================
As expression of MIP-2 and KC by murine splenic B cells has not been previously defined, we initially examined whether murine B cells are capable of producing the CXCL1 chemokines, MIP-2 and KC, upon culture or post cellular activation with LPS, anti-IgM or CD40 mAb in combination with IL-4 for 24 h. Culture supernatants were subsequently examined for MIP-2 and KC expression using ELISA analysis. The results shown in Figure [1A](#F1){ref-type="fig"} demonstrate that while cultured non-stimulated splenic B cells failed to spontaneously produce detectable levels of MIP-2 and KC (\<10 pg/ml), stimulation of B cells with LPS, but not anti-IgM or CD40 mAb in combination with IL-4, resulted in the significant increase in the expressions of both of these chemokines. Interestingly, the quantity of MIP-2 produced in response to LPS was significantly greater than KC. Fig. [1B](#F1){ref-type="fig"} shows representative images of MIP-2 expression patterns in the cytoplasm of LPS-stimulated splenic B cells. Visually, accumulation of cytoplasmic MIP-2 was observed in splenic B cells (IgM^+^) following 24 h incubation with LPS, but was less evident in non-stimulated splenic B cells. Similar results were also obtained examining cytoplasmic KC (data not shown). In agreement with the above findings, MIP-2 and KC mRNA was only detected in LPS- (Fig. [1C](#F1){ref-type="fig"}) but not anti-μ- or anti-CD40 mAb/IL-4-stimulated (data not shown) B cells. Consistent with protein expression, the mRNA levels for MIP-2 were expressed at a greater level than KC. As the engagement of the B cell receptor (BCR) initiates signaling pathways mediated through nonreceptor protein tyrosine kinases, including Fyn, Lyn, Syk, and Bruton\'s tyrosine kinase (BTK), while LPS activate B cells by stimulating signaling through toll-like receptors, more specifically TLR4. The CC chemokines, MIP-1α and MIP-1β, have been shown to be induced and secreted by human B cells in response to BCR signaling \[[@B22]\]. In contrast, the lack of CXCL1 chemokine induction in response to cross-linking of the BCR with soluble anti-IgM antibody here suggests that the BCR signal(s) may not be optimal and/or essential for MIP-2 and KC production. Perhaps additional costimulatory signals or activation pathways may be necessary for CXCL1 production in combination with BCR signaling. These data demonstrate that murine splenic B cells are capable of secreting the CXCL1 ligands upon activation with LPS but not via anti-μ or anti-CD40 stimulation.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Splenic B cells produce MIP-2 and KC in response to LPS.**(A) Purified splenic B cells (1 × 10^6^cells/ml) from 4 month-old C57BL/6 mice were stimulated with 10 μg/ml of LPS, anti-IgM or anti-CD40 plus IL-4 for 24 h. Then, cell-free supernatants were collected and assayed by ELISA for MIP-2 and KC secretion. Data are mean of triplicate ± SD of one representative of two experiments. The value was significantly different from non-stimulated control. (\*\*, *P*\< 0.01; \*\*\*, *p*\< 0.005) (B) Immunofluorescence visualization of MIP-2 expression in the cytoplasm of 24 h LPS-stimulated splenic B cells. Cells were incubated with biotinylated goat anti-mouse MIP-2 antibody followed by SA-Oregon green-488 (green), PE-conjugated anti-IgM antibody (red) and the DNA dye DAPI (blue not shown) and subjected to cytospin (5 × 10^5^cells/microscope slide). Magnification: +100. (C) Total RNA was isolated from non- and LPS-stimulated cells and levels of MIP-2 and KC mRNA were examined by RT-PCR. The housekeeping gene β-actin was amplified as an internal control. The data are representative of two experiments.
:::
:::
Several reports have demonstrated that the proliferative response of whole spleen cells to LPS declines with age \[[@B32],[@B33]\]. The results in Figure [2A](#F2){ref-type="fig"} demonstrate that purified splenic B cells derived from aged mice also display a significantly diminished proliferative response when cultured with LPS for 72 h when compared to B cells derived from younger mice. However, no significant differences in cellular proliferation were observed between young and aged mice after 24 h of culture, which is in accordance with the previous report \[[@B34]\].
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**Effect of aging on LPS-induced B cell proliferation and MIP-2 and KC production by splenic B cells.**(A) *In vitro*proliferation of splenic B cells stimulated with LPS. Purified splenic B cells (1.25 × 10^5^/ml) were cultured with or without LPS. Proliferation was measured by \[^3^H\] thymidine uptake after 24 and 72 h of culture. Data are means ± SD of three mice in each group. The value was significantly different from that of aged mice. (\*\* P \< 0.01) (B) Splenic B cells from three to five young and aged mice were stimulated with 10 μg/ml of LPS for 4 and 24 h. After stimulation, cell-free supernatants were collected and assayed by ELISA for MIP-2 and KC secretion. One representative experiment out of three is shown. (C) 24 h LPS-stimulated splenic B cells were subjected to immunofluorescence staining with anti-MIP-2 and anti-KC antibodies and DNA dye DAPI as described in Figure 1. One representative experiment out of three is shown. Control anti-goat IgG staining in both young and aged B cells are shown as inserts in this panel. (D) After stimulation, cells were harvested, and RNA was prepared for MIP-2 and KC specific RT-PCR. The housekeeping gene β-actin was amplified as an internal control. Data shown are representative of two independent experiments. (E) MIP-2 and KC mRNA levels were measured by real time RT-PCR and normalized to threshold cycle (*Ct*) values of the co-amplified house-keeping gene GAPDH. Normalized values were calibrated to the value derived from non-stimulated controls and shown as fold change of mRNA expression. Data shown are representative of two independent experiments. Value were significantly different from those in aged mice (\*\* P \< 0.01; \*\*\* P \< 0.005)
:::
:::
Despite no significant differences in LPS proliferative responses between young an aged mice at 24 h, LPS-stimulated B cells derived from young mice demonstrated approximately 4 times greater levels of MIP-2 in the culture supernatants than aged B cells during this same time period. Similarly, reduced levels of KC expression were also detected in the 24 h culture supernatants LPS-stimulated B cells derived from aged mice when compared to younger animals (Fig. [2B](#F2){ref-type="fig"}). Moreover, as shown in Fig. [2C](#F2){ref-type="fig"}, splenic B cells from aged mice demonstrate significantly less expression of intracytoplasmic MIP-2 and KC upon stimulation with LPS when compared to younger B cells. These results were supported by conventional RT-PCR analysis where MIP-2 and KC mRNA signals were barely detectable in both freshly isolated and non-stimulated splenic B cells from young or aged mice. The mRNA levels of MIP-2 and KC chemokines in LPS-stimulated B cells from aged mice were dramatically lower (\> 2-fold) than those in younger mice (Fig. [2D](#F2){ref-type="fig"}). Unlike MIP-2, the KC mRNA signal was only detected after 24 h of stimulation with LPS and demonstrated similar patterns of age-associated expression as MIP-2. Furthermore, real time RT-PCR analysis also demonstrated similar age-related alterations in B cell-derived MIP-2 and KC mRNA expression, albeit after 24 h of LPS stimulation (Fig. [2E](#F2){ref-type="fig"}).
Similar results were obtained using an *in vitro*B cell culture system in which murine naïve B cells can be activated and induced to proliferate and differentiate into Ab-forming cells. The EL-4 system has been described as a potent *in vitro*culture system for B activation, proliferation and differentiation \[[@B35]-[@B37]\]. Naive B cells cultured in the EL-4 system differentiate to Ig-secreting cells expressing switched isotypes and a plasma cell phenotype (Fig. [5](#F5){ref-type="fig"}; \[[@B38],[@B39]\]). The presence of both LPS-dependent and T cell-dependent stimulation (e.g., CD40-CD40L) contribute to the plasma cell differentiation in this model system. Here, splenic B cells derived from young and aged mice were co-cultured with irradiated EL-4 thymoma cells in presence of LPS and a macrophage-derived culture supernatant. MIP-2 and KC levels were assessed in the supernatants of those cultures via ELISA on days 3 or 5. As shown in Fig. [3A](#F3){ref-type="fig"}, splenic B cells cultured in this EL-4 culture system yielded similar results to those observed in Fig. [2](#F2){ref-type="fig"}. Briefly, a considerable reduction of MIP-2 production by aged splenic B cells was observed in comparison to B cells derived from younger animals. Interestingly, no KC expression was detected in any of the aged B cell cultures while significant KC production was observed in the 3 and 5-day culture supernatant of young B cells. Furthermore, real time RT-PCR also demonstrated a significant reduction in the mRNA expression of MIP-2 and KC in LPS-activated aged B cells when compared to younger B cell cultures (Fig. [3B](#F3){ref-type="fig"}). Moreover, given our data in Figure [1](#F1){ref-type="fig"} demonstrating that cross-linking of CD40 by anti-CD40 antibody and IL-4 failed to elicit CXCL1 chemokine secretion by B cells, the B cell-derived MIP-2/KC secretion in EL-4 system may be attributed to the effect of LPS-dependent signals rather than CD40 cross-linking signals and/or other cytokines/lymphokines presented in the culture. Taken together, these results demonstrate that MIP-2 and KC expression by murine splenic B cells is significantly altered with increasing age.
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**Effect of aging on MIP-2 and KC production by splenic B cells co-cultured with irradiated EL-4 cells.**(A) Decreased amounts of MIP-2 and KC proteins in supernatants of splenic B cells cultured in EL-4 culture system. Splenic B cells (3 × 10^4^/well) from three to five young and aged mice were cultured with irradiated EL-4 thymoma cells in the presence of LPS and macrophage supernatant for 3 or 5 days. Culture supernatants were collected and analyzed by ELISA for MIP-2 and KC secretion. These values were significantly different from those in aged mice (\*\* P \< 0.01). (B) Real-time RT-PCR analysis of MIP-2 and KC mRNA expression in activated splenic B cells from young and aged mice. After 3 days of culture, total RNA was isolated from cultured cells and levels of MIP-2 and KC mRNA were measured by real time RT-PCR and normalized to threshold cycle (*Ct*) values of the co-amplified housekeeping gene GAPDH. Normalized values were calibrated to the value derived from EL-4 only controls and expressed as fold induction of mRNA. One representative experiment out of two is shown.
:::
:::
Splenic B cells derived from adult mice are comprised of several distinctive subpopulations based on their surface marker expression \[[@B40]\]. To assess if the CXCL1 ligand expression differences observed between young and aged mice are due to differences in B cell subsets, MZ, FO, and NF B cells as well as total IgM^+^B cells were sorted by FACS based on their surface IgM, CD21, and CD23 markers (Fig. [4A](#F4){ref-type="fig"}). Sorted individual B cell subpopulations were co-cultured with EL-4 cells for 5 days. Similar to our above results, total IgM^+^splenic B cells derived from aged mice expressed significantly reduced levels of MIP-2 compared to young B cells. Moreover, although MIP-2 secretion by NF B cells derived from aged mice did not significantly differ from young NF B cells, the levels of MIP-2 expressed by aged FO B cells were substantially diminished in comparison to B cells derived from their younger counterparts. Furthermore, unlike MZ B cells derived from young mice, MIP-2 production was almost undetectable in MZ B cells obtained from aged mice (Fig. [4B](#F4){ref-type="fig"}). In support of these data, MIP-2 mRNA expression was significantly reduced in aged FO and MZ B cells but not in aged NF B cells when compared to B cells derived from younger mice (Fig. [4C](#F4){ref-type="fig"}). Similar results were observed on day 3 (data not shown). Unfortunately, in several repeated subset studies, we were unable to detect KC in the culture supernatants of B cell subsets derived from young and aged mice. We believe this may be due to the low level of KC produced by the splenic B cell subsets compared to non-sorted, non-fractionated splenic B cells, which were also quite low albeit detectable. B cells stimulated with EL-4 T cells behave differently than LPS-stimulated primary B cells. B cells co-cultured with EL-4 cells demonstrate better viability and more plasmocytic differentiation than LPS alone. Thus, B cells are in different states of activation in these two culture systems and may account for the differences in KC expression.
::: {#F4 .fig}
Figure 4
::: {.caption}
######
**Ability of distinct splenic B cell subpopulations from young and aged mice to produce MIP-2 and KC chemokines.**(A) Spleen cells were isolated from three young and aged mice, and then stained with anti-IgM, anti-CD23 and anti-CD21 Abs. Subsequently, NF, FO and MZ B cell subpopulations were sorted from IgM^+^gated cell population. (B) Amounts of MIP-2 protein in supernatants of total IgM^+^B cells and distinct splenic B cell subpopulations cultured in EL-4 system. NF, FO and MZ B cells within the respective gates shown were directly sorted into individual wells of 96 well plates (1,000 cells/well). Each well had 200 μl of medium containing irradiated EL-4 cells, LPS and macrophage supernatant. After 5 days of culture, supernatants were collected and analyzed by ELISA for MIP-2 and KC secretion. These values were significantly different from those in aged mice (\* P \< 0.05; \*\* P \< 0.01; \*\*\* P \< 0.005). (C) Real time RT-PCR analysis of MIP-2 and KC mRNA expression in 5 days-cultured B cells from young and aged mice. MIP-2 and KC mRNA levels in total IgM^+^, NF, FO and MZ B cells were measured by real time RT-PCR and normalized to threshold cycle (*C*~*t*~) values of the co-amplified housekeeping gene GAPDH. Normalized values were calibrated to the value derived from EL-4 only controls and expressed as fold induction of mRNA. One representative experiment out of two is shown. These values were significantly different from those in aged mice (\* P \< 0.05; \*\* P \< 0.01; \*\*\* P \< 0.005).
:::
:::
Given the possibility that the reduced MIP-2 and KC expression by aged B cells in these EL-4 cultures might reflect a diminished capacity of these cells to proliferate and/or differentiate, we examined the proliferative response as well as the generation of class switched B cells in the culture of those B cells. By culturing MZ, FO and NF B cell subpopulations as well as IgM^+^B cells on EL-4, no defect in the cell proliferations was found among those distinct B cell subsets in aged mice on day 3 (Fig. [5A](#F5){ref-type="fig"}) and day 5 (data not shown) of culture. It should be noted that the EL-4 cells utilized in these assays were thymidine kinase deficient \[[@B41]\] and thus, the background thymidine incorporation was quite low (\<200 cpm).
On the other hand, analysis of both percentages (Fig. [5B](#F5){ref-type="fig"}) and the absolute number of IgG1^+^ B cells (Fig. [5C](#F5){ref-type="fig"}) revealed that the frequency of IgG1^+^B cells presented in the aged B cell culture were comparable to those generated in the young B cell culture in response to LPS. Furthermore, ELISA analysis of Ig levels in culture supernatants demonstrated no difference in the level of IgG produced by young and aged splenic B cells in response to LPS (Fig. [5D](#F5){ref-type="fig"}). These studies suggest that the reduced MIP-2 and KC production observed in EL-4 B cell subset cultures are not due to significant alterations in cellular proliferation and/or differentiation.
::: {#F5 .fig}
Figure 5
::: {.caption}
######
**Similar proliferation and differentiation between young and aged splenic B cells cultured in EL-4.**FACS-sorted distinct B cell subsets (1000/well) from three young and aged mice were cultured in EL-4 culture system. Proliferation was measured by \[3H\] thymidine uptake after 3 days culture. Data represent the mean and variations (SD) from triplicate cultures. The data presented are representative of two independent experiments (A). Purified splenic B cells (4 × 10^4^/well) from three young and aged mice were cultured with irradiated EL-4 thymoma cells in the presence of LPS and macrophage supernatant for 3 and 5 days. The cultured cells were stained with anti-IgG1 and anti IgM Abs. Percentages of IgG1^+^IgM^+^B cells are indicated (B). Absolute numbers of IgG1^+^B cells in culture. Total numbers of IgG1^+^B cells per well were calculated from a mixture of 20 wells in each group (C). Culture supernatants were measured by ELISA for IgG production. Data are representative of two independent experiments (D).
:::
:::
To assess the potential functional relevance of MIP-2 and KC expression by LPS-stimulated splenic B cells, splenocytes derived from young mice were labeled with Hoechst and examined for their ability to migrate in response to culture supernatants derived from LPS-stimulated young and aged cells. The results in Fig. [6A](#F6){ref-type="fig"} show that culture supernatants of LPS-stimulated splenic B cells from either young and aged mice induced significant responder cell migration, as compared with non stimulated B cells-derived supernatants. More importantly, the chemotactic activity of culture supernatants derived from LPS-activated younger B cells was significantly higher than the activity observed in response to supernatants derived from LPS-stimulated aged B cells. Interestingly, the addition of anti-MIP-2 Ab to the B cell cultures resulted in an approximate 5% reduction of the migratory capacity of the young responder cells to migrate in response to the LPS-derived supernatants of young B cells but this antibody addition failed to exhibit any significant inhibition of migration induced by aged B cell supernatants (Fig. [6B](#F6){ref-type="fig"}). Neutralization of responder cell migration was also significantly blocked with anti-KC Ab (\~20%) in the young but not aged B cell supernatants. Additional neutralization studies using a panel of anti-chemokine antibodies revealed that several CC chemokines are also being made by B cells and are playing a role in the remaining chemotaxis observed using these young responder cell populations (data not shown). Overall, these results suggest that B cell-derived MIP-2 and KC may play a possible role in leukocyte trafficking and that the MIP-2 and KC derived from these B cell cultures are biologically active.
::: {#F6 .fig}
Figure 6
::: {.caption}
######
**B cell-derived MIP-2 and KC induce splenocyte migration.**(A). Purified splenic B cells (1.5 × 10^6^/ml) from three young and aged mice were added to the lower chamber of Transwell plates and stimulated with and without LPS. 24 hr after LPS stimulation, spleen cells from 4 month old C57BL/6 mice were preincubated with Hoechst and subjected to chemotaxis through 5-μm pore size Transwell filters (upper chamber) to the supernatants in the lower chambers. Hoechst fluorescence of accumulated cells in the lower chamber was measured. Data are the mean of triplicate cultures ± SD of one representative of two experiments. The value was significantly different from that of control. (\* P \< 0.05; \*\* P \< 0.01) (B). For the neutralization, Abs against MIP-2 and KC were added to the supernatants in the lower chambers at the beginning of culture. The percent inhibition was calculated as follows: 100 - 100 × (chemotaxis with neutralizing Ab/chemotaxis without neutralizing Ab). Data are the mean of triplicate cultures ± SD of one representative of two experiments. The value was significantly different from that of control (\* P \< 0.05; \*\* P \< 0.01).
:::
:::
In the current study, we report that endotoxin-activated murine B cells express and secrete the CXC chemokines, MIP-2 and KC. These results are in accordance with the previous studies, which demonstrate that activated human B cells express and secrete CXCL8 \[[@B21]-[@B23]\]. We also demonstrate that the expression of CXCL1 chemokines, MIP-2 and KC, decline with age in murine splenocytes and B cells, particularly evident in MZ and FO B cell subsets (Fig. [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}). This could not be attributed to impaired numbers of MZ and FO B cells as judged by flow cytometric analysis as young and aged B cells demonstrated comparable numbers of these populations (data not shown). In addition, these differences could also not be attributed to the alterations in proliferation (Fig. [2A](#F2){ref-type="fig"} and [5A](#F5){ref-type="fig"}; \[[@B34]\]) or terminal differentiation of these cells to IgG plasma cells (Fig. [5B,5C](#F5){ref-type="fig"} and [5D](#F5){ref-type="fig"}). Thus, the downregulation of MIP-2/KC chemokine production in response to LPS by MZ and FO B cells from aged in relation to young mice appears to be due to an age-dependent signaling difference in response to LPS. In this respect, the expression and function of the LPS receptor TLR4 has been shown impaired in aged animals \[[@B42]\]. In addition, aging can affect gene regulation and some transcription factors, such as nuclear factor-kappa B (NF-kappa B) are required for induction by LPS of MIP-2/KC expression through TLR4 \[[@B43]\]. Thus, further studies will be necessary to elucidate the relevance of age-related alterations in TLR4 expression and/or function as well as NFκB-dependent transcriptional control in the age-related decline of CXCL1 expression in murine B cells.
The accumulation of MIP-2 and KC in tissues is known as an important event in early host defense against bacteria infection. Moreover, evidence indicates the MZ B cells are involved in the early stages of immune response against TI type 2 (TI-2) Ags derived from a number of encapsulated bacteria, including *Streptococcus pneumoniae*, *Neisseria meningitides*, and *Haemophilus influenzae*\[[@B44],[@B45]\]. MZ B cells generate an early IgM producing plasma response after *in vivo*stimulation with TI antigen \[[@B29]\]. The remarkable correlation between the ability of MZ B cells to mount an immune response against bacterial-associated antigens and our observation showing that these cells produce neutrophil-attracting chemokines MIP-2/KC in response to LPS indicate a potential dual role for MZ B cells in preventing host from bacterial infection. Of particular significance for the functionality of MZ B cells, we found that MZ B cell-derived MIP-2 and KC expression was impaired in aged mice. Although TI-2-specific Ab immune responses were not found to be significantly altered with age \[[@B46]\], the diminished capacity of aging immune system to mount an optimal antibody response to encapsulated microbes could be attributed, at least in part, to the diminished capacity of lymphocytes to express inflammatory cytokines and chemokines, such as MIP-2 and KC. Alterations in CXCL1 chemokine production by aged B cells may also have implications in the secondary recruitment of granulocytes to local immune responses at peripheral sites or even within secondary lymphatic organs such as the mucosal immune system in the aged host.
Murine MIP-2 and KC exhibit similar expression patterns and functional activities to that of human IL-8 in inflammatory response. For example, it has been previously shown that a significant increase of IL-8 or MIP-2 in human and mice, respectively, occurs in the grain dust-induced inflammation of the lower respiratory tract \[[@B47]\]. Although age-related alterations in IL-8 production by human B cells have not yet been described, diminished expression of IL-8 has been observed in cultured IL-2-stimulated NK cells \[[@B25]\] and LPS-stimulated monocytes \[[@B27]\] derived from elderly subjects. These data suggest that an age-related MIP-2 or IL-8 production could be consequence of a defective functional activity of B cells in aging.
It should also be noticed that human IL-8 has been shown to be a potent chemoattractant for human B cells \[[@B23],[@B24]\]. In the present study, we demonstrate that chemotactic activity of culture supernatant from young B cells was dramatically higher than that from aged B cells (Fig. [6A](#F6){ref-type="fig"}). Neutralization of these chemokines effects with addition of anti-MIP-2 and anti-KC Abs to the cultures resulted in a significant reduction of the migratory capacity of spleen cells to the culture supernatant from young, but not from aged B cells (Fig. [6B](#F6){ref-type="fig"}). This may be due to the low levels of MIP-2 and KC present in the culture supernatants of aged B cells that were not sufficient to induce significant migration of spleen cells to them. In addition, antibodies specific to KC and MIP-2 partially blocked the young splenic B cell-derived chemotactic activity toward splenocytes for approximately 20% and 5%, respectively, although high level of MIP-2 was present in the culture supernatants. The low ability of neutralizing anti-MIP-2 Ab to alter splenocyte migration suggests that MIP-2 produced by B cells may be a weaker migratory factor for murine splenocytes in relation to KC. As MZ B cells in spleen produce both MIP-2 and KC, one could hypothesize that these chemokines may facilitate B cell migration into and within the MZ area and/or amplify their activity and thus contributing to host defense.
Conclusion
==========
In summary, we demonstrate for the first time that murine splenic B cells are highly efficient in producing ELR-positive CXC inflammatory chemokines, in particular MIP-2, upon activation by LPS stimulation. Moreover, MIP-2 production, particularly by MZ B cells, was found to decline with age. Our finding suggests a possible linkage between functional activity of MZ B cells in production of neutrophil-attracting inflammatory chemokines and host defense. However, detailed and well-controlled *in vivo*studies will be necessary to assess these various possibilities and the significance of this CXCL1 production defect by aged B cells.
Methods
=======
Mice
----
Specific pathogen-free 3--5 months (young) and 24--29 months (aged) C57BL/6 mice were purchased through the Office of Biological Resources and Resource Development of the National Institute on Aging (Bethesda, MD). All mice were maintained in an AAALAC-certified barrier facility and were acclimated for 2 weeks prior to use. All mice were fed autoclaved food and water *ad libitum*. All mice with evidence of disease (e.g., enlarged spleen, gross tumors) were not utilized in these studies.
Preparation of splenic B cells
------------------------------
Splenic B cells were negatively selected via depletion of non-B lineage cells from spleen cells using a MACS system. Briefly, spleen cells were incubated with magnetic microbeads coated with anti-CD43 antibody and anti-CD11b antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 15 min, after which the cells were passed over a MACS apparatus. The purity of splenic B cells was consistently \>95% as routinely checked by FACS analysis.
ELISA analysis
--------------
Supernatants were collected from cultures and were then frozen at -80°C. The frozen supernatants were thawed at room temperature and chemokine levels were measured with commercial ELISA assay kits for MIP-2 and KC (R and D Systems, Minneapolis, MN) and immunoglobulins (Igs) (Bethyl, Montgomery, TX) according to the manufacturers\' instructions.
Immunofluorescence staining
---------------------------
Purified splenic B cells derived from young and aged C57BL/6 mice were cultured in the presence of absence of LPS (10 μg/ml) for 24 h at 37°C in 5% CO~2~. After culture, the cells were harvested, washed, fixed and permeabilized using 3.7% paraformaldehyde and 0.1% Triton X-100 for 15 min. After thoroughly washing these cells, non-specific binding sites were blocked using a 2% BSA solution containing 1% goat, rabbit serum and normal mouse IgG for 15 min at room temperature. Post incubation, these cells were incubated overnight at 4°C in presence of biotinylated mouse anti-MIP-2 and -KC antibodies (1 μg/ml) (R & D Biosystems, Minneapolis, MN). Streptavidin-conjugated Oregon green-488 (Molecular Probes, Eugene, OR) was utilized to label these cells the following day at a concentration of 1:250 for a period of 45 min at room temperature. After washing, cells were then labeled with PE-conjugated anti-IgM antibody (331,12; PharMingen) for 30 min., followed by the stains of cellular nuclei with DAPI (Molecular Probes, Eugene, OR) at concentration of 1 μg/ml for 10 min. These cells were subsequently placed into cytospin funnels and spun onto glass slides using a cytospin centrifuge (Shandon, Pittsburgh, PA) at 1200 rpm for 5 minutes. After being mounted in Immuno Fluor medium (ICN Biomedicals, Aurora, OH), images were acquired by Spot Advanced software on a Zeiss Axiovert S100 microscope under 100X objective (Carl Zeiss, Thornwood, NY).
RT-PCR analysis
---------------
For conventional RT-PCR analysis, total RNA was extracted from cells using the RNeasy Mini kit (Qiagen, Valencia, CA) and cDNA was prepared from 1 μg of total RNA transcribed by the SuperScript First-strand Synthesis system for the RT-PCR procedure (Invitrogen, Carlsbad, CA) according to the manufacture\'s instructions. The mouse MIP-2 and KC primers (Sigma Genosys, Woodlands, TX) utilized in these studies were: MIP-2 sense 5\'-TGCCGGCTCCTCAGTGCTG-3\' and MIP-2 antisense 5\'-AAACTTTTTGACCGCCCTTGA-3\'; KC sense 5\'-CGCTCGCTTCTCTGTGCA-3\'and KC antisense 5\'-ATTTTCTGAACCAAGGGAGCT-3\' as described previously \[[@B48]\]. The cycling conditions for PCR were 95°C for 4 min for denaturation, followed by 30 cycles at 95°C for 30 s, annealing at 57°C for 45 s plus extension at 72°C for 45 s and a final 10 min at 72°C. After 30 cycles of the PCR, 10 μl of the PCR products were separated on a 1.8% agarose gel, stained with ethidium bromide, and photographs were taken. Their densities were quantified by using the image-analysis system FluorChem (Alpha Innotech Corporation, San Leandro, CA) and normalized using β-Actin housekeeping gene in the same sample.
Real-time PCR analysis
----------------------
Approximately 1 μg of total RNA was reverse transcribed by using SuperScript First-strand Synthesis system (Invitrogen, Carlsbad, CA) as described above. Real-time primers for murine MIP-2 and KC were designed using Primer Express software (Applied Biosystems) using the sequences from GenBank (MIP-2, accession no. X53798; KC, accession no. J04596; and GAPDH, accession no. NM\_008084). Primers were constructed as follows: MIP-2 (forward primer, AGTGAACTGCGCTGTCAATGC; reverse primer, AGGCAAACTTTTTGACCGCC), KC (forward primer, TGCACCCAAACCGAAGTCAT; reverse primer, TTGTCAGAAGCCAGCGTTCAC), and GAPDH (forward primer, TGCATGGCCGTTCTTAGTTG; reverse primer, AGTTAGCATGCCAGAGTCTCGTT). Reverse-transcribed cDNA was amplified with primer sets for murine MIP-2, KC and GAPDH as indicated above using SYBR Green PCR core reagents and the GeneAmp 5700 Sequence Detection System (PE Applied Biosystems) following the manufacturer\'s instructions. No PCR products were generated from genomic versus cDNA template. Fold induction of mRNA was determined from the threshold cycle (*Ct*) values normalized for GAPDH expression and then normalized to the value derived from controls.
EL-4-based B cell culture
-------------------------
The splenic B cell culture was performed using the EL-4-based B cell culture system as previously described \[[@B38]\]. Briefly, individual wells of 96-well flat-bottom plates were loaded with 5 × 10^4^irradiated (5,000 cGy) murine EL-4 thymoma cells (clone B5) in 200 μl of RPMI 1640 medium supplemented with 10% FCS, 10^-5^ M 2-mercaptoethanol (2-ME), 25 mM HEPES buffer, penicillin (100 U/ml), streptomycin (100 μg/ml), 10 μg/ml LPS, and 10% supernatant from culture of J774A.1 macrophage cells (no. TIB-67; American Type Culture Collection, Manassas, VA). MACS purified splenic B cells or FACS sorted B cells were seeded directly onto a feeder layer of irradiated EL-4 cells and cultured at 37°C in 5% CO2. Moreover, we have previously shown that approximately 97% of wells contained sorted single cells using the same outfitted FACStar Plus. These results were verified based on the resulting sequencing histograms demonstrating evidence of only one V6 light chain sequence in the amplified cDNA \[[@B38]\]. Therefore, any differences in the levels of CXCL1 chemokine secretion among B cell subsets and age should not be a consequence of unequal number of cells sorted in the culture plates. The culture supernatants were collected on day 3 or day 5 and tested for chemokine secretion by ELISA. These time points were selected based on optimal cell viabilities and time to permit cellular activation and proliferation.
Flow cytometric analysis and cell sorting
-----------------------------------------
The monoclonal Abs utilized for the cell surface staining were FITC-anti-CD21 (clone 7G6; PharMingen, San Diego, CA), -anti-IgM (331,12; PharMingen), PE-anti-CD23 (PharMingen), APC-anti-IgM (clone II/41; PharMingen), and/or biotinylated anti-IgG1 (PharMingen). Biotin-labeled antibody binding was visualized using UltraAvidin-R-Phycoerythrin (Linco Technologies, St. Louis, MO). For each staining, 10^6^ cells were pre-incubated with the blocking antibodies, the anti-Fc receptor (24G2), for 30 minutes on ice and then incubated with a mixture of mAbs for an additional 15 min on ice. Post washing, the cells were subsequently incubated with PE-streptavidin for 15 min on ice after which the cells were washed with 5% FCS/HBSS. These stained cells were subsequently analyzed on a Becton Dickinson FACScan flow cytometer using CellQuest software.
The B cell subsets, NF, FO and MZ, were isolated through the use of cell sorting using the combination of anti-IgM-APC, anti-CD23-FITC and anti-CD21-PE mAbs. Single spleen cell suspensions were stained with the aforementioned Abs and the MZ, FO and NF B cells within the gate of IgM^+^cell population were sorted based on their differential expression of CD21 and CD23 using a FACStar Plus (Becton Dickinson, San Jose, CA). The purity of each sorted population was consistently \>95%.
Proliferation assay
-------------------
MACS-purified splenic B cells (1.25 × 10^5^/well) were cultured with 10 μg/ml of LPS for 1 and 3 days or FACS-sorted distinctive B cell subpopulations (1000/well) were cultured in EL-4 culture system for 3 and 5 days at 37°C in a 5% CO2. Cultures were pulsed with 1 μCi \[^3^H\] thymidine (NEN, Boston, MA) for the final 18 h. Cells were harvested on fiberglass paper. \[^3^H\] thymidine uptake was measured in a liquid scintillation counter (Beckman, Fullerton, CA).
Chemotaxis assay
----------------
In the lower chambers of Transwell plates (Costar, Cambridge, MA), 1.5 × 10^6^/ml purified splenic B cells derived from young and aged mice suspended in 600 μl of RPMI 1640 medium supplemented with 2% FCS, 10-^5^ M 2-mercaptoethanol (2-ME), penicillin (100 U/ml), streptomycin (100 μg/ml) were cultured in the presence and in the absence of LPS (10 μg/ml) and incubated at 37°C in 5% in CO~2~for 24 hr. After incubation, the cells and supernatants in the lower chambers were assessed for chemotactic activity using young splenocyte responder cells. These splenocytes were isolated from 4 month old C57Bl/6 mice after which they were preincubated with 10 μM Hoechst 33342 (Molecular Probes, Eugene, OR) in RPMI 1640 supplemented with 10% FCS for 30 min at 37°C. Subsequently, the Hoechst-stained splenocytes (1 × 10^6^) were washed with RPMI 1640 supplemented with 2% FCS, resuspended in 100 mL of medium, and then added to the upper chamber, containing a 6.5-mm diameter polycarbonate Transwell culture insert with 5 μm size pore. Each expected group was performed in duplicates with supernatants in the lower chambers for 8 to 10 h at 37°C. In certain assays, neutralizing antibodies specific for MIP-2 and KC at (1 μg/ml) were added to the low chamber containing the LPS-stimulated B cell supernatants. The transmigration of the Hoechst-labeled cells into the lower chamber were measured in a fluorescent spectrophotometer at 350 nm (excitation)/460 nm (emission). The results are expressed as fluorescent units or as the percentage inhibition of Hoechst-labeled splenocyte migration.
Statistical analysis
--------------------
Statistical evaluation of significance between the experimental groups was determined by Student\'s t test.
List of abbreviations
=====================
Ag, antigen; MIP-2, Macrophage inflammatory protein-2; ELR, glutamate-leucine-arginine; GRO, growth-related oncogene; HSV-1, herpes simplex virus type 1; KC, cytokine-induced neutrophil chemoattractant; MZ, marginal zone; FO, follicular; NF, newly formed; TI, Thymus-independent; TD, Thymus-dependent
Authors\' contributions
=======================
LH, VMC, and VDD performed the experiments. LH and DDT prepared the figures and wrote the paper. DDT also supervised the work and edited the manuscript. All authors have read and approved the final manuscript.
Acknowledgments
===============
We thank Drs. Ashani Weeraratna and Eric Schaffer for helpful discussions and for critical review of the manuscript, Bob Pyle and Ana Lustig for their technical assistances, and GRC Flow Cytometry Facility for their excellent cell sorting services.
| PubMed Central | 2024-06-05T03:55:46.658979 | 2004-7-26 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509242/",
"journal": "BMC Immunol. 2004 Jul 26; 5:15",
"authors": [
{
"first": "Lina",
"last": "Hu"
},
{
"first": "Vishwa Deep",
"last": "Dixit"
},
{
"first": "Valeria",
"last": "de Mello-Coelho"
},
{
"first": "Dennis D",
"last": "Taub"
}
]
} |
PMC509244 | Background
==========
The epidermis of the skin serves as a mechanical and water permeability barrier essential for terrestrial life (reviewed in \[[@B1]\]) and is composed primarily of epidermal keratinocytes. These keratinocytes stratify to form several layers. The deepest layer, the stratum basalis or basal layer comprises proliferating cells that continuously divide to regenerate cells lost to the environment. As the cells migrate upward into the first differentiated layer, the stratum spinousum or spinous layer, they cease proliferating and begin to express the intermediate filament proteins, the mature keratins 1 and 10. This early differentiation is followed by a late differentiation program in the stratum granulosum or granular layer, which is marked by the expression of other structural proteins, such as filaggrin and loricrin, and by increased activity of the enzyme, transglutaminase, which forms highly durable γ-glutamyl-ε-lysyl bonds to cross-link the proteins into a tough and resistant shell underneath the plasma membrane. At the boundary of the granular layer and the outermost stratum corneum, or cornified layer, the keratinocytes terminally differentiate, degrading their nuclei and other organelles and releasing lamellar bodies, the lipid contents of which form a water-impermeant barrier. The squames, the flattened remnants of the keratinocytes, and the lipids from the lamellar bodies form a sort of brick and mortar, to prevent water loss, microbial invasion and/or other mechanical insults (reviewed in \[[@B2]-[@B4]\]).
1,25-Dihydroxyvitamin D~3~\[1,25(OH)~2~D~3~\] is a known regulator of this process of keratinocyte growth and differentiation (reviewed in \[[@B2],[@B5]\]). *In vitro*, 1,25(OH)~2~D~3~inhibits keratinocyte proliferation and stimulates the expression of numerous keratinocyte differentiation markers (reviewed in \[[@B2],[@B6]\]). *In vivo*a physiologic role for 1,25(OH)~2~D~3~in regulating keratinocyte differentiation is suggested by several lines of evidence: (1) keratinocytes express both the 25-hydroxylase and the 1α-hydroxylase which converts inactive vitamin D~3~to its active 1,25-dihydroxy metabolite (reviewed in \[[@B2],[@B6]\]); (2) receptors for 1,25(OH)~2~D~3~are present in the skin and in epidermal keratinocytes *in vitro*\[[@B7]-[@B11]\]; and (3) Vitamin D receptor null mice exhibit altered skin function, characterized by abnormal hair follicles and reduced expression of several keratinocyte differentiation markers \[[@B12]\]. Furthermore, 1,25(OH)~2~D~3~and its structural analogs have been used as effective treatments for psoriasis, a human skin disease characterized by inflammation and by hyperproliferation and abnormal differentiation of keratinocytes (reviewed in \[[@B13],[@B14]\]).
8-Chloro-cyclic-adenosine monophosphate (8-Cl-cAMP) is known to inhibit growth and to induce apoptosis in a variety of cancer cells \[[@B15]-[@B18]\], suggesting its potential utility as an anti-cancer drug. Indeed, phase I trials with 8-Cl-cAMP have been performed (\[[@B19],[@B20]\] and reviewed in \[[@B21]\]) and phase II trials are in progress \[[@B22]\]. However, the mechanisms by which this agent acts are incompletely understood, and several investigators have proposed that an 8-Cl-cAMP metabolite, 8-chloro-adenosine (8-Cl-Ado) is the active anti-proliferative compound \[[@B16],[@B23]\]. Indeed, 8-Cl-Ado has been shown to inhibit growth in a variety of cell types \[[@B24]-[@B28]\].
Previously, we demonstrated that 8-Cl-Ado arrests the growth of primary mouse epidermal keratinocytes without triggering differentiation \[[@B29]\]. Thus, 8-Cl-Ado functions in an analogous fashion to transforming growth factor-β (TGF-β), which also triggers growth arrest, but not differentiation,, of keratinocytes (reviewed in \[[@B30]\]). In contrast with a polypeptide such as TGF-β, 8-Cl-Ado, as a small molecule rather than a protein, could potentially be taken orally or applied topically to skin. Thus, 8-Cl-Ado may represent a novel therapy for treatment of skin disorders, such as psoriasis, actinic keratoses and basal and squamous cell carcinomas, characterized by hyperproliferation of keratinocytes. One potential problem, however, is that TGF-β also inhibits the expression of differentiation markers elicited by other differentiating agents \[[@B31]\]. Since another characteristic typical of hyperproliferative skin diseases such as psoriasis is impaired differentiation \[[@B32]\], a therapy that inhibits both proliferation and differentiation would be less than ideal.
To determine whether 8-Cl-Ado, as a potent keratinocyte growth arrestor, could potentially be used to treat hyperproliferative skin diseases in combination with a current treatment, we investigated the effect of 8-Cl-Ado on 1,25(OH)~2~D~3~-induced inhibition of keratinocyte proliferation and stimulation of keratinocyte differentiation. We found that low concentrations of 8-Cl-Ado acted additively with 1,25(OH)~2~D~3~to inhibit DNA synthesis, without affecting the ability of 1,25(OH)~2~D~3~to enhance keratin 1 expression, a marker of early differentiation, or transglutaminase activity, a marker of late differentiation. Thus, our results suggest that a combination therapy with 1,25(OH)~2~D~3~and 8-Cl-Ado could potentially be an effective treatment for hyperproliferative skin disorders including psoriasis, actinic keratosis and non-melanoma skin cancers.
Results and discussion
======================
To determine if 8-Cl-Ado could function with the growth inhibiting agent 1,25(OH)~2~D~3~to enhance its antiproliferative effect, we incubated primary epidermal keratinocytes for 24 hours with various concentrations of 8-Cl-Ado in the presence and absence of low concentrations of 1,25(OH)~2~D~3~prior to assessing effects on de novo DNA synthesis as measured by \[^3^H\]thymidine incorporation into DNA. As shown in Figure [1A](#F1){ref-type="fig"}, 8-Cl-Ado inhibited \[^3^H\]thymidine incorporation at concentrations of 5--25 μM with an estimated half-maximal inhibitory concentration (IC~50~) of 5 μM. This value agrees well with our previously determined IC~50~of 7.5 μM \[[@B29]\]. In agreement with previous reports \[[@B33],[@B34]\], 1,25(OH)~2~D~3~also inhibited DNA synthesis at concentrations of 1 to 100 nM with an estimated IC~50~of approximately 4 nM (Figure [1B](#F1){ref-type="fig"}). As shown in Figure [2](#F2){ref-type="fig"}, when the two agents were combined, their effect on DNA synthesis appeared to be additive, as evidenced by the comparable slopes of the \[^3^H\]thymidine incorporation curves at the three different concentrations of 0 (a portion of which is replotted from Figure [1](#F1){ref-type="fig"}), 1 and 10 nM 1,25(OH)~2~D~3~. The combination of 1 or 5 μM 8-Cl-Ado with 10 nM 1,25(OH)~2~D~3~yielded a greater inhibition than 8-Cl-Ado alone, and conversely, the combined effect of 5 and 10 μM 8-Cl-Ado with 1 nM 1,25(OH) ~2~D~3~was significantly larger than 1 nM 1,25(OH)~2~D~3~alone. Importantly, the combination of 10 nM 1,25(OH)~2~D~3~with 10 μM 8-Cl-Ado produced an inhibition of \[^3^H\]thymidine incorporation that was significantly greater than that elicited by either agent alone. Indeed, the inhibition elicited by 10 μM 8-Cl-Ado and 10 nM 1,25(OH)~2~D~3~was comparable to the inhibition produced by 100 nM 1,25(OH)~2~D~3~alone (compare Figures [1B](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}). Thus, our results suggest that not only does 8-Cl-Ado [not]{.underline} prevent the growth inhibitory action of 1,25(OH)~2~D~3~, but, in fact, the two agents seem to act in an additive fashion to more effectively inhibit keratinocyte proliferation.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**8-Cl-Ado and 1,25(OH)~2~D~3~Inhibit Keratinocyte Proliferation.**Near-confluent primary mouse epidermal keratinocytes were treated with the indicated concentrations of (A) 8-Cl-Ado or (B) 1,25(OH)~2~D~3~for 24 hours, and \[^3^H\]thymidine incorporation was determined as indicated in Materials and Methods. Data represent the mean ± SEM of five experimentsperformed in triplicate; \*p \< 0.05, \*\*p \< 0.01 versus the control value.
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**8-Cl-Ado and 1,25(OH)~2~D~3~Act Additively to Inhibit Keratinocyte Proliferation.**Near-confluent primary mouse epidermal keratinocytes were treated with the indicated concentrations of 8-Cl-Ado in the presence of no (closed circles), 1 nM (open squares) or 10 nM (open triangles) 1,25(OH)~2~D~3~for 24 hours, and \[^3^H\]thymidine incorporation was determined as indicated in Materials and Methods. Data represent the mean ± SEM of five experiments performed in triplicate; \*p \< 0.05, \*\*p \< 0.01 versus the control value, †p \< 0.01 versus the corresponding concentration of 1,25(OH)~2~D~3~alone, §p \< 0.01 versus the corresponding concentration of 8-Cl-Ado alone.
:::
:::
TGF-β, another agent that, like 8-Cl-Ado, induces growth arrest but not differentiation of keratinocytes (\[[@B31]\] and reviewed in \[[@B30]\]), can inhibit the ability of differentiating agents to elicit keratinocyte differentiation \[[@B31]\]. However, for an agent to have therapeutic potential as a treatment for hyperproliferative skin disorders, such an inhibition of differentiation would be counterproductive to its efficacy as a medication. To determine if 8-Cl-Ado also inhibited keratinocyte differentiation, we investigated whether 8-Cl-Ado inhibited the ability of 1,25(OH)~2~D~3~to induce the late differentiation marker, transglutaminase activity. For this experiment we chose the concentrations of 8-Cl-Ado (10 μM) and 1,25(OH)~2~D~3~(10 nM) shown in Figure [2](#F2){ref-type="fig"} to produce a greater growth inhibition than either agent alone. As illustrated in Figure [3](#F3){ref-type="fig"}, 10 μM 8-Cl-Ado alone had little or no effect on transglutaminase activity, as reported previously \[[@B29]\]. On the other hand, 10 nM 1,25(OH)~2~D~3~significantly elevated transglutaminase activity by approximately 75%. The combination of 8-Cl-Ado and 1,25(OH)~2~D~3~was not significantly different from 1,25(OH)~2~D~3~alone, with a significant approximate 60% increase relative to the control value. Thus, our results indicate that 8-Cl-Ado did not prevent the differentiative effect of 1,25(OH)~2~D~3~, suggesting that these two agents might be combined to treat keratinocyte hyperproliferative disorders, such as psoriasis.
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**8-Cl-Ado Has No Effect on 1,25(OH)~2~D~3~-Stimulated Transglutaminase Activity.**Near-confluent primary mouse epidermal keratinocytes were treated with and without 10 μM 8-Cl-Ado in the presence and absence of 10 nM 1,25(OH)~2~D~3~for 24 hours, and transglutaminase activity was determined as indicated in Materials and Methods. Data represent the mean ± SEM of four experiments performed in triplicate; \*p \< 0.01 versus the control value.
:::
:::
Transglutaminase activity is a marker of late keratinocyte differentiation. We also examined the effect of 8-Cl-Ado on a marker of early keratinocyte differentiation, namely keratin 1 protein expression, using an even higher concentration of 8-Cl-Ado (25 μM). Western analysis demonstrated that 1,25(OH)~2~D~3~induced an approximate 45% increase in keratin 1 protein levels with the combination of 1,25(OH)~2~D~3~and 8-Cl-Ado producing a comparable 46% increase (Figure [4](#F4){ref-type="fig"}). Thus, early differentiation in response to 1,25(OH)~2~D~3~also was not affected by 8-Cl-Ado. Interestingly, however, in contrast to previous results \[[@B29]\], in these experiments 8-Cl-Ado alone elicited a small but significant increase in keratin 1 protein expression (32%). The reason for this disparity is unclear but may result from differences in the lot of anti-keratin 1 antibody used in the western analysis and/or the increased sensitivity of the method used for detecting and quantifying immunoreactive protein in this work.
::: {#F4 .fig}
Figure 4
::: {.caption}
######
**8-Cl-Ado Has No Effect on the 1,25(OH)~2~D~3~-Induced Increase in Keratin 1 Protein Levels.**Near-confluent keratinocytes were incubated for 24 hours with and without 25 μM 8-Cl-Ado in the presence and absence of 20 nM 1,25(OH)~2~D~3~and were then processed for western analysis. (A) A representative immunoblot is shown. (B) Keratin 1 levels were quantified, corrected for background and normalized for loading, as described in Materials and Methods. Data represent the mean ± SEM of four experiments performed in duplicate; \*p \< 0.05 versus the control value.
:::
:::
Most current treatments for psoriasis suffer from one or more disadvantages including lack of efficacy, contraindications due to deleterious side effects and/or aesthetic deficiencies (\[[@B35]\] and reviewed in \[[@B36]\]). Indeed, monotherapies tend to be less efficacious than combination therapies with two or more agents used concurrently, sequentially or in a rotational fashion (reviewed in \[[@B36]\]). Treatment with 1,25(OH)~2~D~3~and its analogs has proven successful, although the possibility of toxicity as the result of 1,25(OH)~2~D~3~\'s ability to affect calcium metabolism has led to the search for topically effective analogs with little or no effect on serum calcium levels (reviewed in \[[@B32]\]). If the amount of 1,25(OH)~2~D~3~(or its analog) required for treatment could be reduced, this decrease in dosage would presumably minimize systemic effects on calcium, which is the primary dose-limiting factor in the use of 1,25(OH)~2~D~3~analogs in the treatment of psoriasis \[[@B32]\]. Thus, our results indicating that 8-Cl-Ado enhances the growth inhibitory effect of 1,25(OH)~2~D~3~, a known keratinocyte differentiating agent and possible treatment for psoriasis \[[@B32]\], suggests the potential for combination therapy. Moreover, the fact that 8-Cl-Ado does not interfere with the promotion of differentiation by 1,25(OH)~2~D~3~further supports the possible combined use of these two agents for treatment of hyperproliferative skin disorders.
Several lines of evidence suggest that 8-Cl-Ado is not simply acting through cyototoxicity to inhibit keratinocyte growth. First, we have previously shown that 8-Cl-Ado growth arrests keratinocytes in the G~0~/G~1~phase of the cell cycle with no increase in the sub-G~0~/G~1~(apoptotic) population of cells \[[@B29]\]. Second, we also showed that the effect of 8-Cl-Ado to inhibit proliferation is reversible in that washout of the compound returned DNA synthesis essentially to basal (untreated) levels \[[@B29]\]. Finally, in this report we demonstrate that 8-Cl-Ado did not inhibit the 1,25(OH)~2~D~3~-stimulated increase in transglutaminase activity (Figure [3](#F3){ref-type="fig"}) or keratin 1 protein expression (Figure [4](#F4){ref-type="fig"}). Together, these results indicate that 8-Cl-Ado is acting in a specific manner to decrease keratinocyte proliferation.
Nevertheless, the mechanism by which 8-Cl-Ado exerts its growth inhibitory effects in keratinocytes is not clear. Our previous results indicate that 8-Cl-Ado must enter the cells to trigger growth arrest, since inhibiting uptake with an adenosine transporter, NBTI, prevented the arrest in the G~0~/G~1~phase of the cell cycle \[[@B29]\]. We also reported in a prior publication that 8-Cl-Ado induced the expression of the cyclin-dependent kinase inhibitor, p21 \[[@B29]\], which is known to contribute to growth arrest in keratinocytes and other cell types (\[[@B37]\] and reviewed in \[[@B30]\]). However, other investigators have reported 8-Cl-Ado-mediated inhibitory effects on RNA synthesis and the levels of cellular ATP \[[@B16]\]. Clearly, further research is necessary to define the pathways used by 8-Cl-Ado to regulate keratinocyte proliferation.
Conclusions
===========
In summary, our data show that 8-Cl-Ado functions with the keratinocyte-differentiating agent 1,25(OH)~2~D~3~to inhibit keratinocyte proliferation without altering the ability of 1,25(OH)~2~D~3~to induce differentiation. Thus, our results support the possibility of using 8-Cl-Ado alone or in combination with differentiating agents such as 1,25(OH)~2~D~3~or its analogs to treat hyperproliferative keratinocyte disorders including psoriasis.
Methods
=======
Materials
---------
Tissue culture reagents were obtained from standard suppliers as indicated in a previous publication \[[@B29]\]. 1,25(OH)~2~D~3~was a generous gift of Dr. Maurice Pechet (Research Institute for Medicine and Chemistry, Cambridge, MA). 8-Cl-Ado was obtained from Biolog (La Jolla, CA). \[^3^H\]Thymidine and \[^3^H\]putrescine were purchased from Dupont/NEN (Boston, MA). Dimethylated casein was obtained from Sigma (St. Louis, MO). All other reagents were from standard suppliers.
Keratinocyte culture
--------------------
Primary cultures of mouse epidermal keratinocytes were prepared from neonatal ICR CD-1 mice and cultivated in a 25 μM calcium-containing serum-free keratinocyte medium as in \[[@B29]\].
Measurement of DNA synthesis
----------------------------
For measurement of \[^3^H\]thymidine incorporation into DNA, as in \[[@B29]\], near-confluent cultures were refed with SFKM containing various concentrations of 8-Cl-Ado with or without different concentrations of 1,25(OH)~2~D~3~. After 24 hours, cells were labeled with 1 μCi/ml \[^3^H\]thymidine for an additional hour in the continued presence of 8-Cl-Ado and/or 1,25(OH)~2~D~3~. Cultures were washed twice with phosphate-buffered saline without calcium or magnesium (PBS^-^) and macromolecules were precipitated using ice-cold 5% trichloroacetic acid (TCA). After additional washing with 5% TCA and distilled water, cells were solubilized in 0.3 M NaOH, and the amount of \[^3^H\]thymidine incorporated into DNA was determined by liquid scintillation counting.
Measurement of transglutaminase activity
----------------------------------------
Transglutaminase activity was assessed essentially as described in \[[@B33]\]. Briefly, near-confluent keratinocytes were incubated for 24 hours with the indicated agents in SFKM. The cells were scraped into homogenization buffer (0.1 M Tris-acetate, pH 7.8, 2 μg/ml aprotinin, 2 μM leupeptin, 1 μM pepstatin A, 0.2 mM EDTA and 0.2 mM PMSF), collected by centrifugation and subjected to one freeze-thaw cycle prior to disruption by sonication. Aliquots of the homogenate were removed for determination of protein content and transglutaminase activity. Transglutaminase activity was measured as the \[^3^H\]putrescine radioactivity incorporated into casein after an overnight incubation at 37°C. Casein was precipitated with TCA, collected onto glass fiber filters and counted by liquid scintillation spectrometry. The cellular protein content of the samples was determined using the Bio-Rad DC protein assay system (Bio-Rad, Hercules, CA), with BSA as standard, and transglutaminase activity was expressed as cpm/μg protein.
Western analysis of keratin 1 protein levels
--------------------------------------------
Keratinocytes were treated and solubilized in sample buffer (31.2 mM Tris, pH 6.8, 1% SDS, 12.5% glycerol). Equal sample volumes were separated by SDS polyacrylamide gel electrophoresis on an 8% gel and transferred to Immobilon PVDF membrane (Millipore, Billerica, MA). Membranes were blocked with Odyssey blocking buffer (Licor Biosciences, Lincoln, NE), probed with a rabbit polyclonal anti-keratin 1 antibody (Covance, Princeton, NJ) and a mouse monoclonal anti-actin antibody (Sigma, St. Loius, MO). Immunoreactive proteins were visualized with IRDye800-coupled donkey anti-rabbit IgG (Rockland Immunochemicals, Gilbertsville, PA) or IR Alexa Fluor 680-coupled goat anti-mouse IgG (Molecular Probes, Eugene, OR) on a Licor Odyssey Infrared Imaging System. Keratin-1 protein levels were corrected for background and normalized using background-corrected actin levels.
Statistical analysis
--------------------
Significance of differences was determined with the computer program InStat (Graphpad Software, San Diego, CA) using ANOVA with a Student-Newman-Keuls post-hoc test.
Abbreviations
=============
1,25(OH)~2~D~3~, 1,25-dihydroxyvitamin D~3~; 8-Cl-Ado, 8-chloro-adenosine; 8-Cl-cAMP, 8-chloro-cyclic-adenosine monophosphate; IC~50~, half-maximal inhibitory concentration; TGFβ, transforming growth factor-beta
Authors\' contributions
=======================
WBB conceived of the study, planned the experiments, analyzed the data and drafted the manuscript; XZ and SJ planned, conducted and analyzed the keratin 1 expression experiments.
Acknowledgements
================
The authors gratefully acknowledge Dr. Maurice Pechet for his generous gift of 1,25(OH)~2~D~3~. The authors also thank Ms. Sagarika Ray and Mr. Brian Shapiro for their expert technical assistance.
| PubMed Central | 2024-06-05T03:55:46.662956 | 2004-7-27 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509244/",
"journal": "BMC Pharmacol. 2004 Jul 27; 4:13",
"authors": [
{
"first": "Wendy B",
"last": "Bollag"
},
{
"first": "Xiaofeng",
"last": "Zhong"
},
{
"first": "Sarah",
"last": "Josephson"
}
]
} |
PMC509245 | Background
==========
Atrial fibrillation (AF) has been recognised as an important independent risk factor for thromboembolic disease, particularly stroke with which it is associated with a five fold increase in risk \[[@B1]\]. There are few data on the prevalence of AF in the United Kingdom. Local data derived from the Echocardiographic Heart of England Screening (ECHOES) study suggested a prevalence of AF in people over the age of 65 of 3.8% (95% CI: 2.5--5.1) \[[@B2]\]. A review of four large community based studies of AF suggested that the overall community prevalence in the United States is 0.89% \[[@B3]\]. In these studies, the prevalence increased sharply with age: 2.3% of people aged 40 or over; 5.9% of people aged over 65 (higher than the local estimate), and 10% of those over 80. The vast majority (84%) of people with AF are over the age of 65. AF is a particularly important risk factor for stroke in the elderly -- while 15% of all strokes are associated with the arrhythmia, it is associated with 36% of strokes in people over the age of 80. The incidence of new cases of AF in people over the age of 65 is of the order of 1% per annum \[[@B4]\].
Screening for AF in the elderly fulfils many of the Wilson-Jungner criteria for a screening programme \[[@B5]\]. It is a common and important condition which can be diagnosed by means of a simple test, and the risk of serious sequelae such as stroke can be dramatically reduced by treatment.
One UK study has compared systematic nurse-led screening with prompted opportunistic case finding for AF in primary care \[[@B6]\]. This small scale study (four practices, n = 3001) demonstrated that systematic nurse-led screening detected more cases than opportunistic case finding, however most of those cases detected were already diagnosed. Two further single practice based studies have investigated the role of practice nurses in the screening process \[[@B7]\], and whole population screening \[[@B8]\]. 5% of total NHS expenditure can be attributed to stroke, and there would be expected to be about 1,000 new cases of stroke per annum in a typical health authority of a half million population. Therefore, any programme that might lead to an important reduction in stroke incidence needs serious consideration, both because of the potential for health gain, and the potential for reduced overall NHS expenditure. Screening for AF might be one such programme since, in population terms, AF is an important risk factor for stroke and anticoagulation provides a highly effective treatment to reduce this risk. A meta-analysis of randomised controlled trials has shown a 68% relative risk reduction in patients\' with AF receiving oral anticoagulation \[[@B9]\]. It has been estimated that optimal treatment of AF in the population might reduce the overall incidence of stroke by 10%. However, before implementing screening programmes, unresolved questions over how the screening should be conducted must be answered.
The appropriate screening strategy to be employed
-------------------------------------------------
### Opportunistic screening
The simplest strategy was *opportunistic case finding*, where a health care professional took the opportunity to feel a patient\'s pulse during a consultation. If the pulse is irregular, they might make a clinical diagnosis of AF, or request/perform an electrocardiogram (ECG) as a confirmatory test. However, opportunistic case finding is likely to miss a significant proportion of people who would otherwise have benefited from treatment. For example, detection of hypertension in general practice was traditionally detected in an opportunistic way until the introduction of health checks with the 1990 GP contract. The Health Survey for England shows that in 1991, 42% of the population over the age of 75 had hypertension for which they were not taking any medication \[[@B10]\]. This figure had fallen to 31% by 1994, after the GP contract had taken effect.
### Targeted screening
One possible approach was to screen patients who are at higher risk of AF -- a *targeted screening programme*. Cardiac failure, hypertension and rheumatic heart disease are important precursors of AF \[[@B7]\]. AF is more common in people with a history of myocardial infarction, angina, diabetes mellitus, hyperthyroidism, stroke or transient ischaemic attack (than in people without these conditions) \[[@B11]\]. Most general practices were computerised, and some have disease registers. A targeted screening programme could exploit these to identify such high risk patients, either through disease registers, or through prescribing information on the computerised records.
### Whole population screening
Another approach was to screen everyone 65 and over (65+) for AF -- a *whole population screening programme*.
A modelling exercise using decision analysis to inform on the methodology for this study indicated that there were not sufficient primary data available to recommend which of these (targeted or whole population) would be the optimum policy
### The most appropriate screening test for AF
12-lead ECG is recognised as the gold standard test, but this test is time consuming (taking at least 15 minutes to perform in an outpatient setting). Therefore, it is important to consider simpler tests. This study assessed simpler methods compared to the gold standard, both in terms of accuracy, time taken and patient acceptability. These include taking the pulse, and simpler ECGs.
### Interpreting the ECG
Cardiologists offer the most accurate readings of ECGs, but can satisfactory interpretations be obtained by the GP, the practice nurse, or computerised diagnostic software? This study assessed the accuracy of these different approaches to interpreting the ECG.
### The value of echocardiography
The main treatment options to reduce risk of stroke in patients\' with AF are currently warfarin or aspirin. Aspirin is much less effective than warfarin -- it achieves a barely significant 21% reduction in stroke risk \[[@B9]\]. However, it is safer to use, since it confers a lower risk of serious haemorrhage. Therefore, in practice, the clinical decision as to which treatment to use depends upon the balance of risks and benefits for the individual patient. Thromboembolic risk is currently determined primarily on clinical criteria. Data from the SPAF study \[[@B12]\] suggested that echocardiography (Echo) may inform on risk stratification, assisting in therapeutic decision making. The role of routine Echo for patients with AF identified in the community remains to be proven. Data also needs to be quantified regarding the cost effectiveness of Echo versus clinical impression alone. Studies have suggested that the clinical utility in people aged over 74 is poor \[[@B13],[@B14]\]. Therefore this study focused on patients aged 65--74. Once somebody has been identified as having AF, should they also receive an echocardiogram to assess their risk of stroke, or is clinical assessment of risk adequate?
### Optimum strategy
This study, by providing answers to these questions, allowed the optimum strategy for introducing a screening programme for AF in the over 65s to be determined. However, before a decision is made as to whether to institute a screening programme, not only must the question of the best strategy be considered, but also, the question of whether any screening programme at all should be introduced. This study provided data to assist in answering this fundamental question by providing:
i\) An accurate estimate of the community prevalence and incidence of AF in over 65s;
ii\) An assessment of the health economic implications of screening for AF;
iii\) An assessment of the service provision implications of implementing such a programme;
iv\) An assessment of the impact on patient quality of life and anxiety after various screening methodologies.
### Health economics of screening
Although the cost effectiveness of different approaches to screening is often put in terms of the average cost per case detected, such an approach ignores the sensitivity and specificity of the screening test. This is because average cost per case detected focuses entirely on true positives, paying no attention to false positives, false negatives and true negatives. False positives and false negatives impose costs on patients and health services which would be neglected if the focus was confined to true positives \[[@B15]\]. An undue emphasis on the average cost per case detected could justify opportunistic screening of a small number of high risk patients who present, with no consideration of the number of cases missed.
This study compared the incremental cost per case detected for different methods of AF screening. This refers not to the average cost but rather approximates the incremental cost per case detected in moving from one of the screening options to another. Use of incremental cost per case detected by option shows how the cost per additional case detected is likely to increase as the intensity of screening increases. This method has been used to deal with similar uncertainties about the cost effectiveness of screening for other diseases, including breast and colorectal cancer and has been recommended by the US guidelines \[[@B16]\].
Objectives
----------
### Primary objective
• To determine baseline prevalence and the incidence of AF based on a variety of screening strategies and in doing so to evaluate the incremental cost-effectiveness, in terms of cost per case identified, of the different screening strategies (targeted or whole population screening) compared with routine clinical practice for detection of AF in people aged 65 and over.
### Secondary objectives
• To evaluate the relative cost-effectiveness of screening methods for AF diagnosis, comparing 12 lead ECG (gold standard) with pulse taking, lead II rhythm strip from standard ECG limb leads alone and single lead thoracic placement ECG.
• To evaluate the most cost-effective method of test interpretation, comparing cardiologist (gold standard), with GP, practice nurse, or computerised diagnostic software.
• To assess the differing combinations of screening strategies and procedures in terms of patient acceptability and impact on patient quality of life, including any psychological effects of screening.
• To determine the community prevalence of AF in people 65+.
• To evaluate the value of clinical assessment and echocardiography as additional methods of risk stratification for thromboembolic disease in patients with AF.
• To evaluate the service provision implications should screening for AF become a national programme, and identify the optimum screening algorithm for identification of patients with AF.
Outcome measures
----------------
### Primary outcome
• The incidence of AF according to a variety of screening strategies
• The associated costs providing an incremental cost per case detected. The cost data was collected from an NHS and patient perspective. It has focused on resources required to establish screening, time taken to complete screening and the cost of the equipment.
### Secondary outcomes
• Cost effectiveness of 4 different methods of screening for AF. The cost data focused on the difference in the cost of the equipment and the time taken for each of the different methods of screening to be completed. This was from both an NHS and patient perspective.
• Cost effectiveness of 4 different methods of ECG interpretation. The cost data focused on the difference in the cost of the grade of staff interpreting the ECG and the accuracy of their interpretation.
• Overall community prevalence and incidence of AF
• Patient acceptability to AF screening was measured using an adapted version of the screening specific questionnaire used in the Colorectal Screening Programme \[[@B18]\]. Patient uptake of screening was also monitored. The impact on quality of life was assessed using EQ-5D \[[@B24],[@B25]\]. Patient anxiety was measured using the Spielberger 6 item Anxiety Questionnaire \[[@B17]\].
• Modelling techniques were used to identify the implications of AF screening on health service provision nationally. This included the effect on echocardiography and anti-coagulation clinic provision.
Methods
=======
This was a multi-centre randomised controlled trial. The study schema is shown in figure [1](#F1){ref-type="fig"}.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Study schema for the multi-centre randomised controlled trial.
:::
:::
50 computerised general practices within the West Midlands were recruited through MidReC (Midlands Research Practices Consortium). This was undertaken by writing to all practices in the West Midlands and surrounding counties explaining the study and asking whether they were interested in participating. Practices showing an interest were given further information about the study and invited to attend an investigators meeting. Following the investigator meetings sixty practices interested in participating in the project were randomised (stratified based on Townsend score and practice list size): 25 as intervention, 25 as control practices with 10 reserve practices.
A computerised list of all patients aged 65+ was obtained from each practice, and from this a random sample of 10,000 patients from the intervention practices (representing approximately ^1^/~3~^rd^of the total population of patients 65+ in this group), and 5000 from the control practices (representing approx. ^1^/~6~^th^of the total population of patients 65+ in this group) were identified.
Patients from intervention practices were randomised, by patient, to opportunistic or systematic groups. All patients within the systematic screening arm, including those with a history of AF, were invited by post to attend a screening clinic.
For patients in the opportunistic arm, their notes were flagged within the practice to encourage practice staff to undertake pulse recording. Patients with an irregular pulse were invited to attend a screening clinic. Once this process had been undertaken, the flag was removed from the notes and returned to the research team.
The screening clinic was run by practice nurses. Patients gave informed consent. Data collected was baseline information, past medical history (including any previous diagnosis of AF), radial pulse and a 12 lead ECG. The 12 lead ECG was performed using an electronic ECG machine which allowed print-out of single lead thoracic placement ECG and a rhythm strip of lead 2 using limb leads from standard ECG.
All 12 lead ECGs were sent to two cardiologists for reporting (GL, MD). Where there was disagreement over the diagnosis a third cardiologist was used to decide. The cardiologists were asked to state whether the ECG showed AF or not, and to state whether there were any other significant abnormalities. Patients were informed of the result within two weeks. Patients with normal ECGs were informed of this, patients with any abnormality were asked to make an appointment with their GP. At the GP appointment patients with AF aged 65 -- 74 were offered echocardiography. GPs were asked to make a clinical decision as to thromboprophylaxis both before and after the echocardiogram. Patients with other ECG abnormalities were managed as clinically indicated.
At the end of the screening process, GPs and Practice Nurses from both intervention practices (who had received education on ECG interpretation) and control practices (who had received no education) were sent ECGs to interpret for the presence or absence of AF. All ECGs recorded within the study were printed off as either 12 lead, single lead thoracic placement or limb lead recordings. Allocation to ECG type was random and resulted in three equal ECG groups. In order for each interpreter to read all three types of ECG, batches of ECGs were collated with the same numbers of each type of ECG. Allocation to a batch was also random. In total, there were 25 batches of ECGs to match the number of practices in each arm. The GP and Practice Nurse from the same practice read the same batch of ECGs and each batch was read by one control practice and one intervention practice. Therefore each ECG was read by two GPs and two Practice Nurses. All ECGs were anonymised, and practices did not receive any ECGs from their own practice. The interpreters were given a sheet to fill in to indicate for each ECG the presence or absence of AF. All ECGs (as 12 lead) were also analysed by the specific software package accompanying the electronic ECG and results recorded.
Patient acceptability and quality of life for different screening strategies were established using EuroQol (EQ-5D) combined together with the Speilberger 6-item Anxiety Questionnaire. EQ-5D allowed the measurement of broad aspects of quality of life. The shortened Speilberger anxiety questionnaire also has proven validity and is more specific to anxiety than is the SF-12 \[[@B17]\]. An adapted version of the screening-specific tool used in the Colorectal Screening Programme \[[@B18]\] was used to assess the acceptability of the screening process.
A random sample of 750 patients (375 screened patients and 375 opportunistically screened patients) were sent postal versions of the psychological instruments (EQ-5D and Spielberger) on entry to the study (i.e. before the intervention group has received their invitation to attend for screening). One reminder was sent a month later to non-responders. The same questionnaires were sent to the same groups plus those patients who had screened postive at the end of the screening period, approximately 17 months later. This allowed a non-randomised comparison between the effects on quality of life and anxiety in screen positive and screen negative patients.
In addition, all patients who were screened were asked to complete the acceptability and Spielberger questionnaire immediately after screening. The patient acceptability questionnaire was also administered to all patients who proceeded for echo.
The value of clinical assessment and echocardiography in risk stratification were determined in patients aged 65--74. This compared GP assessment based on the Birmingham guidelines for thromboprophylaxis in AF with any changes in recommendations for treatment once echocardiography results were available to the GP.
Sample size and power calculations
----------------------------------
The assumptions for the power calculations were that patients aged 65 and over represent 17% of the total population; that 40% of study population will be in the high risk group.
Also assuming that:
1\. Minimum worthwhile change in detection rate was 1% for targeted screening versus routine practice. It is estimated that this change would equate to £10,000 per life year gained. This is based on the following assumptions:
a\) 60% of new cases of identified AF would be suitable candidates for warfarin
b\) Annual risk of stroke in this population was 5%, reduced by 60% to 2% if treated
c\) Costs: £25 to screen a patient; £100 to treat with warfarin pa; £6,000 NHS costs to treat a stroke
2\. 50% of patients with AF will be already known to their general practitioner (estimates range from 30% \[[@B19]\] to 76% \[[@B20]\])
3\. Community prevalence of AF in this population was 6% \[[@B2]\]
See figure [1](#F1){ref-type="fig"}. It was assumed that the baseline prevalence of AF known to the practice (A1) would be 3% (i.e. half of real prevalence of 6%) and that the prevalence of known AF in the control practices would remain constant over the screening period. Thus, the change in the prevalence of known AF in the control practices between baseline to follow up (C2-C1) should be approximately 0%. The change in the GP educated arm (B2-A1) should be marginally higher and is assumed to be between 0 and 1%. The change in the systematic screening arm should, on average, be between 0 and 3% and was assumed to be approximately 2% for the total screening arm (A3-A1) and in the high risk arm (A2-A1) was approximately 3%.
All sample size calculations were for 90% power and 5% significance levels unless otherwise stated.
*a) To detect a 1% difference in detection rate between intervention (GP educated) and control practices (B2-A1) vs (C2-C1).*This requires 1,236 patients. However, since this is a difference based at the practice level of randomisation, it needed to be inflated by the design factor. Based on AF prevalence data from the EcHoES (Echocardiographic Heart of England Screening) Study \[[@B2]\], the between practice variance is 3.7 and the within practice variance is 246. This gave an intra-cluster correlation coefficient of 0.015. The most efficient design in this circumstance would be a cluster size of 200, which gives a design factor of 4. Therefore, 5,000 patients would be needed in 25 practices in both intervention (GP educated) and control groups.
*b) To detect a 1% difference in detection rate between intervention (Systematic screening **total** arm) and control practices (A3-A1) vs (C2-C1)*. This requires 1,236 patients but when scaled by the design factor of 4 required 5,000 patients.
*c) To detect a 1.8% difference in detection rate between intervention (Systematic screening **high risk** arm) and control practices (A2-A1) vs (C2-C1)*. This requires 684 patients. However, since this is a difference based at the practice level of randomisation, it also needed to be inflated by the design factor. This meant that 2,736 patients would be needed in each arm. Since the ratio of patients in the two arms is 2:5 this means that 1,916 patients would be needed in the high risk arm and 4,789 in the control arm. With the 2,000 patients expected to be at high risk in this arm -- resulting from the 5,000 needed for the previous comparison there were more than enough patients to detect the required difference.
Although comparison b) required fewer patients to detect the expected difference (2%) stated in the assumptions, it would be possible to detect differences as low as 1%, should the detection rate not be as high as expected.
The a), b) and c) comparisons are all at practice level randomisation.
*d) To detect a 1% difference in detection rate between high risk screening strategy and routine practice prompted by education (opportunistic arm) (A2-A1) vs (B2-A1).*This requires 1,236 patients in both the high risk systematic screening and the GP educated (opportunistic) screening arms of the intervention practices assuming the high risk screening detects a 1% increase and opportunistic screening detects 0% increase. Should the increased detection rates be higher in each arm (1.7% in the high risk arm and 0.7% in the opportunistic arm) then this could require 2,686 patients in each arm. However, since there is a ratio of 2:5 patients in these arms there will be sufficient patients as only 1,880 are needed in the high risk arm and 4,700 in the opportunistic arm to be able to detect this 1% difference*.*
e\) To detect a 1% difference in detection rate between total screening strategy and routine practice prompted by education (opportunistic arm) (A3-A1) vs (B2-A1).This requires 3,300 patients in both the total screening and the GP educated (opportunistic) screening arms of the intervention practices.
*f) To detect a relative risk (RR) of 2 (1% detection rate difference) between total population and high risk screening (A3 vs A2).*It was assumed that 40% of the study population fall into the high risk group, and the prevalence of undetected AF is 3%. This meant that 1,434 patients would be needed in each of the two risk groups to detect a two fold difference in risk (i.e. RR of AF in high risk as compared to low risk group is 2). This RR of 2 equates to an increase in AF detection rate from 3% in the total population arm to 4% in the high risk arm. Since there was a 40:60 split in the two risk groups unequal sample size calculations only require a minimum of 1,200 patients in the high risk group and 1,800 in the moderate/low risk group. This was achievable with a screening arm of 5,000 patients, as there would actually be 1,320 in the high risk group and 1,980 in the moderate risk group if a 66% screening acceptance rate was assumed.
Sample size for quality of life assessment
------------------------------------------
Although some of the variances are from North American populations we have no reason to suspect that the variation will be different in a British population since data from the ECHOES study on the SF36 gives variations very similar to the North American norms.
Spielberger
-----------
The shortened (6-item) version of the Spielberger state anxiety questionnaire has been validated and used in populations different from that under consideration in SAFE, namely it tends to have been used in young and mostly female populations \[[@B17],[@B21],[@B22]\]. The variance obtained from these papers appears to be approximately 144 for Marteau \[[@B17]\] but higher for the Ubhi \[[@B22]\] paper. However, the women in the latter paper were being informed of major illness outcomes (either benign or malignant breast cancer). A full Spielberger on people undergoing physiological tests also gave a variance of the order of 144 \[[@B23]\]. The full version of the Spielberger state anxiety when used with an elderly population also seems to give a variance that is not too far from the previously mentioned papers being 188.8 \[[@B23]\]. Taking this latter value as being the nearest to our population we can detect a 4 point difference in the mean values obtained with 249 patients in each arm.
EQ5D
----
### The VAS scale
The VAS variance as reported for an elderly population aged 75 and over was 365 \[[@B24]\] but for a group of recovered stroke patients (ages not given) it was approximately 100 \[[@B25]\]. Taking the former value as a worst case this means it will be possible to detect a 6% difference between groups on the VAS with 213 patients within each group.
### The Utility index
This was reported in different ways in the Johnson \[[@B24]\] and Dorman \[[@B25]\] papers. Using the utility values from the Dorman et al paper the variance is approximately 0.066 and this allows us to detect a 0.1 difference with 139. Using the Johnson paper the variance is approximately 576 and using this means that we can detect a mean change of 7 with 247 patients in each arm.
Statistical analysis
--------------------
Intention to treat analysis will be used.
Any previously known Atrial Fibrillation cases will be subtracted from the totals obtained at the end of the study to ensure there is no double counting in the incidence figures.
Chi-squared, independent t-tests and log-linear models will be used to describe demographic data. If there are differences between the groups this may need to be adjusted for in later analyses.
Primary objective: to determine baseline prevalence and incidence of AF on a variety of screening strategies
------------------------------------------------------------------------------------------------------------
Proportions and rates will be used as the measures of prevalence and incidence.
The independent t-test and ANOVA with random effects (as appropriate) will be used to examine the detection rate differences between the intervention and control screening strategies. Should the data be strongly non-normal a non-parametric equivalent will be used.
Secondary objectives
--------------------
a\) to assess patient acceptability and impact on QoL of different screening strategies
The independent and related t-test and ANOVA will be used to examine the differences between the intervention screening strategies on the Spielberger and EuroQol EQ5D. Should the data be strongly non-normal a non-parametric equivalent will be used. Chi squared tests will be used on the screening tool.
b\) to assess the value of echocardiography in risk stratification for thromboembolic disease in patients with AF
McNemar\'s test will be used to see whether there is any significant change in the doctor opinion on risk of CVA and treatment decision before and after echo screening.
c\) to evaluate the most cost effective method of test interpretation
The cost effectiveness will be covered in the economic section. However, the use of sensitivity, specificity, Cohen\'s κ and conditional logistic modelling will allow for comparison of the various methods for detecting AF between the GPs, nurses and consultants.
d\) to evaluate the most cost effective method of screening
This will be covered in the economic analysis section.
Multivariate and logistic modelling analyses will be undertaken in order to determine which markers might be the best predictors of the presence of AF. This will act as confirmatory analysis for the risk factors used in the screening strategy to define a high risk patient.
Economic analysis
-----------------
### Framework for the economic analysis
This trial evaluated a large number of alternative screening scenarios for identifying atrial fibrillation (2 screening strategies i.e. target v population; 6 screening methods i.e. pulse plus 3 types of ECG if pulse abnormal, and 3 types of ECG regardless of pulse; and 4 screening test interpretations, making 2\*6\*4 = 48 plus control and opportunistic screening = 50). The study has been powered to detect a difference in the targeted versus population arms, and in systematic screening versus routine clinical practice. However, the economic analysis will compare the cost-effectiveness of all alternative screening approaches using a modelling framework, whereby data will be drawn from the trial (where appropriate and available) and from external sources.
The use of a modelling approach allows the timescale for the economic analysis to be extended beyond the follow-up period allowed for in the trial. The use of such extrapolation will enable estimation of the incremental cost-effectiveness ratios (ICERs) for each approach:
Incremental cost per case detected
Incremental cost per life year gained
Incremental cost per quality-adjusted life year (QALY) gained
### Data on consequences
The use of EQ-5D allows the measurement of broad aspects of quality of life. EQ-5D allows changes in health status to be measured but also valued, using the University of York Measurement & Valuation of Health general population survey tariff \[[@B27]\].
### Cost data
The cost analysis adopted a broad perspective to include costs incurred within the health sector and by patients and carers. Data collection was undertaken on all trial patients in order to allow a stochastic cost analysis to be conducted. The focus of the data collection will be upon the key cost drivers which will include:
a\) the resources required to establish screening (invitation to patient, follow ups, communication of results),
b\) the time taken to carry out the various tests, and
c\) the cost of the equipment (expressed as cost per test).
a), b) and c) will be based on data collected in the study.
The analysis adopted an incremental approach such that data collection concentrated on resource use differences between alternative screening scenarios. The process of collecting data on resource use was undertaken separately from data collection on unit costs. Resource use data on the screening process was principally collected within the trial. Unit costs were collected from published sources and a representative sample of NHS providers in order to increase generalisability. The methods used in collecting data will include patient questionnaires (see above) and review of patient records (both GP and hospital). Data on private costs were collected from a survey of a sub-cohort of the trial population.
### Cost effectiveness analysis
The plan for the analysis is:
1\. Report a cost consequence analysis, which will involve providing a full description of all important results relating to costs and consequences.
2\. Conduct both a cost-effectiveness analysis and a cost utility analysis (using data on true positive cases detected as the measure of effect and data on EQ-5D to estimate QALYs).
An incremental approach will be used in order to compare the large number of alternative screening strategies.
We are interested in comparing the mean costs per patient since our concern is with predicting overall programme costs. However, the data on costs are likely to have a skewed distribution. Therefore, the plan for the analysis of costs is:
1\. To explore the nature of the distribution of costs
2\. If required, to use non-parametric comparison of means (e.g. bootstrapping)
3\. If the distribution of the data is approximately normal, parametric methods will be used.
This approach is in line with recent recommendations \[[@B26]\].
If missing data are a problem at the economic analysis stage, then imputation techniques will be employed.
Longer term costs and consequences will be explored by extrapolating beyond the end of the trial using a modelling framework using data from a range of trial and non-trial sources. The precise form of modelling is yet to be determined, but is likely to be either Markov or Discrete Event Simulation, depending upon the extent to which the Markov assumptions are justified. An advantage of using such an approach is that it will allow the additional costs of increasing survival to be explicitly incorporated into the analysis. In particular, modelling will provide estimates of the optimal frequency of screening for AF, based on the estimates of incidence and prevalence from the trial.
Identifying untreated patients with AF will have implications for service provision. These will depend upon the prevalence of atrial fibrillation, the screening mechanism employed, the use made of echocardiography, and the additional requirements for anticoagulation monitoring. The separate parallel study, BAFTA, will provide empirical data that will allow such implications for service provision to be assessed. The modelling exercise, which will draw on both SAFE and later BAFTA results, will combine best estimates of both screening and anticoagulation options.
The robustness of the results of the economic analysis will be explored using sensitivity analysis \[[@B27]\]. This will explore uncertainties in the trial based data itself, the methods employed to analyse the data and the generalisability of the results to other settings. Uncertainty in the confidence to be placed on the results of the economic analysis will be explored by estimating cost-effectiveness acceptability curves. These plot the probability that the intervention is cost-effective against threshold values for cost-effectiveness.
Inclusion and exclusion criteria
--------------------------------
### Inclusion criteria
Patients aged 65 years or over (65+).
### Exclusion criteria
Patients who were terminally ill.
### Randomisation
Randomisation of practices and patients was performed by statisticians from the Department of Primary Care and General Practice at The University of Birmingham. Cluster randomisation of practices to intervention or control was stratified by Townsend quartiles and practice size. Computer searches were carried out to identify cases of known AF, within the sample of patients identified above, using a published strategy \[[@B15]\]. The randomisation of patients within the intervention practices ensured that the study patients in each practice were divided equally between systematic and opportunistic screening arms and also that there was an even distribution of patients with known AF between the two arms. Patients within the systematic screening arm were identified by computerised record searching as either being at high risk (target population) or moderate risk (non-target population) of AF by recognised criteria \[[@B28],[@B11]\].
### Cleaning of lists post randomisation
Following initial sampling of the total population the list of patients from each practice were returned to the practices who were asked to remove any patients who had died, moved or were terminally ill. Patients removed following this process were replaced with patients from a reserve list, which had been randomised at the same time as those on the initial lists.
### Patient information and consent
Patients aged 65+ who were selected to the systematic screening arm received an information sheet with an invitation letter to attend the ECG clinic. Entry to the trial was discussed with the practice nurse at the clinic. The practice nurse then obtained written consent from those patients who were willing to participate.
Study patients found opportunistically to have an irregular pulse were given an information sheet and invited to attend the screening clinic.
### Practice staff education and ECG training
GPs and other members of the primary health care team in the intervention practices attended investigator days at which they were given educational materials informing them of the importance of detection of AF, and the treatment options that are available. The materials encouraged them to consider opportunistic screening of patients.
Members of the primary health care team in control practices received no educational input from the research staff.
Practice nurses attended an ECG training day prior to starting the ECG screening clinics. Training included how to perform an ECG (using the Biolog) to ensure a standardised high quality tracing and basic ECG interpretation (specifically how to identify AF).
Computerised and note searches of GP records
--------------------------------------------
### Prevalence and incidence data
Computer searches were carried out to identify cases of probable AF in the 15,000 study patients using a published strategy \[[@B15]\]. Searches were tailored towards the information that is held on computer in each practice. If practices hold AF registers, or use READ diagnosis coding, then these were used. In addition, a search was carried out to identify prescriptions of digoxin, a beta-blocker, a class 1,3 or 4 anti-arrhythmic agent, aspirin or warfarin. This information was recorded into computerised case report forms.
Case notes of patients identified as \'known\' or \'probable\' AF in any of these computer searches were reviewed for mention of a diagnosis of AF. AF diagnosis were drawn from hospital letters stating the existence of the condition or ECG recordings from the last 5 years.
An additional 5% random sample of case notes of patients not identified as \'known\' or \'probable\' AF by computer searching were reviewed (750 in all) to estimate how many other patients who are known to have AF were not identified by the computer searches. If this had revealed a significant number of extra cases of known AF, then the sample size for manual searching would have been increased to allow a precise estimate of the baseline rate of known AF. Unidentified extra AF cases were not found to be significant so no additional note search was required.
The same computer searches on both intervention and control practice patients notes were performed prior to, and 12 months after, commencement of screening.
### Screening clinics
All patients in the systematic screening arm and those found to have an irregular pulse in the opportunistic screening arm of the study were invited to attend an ECG screening clinic. At the clinic the practice nurse explained the aims of the study and answered any questions about the study. Written informed consent to participation in the study was obtained from the patient. The nurse then recorded baseline information on age, sex, present smoking and alcohol status and past medical history, including previous diagnosis of AF, and any treatment the patient may be receiving for AF. Radial pulse rate, and whether regular or irregular, was noted. A 12 lead ECG, the gold standard by which other traces were compared, was then recorded using the Biolog machine, which was also able to produce a trace corresponding to the single lead thoracic placement and a rhythm strip of lead II. Finally, the patient was asked to complete an acceptability questionnaire.
Discussion
==========
This study will identify the most cost-effective strategy for identifying atrial fibrillation in patients aged 65 and over. The policy implications will be dependent on the findings and one of the strengths of the current study is the utilisation of modelling techniques to investigate the implications of different screening strategies and frequency of screening within different health care environments. The initial draft of the report has been submitted to the Department of Health and publication of the results should be expected later this year (2004).
Competing interests
===================
None declared.
Authors\' contributions
=======================
RH and DF were principal investigators. EM and SJ were project managers. DS collected data and prepared the first draft of the paper. JM, EM, JR, SB, MD and GL all contributed to the project design. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2261/4/12/prepub>
Acknowledgements
================
Funding: NHS R&D HTA programme, Grant number: 96/22/11
DA Fitzmaurice is funded by an NHS Career Scientist Award
ET Murray is funded by an MRC Health Services Research Fellowship
| PubMed Central | 2024-06-05T03:55:46.664835 | 2004-7-29 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509245/",
"journal": "BMC Cardiovasc Disord. 2004 Jul 29; 4:12",
"authors": [
{
"first": "Dawn",
"last": "Swancutt"
},
{
"first": "Richard",
"last": "Hobbs"
},
{
"first": "David",
"last": "Fitzmaurice"
},
{
"first": "Jonathan",
"last": "Mant"
},
{
"first": "Ellen",
"last": "Murray"
},
{
"first": "Sue",
"last": "Jowett"
},
{
"first": "James",
"last": "Raftery"
},
{
"first": "Stirling",
"last": "Bryan"
},
{
"first": "Michael",
"last": "Davies"
},
{
"first": "Gregory",
"last": "Lip"
}
]
} |
PMC509246 | Background
==========
Warfarin therapy is an effective anticoagulant indicated for the prophylaxis and/or treatment of venous thrombosis and atrial fibrillation (AF), the most common cardiac arrhythmia in older individuals \[[@B1]\]. Oral anticoagulation with warfarin is known to reduce the risk of disabling stroke; indeed, the benefits of oral anticoagulation have been demonstrated in a number of systematic reviews, providing high evidential support for prophylaxis \[[@B2],[@B3]\]. Published guidelines on the management of AF emphasize the importance of warfarin therapy for the prevention of stroke \[[@B4]\]. Likewise, there is convincing evidence that long-term warfarin therapy is a highly effective method of preventing recurrent venous thromboembolism \[[@B5]\].
Despite the strong evidence base and the endorsement of warfarin therapy by authoritative guidelines, current prescribing patterns of warfarin remain something of a puzzle. Bungard and associates have described the \'real world\' use of warfarin therapy as \"sub-optimal\" \[[@B6]\] and have estimated that only 15% to 44% of patients eligible for anticoagulation are actually prescribed warfarin \[[@B7]\]. Hart concurs that the use of warfarin is poor, noting that \"it is often given to patients who benefit minimally, while those patients who would benefit most are not treated\" \[[@B8]\].
Bungard and his colleagues recently conducted a systematic review of the reasons for the underuse of warfarin, identifying patient, provider, and system factors as well as identifying limitations in research studies and arguing for further research into these factors \[[@B6]\]. Studies employing trade-off methods to determine the risk/benefits threshold where therapy becomes acceptable have demonstrated that patients are more willing to assume risk when better informed about the medication \[[@B9]\]. Physicians, however, are reluctant to prescribe warfarin to elderly patients owing to concerns regarding compliance, a perceived risk of falls, and the lack of randomized controlled trial evidence in this patient population \[[@B7]\]. A recent study reporting the findings of interviews with individuals with a history of AF indicated that patients\' health beliefs and attitudes toward death play an important role in their decision-making \[[@B10]\].
Clearly, the decision to initiate warfarin therapy is a complex interaction of many variables, involving patient, provider, and system factors. Most studies examining anticoagulation practices in primary care have used surveys or other forms of quantitative methods; however, given the inherent complexity of this subject matter, it is likely that qualitative research methods could provide significant additional insight. A comprehensive literature search failed to find a qualitative investigation of patient perspectives and experiences taking warfarin. The aim of this study, therefore, was to employ qualitative methods to examine the experience and perspective of individuals on long-term warfarin therapy for atrial fibrillation as a means to assess the extent to which the physician-identified barriers reported by Bungard \[[@B6]\] are in concordance with the unique views of patients.
Methods
=======
Participants and setting
------------------------
This research was undertaken in a family practice clinic situated in a large, tertiary care teaching hospital. The clinic employs 12 physician full-time equivalents working in three teams; each four-physician team is supported by two registered nurses. The practice, which serves a medium-high socioeconomic status population, has a large proportion of elderly patients and the prevalence of AF is higher than reported elsewhere \[[@B1]\]. The prevalence of atrial fibrillation in our population as a whole is 3.9 percent. When considering different age groups, the prevalence rises as high as 18.2 percent and 18.5 percent for patients aged 80--89 and 90--99 years, respectively. A recent chart audit study found that the majority of eligible AF patients in the clinic (78%) are being treated with warfarin for stroke prevention \[[@B11]\]. Comprehensive anticoagulation services are provided and the clinic offers access to physicians on-call 24 hours a day. The nurses maintain an historical record of International Normalized Ratio (INR) results and warfarin dosage changes for each individual patient and, in consultation with the physicians, inform patients of prescribed warfarin dosage changes in a timely fashion, usually on the same day as the INR reading.
Potential participants were identified by the clinic nursing staff. The inclusion criteria stated that the patient must currently be on warfarin therapy and have been so for a minimum period of six consecutive months. Patients were excluded from the study if a significant co-morbidity prevented their participation, if they were unable to converse in English, or if they were unwilling/unable to provide informed consent. From a pool of approximately 60 eligible candidates, the nurses purposively sampled in order to achieve an even gender split as well as an equivalent number of patients who were both normally in-range and out-of-range on their INR tests. A total of 24 patients were invited to participate in the study. Three patients declined to take part, two of whom expected to be unavailable during the interview phase, whereas the third declined due to lack of interest. All participants signed informed consent forms in advance of their participation in the study, which was approved by the Research Ethics Board of the host institution. A demographic profile of the sample is presented in Table [1](#T1){ref-type="table"}. The mean age of participants was 74 years; there were 12 males, 9 females. The majority were both married (86%) and retired (86%). The mean length of time participants had been on warfarin therapy was 4.6 years (range = 1 year to 10 years).
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Demographic profile of participants
:::
**Code** **Age (years)** **Sex** **Marital Status** **Employment Status** **Years on Warfarin** **INR in Range?**
---------- ----------------- --------- -------------------- ----------------------- ----------------------- -------------------
P1 67 F Widowed Retired 5 Yes
P2 73 M Married Retired 10 Yes
P3 84 M Widowed Retired 5 Yes
P4 83 F Married Retired 4 No
P5 81 F Married Retired 1 Yes
P6 76 F Married Retired 3 Yes
P7 75 M Divorced Retired 2 No
P8 60 M Married Working 3 No
P9 79 F Married Retired 5 No
P10 67 F Married Retired 2 No
P11 76 M Married Retired 5 No
P12 80 M Married Retired 2 No
P13 53 M Married Working 8 No
P14 71 M Married Retired 3 Yes
P15 69 M Married Retired 4 Yes
P16 77 F Married Retired 5 Yes
P17 78 M Married Retired 1 No
P18 80 M Married Retired 10 Yes
P19 71 M Married Working 7 No
P20 71 F Married Retired 6 No
P21 82 F Married Retired 5 Yes
:::
Data collection and analysis
----------------------------
We utilized a semi-structured interview guide that was developed on the basis of salient issues identified in the scientific literature, specifically the various barriers to the prescription of warfarin for atrial fibrillation as reported by Bungard et al \[[@B6]\]. Interviewees were asked to share their experiences with warfarin in relation to four specific content areas or themes: decision-making, knowledge and education, impact on daily life, and patient satisfaction. Throughout the course of the interview, participants were provided several opportunities to raise issues or to describe experiences that had not been specifically addressed. Standard demographic information was also collected. Three of the authors (GCD, JM, and BT) shared the task of conducting the interviews. The protocol for assignment of each individual participant to an interviewing author ensured that there had been no previous clinical contact between the two parties; moreover, interviewers were blind to interviewees\' INR status. The interviews lasted an average of 45 minutes and took place either in the clinic (n = 13) or in participants\' homes (n = 8), in accordance with each participant\'s preference. Data collection ceased when, in the consensus of the research team, saturation had been reached; that is, no new ideas or perspectives were emerging. All interviews were audiotaped and transcribed verbatim.
Given the pre-determined nature of the themes, as described above, we employed a content analysis approach to the analysis of the interview data. Whereas grounded theory is used to develop data-induced themes or hypotheses, content analysis is the better-suited approach in those instances where the codes, categories, or themes of interest to the investigators have been previously discovered and described \[[@B12]\], as is the case in the present study.
Precise criteria were developed for each of the four pre-determined themes in the codebook, namely decision-making, knowledge/education, impact on daily life, and patient satisfaction. The 21 transcripts were then coded according to these criteria. Each transcript was coded by at least two members of the research team using a standardized coding form. Tests for inter-coder reliability indicated a high level of agreement among the coders; instances of disagreement were resolved through a process of discussion and negotiation that included both the fourth author and the principal investigator (CST & REGU). This process yielded a unit-by-variable matrix that allowed for substantive analysis of the data. In order to strengthen the validity of the findings, the analytic processes of coding and interpretation were reviewed by an independent external reader (DG).
Results
=======
Decision-making
---------------
The great majority of participants reported that the decision to initiate warfarin therapy had been made by \"the doctor\" -- a term that was used to refer not only to family physicians and general practitioners, but also to specialists and attending physicians in urgent care settings. Typically, there was little or no patient involvement in the decision-making process (Table [2A](#T2){ref-type="table"}). In most cases, this unilateral decision-making appeared to be related to the high level of trust that patients place in the medical expertise of physicians; indeed, the phrase \"doctor knows best\" was commonly-used in these accounts (Table [2B](#T2){ref-type="table"}). For a smaller number of participants, the specific circumstances surrounding the initial decision to commence therapy served to preclude any degree of significant involvement on their part (Table [2C](#T2){ref-type="table"}).
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Quotations: Decision-making
:::
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
***[A. Minimal patient involvement]{.underline}***
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
My decision \[to take warfarin\]? It was the doctor\'s decision. **(P5)**\
I had nothing to say \[regarding decision to initiate warfarin\]. If the doctor tells me something, I do it. **(P17)**\
Q: What influenced your decision to take the drug?\
A: Well, because I was told to.\
Q: The main reason is that it was the physician\'s recommendation?\
A: Yes. **(P20)**\
I don\'t recall him \[the physician\] saying anything much. He said a lot of things when he examined me first, and he put me up in the ward overnight, then he started with the medications. That\'s all there was to it\...Not really, no \[no much discussion on reasons to start warfarin\]. He just said that, \"*This is what medication we\'re going to put you on for the myopathy.\"*That was it. **(P11)**
***[B. Trust of physicians]{.underline}***
I just figured the doctor knows best\... **(P1)**\
A: No \[trouble to decide to take warfarin\], because I knew nothing about it. My doctor, as far as I know, is very competent so\...\
Q: So you are taking it basically because the doctor told you to?\
A: That\'s right. **(P6)**\
I\'m at this hospital and it\'s got a very good reputation\... Doctor knows best, I guess. They know exactly what you have to do for it, and they did it. **(P14)**\
I can recall that I had no objection. I said, \"*You are the experts, you are the doctors. If I get any help, I mostly will appreciate it*.\"\.... I don\'t think I would trust myself that much \[to make the right decision\]. **(P15)**
***[C. Constraining effect of circumstances]{.underline}***
When I went into the \[clinic\] to see my doctor, they admitted me to the cardiac emergency, and they kept me there all day \... I was in for just about a week. \... and when I was discharged the doctors explained that they were putting me on to certain medications, and Coumadin was one of them. **(P10)**\
I had congestive heart failure, that\'s what I was in hospital for. I don\'t know what I was on when I was in hospital, but when I came out I had a whole slew of medications and Coumadin was one of them. **(P5)**\
I had lymphoma, and then I had a bone marrow transplant for lymphoma, and I had my spleen taken out, and I started getting deep vein thrombosis. Then I had a pulmonary embolism at one point and they started me on it \[warfarin\] then\... I had just about every complication in the book, and this \[thrombosis\] was one of them. I think it was around that time, or within a year after the thromboses started, they gave me warfarin. **(P13)**\
The surgeon said I had to take it, basically. I don\'t like taking pills, so when I went in to get my one valve replaced, they gave me -- they persuaded me -- and I agreed to trying out something new that had just been approved. The reason for doing that was that I would not have to take Coumadin\... Unfortunately, however, one of my other valves blew when I was in there \[during surgery\], so I got two for the price of one, and then there was no question I had to go on a blood thinner. **(P19)**
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
:::
Knowledge and education
-----------------------
The level of knowledge and understanding of the benefits and risks associated with warfarin therapy tended to vary with age. Elderly patients (aged 75+) demonstrated poorer knowledge than their younger counterparts; indeed, the knowledge level among older participants appeared to be quite superficial and scattered (Table [3A](#T3){ref-type="table"}). Whereas elderly patients could not explain with any degree of exactitude the rationale for taking warfarin and the associated risks, for a subset of participants, most of whom were less than 75 years old, the knowledge level was higher and, for a small number, considerably higher (Table [3B](#T3){ref-type="table"}). Overall, less than half of our sample was able to name one specific benefit, risk, and lifestyle change/concern associated with warfarin therapy.
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Quotations: Knowledge and education
:::
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
***[A. Superficial level of knowledge]{.underline}***
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
I don\'t really know what these different pills do for me. **(P3)**\
I\'m assuming these people know what they\'re doing. They\'re not doing this for nothing. They must have good reasons, and they tell me, \"*Hang on there, you\'re doing all right. Keep it up.*\" So I do. I don\'t question them. Very little, if any. I probably wouldn\'t know what they were talking about if they started to explain it all, and what\'s the point of that? **(P12)**\
Q: What do you think Coumadin is doing for you and your health?\
A: It makes the blood sticky, I believe, or thins it. I really don\'t know.\
Q: Do you know why they added the Coumadin?\
A: No idea.\
Q: It doesn\'t much matter to you?\
A: It doesn\'t matter to me.\
Q: Everyone\'s different. Some people like to know all the details.\
A: Oh, I couldn\'t care less, just as long as it keeps me alive. **(P17)**\
I hope it\'s \[warfarin\] keeping everything under control\... Well, the stroke that I had, I don\'t feel sick, I don\'t have any pain, or anything. **(P21)**
***[B. Superior knowledge of risks and benefits]{.underline}***
A 72-year-old male has a 30 percent chance of having a stroke regardless, but if I didn\'t take the Coumadin, it would be a 70 percent chance of having one. So I\'m taking medication to avoid the stroke. **(P14)**\
Nobody really explained to me in full what Coumadin is all about, but I did some reading about it. I know it\'s a blood thinner, an anti-coagulant\... helps with the atrial fibrillation that I have, because apparently blood stays longer than it should in the atrium, and if it thickens it can go to your brain and you can have a stroke. **(P8)**
*[**C. Patient education**]{.underline}*
Q: When you started on the Coumadin, did you receive any education about the medication?\
A: Just that the doctor said to me that it\'s not 100 %, like anything else, but there\'s less chance \[of stroke\]. **(P16)**\
I said to the nurses once, \"*Supposing I stop taking it.\"*They said, \"*Oh, I wouldn\'t advise it, you know, because within a month, you\'d have the most severe stroke, or it would kill you, one or the other.\"*That scared me.**(P3)**\
Q: Do you remember if you received any educational material about Coumadin?\
A: No.\
Q: Or any talk about how it works, the benefits?\
A: Not that I can remember. I cannot recall that, no.\
Q: Any pamphlets, any coloured paper, anything?\
A: No.\
Q: You don\'t recall?\
A: If I did have, I read it, then I dismissed it\... I have an appetite, I can eat and drink, I can sleep, and I still can work. Anything else, to me, is not quite important. It\'s probably wrong. I should read them and pay more attention. **(P15)**\
Then he \[physician\] said, \"*If you go off the Coumadin for your operation, you could get a stroke. You\'ve got a choice: you can either go off the Coumadin or you can stay on it and bleed to death.\"*Not to death, he didn\'t say that. You\'d bleed. The other way, you could have a stroke. That\'s all he said. So I presume that it could happen. **(P9)**\
Q: Is there any sort of educational material that you would like to see on warfarin?\
A: No. I\'ve got a pile of books to read now, and as soon as I start to read, I fall asleep. The pamphlets would fare worse than the books. I don\'t think that would help. **(P12)**\
Q: Did you have many questions about it at the time \[when warfarin was initiated\]?\
A: No. You see, the darn trouble was that my wife would be sitting there, and I\'d say, \"*She knows what it\'s all about; tell her.\"*\... When you have a sit-in nurse, you know, I don\'t worry about that stuff \[getting education on the therapy\]. **(P3)**
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
:::
According to participants\' accounts, educational efforts aimed at informing patients about warfarin were minimal and insufficient (Table [3C](#T3){ref-type="table"}). Those who were able to recall some form of education typically referred to a \"booklet\" or \"sheet\" supplied by either the clinic or a pharmacy. In several cases, spouses were more knowledgeable than patients and appeared to play an important role in monitoring the regime. A number of participants lauded the availability of clinic staff to answer questions; however, two others reported the use of \"scare tactics\" by health care professionals with regard to the need to take warfarin.
Impact of warfarin regime
-------------------------
While there is tremendous range in the perceived impact of warfarin therapy on the lives of these patients, the vast majority reported that they have not experienced complications (e.g., hemorrhage, drug interactions). Typically, the decision to start taking warfarin did not precipitate significant changes in their day-to-day lives; many participants reported experiencing only minor inconveniences (Table [4A](#T4){ref-type="table"}). For these individuals, warfarin is just another pill to be taken everyday; many reported the use of some reminder strategy, such as calendars, dosettes (pill boxes), or taking the pill right before some regular activity, in order to avoid missing a dose. On the other hand, a sizeable proportion (25%) of interviewees reported that adhering to the warfarin regime does impact upon their day-to-day lives. From the perspective of these patients, who were more likely to have multiple co-morbid illnesses and/or were taking multiple medications, the warfarin regime presents a considerable struggle to be managed, particularly when dosages needed to be adjusted. Regular visits to the clinic, restrictions on diet and alcohol intake, and anxiety regarding bleeding and potential drug interactions counted among the most commonly cited impacts (Table [4B](#T4){ref-type="table"}). Only a small number of participants reported experiencing significant complications related to the warfarin regime, with one case involving repeated gastro-intestinal bleeding. These patients demonstrated a very high level of commitment towards their warfarin management and have placed the ritual of taking the medication at the centre of their daily routine (Table [4C](#T4){ref-type="table"}).
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
Quotations: Impact of warfarin regime
:::
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
***[A. Minor impact]{.underline}***
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Coumadin, it\'s just a matter of taking the pills each day and coming for a blood test and adjusting the dose. That\'s all. No other impact, as far as I\'m concerned. **(P13)**\
I had the test \[INR\] done before I went to Europe. I arranged it so that two days before we flew to Europe, I had it tested, and it was 2.3, I think\... The nurse said: \"*You might keep it this same way, and enjoy.*\" And as soon as I got back, I came in and had it checked. **(P15)**\
It\'s inconvenient having to come in every week, but on the other hand, I understand. **(P20)**
***[B. Moderate impact]{.underline}***
I will only drink one glass of wine a day. I like a glass of wine. They say just go easy on the single malt, and stuff like that\...There wasn\'t any special \[instructions regarding diet\]. We like good food, and we eat a good, balanced diet. I like seafood, and I love fish, and I like the odd steak. I try to stay off butter. I\'m taking Becel^®^just now, which I don\'t really like, but I try to stay off the butter and cooking with all the white sauce, and butter sauce, and stuff like that. **(P10)**\
I come here every 4 or 5 weeks to have the bloodwork done for Coumadin. Whenever I have this done, the nurse calls me that afternoon and says, \"S*tay on with the same milligrams\"*or \"*Change to that and that.\"*But I feel fine \[with this routine\]\... The only disadvantage of the Coumadin is the bruising and bleeding on just the slightest touch\... but if that\'s the worst that happens, I\'m not worrying about it. **(P11)**\
I\'m extremely careful with my alcohol intake, although as I said before, I\'m not an everyday drinker. Other than that, the only other thing is I started noticing I have experienced some hair loss. **(P8)**
***[C. Major impact]{.underline}***
Three years ago, I had two very serious stomach bleeds and I do not know, to this day, whether I should attribute it to Coumadin. Once I spent a couple of days in intensive care and then three months later, again a couple more days, this time in critical care. **(P11)**\
It isn\'t worth it to risk a stroke by going off the Coumadin to have the hernia fixed. So Coumadin has played a major part in my life, because this hernia is a daily fact I have to live with\... The fact that I\'m taking Coumadin means that if I want to be operated on, I have to be careful\... They \[the Hernia Clinic\] don\'t take guys like me that require a bit of time and skill and more facilities than they have. But what do guys like me do? **(P14)**\
The thing is, the last 558 times I\'ve taken it, which is right here \[referring to the records he keeps\], I went through this last night, and I found only three miscues the entire time I\'ve been taking it. That works out to 0.005 percent. It\'s a very, very low percentage of goofing taking it. I\'ve never forgotten. For at least two of these incidents, I took it at 10:00 or 10:45, instead of at 6:00. That\'s four hours late. I consider that a goof. Another time I took it in the morning instead of at night, which is a goof. **(P14)**
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
:::
Patient satisfaction
--------------------
The vast majority of interviewees reported a high level of satisfaction with the care they receive from the nurses and physicians in the clinic (Table [5A](#T5){ref-type="table"}). Most participants were also satisfied with the warfarin regime itself (Table [5B](#T5){ref-type="table"}). Only a very small number of participants expressed significant dissatisfaction. The sources of dissatisfaction, which tended to be highly localized to specific concerns, included the cost and inconvenience of attending the clinic for regular INR tests, a lack of information provided to patients, and insufficient awareness of patient history on the part of clinic staff (Table [5C](#T5){ref-type="table"}).
::: {#T5 .table-wrap}
Table 5
::: {.caption}
######
Quotations: Patient satisfaction
:::
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
***[A. Satisfaction with clinic staff]{.underline}***
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Oh, I am more than pleased. I\'m absolutely more than pleased. I think they\'re wonderful. The nurses are wonderful -- you know, taking my blood, and phoning me, and giving me instructions. **(P1)**\
I think they\'ve been 100 percent. From my cardiologist to the family physician and to the pharmacists, because they\'re just amazing. **(P10)**\
My doctor thought my blood was, I guess, too thick. They did an INR and recommended the Coumadin, which I didn\'t want to get on, because I knew it was warfarin and you associate that with rat poisoning. Anyway, she took the time and very patiently explained what the purpose of it was, and highly recommended it. I really like her. I like everybody there. They\'re very caring, supportive people. They\'re just dear. **(P4)**
***[B. Satisfaction with warfarin regime]{.underline}***
This is a pill that keeps your blood thin, and you have to check it out \[INR level\]. I just do as I\'m told and I\'m thrilled that they keep me at 2-point-something\....I go every week, or every other week, or once a month, depending on my stability. It doesn\'t bother me going. **(P1)**\
It\'s worked out well \[the regime\]. I know it has to be done, and I\'m lucky in the fact that it has regulated it. It has totally regulated itself -- I\'m taking 4 mg a day now. I\'m only coming in once a month. **(P18)**\
Oh, yes \[happy with warfarin regime\]. They let me know what the status is each week, whether it\'s going to level out, and whether I\'m going to be able to stay on the same dosage and then stop going up there every week. I started going every week, and now it\'s been levelled to every two weeks. **(P7)**\
I really couldn\'t say anything bad about it \[warfarin regime\]. Apparently I\'ve been outstanding in how steady I would go with it, and I\'ve been going -- normally, for years, I\'ve been going once a month, pretty well. A couple of times it would go up and I\'d come back in two weeks, or something. **(P2)**
***[C. Sources of dissatisfaction]{.underline}***
Nobody tells me anything. That\'s one of my problems with this whole bloody business. Nobody tells me how I\'m doing. All I know is that I\'m supposed to be between 2 and 3 \[INR levels\]. **(P7)**\
I just more or less come when I\'m ordered to. From home it\'s almost an hour on the bus each way, and the parking around here, the cost is wild. They must be financing the place with the parking. No, I would prefer not to come at all. I would prefer to forget the whole deal, but that doesn\'t seem to be in the offing at the moment. **(P12)**\
I haven\'t been coming here that long, about two years I think\... but I wouldn\'t say they\'re fully aware of my history and really understand the depth of it\... Considering my history, I think they should know more. They certainly don\'t have my files. **(P13)**
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
:::
Discussion
==========
The findings of this study provide significant and original insight into the perception and experience of patients taking warfarin. The data indicate that patients tend to have minimal input into the decision to initiate warfarin therapy; many have only a superficial level of understanding of the risks and benefits of warfarin; and the majority retain little from the education they received regarding warfarin therapy. This outcome is balanced, however, by the finding that for these patients there was both a high level of satisfaction with the care provided in the family practice setting and a low level of impact on their day-to-day lives.
The principal strength of this study is the insight into the lived experience of warfarin therapy as gleaned from the unique perspective of family practice patients currently taking warfarin. We view this as a significant and novel contribution to the literature as we could find no other such study. It is important to note, however, that our sample was drawn from the patient population of an academic primary care practice that is both well-educated and of medium-high socioeconomic status. The applicability of these findings in other patient populations may therefore be limited.
Interviewees revealed clear detachment from participation in the decision-making process around initiating warfarin therapy. In some cases, this detachment appeared to stem from the particular circumstances at the time; for instance, if the patient was involved in a medical emergency or was admitted to hospital and had several medications initiated. For others, there was a general belief or understanding that warfarin is a medication without which the patient faced imminent risk of death -- it is therefore not a matter to be discussed or negotiated. This finding may be a function of the age of our participants. As a group, elderly patients tend to prefer a directed rather than shared consultation. Prior research indicates that seniors are more likely to be accepting of medical advice without much questioning, rather than assuming a more active role in the decision-making process \[[@B13]\]. This attitude was reflected in participants\' comments that \"doctor know best\" and \"it\'s the doctor\'s decision.\"
Patient knowledge of risks, benefits, and issues related to diet and alcohol intake was low, although younger patients demonstrated greater levels of understanding than did those over age 75. This finding is consistent with previous investigations. Lip and colleagues have detected lower levels of knowledge among elderly patients; moreover, longer duration of anticoagulation does not appear to ameliorate patient understanding significantly \[[@B14],[@B15]\]. For the most part, we found that retention of instructions pertaining to the warfarin regime was poor. Many participants reported that they simply follow the nurses\' directions and have little interest in learning anything more. The limited knowledge and seemingly low level of interest to learn more could be attributable to the great deal of trust invested in the expertise of the clinic staff as discussed above. The fact that several spouses exhibited greater understanding of the risks and benefits associated with warfarin therapy has implications for educational interventions recognizing the importance of the spousal or care giver role in the monitoring of therapy.
With regard to the impact of warfarin therapy on daily life, our results indicate that warfarin is for the most part well tolerated and does not pose heavy additional burdens or lifestyle changes. The majority of participants were already taking several medications and visiting more than one doctor on a regular basis. This sample also had a low incidence of complications and previous studies have shown that the potential for complication does not on its own result in a significant impact \[[@B16],[@B17]\]. That is, the mere possibility of an adverse side-effect does not bring about substantial anxiety unless the complication is actually experienced. These results do not, however, capture the experiences of individuals who have tried and ceased taking warfarin for whatever reasons. This population represents a high priority for future study. Additional support may be required for individuals with multiple medical problems. Continuity of care and a strong relationship and identification with the primary care team that provides anticoagulation services can overcome potential miscommunication and misunderstanding regarding side effects and drug interaction.
The importance of the association between patient satisfaction and adherence has been established in prior research on anticoagulation. In a case-control study, Arnsten and colleagues \[[@B18]\] found that, among patients with a regular physician, the non-adherent cases were those who expressed dissatisfaction. In our sample, the level of patient satisfaction was high, both with the clinic staff and the warfarin regime itself. Based on participants\' testimonies, the coordination and continuity of care by a trustworthy team of doctors and nurses were key contributing factors to the high satisfaction ratings. In an evaluation of a telephone-based anticoagulation service, Waterman found that patient satisfaction with warfarin management was associated with the timeliness of receiving blood test results from the service provider \[[@B19]\]. The high level of patient satisfaction observed in the present study may also be due in part to the low rate of complications (e.g., hemorrhage, drug interactions), which may serve to reinforce patients\' trust both in the therapy and in the health care team.
Increasingly, theoretical models of the physician-patient encounter advocate the inclusion of patients in the decision-making process \[[@B20]\]. Of course, shared decision-making presupposes an understanding of the benefits and risks on the part of patients. With regard to warfarin therapy, patient preferences would be expected to vary according to expected benefits or awareness of risks of suffering a stroke. Man-Son-Hing et al have demonstrated that the minimal clinically important difference of warfarin therapy is often considerably smaller for patients than that identified by clinicians \[[@B21]\]. Protheroe and colleagues, in an observational study of patient-based decision analysis, noted marked disagreement between patient preferences and guideline recommendations \[[@B22]\]. A patient decision aid was shown to improve knowledge and understanding of the risks and benefits of warfarin for patients with atrial fibrillation, and aided in therapeutic choice \[[@B23]\]. Given the low level of patient knowledge observed in the present study and elsewhere, the vision of shared decision-making \[[@B24]\] remains an as yet unachieved, but laudable goal; indeed, the present results highlight the challenges of shared decision-making and increased autonomy in patients with complex chronic diseases.
Conclusions
===========
In summary, the results of this study suggest that patients tend to have limited input into the decision to initiate warfarin therapy. Moreover, a majority appear to lack a comprehensive understanding of the risks and benefits associated with treatment. These findings, however, were balanced by the minimal impact of warfarin on daily life and the high level of patient satisfaction. Further research is required to assess whether these findings are similar in other patient groups, with different demographic and socioeconomic characteristics, including multi-cultural communities \[[@B14]\]. Investigation of physician views of the underutilization of warfarin therapy would allow for a comparison of the patient and provider perspectives. Clearly, there is a pressing need for innovative methods of continuing patient education in order to communicate the risks and benefits of warfarin therapy in a friendly, non-threatening manner. Also, these results highlight the tremendous influence that care providers exert on the decision-making of patients. The development of decision aids for anticoagulation may help patients make more informed decisions \[[@B23],[@B25]\], but only if care providers know of their existence and take the time to use them, assuming that such tools are feasible in a busy clinical setting.
Competing interests
===================
None declared.
Author\'s contributions
=======================
REGU conceived and initiated the study and will act as guarantor. GCD, JM, and BT conducted the interviews. CST co-ordinated the data analysis process. All authors participated in the analysis of the data and the writing of successive drafts of the manuscript and all have read and approved the final draft.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2296/5/15/prepub>
Acknowledgements
================
We are indebted to our three peer reviewers - Drs. Malcom Man-Son-Hing, Maggie Somerset, and David Armstrong- for their comments and suggestions on an earlier draft of this paper.
This study was funded by research grants from Physicians\' Services Foundation Incorporated and Samcor/Sunnybrook Primary Care Research Trust. Dr. Upshur is supported by a New Investigator Award from the Canadian Institutes of Health Research and a Research Scholar Award from the Department of Family and Community Medicine, University of Toronto.
The authors would like to thank the 21 patients who volunteered to take part in this study and share their experiences with us. We would like to acknowledge the contribution of the nurses in the Family Practice Unit who referred the participants, and that of Dr. Denise Gastaldo who acted as an independent external reader. Special thanks also to Jennie Jones for transcribing the interviews and to Shari Gruman for formatting the paper.
| PubMed Central | 2024-06-05T03:55:46.668854 | 2004-7-21 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509246/",
"journal": "BMC Fam Pract. 2004 Jul 21; 5:15",
"authors": [
{
"first": "Guilherme Coelho",
"last": "Dantas"
},
{
"first": "Barbara V",
"last": "Thompson"
},
{
"first": "Judith A",
"last": "Manson"
},
{
"first": "C Shawn",
"last": "Tracy"
},
{
"first": "Ross EG",
"last": "Upshur"
}
]
} |
PMC509247 | Background
==========
The problem of chronic pain in the elderly
------------------------------------------
Chronic pain is a common problem in the elderly, and is often associated with significant physical disability and psychosocial problems \[[@B1]\]. Estimates of the prevalence of chronic pain problems among community-dwelling older adults range from 58--70% \[[@B1]\]. The most common painful conditions among older adults are musculoskeletal conditions such as osteoarthritis, low back pain, and previous fracture sites \[[@B2]\]. Chronic pain often results in depression, sleep disturbance, decreased mobility, increased health care utilization, and physical and social role dysfunction \[[@B1]\]. Despite its high prevalence, pain in the elderly often is inadequately assessed and treated \[[@B1]\].
As the United States population grows older, the public health problem of chronic pain and its sequelae will worsen. Projections show dramatic increases in this age group; approximately 25% of the population will be age 65 years or older in 2050. Moreover, by 2030 there will be an estimated 8 million people who are 85 years or older \[[@B3]\]. Thus, there is an urgent and growing need for interventions that are effective in decreasing pain, suffering, and pain-related disability in this group.
The role of self-management in the treatment of chronic painful conditions
--------------------------------------------------------------------------
There is substantial empirical evidence that attention to cognitive and behavioral factors, in addition to physiological factors, is necessary for the successful treatment of chronic nonmalignant pain \[[@B4],[@B5]\]. Empirically supported multimodal therapies that incorporate cognitive and behavioral strategies now exist for many chronic pain conditions, including rheumatoid arthritis, osteoarthritis, fibromyalgia, and low back pain \[[@B6]-[@B10]\]. These therapies aim to enhance the ability of patients to successfully self-manage their pain, using a variety of techniques. Related approaches and strategies are described under the rubrics \"cognitive-behavioral therapy\" (CBT), \"psycho-educational\" or \"educational,\" and \"self-management\" or \"self-help.\" Although there are variations among these approaches, they share some or all of the following components: education about pain, instruction in the identification and modification of negative thoughts, exercise, communication skills, relaxation training, and physical therapies. The goal of the therapies is to enhance function, improve mood, and decrease pain intensity by changing the emotional, cognitive, and behavioral responses to pain.
Despite their documented efficacy in young to middle-aged samples \[[@B9]-[@B12]\], cognitive-behavioral and self-management pain therapies have been little-studied in elderly populations. In one of the first examinations of CBT for elderly patients with pain, 69 outpatients with chronic pain were randomly assigned either to immediate treatment or delayed (wait list) treatment \[[@B13]\]. Approximately half of the sample was over 60 years of age, and age was unrelated to outcome. The intervention resulted in significant decreases in pain interference with daily activities and increases in participants\' self-reported ability to cope with pain. Limitations of this study included the fairly small sample size and the lack of intent-to-treat analysis.
Keefe and colleagues \[[@B14]\] evaluated the efficacy of a pain coping skills training (CST) intervention as compared with arthritis education and standard care in decreasing pain and physical and psychological disability among 99 middle-aged to older outpatients with osteoarthritic knee pain. The CST consisted of 10 weekly group sessions focusing on identifying and reducing irrational thoughts, diverting attention away from the pain, and changing activity patterns to manage pain. The CST group showed significantly less pain and psychological disability following treatment as compared with the other two groups. At 6-month follow-up, the CST group showed significantly less physical and psychological disability as compared with the education group and marginally less psychological disability as compared with the standard care group \[[@B15]\]. Although this study provides evidence for the benefits of cognitive-behavioral therapy for older adults, it focused on arthritis patients and not older adults per se. Moreover, the average subject age was 64 years. It is not clear whether these findings would generalize to mid-old (i.e., 75--85 years) and old-old (85 years and older) adults. These groups have been shown to differ from their younger counterparts (i.e., those 65--74 years) in several dimensions, including pain prevalence, physical and cognitive function, involvement in recreational and social activities, and social support \[[@B16]-[@B19]\], that potentially could affect the pain experience and response to pain therapies.
One study that examined a cognitive-behavioral therapy in old-old adults (mean age 77.2 years) evaluated the efficacy of a 10-week CBT intervention (n = 11) versus an attention/support (AS) condition (n = 10) for nursing home residents \[[@B20]\]. The CBT condition incorporated pain education, progressive relaxation, imagery, coping skills training, cognitive restructuring, and attention diversion. CBT participants reported significantly less pain and pain-related disability following the intervention, as compared to the AS group. These significant differences were maintained at the 4-month follow-up. This study provides important evidence that CBT can be successfully applied to old-old adults; however, the results need to be replicated in other, larger samples, including non-institutionalized elderly.
Retirement communities as a study setting
-----------------------------------------
As the U.S. population continues to age, retirement communities have gained popularity. A retirement community allows older adults with varying lifestyles and physical abilities to live in an environment that encourages independence while providing needed access to health and social resources \[[@B21]\]. Although most residents of these communities live independently (some facilities also include assisted living apartments and skilled nursing facilities), the retirement community, on average, represents a mid-old to old-old population that is vulnerable to physical disability, health problems, and social isolation \[[@B22]\]. The growing population in retirement communities, then, is one in which self-management group therapies for chronic pain may hold great promise. Adoption of regular wellness-oriented pain management strategies may contribute to enhanced functioning and prolonged independence.
Study purpose and specific aims
-------------------------------
The primary goal of this study is to evaluate the efficacy of a pain self-management group intervention (SMG), as compared with a control condition (BOOK), in decreasing physical disability, pain, pain-related interference with activities, and depression in older retirement community residents with chronic pain. In addition, we wish to determine the extent to which SMG participation is associated with changes in specific pain-related beliefs and coping strategies, and the extent to which changes in these process variables are associated with changes in outcomes (physical disability, pain intensity, pain-related interference with activities, and depression). We plan to test the following hypotheses:
*1.*At post-treatment and each follow-up, participants assigned to SMG, as compared with participants assigned to BOOK, will report less physical disability (primary outcome), and lower pain intensity, pain-related interference with activities, and depressive symptom severity (secondary outcomes).
*2.*Participants assigned to SMG, as compared with participants assigned to BOOK, will show greater pre- to post-treatment increases in self-efficacy and use of adaptive pain coping strategies and greater decreases in catastrophizing. Significant differences between SMG and BOOK groups in pain-related beliefs and coping strategies will be maintained at 6-month and 1-year follow-ups.
*3.*Pre- to post-treatment changes in specific pain-related beliefs (catastrophizing, self-efficacy) and coping strategies (Chronic Pain Coping Inventory subscales) will be associated significantly with changes in physical and social functioning, pain intensity, and depression over the same period among SMG participants. These changes in beliefs and coping strategies will be maintained at 6-month and 1-year follow-ups.
Figure [1](#F1){ref-type="fig"} depicts the hypothesized relationships among study variables.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Hypothesized relationships among study variables
:::
:::
Methods/Design
==============
Design
------
This is a currently ongoing randomized controlled trial. The study procedures and measures have been approved by the Swedish Medical Center institutional review board. Figure [2](#F2){ref-type="fig"} outlines study procedures and follow-up.
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Study flowchart
:::
:::
Participants
------------
Participants (targeted enrollment n = 273) are recruited from residents living in one of the 34 participating retirement communities in Seattle, Washington and the surrounding area. Study inclusion criteria are: (1) 65 years of age or older, (2) pain \> 3 months duration that interferes with regular activities; and (3) ability to read and complete study questionnaires in English. Exclusion criteria are: (1) current, active cancer and (2) surgery within the past 6 months or surgery planned in the next 6 months.
Recruitment and randomization procedures
----------------------------------------
Participants are recruited using newsletter announcements, flyers, brochures, and informational talks given at each facility. Retirement community residents who are interested in the study are screened to assess eligibility. Eligible residents who provide written informed consent are then asked to complete the baseline measures and to provide the name of their primary care provider (PCP), as well as permission to contact the PCP. PCPs of SMG group participants are sent a letter about the study and asked to contact the research nurse if there is any medical reason to restrict the resident\'s participation in the exercise portion of the study.
After all participants from a facility have completed the baseline questionnaires, the facility is randomized to receive either the BOOK or the SMG. Randomization is done by facility, rather than by individual participant, for several reasons. First, it expands the number of participating facilities by making feasible recruitment from smaller facilities. If approximately 5% of residents were recruited from any one facility, then it would not be scientifically or financially sound to involve facilities with fewer than 200 residents in independent or assisted living. A pilot study indicated that the ideal self-management group size is 5--12 participants. If we randomized within facilities, at least 10 participants would need to be recruited from each facility to allow 5 SMG participants. However, if all participants within a facility are randomized to the same condition, then smaller facilities can participate. If more than 12 residents in a facility randomized to the SMG condition enroll in the study, more than one group is scheduled. A second advantage of randomization by facility is that there is little risk of participants in one condition talking with participants randomized to another condition about their experiences in the study. Thus, there is less treatment contamination and likelihood that participants from different conditions will compare treatments, a situation that can provoke dissatisfaction among participants who do not receive the treatment of their choice.
Pain self-management group (SMG)
--------------------------------
The SMG intervention, which consists of seven weekly 90-minute group sessions, includes the major components of empirically-supported self-management interventions \[[@B23],[@B24]\], refined for use with the elderly (see Table [1](#T1){ref-type="table"}). For example, we include a discussion of myths about pain in older adults (e.g., pain is an inevitable part of aging) and focus on exercises that are effective and safe for older adults with musculoskeletal pain. The intervention is designed to decrease participants\' physical disability and pain intensity; increase participation in home, social, and recreational activities; and enhance participants\' self-efficacy for managing chronic pain. To accomplish these objectives, the intervention provides basic information about pain management, teaches problem-solving and relaxation skills, and provides practice with a variety of pain management techniques. Participants receive a class syllabus, relaxation tape, Theraband^®^tubing for the performance of selected exercises, and two hot/cold gel packs.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Summary of the self-management group intervention
:::
**SESSION NUMBER**: TOPICS MAJOR CONTENT AND ACTIVITIES
----------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Session 1**: Introduction; Basic principles of pain Review purpose of the program/study. Review definition, types, & mechanisms of pain. Discuss myths about pain in older adults. Emphasize goals of chronic pain management. Discuss signs/symptoms that require medical attention. Introduce problem-solving techniques for pain management.
**Session 2**: Role of exercise & physical activity in pain management Discuss exercise in pain management: problem of de-conditioning, types of exercise, tips for starting exercise program. Demonstrate & practice specific exercises. Introduce relaxation and breathing techniques as effective pain management strategies. Practice progressive muscle relaxation & abdominal breathing.
**Session 3**: Engaging in pleasant, meaningful activities; pacing activities Discuss ways in which chronic pain may be limiting participation in enjoyable or meaningful activities Use problem solving to develop individualized plans for increasing these activities. Discuss strategies for activity pacing and rationale for avoiding guarding and inactivity. Practice relaxation.
**Session 4**: Challenging negative thoughts; Dealing with pain flare-ups and setbacks Discuss critical role of thoughts and appraisals about pain in determining affective and behavioral responses to pain. Help participants to identify negative thoughts that they may have in response to pain. Practice challenging negative thoughts with positive thoughts about effective ways to manage pain. Discuss strategies for dealing with setbacks and pain-flare-ups. Practice relaxation.
**Session 5**: Non-drug pain therapies; Heat & cold; Dealing with pain flare-ups and setbacks (continued) Describe rationale for using nondrug pain therapies. Describe and practice application of heat and cold; review precautions in using heat and cold for pain Continue discussion about coping with pain flare-ups & setbacks in pain management. Practice relaxation.
**Session 6**: Pain medications & complementary therapies Describe the role of medications for pain management. Discuss the major types of pain medications. Describe the use of complementary therapies in pain management. Discuss steps in making informed decisions about all pain therapies.
**Session 7**: Pain management plan; Wrap-up Discuss maintenance of gains made through the program. Review coping with set backs & pain flare-ups. Revise written individualized maintenance plans for each participant.
:::
A key component of this self-management group is the development of personalized pain management plans. Participants begin developing a plan during the first class and revise it each week as they learn and practice additional pain management skills. With the assistance of the facilitator and, at times, other group members, participants review pain control strategies that they have learned and practiced and choose one or several strategies that best meet their individual needs and interests. Participants identify specifically what they will do (in measurable terms) and define the parameters (e.g., how many times per week, how far they will walk, how many repetitions of each exercise they will do). Although each person develops his or her own plan, the plans incorporate the same repertoire of activities that are taught in the class. These plans are monitored weekly during the classes and during follow-up phone calls (described below).
Educational book control condition (BOOK)
-----------------------------------------
Participants who are assigned to the BOOK condition receive a copy of *The Chronic Pain Workbook, 2*^*nd*^*Edition*\[[@B25]\]. Facilitators telephone participants 1 and 4 weeks after participants receive the workbook. The BOOK condition was designed to control for attention and information. In these calls, facilitators inquire about participants\' current pain and functioning, and ask about use of pain therapies and self-management techniques. There is no specific therapeutic component in the phone calls and facilitators do not help BOOK participants identify goals or develop a pain management plan.
Booster and follow-up phone calls
---------------------------------
The SMG group facilitator telephones each participant at 12, 16, 22, and 30 weeks after the final group session. During the booster phone calls, facilitators inquire about pain and functioning, current pain management plans, and successes and obstacles in meeting pain management goals, as well as provide encouragement and assistance in problem-solving obstacles encountered in pain management. BOOK participants receive follow-up phone calls at the same intervals to control for attention.
Steps taken to ensure and monitor group facilitator adherence
-------------------------------------------------------------
Group facilitators are nurses and psychologists with expertise in geriatrics and/or pain management and experience in facilitating therapeutic groups. All are specifically trained according to the treatment protocol. We monitor group facilitator adherence to the self-management group protocol, as recommended by Waltz et al. \[[@B26]\]. All facilitators receive and review a facilitator\'s syllabus that contains a detailed protocol describing the goals, contents, and activities for each of the 7 sessions. Group facilitators have met 3 times to discuss protocol and treatment integrity issues. Finally, each session for each treatment group is audiotaped. Twenty percent of the audiotapes are randomly chosen and reviewed by a trained research nurse who is not involved in any other aspects of the study. The research nurse listens to the tapes and evaluates the degree to which the group sessions are conducted according to the protocol using a checklist developed for this purpose.
Measures
--------
Study measures were chosen based on psychometric properties, including sensitivity to change; brevity; and appropriateness for use with community-dwelling, older adults with chronic pain. They are described below and summarized in Table [2](#T2){ref-type="table"}.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Measures and assessment times
:::
**CONSTRUCT/MEASURE** **SCREENING/BASELINE** **POST-INTERVENTION** **6-MONTH FOLLOW-UP** **12-MONTH FOLLOW-UP**
------------------------------------------------------------------------------ ------------------------ ----------------------- ----------------------- ------------------------
**Physical Functioning** √ √ √ √
Roland-Morris Disability Questionnaire
Brief Pain Inventory (BPI) -- pain interference subscale
**Pain Intensity** √ √ √ √
Brief Pain Inventory -- pain intensity subscale
**Mood Disturbance/ Social functioning** √ √ √
Geriatric Depression Scale
**Pain Beliefs and Coping** √ √ √
Chronic Pain Coping Inventory -- (*includes pain medication use)*
Coping Strategies Questionnaire -- catastrophizing, praying/hoping subscales
Self-efficacy Scale
**Pain Knowledge** √ √
**Demographics, Medical Conditions, Medications** √
Screening & Intake Questionnaire
Adapted Charlson Index
**Cognitive Functioning** √
Folstein Mini-Mental State Examination
**Pretreatment Expectations** √
**Adherence to Treatment** √ √
Attendance at classes
Completion of reading assignments
Attainment of goals (Personal Pain Management Plan)
:::
### Descriptive measures
The following measures are administered at baseline to describe the sample and to explore whether these variables are associated with treatment response. We will compare the two study groups on these measures to determine whether they are comparable at baseline.
#### Screening and intake interview schedule -- demographic information and pain history
During the screening process and baseline assessment, participants are asked a series of questions to elicit demographic and pain history variables, including age, race, ethnicity, gender, marital status, education level, sites and duration of pain, and prior and current pain treatments.
#### Folstein mini-mental state examination (MMSE) \[[@B27]\]
The MMSE is a measure that is widely used to assess cognitive function, particularly in older adults. It consists of 30 items, and requires 5--10 minutes to administer. Items assess orientation, memory, attention, and calculation. The MMSE has been demonstrated to be valid and to have good test-retest reliability \[[@B28]\].
#### Charlson index of comorbidity (CI)
The CI is an extensively used, valid, and reliable measure of comorbid medical conditions \[[@B29]\]. The CI uses 19 categories of comorbidity; each category is weighted and scored according to an algorithm \[[@B29]\]. Higher scores indicate greater health burden from comorbid causes. In this study, we are using a self-report version of the CI demonstrated to be reliable and valid in a group of older adults \[[@B30]\]. Because comorbid conditions may be associated with pain appraisal, coping, and outcomes \[[@B31]\], we will examine the association between comorbid conditions and response to therapy.
### Process measures
#### Self-efficacy scale (SES)
Participants complete the 8-item version of Lorig et al.\'s Self- Efficacy Scale \[[@B32]\], which assesses confidence in ability to manage pain and associated problems such as fatigue and negative mood \[[@B33],[@B34]\]. Previous studies have supported the reliability and validity of this measure \[[@B32],[@B34],[@B35]\]. The SES has been tested and used in studies of older adults \[[@B36]\].
#### Coping strategies questionnaire (CSQ) \[[@B37]\]
The CSQ is one of the most widely used measures of pain coping and catastrophizing \[[@B38],[@B39]\]. Measures derived from the CSQ have been shown to be associated with various measures of functioning among patients with different pain conditions \[[@B38],[@B40]-[@B43]\]. The CSQ has demonstrated reliability and validity in several samples of older adults, including those who are older than 75 years \[[@B44]\]. For this study, only the catastrophizing and praying/hoping subscales are used. Catastrophizing is included because prior studies have shown that this variable is associated with pain intensity, depression, and disability \[[@B45]\]. The praying/hoping subscale was included because this coping strategy has been found to be associated with the pain experience of older persons\[[@B46]\].
#### Chronic pain coping inventory (CPCI)
The CPCI measures cognitive and behavioral coping strategies used by people to manage chronic pain. It contains 9 subscales: guarding, resting, asking for assistance, relaxation, task persistence, exercise/stretch, seeking support, coping self-statements, and medication use \[[@B47]\]. The CPCI scales have been shown to have acceptable internal consistency and test-retest reliability, and to be associated significantly with physical disability and depression \[[@B47]-[@B49]\]. Additional development and psychometric testing have supported the reliability and validity of an additional activity pacing subscale \[[@B49]\].
#### Pretreatment expectations
Prior to learning the study condition to which they are randomized, participants are asked the degree to which they believe that each study condition will be helpful to them. They respond using a 0 to 10 scale, with 0 indicating \"not helpful at all\" and 10 indicating \"extremely helpful.\"
#### Treatment adherence
1\. Class attendance. Group leaders document weekly class attendance. Total attendance is assessed as a percentage of classes the participant attended (out of 7).
2\. Reading log/usefulness. Both BOOK and SMG participants complete a form in which they report the amount read on each topic using a 0--5 scale (\"I did not look at the section at all\" to \"I read the section thoroughly\"). They also rate the usefulness of each section using a 0--5 scale (\"Not at all useful\" to \"Very useful\").
3\. Goal attainment. Attainment of SMG participants\' pain management goals is assessed using the Personal Pain Management Plan (PPMP). Each week, participants in the SMG group are asked to document the type and frequency of each activity they have chosen to utilize in the management of their chronic pain. They monitor and document the pain management activities that they actually performed over the week. Participants also document obstacles that they have encountered in trying to meet their goals and the solutions they have identified to overcome those obstacles. This form is printed on 2-page paper. The top copy is turned in each week and participants keep the bottom copy for their own records. The PPMP serves several purposes: 1) to assist participants to identify and follow through on their personalized goals; 2) to assess treatment adherence; and 3) to cross-validate data that are collected using the Chronic Pain Coping Inventory.
### Outcome measures
#### Primary outcome
*Roland-Morris disability questionnaire (RMDQ):*The RMDQ \[[@B50]\] is widely used to assess physical disability associated with low back pain. The RMDQ has been demonstrated to be valid, reliable, and responsive to change \[[@B50]-[@B55]\]. Although developed as a measure of physical disability related to back pain, the RMDQ, re-worded without reference to the back, has been found to be a reliable and valid measure of physical disability for patients with other chronic pain problems as well \[[@B52]\]. The RMDQ is scored from 0--24, with higher scores indicating more severe physical disability. Physical disability, as measured by the RMDQ, is the primary study outcome.
#### Secondary outcomes
*Brief pain inventory (BPI):*The BPI is a widely-used, reliable, valid instrument that assesses pain history, location, intensity, and activity interference \[[@B56],[@B57]\]. For this study, pain intensity is measured by calculating the mean of four items in which respondents are asked to rate their average, current, least, and worst pain during the past week, using a scale of 0 (\"No pain\") to 10 (\"Pain as bad as you can imagine\"). \[[@B58]\].
Pain-related interference is a composite measure of the degree to which pain limits a person\'s general function \[[@B57]\]. This variable is calculated as the mean of ratings of pain interference with general activity, mood, walking, work (including housework), relations with others, sleep, and enjoyment of life. Each item is rated on a scale of 0 (\"Does not interfere\") to 10 (\"Completely interferes\").
*Geriatric depression scale (GDS):*The GDS \[[@B59]\] is a 30-item self-report measure specifically designed to assess depressive symptoms in older persons. Scores of 11 or higher are considered indicative of depression in older adults. Good sensitivity and specificity for detecting depression in geriatric psychiatric and medical outpatients has been demonstrated (84--100% sensitivity; 73--96% specificity) \[[@B60],[@B61]\]. The GDS was selected over other available depression measures because of its screening efficiency with geriatric outpatient populations, its focus on affective rather than physical symptoms, and its true/false scoring format, which studies have found to be simpler for older adults to complete \[[@B61]\].
Sample size calculations and statistical analyses
-------------------------------------------------
### Power analysis/sample size calculations
A mixed effects model will be used to analyze data using the participant as the unit of analysis and controlling for baseline value of the outcome as a covariate. A reasonably accurate approximation to this analysis could be obtained by the following procedure: first compute change scores (pre to post) for each person, then collapse to get the mean change score within each site, then do t-tests on these means. This simpler model was used for power calculations, since it allows standard software to be used.
The plan to randomize by site, rather than by individual participant, required additional considerations in calculating statistical power. With this group-randomized design, power depends on the correlation of people within sites, or the *intra-class*correlation. Effect size is defined as the mean change score of all individuals in the intervention group minus the mean change score of all individuals in the control group, divided by the standard deviation of change score within groups. Power calculations for the proposed study are based on estimates of 34 sites (17 intervention and 17 control), 6.4 participants per site (N = 218) providing data at 12 months (20% attrition rate).
Table [3](#T3){ref-type="table"} shows how power (the probability of detecting a difference) varies with the correlation of individuals within site and the effect size. The second column of this table shows the \"effective sample size,\" meaning that the study would have the same power as a study with this sample size and no clustering. If the correlation is zero, the effective sample size is 272, the actual sample size. A correlation of 1 would indicate that all individuals in each site have exactly the same outcome (i.e., no different from having one person per site), so the effective sample size would be 27. Analysis of data from a pilot study showed an intra-class correlation (ICC) of .07 \[[@B62]\]. Although this estimate should be interpreted cautiously because of the limited number of sites in the pilot study, it indicates that the intra-class correlation will probably be fairly small, perhaps 0.05 to 0.1. Our target sample size of 273 enrolled and 20% attrition at 12-month follow-up (yielding a final sample size of 218), assuming ICC=.1, will result in 84% power for detecting an effect size of .5, which Cohen \[[@B63]\] refers to as a \"moderate\" effect size.
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Power for detecting a difference between the intervention and control group, depending on effect size and intra-class correlation (34 sites, average 6.4 subjects per site)
:::
Intra-class correlation Effective sample size Effect size (the difference in means between the two groups, divided by the within-group standard deviation)
------------------------- ----------------------- -------------------------------------------------------------------------------------------------------------- --------- --------- ----------
0.00 218 **84%** **96%** **99%** **100%**
0.05 171 74% **90%** **97%** **100%**
0.10 141 65% **84%** **94%** **98%**
0.20 105 52% 71% **86%** **94%**
0.30 83 43% 61% 77% **88%**
0.50 59 32% 46% 61% 75%
1.00 34 20% 29% 40% 51%
:::
### Statistical analysis
The test of hypothesis 1 compares the SMG and BOOK participants on the primary outcome (physical disability) and secondary outcomes (pain intensity, pain-related interference with activities, and depressive symptom severity) at each of the 3 follow-up assessments. The analytic method that we will use to evaluate this hypothesis is the mixed effects analysis of covariance (ANCOVA), as proposed by Laird and Ware \[[@B64]\] and implemented in the SAS PROC MIXED procedure \[[@B65],[@B66]\]. This model will have two random effects, *site*and *person nested within site*. *Group*(i.e., treatment or control) and *Time*will be fixed effects. The repeated measurements of physical disability at post-intervention, 6 months, and 1 year will be the outcome measure. The baseline value of physical disability will be included in the model as a covariate. Any baseline variables that are correlated with the outcome variable and/or differ between the two treatment groups (e.g., gender, age, comorbidity) will also be included as covariates in the analysis. If the main effect for *group*is significant, contrasts within this model will be used to test for treatment effect separately at each of the three outcome times. Secondary analyses will be similar, fitting a mixed effects model that uses one of the secondary outcomes (e.g., pain intensity, pain-related interference, and depression) in place of physical disability. The analysis of Hypothesis 1 will be by intent-to-treat.
Hypothesis 2 involves comparing the SMG and BOOK groups on changes in process variables (pain-related beliefs and coping strategies). A mixed effects model, as described under hypothesis 1, will be used for these analyses. As for hypothesis 1, baseline variables that are predictive of outcome and/or differ between groups will be included as covariates in the analyses for hypothesis 2.
Hypothesis 3 involves the correlation of changes in beliefs and coping to changes in the outcome variables. For each assessment time, change from baseline will be computed and scatter plots will be used to describe relationships, with Pearson and/or Spearman correlation coefficients used to summarize the strength of the association. Although we hypothesize that significant associations in changes will occur only in the SMG group, we also will perform exploratory analyses in the BOOK group to assess for these associations.
In addition to performing major analyses to test study hypotheses, we will also perform exploratory analyses to examine whether there are subgroups of participants in whom the intervention had a particularly strong or a particularly weak effect. For example, we will explore whether: (1) there is a difference in response to therapy based on age group (young-old, mid-old, old-old); (2) men respond differently to therapy than women; and (3) pain severity at baseline is related to strength of treatment effect. These analyses will be performed using the ANCOVA described above, augmented by adding, for example, an indicator for female gender and the interaction term between gender and treatment group.
Discussion
==========
Persistent pain is a common problem in older adults that can be debilitating. Self-management strategies that incorporate physical and psychosocial pain coping skills are effective in decreasing pain and improving function and mood in younger adults. Little is known, however, about the efficacy of this therapy for older adults, especially those in the mid-old to old-old range. Our randomized controlled trial assesses the efficacy of such a treatment program, as compared with a control condition, in decreasing pain and improving physical and psychosocial functioning in elderly retirement community residents with chronic pain.
Competing interests
===================
None declared.
Authors\' contributions
=======================
ME and JAT developed the intervention and conducted the pilot test of the self-management groups. KCC developed the analysis plan, conducted the power calculations, and wrote the related sections of the paper. CAK assisted in refining the intervention. ME and JAT wrote the initial description of the intervention and this article. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2318/4/7/prepub>
Acknowledgements
================
Support for the preparation of this article was provided by grant \#R01 NR007787 from the National Institute of Nursing Research, National Institutes of Health.
| PubMed Central | 2024-06-05T03:55:46.673039 | 2004-7-30 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509247/",
"journal": "BMC Geriatr. 2004 Jul 30; 4:7",
"authors": [
{
"first": "Mary",
"last": "Ersek"
},
{
"first": "Judith A",
"last": "Turner"
},
{
"first": "Kevin C",
"last": "Cain"
},
{
"first": "Carol A",
"last": "Kemp"
}
]
} |
PMC509248 | Background
==========
Cystic fibrosis (CF) is a common, often fatal disease with a well-defined genetic cause, so that it is now recommended in many countries in Europe and the United States to offer genetic screening for CF mutations to identify carriers among adults with a positive family history of CF, partners of individuals with CF, couples planning a pregnancy, couples seeking prenatal care and, recently, neonatal screening. Because of the mutational heterogeneity and the rarity of many mutations, most clinical DNA laboratories offer tests that aim to detect 75--95 % of CF alleles depending on the ethnic and geographic backgrounds of the population, using available commercial kits motsly including 20 to 31 mutations selected on the basis of their frequency as CF-causing mutations. A few laboratories (most often national reference laboratories) have developed the scanning of coding/flanking CFTR sequences to detect unknown mutations. As of February 2004, about 1200 disease-causing mutations have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene <http://www.genet.sickids.on.ca/cgi-bin/webobjects/MUTATION> Frameshift, splice-site, nonsense, and in-frame but nonfunctional deletions (such as p.F508del) are disease-causing mutations. By contrast, the status of some missense mutations is extremely difficult to assess and functional studies are not available to diagnostic laboratories. Missense mutations may represent, depending on the populations, up to 45% of mutations responsible for CF or CBAVD (congenital bilateral absence of vas deferens). Moreover, due to improved scanning strategies, a growing number of complex alleles (several sequence changes on the same gene) are thought to affect the expression of the disease phenotype by modulating the effect of a mutation \[[@B1]-[@B5]\]. The most striking exemple is the length of the intron 8 polythymidine tract (7, 9, or 5 thymidines) on exon 9 splicing as a genetic modifier of the severity of the p.R117H mutation \[[@B1]\]. Another exemple is the revertant mutation p.R553Q which, when carried on the same gene as p.F508del, is associated with a CF phenotype with normal chloride concentration in sweat test \[[@B6]\] and which, when expressed in heterologous cells, can partially correct the processing and Cl- channel gating defects caused by the p.F508del mutation \[[@B7]\].
With the start of population screening for CF carriers, new data on the prevalence of some missense mutations have been provided, questioning their involvement as disease-causing mutations. In North American populations, missense mutations p.I148T and p.D1270N were found \>100 times and \>200 times, respectively, more frequently in carrier screening than in CF patients \[[@B8],[@B9]\]. Moreover, we and others have found that individuals affected with CF or CBAVD carry p.D1270N associated with p.R74W on the same allele \[p.R74W;p.D1270N\] \[[@B10],[@B11],[@B5]\]. Similarly, p.I148T has been shown to be associated with a CF phenotype only as a complex allele, i.e. when associated with mutation c.3199del6 on the same gene \[[@B8]\]. A completely asymptomatic male individual who is a compound heterozygote for p.D1270N and p.I148T has been recently identified \[[@B9]\]. These findings provided evidence that these missense changes may not be the true mutations and prompted us to reanalyze all the patients in our CF or CBAVD cohort who had been originally diagnosed as compound heterozygotes for either p.I148T or \[p.R74W;p.D1270N\] and another mutation on the other allele. The result of full scanning of CFTR sequences showed that a second mutation (c.3199del6 or the novel mutation c.3395insA) was associated in *cis*with p.I148T in all individuals with a CF phenotype, and that a third missense mutation (p.V201M) was associated in *cis*with complex allele \[p.D1270N;p.R74W\] in patients with a CBAVD phenotype in this series.
Methods
=======
CFTR scanning for individuals with p.I148T or \[p.D1270N;p.R74W\]
-----------------------------------------------------------------
From 1990 to 2003, we have analysed for CFTR mutations genomic DNA from 437 families with CF and 170 with isolated azoospermia caused by CBAVD, using a combination of mutation screening for known and scanning for unknown mutations. The first step was the search of 31 CF mutations detected by the ABI oligonucleotide ligation assay and 3 common intronic mutations by using restriction analysis. The second step was the scanning of coding/flanking sequences by DGGE (Denaturing Gradient Gel Electrophoresis) using 32 GC-clamped amplimers (which in our experience detected 98% of CFTR mutants), followed by sequencing to resolve abnormal PCR products (BigDye terminator cycle sequencing on ABI 310 automate sequencer). Whenever possible, family members were assayed for the mutations and associated polymorphisms. We detected 160 different mutations in the CF group accounting for 97 % of CF alleles, and 64 different mutations in the CBAVD group accounting for 85% of CBAVD alleles, which represents one of the highest allelic heterogeneity reported so far. Usually, mutation scanning is stopped when two mutations are found to be in *trans*.
In this study, we analyzed by DGGE the entire coding and flanking regions of the CFTR gene of individuals who had been previously found to carry p.I148T or the complex allele \[p.R74W;p.D1270N\] and assayed their relatives for the additional sequence changes identified. We also re-analyzed the CFTR gene of a CF patient who had been originally described with c.394delTT in *trans*of c.3195del6 \[[@B12]\], now renamed c.3199del6 (see the Results). Studies to determine the frequency of each sequence alteration described in this report were performed on 600 chromosomes from our general population (Southern France). In addition, we also reanalyzed two additional CF patients previously found to carry p.I148T in the Lyon genetic center. The study was approved by the institutional ethical committees and informed consent was obtained from families.
Nomenclature
------------
Gene variants and mutants are described using DNA and protein designation: intronic changes, deletions, insertions and frameshifts are reported at the cDNA level (c.) and amino acid changes at the protein level (p.), as recommended in the Human Genome Variation Society web page <http://www.genomic.unimelb.edu.au/mdi/>.
Results
=======
A CF mutation (c.3199del6 or c.3395insA) is associated in *cis*with p.I148T in CF patients
------------------------------------------------------------------------------------------
Two out of 437 CF patients analyzed in Montpellier and two out of 801 CF patients analyzed in Lyon were found to carry p.I148T, which was initially thought to be one of the two mutations responsible for CF in these patients. However, thorough re-analysis of the entire CFTR sequence determined that a CF mutation (c.3395insA or c.3199del6) was present on the same gene in both cases (table [1](#T1){ref-type="table"}).
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
CFTR haplotypes associated with mutations found in CF patients carrying p.I148T in *cis*with c.3395insA or c.3199del6 and in one CF patient carrying c.3199del6 alone
:::
Indiv No. Age at Diagnosis Phenotype CFTR Mutations CFTR haplotype
----------- ------------------ ----------- ---------------------------- ---------------- -------- -------- ------- ------- -------- -------- --------
IVS1 IVS8 IVS8 IVS8 470 IVS17B IVS17B EGH^a^
CA CA TGm Tn TA CA
CF1 7 yrs CF-PI c.394delTT 21 23 10 9 M 36 13 B
c.3199del6 22 16 11 7 V 7 17 C
CF2 10 yrs CF-PS **\[c.3395insA;p.I148T\]** **21** **23** **10** **9** **M** **7** **17** **B**
p.R334W 22 17 11 7 V 46 13 A
CF3 6 ms CF-PI **\[c.3199del6;p.I148T\]** **21** **23** **10** **9** **M** **7** **17** **B**
p.F508del 21 23 10 9 M 31 13 B
CF4 3 yrs CF-PI **\[c.3199del6;p.I148T\]** **22** **23** **nd** **9** **M** **7** **17** **B**
p.F508del 22 17 nd 9 M 31 15 B
CF5 6 ms CF-PI **\[c.3199del6;p.I148T\]** **22** **23** **nd** **9** **M** **7** **17** **B**
p.F508del 22 23 nd 9 M 31 13 B
^a^EGH, extragenic haplotype XV2c/*TaqI*, KM19/*PstI ;*nd, not determined Patients CF1-3 were from the cohort of Montpellier (n = 437), patients CF4-5 were from the cohort of Lyon (n= 801).
:::
Mutation c.3199del6 can also occur alone as a CF-causing allele
---------------------------------------------------------------
Mutation c.3199del6 was found to be carried without p.I148T in a young CF male with 394delTT on the other allele, diagnosed at the age of 7 years on the basis of typical pulmonary disease, pancreatic insufficiency, poor growth and positive sweat test \[[@B12]\]. Mutation c.3199del6 was initially described by us in 1994 in this patient as c.3195del6 \[[@B12]\], in accordance with the first draft of mutation nomenclature \[[@B13]\]. However, it occurred in the same palindromic sequence in exon 17a than mutation c.3199del6 reported in 1998 by Bozon *et al*. \[[@B14]\]. Both mutations are expected to delete either amino acids Val1022 and Ile1023, or Ile1023 and Val1024 from the CFTR protein. As it is impossible to determine at the genomic level in which part of the palindrome each of them occurred, the most 3\'copy of the repeat is arbitrarily assigned to have been mutated, according to the current rule \[[@B15]\]. Consequently, mutations c.3195del6 and c.3199del6 should be considered as identical and reported as c.3199\_3204del. The familial segregation analysis of polymorphisms covering the CFTR gene showed that p.I148T, when present in individuals with a CF phenotype, occurred on a unique haplotype carrying IVS8-9T whatever the mutation in *cis*, c.3395insA or c.3199del6 (table [1](#T1){ref-type="table"}). By contrast, c.3199del6 without p.I148T occurred on a different haplotype carrying IVS8-7T.
Mutations p.I148T, c.3199del6 and c.3395insA were not found on 600 chromosomes from our general population.
Triple-mutant allele \[p.R74W;p.V201M;p.D1270N\] is found in males with CBAVD whereas double-mutant allele \[p.R74W;p.D1270N\] is found in asymptomatic individuals
-------------------------------------------------------------------------------------------------------------------------------------------------------------------
Re-analysis of the CFTR gene in families carrying \[p.R74W;p.D1270N\] identified a third mutation (p.V201M) on the same chromosome in three unrelated individuals with CBAVD (table [2](#T2){ref-type="table"}). Only the double-mutant p.R74W-p.D1270N was present in the two unaffected individuals who were found with these changes in our sample. The first case was a young boy who had been initially suspected of having CF at age 4 years because of allergic rhinitis but for whom the diagnosis of CF was later ruled out; no other CFTR sequence alteration could be identified and the sweat tests were negative (chloride values \<40 mM). The second individual was the mother of a CF girl who was compound heterozygous for p.F508del and p.P67L. This woman, who was carrying p.P67L on one CFTR gene and \[p.R74W-p.D1270N\] on the other (table [1](#T1){ref-type="table"}), was completely asymptomatic at age 45 years and displayed three negative sweat tests (chloride values \<20 mM). The triple and double mutant alleles seem to have occurred on the same haplotype TG11-T7-V470.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
CFTR sequence changes found in individuals carrying missense alterations p.R74W, p.D1270N, or p.V201M
:::
Mutations Haplotype
------------------------------------------- --------------------------------- ----------- -------- -------- ------- ------- -------- --------
CBAVD1 p.R1066C 22 16 11 7 V 30 13
**\[p.R74W;p.V201M;p.D1270N\]** **22** **16** **11** **7** **V** **31** **13**
CBAVD2 p.M952I 26 17 10 7 M 7 17
**\[p.R74W;p.V201M;p.D1270N\]** **22** **16** **11** **7** **V** **31** **13**
CBAVD3 **\[p.R74W;p.V201M;p.D1270N\]** **22** **16** **11** **7** **V** **31** **13**
**\[p.R74W;p.V201M;p.D1270N\]** **22** **16** **11** **7** **V** **31** **13**
Individual non affected with CF
No mutation 21 nd 10 7 M 7 17
**\[p.R74W;p.D1270N\]** **22** nd **11** **7** **V** **30** **13**
Asymptomatic mother of a CF affected girl
p.P67L 23 16 10 7 M 7 17
**\[p.R74;p.D1270N\]** **22** **16** **11** **7** **V** **31** **13**
:::
Discussion
==========
p.I148T is a low penetrance CF mutation or a neutral polymorphism
-----------------------------------------------------------------
Since its initial description in a CF Canadian patient with pancreatic insufficiency \[[@B16]\], the mutation p.I148T, which changes a conserved amino acid and occurs in the first cytoplasmic loop of the CFTR protein, has been considered as a severe CF allele in many countries. It was thought to be the second most common CF mutation in the French Canadian population, accounting for 9.1% of the French Canadian chromosomes \[[@B17]\], whereas in France, p.I148T accounted for only 0.11 % of the CF alleles in a sample of 3,710 patients affected with the disease \[[@B5]\]. p.I148T can now be detected by several commercially available kits developed for routine screening of CF carriers and for CF neonatal screening, and recently it has been included in the core panel of 25 CF mutations recommended by the American College of Medical Genetics (ACMG) \[[@B18]\]. Thousands of individuals are being screened for this mutation worldwide and it is possible that several prenatal diagnosis have been or will be performed. However there are now several lines of evidence that question the role of p.I148T by itself in causing disease. First, compound heterozygosity for p.I148T and a severe CF mutation was recently identified in several healthy individuals \[[@B9],[@B19]\]. When affected and unaffected individuals carrying apparently the same mutational genotype were re-analyzed for additional changes that could explain the different phenotypes, p.I148T was found to be associated in *cis*with another mutation, c.3199del6, in patients with a classic CF phenotype, whereas healthy adults who were compound heterozygous for p.I148T and a severe CF mutation or homozygous for p.I148T did not carry the deletion \[[@B8]\]. In a recent study, the p.I148T mutation has been further documented to be linked with the 3199del6 mutation in all 24 CF patients of French Canadian descent originally identified as compound heterozygous for the p.I148T mutation and a second severe CFTR mutation \[[@B20]\]. Second, p.I148T was found to be over 100 times more common in two independent U.S. carrier screening programmes than in CF patients: it accounted for 6.4 to 7.7% of chromosomes detected in the screened populations versus 0.06 to 0.068% of CF chromosomes in CF patients \[8. 9\]. This discrepancy suggests that p.I148T is either a poorly penetrant mutation or a neutral polymorphism. Third, when transiently expressed in epithelial cells, p.I148T mutant protein is normally processed and is able to mediate normal chloride transport with properties identical with those of wild-type cells \[[@B21]\]. As the mutant seems to suppress the ability of CFTR to support HCO3- transport, it has been hypothesized that p.I148T may contribute to disease through Cl- coupled HCO3- altered transport; however, the major CFTR functions are retained by the mutant \[[@B21]\]. Fourth, we show in this study for the first time that p.I148T can be associated with a frameshift mutation c.3395insA in exon 17b instead of in-frame deletion c.3199del6 in exon 17a. Insertion c.3395insA (designated as c.3395\_3396insA) is a previously unreported mutation that is predictive of premature termination of translation at amino acid residue 1155. The truncated protein lacking the 325 last amino acids is believed to be not functional and be degraded rapidly, generating no detectable protein. A CFTR alteration producing a premature termination signal is a class I mutation, considered severe enough to cause CF by itself and exclude the contribution of any other sequence change on the same allele. Fifth, in contrast with other studies that stated that only the complex allele \[p.I148T;9T;c.3199del6\] appeared to be associated with a classic CF phenotype \[[@B8]\], we demonstrate that c.3199del6 is associated with a CF phenotype even if the deletion occurs on a chromosome that does not carry p.I148T, which adds further value to the consideration that p.I148T is not a true mutation but simply a polymorphism. Although no functional test was performed to prove its contribution to the severe phenotype, mutation c.3199del6 has been considered as a defective allele as it results in the loss of two amino acid residues in the TM10 domain of the CFTR protein and has not been detected in non-CF alleles. Our data fully support the recent recommendation that p.I148T should not be included in the mutation panel selected for prenatal screening strategy \[[@B22]\].
The complex allele \[p.R74W;p.D1270N\] may be not enough to cause disease
-------------------------------------------------------------------------
We and others had initially described p.R74W \[[@B23]\] and p.D1270N \[[@B24]\] in isolation but they have since been found in association in many CBAVD or CF patients \[[@B10],[@B11]\] and these two changes were thought to be deleterious, alone or in combination. A few complex alleles have been expressed in heterologous systems to evaluate the impact on CFTR processing and Cl- channel activity and better understand the contribution of each missense mutation on phenotype. When expressed in HeLa cells, mutant p.R74W, p.D1270N and \[p.R74W;p.D1270N\] did not affect CFTR processing, however a lower cAMP-responsive anion conductance was observed with the double mutant \[p.R74W;p.D1270N\] \[[@B3]\]. The assay suggested that p.R74W alone should be considered as a polymorphism, p.D1270N alone could generate a CBAVD phenotype while the complex allele could produce a more severe phenotype as p.R74W could enhance the effect of p.D1270N \[[@B3]\]. However these findings have not yet been confirmed by other studies.
We have found here that a triple-mutant \[p.R74W;p.V201M;p.D1270N\] allele was carried in all three patients with CBAVD whereas only the double mutant \[p.R74W;p.D1270N\] allele was present in two asymptomatic individuals including an obligate carrier who was compound heterozygous for a CF mutation. Another mother carrying \[p.R74W;p.D1270N\] *in trans*of a CF mutation has been described previously; despite two positive sweat tests she was absolutely asymptomatic \[[@B25]\]. Missense p.V201M in exon 6a changes a valine for a methionine in the third transmembrane domain; it was initially reported alone in a French patient with CBAVD \[[@B26]\], then in Brazilian patients with CF \[[@B27]\].
Recent large scale screening for CF carrier showed that p.D1270N was present 205 times more commonly in the screened population than in the CF patients (frequency of 14% versus 0.068%); in addition, a completely asymptomatic adult compound heterozygote for p.D1270N and p.I148T has been identified \[[@B9]\]. Although it is not known whether these alleles are associated or not with the third change p.V201M, there are now enough evidence to question the role of the complex allele \[p.R74W;p.D1270N\] as being a CF or CBAVD mutation. Further experimental and genetic investigations will be necessary to demonstrate the role of p.V201M in causing disease.
Conclusions
===========
This report further corroborates the recent hypothesis \[[@B9]\] that p.I148T and p.R74W-p.D1270N may not be true CF/CBAVD mutations. If these observations are further confirmed by a large multicentric study, they will have important implications for genetic counseling of patients and couples found to carry p.I148T or \[p.R74W;p.D1270N\]. They also pinpoint several important points in genetic testing for CF : first, the necessity of scanning the whole regions of the CFTR gene for diagnosis purposes, whatever the cost; second the necessity to better standardize mutation nomenclature, and third the usefulness of confirming inheritance of mutations from both parents whenever possible to avoid the risk for erroneously reporting changes in *trans*that are in fact complex alleles.
Competing interests
===================
None declared.
Authors\'contributions
======================
JPA, CG, CT and FC carried out the molecular genetic studies. MDG coordonated the molecular analysis. MC conceived the study and drafted the manuscript. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2350/5/19/prepub>
Acknowledgements
================
We thank the CHU of Montpellier and the French Association against CF for their support, and the reviewers for their helpful comments.
| PubMed Central | 2024-06-05T03:55:46.676762 | 2004-8-2 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509248/",
"journal": "BMC Med Genet. 2004 Aug 2; 5:19",
"authors": [
{
"first": "Mireille",
"last": "Claustres"
},
{
"first": "Jean-Pierre",
"last": "Altiéri"
},
{
"first": "Caroline",
"last": "Guittard"
},
{
"first": "Carine",
"last": "Templin"
},
{
"first": "Françoise",
"last": "Chevalier-Porst"
},
{
"first": "Marie Des",
"last": "Georges"
}
]
} |
PMC509249 | Background
==========
Adenoid cystic carcinoma (ACC) is a rare malignant neoplasm of the salivary gland. Salivary gland neoplasms constitute 3% of cancers of all sites, of which, 10--15% are malignant \[[@B1],[@B2]\]. Though ACC is the most common malignant tumor of the submandibular, sublingual and minor salivary glands, it accounts for only 15% of parotid cancers \[[@B3]\]. They are generally slow growing and spread relentlessly to adjacent structures. Hematogenous spread is more common than lymphatic spread, the common sites of metastasis being the lung, bone and viscera \[[@B4],[@B5]\]. We present a case of multiple liver metastases occurring 3 years after surgery for ACC of the parotid gland. The primary therapy, metastasis and outcome of ACC are discussed.
Case presentation
=================
A 60 year old woman presented with a small swelling beneath the right ear lobe of 4 months duration. The swelling measured 2 × 1 cm placed in the superficial part of the parotid and was not fixed. There was no facial nerve palsy or palpable cervical nodes. A fine needle aspiration cytology (FNAC) was carried out which showed the lesion to be ACC \[[@B6]\]. The clinical staging was T1, N0, M0. The patient underwent a total conservative parotidectomy after metastatic work up. Histopathology revealed ACC with cribriform pattern and perineural invasion (Figure [1](#F1){ref-type="fig"}). 60 Gy adjuvant external beam radiotherapy was administered post-operatively to the parotid area and the neck. Patient was placed on regular follow-up. Three years after primary surgery, patient presented with heaviness and pain in the right hypochondrium of 15 days duration. Patient was anicteric and abdominal examination revealed firm nodular and non tender enlargement of the liver. There was no ascites. Surgical site and neck were clinically normal. Chest roentgenogram was normal. Ultrasonography (US) of the abdomen revealed multiple metastatic lesions scattered in both lobes (Figure [2](#F2){ref-type="fig"}). Liver function tests were normal and an US guided FNAC revealed metastatic ACC (Figure [3](#F3){ref-type="fig"}). Since the lesions were multiple and scattered over both lobes of liver, surgical option was not considered and the patient was offered palliative chemotherapy which she declined. She developed pedal edema and abdominal distention 20 months after detection of liver metastasis. On clinical examination, patient was anicteric but liver had increased in size and abdomen showed evidence of a little free fluid. Chest CT scan was normal. Bone scan did not suggest any metastatic focus. Patient died a month later still without evidence of local recurrence or pulmonary metastasis.
Conclusions
===========
Although ACC is the second most common malignant salivary gland neoplasm and constitutes approximately one third of all salivary gland malignancies it constitutes only 2% of parotid neoplasms \[[@B3]\]. As ACC is neurotropic, frozen section analysis of nerve margins is suggested specially when nerve is grossly involved by the tumor \[[@B7]\]. A total conservative or a radical parotidectomy is advocated for ACC though the main intent is to obtain a tumor free area of at least 1 cm \[[@B8]\]. ACC, with its often unusually slow biologic growth, tends to have a protracted course and ultimately a poor outcome, with a 10-year survival reported to be less than 50% for all grades \[[@B9],[@B10]\]. These carcinomas typically show frequent recurrences and late distant metastases \[[@B11]\]. In a retrospective review of 92 cases, a tumor size greater than 4 cm was associated with an unfavorable clinical course \[[@B12]\]. Cribriform and solid patterns seen histologically were thought to predict more biological aggressiveness while tubular pattern represented more differentiated pattern of ACC. Over long periods of patient follow-up such grade based prognostication is less valid. Currently, stage and tumor location are the only factors considered prognostically significant \[[@B13]\].
Radiotherapy has been used as a primary modality for patients with surgical contraindications and in those with unresectable neoplasms. Though no improvement in survival is reported, the use of adjuvant radiation improves locoregional control and disease free survival. This patient received adjuvant radiation and did not have any locoregional recurrence.
Regional metastasis is less common occurring in about 17% while systemic failure occurs in 33 to 50% of the patients \[[@B4],[@B8]\]. Though involvement with distant metastases are unpredictable, organs involved in the order of decreasing frequency are lung, bone, brain and the liver \[[@B3]\]. Other rare metastatic sites of parotid and non parotid ACC include stomach, toe, choroids, brain and skin \[[@B14]-[@B18]\]. The initial site of metastasis is usually the organ containing the first capillary bed (first filter) and hence lungs would be the common site of metastasis \[[@B19]\]. Clinical observations from various malignancies have indicated that metastasis from certain types of tumor tend to occur in specific target organs leading to the famous \'soil and seed\' hypothesis where metastatic cells \'home\' to the organ \[[@B20]\]. Though liver metastasis has been reported, most of the liver metastases reported are of non parotid ACC \[[@B3],[@B5]\]. The occurrence is usually metachronous or synchronous with metastasis to other organs like the lung as it is the first filter. In the series of Spiro, of the 74 patients developing metastasis from salivary gland ACC, 23 did so without loco-regional recurrence while 5 had isolated bone metastasis \[[@B5]\]. Sung and colleagues found metastases in 46 out of 94 head and neck ACC \[[@B21]\]. In that study, only one patient developed liver metastasis and that patient had metastasis to both lung and bone. In this case, the patient manifested with multiple metastatic foci in the liver as the first and only metastatic organ which is very unusual. Surgical options of metastatectomy were not explored as patient had multiple metastases involving both lobes of liver. Although the patient lived with disease for a further two years, she did not show any evidence of lung metastasis or loco-regional recurrence. In this patient, the liver metastasis could have occurred prior to treatment as an organ of preference; evidenced by the fact that there were no other organs showing metastasis nor was there a loco-regional recurrence later. Studies in ACC have shown long tumor doubling times of pulmonary metastasis and late recurrences up to 10 years after primary treatment \[[@B22]\]. The estimated doubling time of lung metastasis in ACC ranges from 200 to 600 days \[[@B22]\]. There is even a suggestion that metastasis at the cellular level could occur many years prior to clinical presentation of primary tumor. FNAC was done to prove the metastatic foci in this patient and can be a useful tool for diagnosis \[[@B23]\]. Although this patient declined chemotherapy, chemotherapeutic responses have been reported in ACC \[[@B24]\]. ACC carries a mortality of 75--80% over a 30 year period and most patients who die of their disease do so between 5 and 10 years after initial treatment \[[@B10]\]. Our patient died 5 years from diagnosis with metastatic disease that developed 3 years after initial treatment.
ACC is a rare malignant tumor of parotid gland. Metastasis can manifest very late and hence a long term follow-up and a high index of suspicion is necessary to diagnose them early. An annual ultrasound study of abdomen would be desirable on follow-up. Unlike metastasis from other malignancies, these grow indolently and long term survival can be expected even with multiple metastases as also evidenced in the present case. Chemotherapy could be considered in selected patients as a therapeutic option in metastatic disease.
List of abbreviations used
==========================
**ACC:**Adenoid cystic carcinoma
**FNAC:**Fine needle aspiration cytology
**Gy:**Gray
Competing interests
===================
None declared
Authors\' contributions
=======================
**KH**was the principal clinician who planned the evaluation and procedure, in addition to conceptualizing and drafting the article.
**SRMG**was the pathologist.
Both the authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2407/4/41/prepub>
Acknowledgments
===============
Since the patient has expired, the legal heir was informed that the case would be published and his consent was obtained.
Figures and Tables
==================
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Photomicrograph showing small darkly stained cells with scanty cytoplasm arranged in nests fenestrated by round or oval spaces -- cribriform pattern, H & E (× 100)
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Abdominal ultrasound showing multiple hypoechoiec (short arrow) and hyperechoeic lesions (long arrow) in the liver
:::
:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
FNAC liver showing metastatic ACC, H & E (× 400)
:::
:::
| PubMed Central | 2024-06-05T03:55:46.679588 | 2004-7-30 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509249/",
"journal": "BMC Cancer. 2004 Jul 30; 4:41",
"authors": [
{
"first": "K",
"last": "Harish"
},
{
"first": "SR",
"last": "Mangala Gouri"
}
]
} |
PMC509250 | Background
==========
Public health surveillance is defined as the \"ongoing systematic collection, analysis, and interpretation of data essential to the planning, implementation, and evaluation of public health practice, closely integrated with the timely dissemination of these data to those who need to know\"\[[@B1]\]. Reasons for conducting public health surveillance can include the need to assess the health status of a population, establish public health priorities, and reduce the burden of disease in a population by appropriately targeting effective disease prevention and control activities \[[@B2]\].
Timeliness is a key surveillance system metric and should be periodically evaluated \[[@B3],[@B4]\] because it can reflect the time delay between any number of response steps in the public health surveillance process. Surveillance system timeliness depends on a number of factors and its assessment should include a consideration of how the data will be used and the nature of the condition under surveillance (e.g., for infectious diseases, this includes the communicability of the disease) \[[@B3]\]. If the data are to be used to implement immediate disease control and prevention activities for infectious diseases that are acute, severe, and highly transmissible, timeliness is critical. Timeliness requirements for a surveillance system might vary by different levels of public health system (e.g., local, state, or national), on the basis of the intended uses of the surveillance data at that level (Table [1](#T1){ref-type="table"}). For example, timely data are needed within a state for identifying cases or clusters of disease that will prompt an immediate public health response. Timely national surveillance data aggregated from a number of jurisdictions may be used for identifying multistate outbreaks or disease clusters and enable the federal public health system to assist the states in performing and coordinating their prevention and control activities. In reportable disease surveillance systems, health care providers and diagnostic laboratories usually report information regarding persons with notifiable conditions to the local public health system. Then, reporting proceeds in a hierarchical fashion to the state and then to the national level. Health care provider and public health system actions at each successive level of the reporting hierarchy contribute to reporting timeliness delays at the national level.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Potential uses of infectious disease surveillance data, by level of the public health system
:::
Intended Uses Used at which level(s) of the public health system?\*
------------------------------------------------------------------------------------------------------------ -------------------------------------------------------
Identify individual cases or clusters in a jurisdiction to prompt intervention or prevention activities Local, State (National)
Identify multi-state disease outbreaks or clusters. State, National
Monitor trends to assess the public health impact of the condition under surveillance. State, National (Local)
Demonstrate the need for public health intervention programs and resources, as well as allocate resources. State, National (Local)
Monitor effectiveness of prevention, control, and intervention activities. State, National (Local)
Formulate hypotheses for further study. National (State)
\*Public health system level in parentheses represents secondary use of the data for that purpose.
:::
State and national surveillance processes
-----------------------------------------
Before data can be used for public health action, health-related data must be collected by the public health system, analyzed, and disseminated to those responsible for taking action (Figure [1](#F1){ref-type="fig"}). Within a state (Steps 1--7), the public health system can use surveillance data for a number of purposes, including outbreak detection and intervention planning and implementation (Table [1](#T1){ref-type="table"}). The number and sequence of actions a state conducts before reporting data to the national public health system might vary by state, depending on state policies and protocols (Figure [1](#F1){ref-type="fig"}). For example, for nationally notifiable infectious disease reporting, CDC recommends that states report as soon as they first receive information about a suspect, probable, or confirmed case. However, some states only report confirmed cases, which usually requires laboratory confirmation, and decreases reporting timeliness at the national level.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Sequence of actions needed to gather and use health-related information for public health purposes
:::
:::
Each week, states and the U.S. territories report case information on persons suspected of having or diagnosed with a nationally notifiable infectious disease to the Nationally Notifiable Diseases Surveillance System (NNDSS), maintained by the Centers for Disease Control and Prevention (CDC) \[[@B5]\]. A nationally notifiable disease is one for which \"regular, frequent, and timely information regarding individual cases is considered necessary for prevention and control of the disease\" \[[@B6]\]. At the national level, NNDSS data are used for monitoring trends, program planning, evaluation, policy development, research, and monitoring the effectiveness of prevention and control activities. Although NNDSS reporting timeliness for these long-range goals and objectives is not critical, the threat of terrorism prompted consideration of whether NNDSS could be enhanced in the future to support public health response for either naturally occurring diseases or terrorism preparedness and response efforts. Therefore, the timeliness of NNDSS data was evaluated to determine if NNDSS could support timely notification and state response to multistate outbreaks. To provide a context for the evaluation of NNDSS timeliness, published studies reporting timeliness measures for infectious disease surveillance systems in the United States were reviewed.
Methods
=======
Literature review
-----------------
Infectious disease surveillance evaluation studies reporting timeliness measures that were published between January 1970 and March 2003 in biomedical and public health literature were reviewed. English-language papers were identified by using the Medline database (U.S. National Library of Medicine). The search strategy used various combinations of the following key words \"timeliness,\" \"reporting delay,\" \"time delay,\" \"lag time,\" \"disease surveillance,\" \"disease outbreaks,\" \"communicable diseases,\" and \"infectious diseases.\" Reference lists of the studies identified through the Medline search and studies citing CDC\'s surveillance evaluation guidelines were also reviewed \[[@B3],[@B7]\]
Reports were included if they evaluated a public health surveillance system operating in the United States and provided a quantitative estimate of disease-specific timeliness (e.g., interval in days). Studies without quantitative timeliness estimates or that reported a quantitative estimate for a group of infectious diseases (versus a disease-specific estimate) were excluded. In addition, studies describing the timeliness of syndromic surveillance systems were excluded.
Information abstracted for the review included the disease(s) under surveillance, the geographic area and time period studied, the purpose of the surveillance evaluation, the surveillance time interval measured, the surveillance processes or actions (steps in Figure [1](#F1){ref-type="fig"}) covered within the measured time interval, the timeliness measure, and the study\'s assessment of whether surveillance data timeliness met the surveillance goals.
NNDSS timeliness
----------------
Information available for assessing NNDSS reporting timeliness includes the *Morbidity and Mortality Weekly Report*\[*MMWR*\] week number the state assigns to each case and one of the following earliest known dates associated with the incidence of this disease (earliest known date) from the following list of hierarchical date types: onset date, diagnosis date, date of laboratory result, or date of first report to the community health system. National reporting delay was calculated as the difference in days between the midpoint of the *MMWR*week and the earliest known date reported in association with the case. This time interval reflects various state-specific surveillance intervals in the surveillance process that occur between the occurrence of a health event and the reporting of that health event to NNDSS, but at a minimum it includes Intervals 1--4 (Figure [1](#F1){ref-type="fig"}). National median reporting timeliness was calculated overall for the years 1999--2001, for each disease in our study, by date type and state, and across all states. Median reporting delay was calculated using Proc means in SAS version 8 software for Windows (SAS Institute, Inc., Cary, North Carolina).
To assess whether analysis of NNDSS data could support the timely identification of multistate outbreaks at the national level, the percentage of NNDSS cases reports reported within one to two incubation periods for each of the diseases was determined. Incubation periods were used as a surrogate measure for period of communicability which is critical to consider when implementing effective, disease-specific prevention and control measures. For this analysis, estimated incubation periods were used for the seven nationally notifiable infectious diseases selected for this study: 7 days for cryptosporidiosis, 4 days for *Escherichia coli*O157:H7 (*E. coli*), 30 days for acute hepatitis A virus infection, 4 days for meningococcal disease, 20 days for pertussis, 1.5 days for salmonellosis, and 3 days for shigellosis \[[@B8]\]. These diseases were selected because they were confirmed on the basis of laboratory criteria; they have the potential to occur in epidemics; they were designated nationally notifiable five years or more before the study period began; and the magnitude of reported disease incidence supported this analysis.
Only finalized case-specific data reported from U.S. states and two autonomous reporting entities (New York City and Washington D.C., referred to as states, hereafter) that designated the reported condition as notifiable (reportable by law or regulation) and that met NNDSS publication criteria \[[@B9]\] were included in the analysis. Data were analyzed for *MMWR*years 1999, 2000, and 2001.
Results
=======
Literature review
-----------------
Eight papers were identified that met the inclusion criteria for this study (Table 2 - see [Additional file: 1](#S1){ref-type="supplementary-material"}) \[[@B10]-[@B17]\]. Seven of the eight papers met the inclusion criteria resulting from the literature review; an additional paper was identified from the review of reference lists of studies identified through the Medline search and studies citing CDC\'s evaluation guidelines \[[@B3],[@B7]\]. Three of the eight papers in this study assessed national reporting timeliness; the remaining five papers focused on local or state reporting timeliness. The studies of national reporting timeliness focused on the following diseases: acquired immunodeficiency syndrome (AIDS) \[[@B17]\]; *Neisseria meningitidis*and *Haemophilus influenzae*infections \[[@B16]\]; and shigellosis, salmonellosis, hepatitis A, and bacterial meningitis \[[@B11]\]. The studies of local or state reporting timeliness analyzed data for AIDS \[[@B14],[@B15]\], tuberculosis \[[@B13]\], influenza-like illness \[[@B10]\], and meningococcal disease \[[@B12]\]. In seven of the eight papers, timeliness was calculated as the median reporting delay between the date of disease occurrence (e.g., disease onset date, diagnosis date, or laboratory result date) and the date the public health system was notified or as the proportion of cases reported to the public health system in a specific time interval. In one study \[[@B10]\], epidemic curves were compared for two influenza surveillance systems and timeliness was assessed as the time interval between the epidemic peaks noted in each system. In addition, two studies described the factors associated with delayed reporting \[[@B13],[@B15]\].
Seven of the eight studies addressed whether the calculated timeliness measure met the needs of the surveillance process being evaluated \[[@B10],[@B12]-[@B17]\]. Measured timeliness was compared with recommended reporting timeliness in two papers -- a national recommendation for local tuberculosis reporting timeliness \[[@B13]\] and a state mandate for reporting meningococcal disease cases to local public health \[[@B12]\]. The adequacy of the timeliness measure for the surveillance purpose was also assessed in other ways: 1) by comparing the timeliness of the same surveillance interval in an AIDS surveillance system before and after a major revision in the AIDS surveillance case definition \[[@B17]\], 2) by comparing the timeliness of the same surveillance interval across an active and a passive AIDS surveillance system \[[@B14]\], 3) by comparing outbreak detection abilities of an existing sentinel health care provider-based surveillance system for influenza-like illness with a new school-based system monitoring illness absenteeism \[[@B10]\], 4) by assessing whether reporting timeliness for *Neisseria meningitidis*and *Haemophilus influenzae*was adequate to initiate a rapid public health response \[[@B16]\], and 5) by comparing the timeliness of reporting by whether the case-patient\'s initial AIDS-defining condition was included in the 1997 or 1993 AIDS surveillance case definition \[[@B15]\].
The reporting timeliness of AIDS and bacterial meningitis (including meningococcal disease) surveillance systems were more frequently assessed than those for other infectious diseases. The AIDS reporting timeliness studies indicate that local and national AIDS reporting timeliness meets the goals of the AIDS surveillance systems monitoring trends, targeting prevention programs, estimating needs for medical and social services, and allocating resources \[[@B14],[@B15],[@B17]\]. Timeliness of AIDS surveillance improved after the revision of the AIDS surveillance case definition in 1993 \[[@B14],[@B15],[@B17]\]. Evaluation of Tennessee\'s *Neisseria meningitidis*infection surveillance system for 1989--1992 indicated that the lengthy reporting interval limited the usefulness of the system for supporting rapid response for control and prevention \[[@B16]\]. In contrast, a 1991 evaluation of New York State\'s meningococcal surveillance system indicated that the majority of cases (66%) were being reported within the recommended time frame (i.e., within one day of the diagnosis to ensure chemoprophylaxis for exposed persons) and therefore, supported prevention and control efforts \[[@B12]\]. In addition, on the basis of nationally notifiable infectious disease data from 1987, bacterial meningitis had the shortest reporting timeliness (median 20 days) of the other infectious diseases studied \[[@B11]\].
The definition of reference dates used in the timeliness evaluations varied. The initial date associated with the case varied among date of disease onset, date of diagnosis, and date of positive culture result. The ending date for the timeliness studies evaluated was the date the case report was received by the public health system, whether at the local, state, or national level. This time period corresponds to the sum of Intervals 1 and 2 or Interval 2 alone for local or state timeliness studies (Figure [1](#F1){ref-type="fig"}). For national evaluations of timeliness, the time period assessed was the sum of Intervals 1, 2, 3, and 4 or only Intervals 2, 3, and 4 (with or without inclusion of Intervals 5, 6, 7, and 8, dependent upon state protocol).
NNDSS timeliness
----------------
For *MMWR*years 1999--2001, a total of 9,276 cases of cryptosporidiosis, 12,332 cases of *E. coli*O157:H7 infection, 41,058 cases of hepatitis A virus acute infection, 7,090 cases of meningococcal disease, 22,735 cases of pertussis, 120,688 cases of salmonellosis, and 60,693 cases of shigellosis and were reported to NNDSS. Of those, 7,079 (76.3%) cryptosporidiosis case reports, 9,674 (78.4%) case reports of *E. coli*O157:H7 infection, 32,953 (80.3%) case reports of acute hepatitis A virus infection, 5,580 (78.7%) case reports of meningococcal disease, 19,904 (87.5%) case reports of pertussis, 84,746 (70.2%) case reports of salmonellosis, and 41,643 (68.6%) case reports of shigellosis were eligible for analysis. A total of 72,293 (26.4%) case reports were excluded for one or more of the following reasons: reported as a summary or aggregate record in which individual cases may have different event dates (20,194 cases), unknown or missing date types (20,019 cases), date type coded to *MMWR*report date (11,851 cases), and calculated reporting lag had a value of zero (indicating the event date and midpoint of the *MMWR*week matched) or had a negative value (indicating the event date was later than the mid-point of the *MMWR*week \[67,557 cases\]).
Timeliness of reporting varied by disease and date type (Table [3](#T2){ref-type="table"}). For cases reported with a disease onset date, the median reporting delay across all reporting states varied from 12 days for meningococcal disease to 40 days for pertussis. For cases reported with a laboratory result date, median reporting delay varied from 10 days for both meningococcal disease and shigellosis to 19 days for pertussis. There was also substantial variation in state-specific median reporting delays for each disease (Table [3](#T2){ref-type="table"}). For example, for meningococcal disease cases reported with a laboratory result date, state-specific median reporting delay varied from a median of 2 days in one state to 117 days in another.
::: {#T2 .table-wrap}
Table 3
::: {.caption}
######
Timeliness of reporting of selected nationally notifiable infectious diseases, by date type, NNDSS, 1999--2001
:::
**Date type (Intervals from Figure 1)**
---------------------------------------------------- ----------------------------------------- ---------- -------------- ----------
**Cryptosporidiosis**(7 day incubation period)
Median time interval (days) 22 14 13 26
State-specific reporting range^a^ 2--149 1--73 2--58 1--53
No. cases 4,130 956 1,825 168
No. states 44 24 41 15
\% within 1, 2 incubation periods^b^ 24%, 39% 37%, 50% 35%, 54% 19%, 33%
***E. Coli*O157:H7**(4 day incubation period)
Median time interval (days) 17 21 11 15
State-specific reporting range^a^ 2--81 2--41 1--53 1--49
No. cases 6,891 473 2,206 104
No. states 48 22 39 14
\% within 1, 2 incubation periods^b^ 15%, 27% 13%, 25% 19%, 39% 21%, 33%
**Hepatitis A, acute**(30 day incubation period)
Median time interval (days) 23 18 12 12
State-specific reporting range^a^ 2--54 2--80 2--29,231^+^ 1--126
No. cases 21,570 4,394 6,695 294
No. states 49 36 39 14
\% within 1, 2 incubation periods^b^ 62%, 84% 67%, 83% 82%, 94% 79%, 91%
**Meningococcal disease**(4 day incubation period)
Median time interval (days) 12 13 10 10
State-specific reporting range^a^ 2--56 1--54 2--117 4--62
No. cases 3,804 450 1,255 71
No. states 50 30 39 7
\% within 1, 2 incubation periods^b^ 23%, 39% 26%, 40% 25%, 44% 31%, 42%
**Pertussis**(20 day incubation period)
Median time interval (days) 40 31 19 23
State-specific reporting range^a^ 2--124 1--106 2--190 2--48
No. cases 18,750 289 758 107
No. states 50 26 34 15
\% within 1, 2 incubation periods^b^ 24%, 50% 34%, 60% 53%, 78% 45%, 68%
**Salmonellosis**(1.5 day incubation period)
Median time interval (days) 17 7 12 16
State-specific reporting range^a^ 2--44 1--54 2--61 1--27
No. cases 49,659 5,558 28,172 1,357
No. states 47 35 42 28
\% within 1, 2 incubation periods^b^ 4%, 13% 17%, 43% 6%, 17% 7%, 19%
**Shigellosis**(3 day incubation period)
Median time interval (days) 15 10 10 9
State-specific reporting range^a^ 2--43 1--51 2--34 1--26
No. cases 26,635 2,850 11,603 555
No. states 46 28 41 17
\% within 1, 2 incubation periods^b^ 15%, 22% 33%, 39% 22%, 35% 29%, 41%
\*Source: Control of Communicable Diseases Manual 17^th^Edition \[8\]. ^+^The maximum state-specific median reporting delay for this disease and date type is from a state that reported 19 cases having event years 1919 or 1920. Excluding these cases as data entry errors, the maximum state-specific median reporting delay is 78 days. ^a^State-specific median reporting range (minimum, maximum) in days ^b^% of cases reported within 1 and 2 incubation periods, respectively
:::
For the same date type, NNDSS diseases with longest incubation periods tended to have a higher percentage of cases reported within one or two incubation periods than NNDSS diseases with shorter incubation periods (Table [3](#T2){ref-type="table"}). For example, for acute hepatitis A virus infection, which had the longest incubation period of all the study diseases, more than 60% of cases were reported within one incubation period, for each date type reported. For all other diseases except pertussis, less than 40% of cases were reported within one incubation period for each reported date type. For pertussis, the percentage of cases reported within one incubation period varied from 24% for reports with disease onset date to 53% for case reports with laboratory result dates. In addition, state-specific percentage of cases reported within one or two incubation periods varied for a given disease and date type (data not shown).
Comparison of NNDSS timeliness and literature review results
------------------------------------------------------------
The 1999--2001 NNDSS meningococcal disease median reporting interval between date of disease onset and date of report to CDC in this study was 8 days shorter than a previous study reported \[[@B11]\] using 1987 notifiable disease data for bacterial meningitis (median 20 days); and, the meningococcal disease median reporting delay was 9 days shorter in this study than in a previous study \[[@B16]\] using Tennessee\'s data for the years 1989--1992 for *Neisseria meningitidis*infection (median 21 days). In addition, the median reporting delay between disease onset and the date of report to CDC was shorter in this study than in a previous study (which used 1987 notifiable disease data) by 10 days for hepatitis A, 5 days for salmonellosis, and 8 days for shigellosis \[[@B11]\].
Discussion
==========
Few published studies evaluating surveillance systems presented timeliness measures. When timeliness was evaluated, standard methods were not used. Information collected by public health surveillance systems should support the quantitative assessment of timeliness by various steps in the pubic health surveillance process. Public health programs should periodically assess timeliness of specific steps in the surveillance system process to ensure that the objectives of the surveillance system are being met. A more structured approach to describing timeliness studies should be considered.
Published papers describing local or state surveillance system reporting timeliness generally do not explicitly describe the surveillance system processes contributing to the timeliness measure, such as processing and analyzing the data or implementing a public health action before data are reported from a state to CDC. To facilitate future comparisons of reporting timeliness across jurisdictions, studies should include an explicit description of the public health surveillance reporting process and the surveillance process interval being measured. Additionally, surveillance information systems must support the collection of appropriate reference dates to allow the assessment of the timeliness of specific surveillance processes.
A more structured approach to describing timeliness studies could include a description of the following characteristics: 1) the level of the public health system being assessed (e.g., local, state, or national), 2) the purpose of the surveillance evaluation, 3) goals of the surveillance system, 4) the surveillance interval being measured and a description of the reference dates that define the upper and lower boundaries of the surveillance interval, 5) the surveillance steps (processes or activities) that contribute to the surveillance interval being measured, 6) whether the measured timeliness met the needs of the surveillance step being evaluated, and 7) whether the timeliness met the goals of the surveillance system. No single timeliness measure will achieve the purpose of all evaluations or meet all the goals of the surveillance system. In addition, if the goal of the surveillance evaluation is to identify ways to improve timeliness, the analysis should identify factors associated with delayed reporting, such as the role of specific case ascertainment sources.
The 1999--2001 national notifiable diseases data were timely enough to support the following surveillance objectives: monitoring trends over time, informing allocation of public health resources, monitoring the effectiveness of disease control, identifying high risk populations, and testing hypotheses. If NNDSS data are to be used to support timely identification of and response to multistate outbreaks at the national level, the timeliness of reporting needs to be enhanced for all diseases, but especially for diseases with the shortest incubation periods (e.g., cryptosporidiosis, *E. coli*O157:H7, meningococcal disease, salmonellosis, and shigellosis). Until reporting timeliness is enhanced, the application of aberration detection analytic methods to NNDSS data to aid in the identification of changes in disease reporting that may indicate a multistate outbreak in time to alert states for the purposes of disease control and prevention may be of limited use. Future work to improve reporting timeliness will need to address the substantial variation across states. As states enhance their reporting mechanisms with the use of automated electronic laboratory reporting systems \[[@B18]\], there may be less variation in state-specific reporting timeliness, but this should be assessed.
NNDSS timeliness improved compared to timeliness of notifiable infectious diseases measured in previous reports \[[@B11],[@B16]\]. However, the methods or variables used in these analyses were different. A few factors may have contributed to improvements in timeliness seen in this study. Since 1992, states have been routinely transmitting electronic case-specific records intended to improve reporting procedures and protocols. In addition, the use of automated electronic laboratory reporting to enhance infectious disease case reporting may have contributed to increased timeliness.
Our study findings are subject to several limitations. The variables available for assessing NNDSS reporting timeliness are based on the *MMWR*week numbers that are assigned by states and the earliest known date reported in association with the case. While these variables might provide an estimate of national reporting timeliness, NNDSS data do not include a fixed date defining when a case report was initially transmitted to CDC or received at CDC, which would provide a more precise measure of national reporting timeliness. NNDSS data management protocols should be modified to permit direct calculation of national reporting timeliness. If the ability to support outbreak detection at the national level using NNDSS data is generally viewed as an important and sustainable enhancement for the NNDSS, states and CDC programs should facilitate reporting that more closely approximates real-time and define reporting protocols and data requirements to ensure that reporting timeliness can be improved and accurately monitored. The current NNDSS practice of weekly reporting and data processing limits reporting timeliness to CDC. Lastly, 72,293 (26.4%) cases were excluded from our analysis because the information contained in the database would not permit calculation of timeliness and this exclusion may have resulted in our study results either falsely overestimating or underestimating the magnitude of NNDSS reporting lags.
The reporting timeliness variations across states may result from different reporting protocols in the states (e.g., centralized versus distributed reporting within the state\'s public health system) or from variations in how states assign *MMWR*week numbers. Other factors that might have contributed to reporting delay in our study included: the patient\'s recognition of symptoms; the patient\'s acquisition of medical care; the use of confirmatory laboratory testing; reporting by the health care provider or the laboratory to the local, county, or state public health authority; the volume of cases identified in the state; case follow-up investigations to verify the case report or to collect additional case information; periods of decreased surveillance system activity due to variable staffing levels; computer system down-time for maintenance, upgrades, or new application development; and data processing routines, such as data validation or error checking. Following a structured approach to evaluation of timeliness by specifying the surveillance objectives and the process(es) being measured may allow better definition of the factors that contribute to reporting delay. It was beyond the scope of this study to assess how these factors contribute to NNDSS reporting timeliness.
In addition to reporting timeliness, other surveillance system attributes are important to assess (e.g., completeness of reporting). Completeness of notifiable infectious diseases reporting in the United States varies from 9% to 99% \[[@B7]\]. Six of the eight papers reviewed for this study assessed completeness of reporting \[[@B12]-[@B17]\]. One paper \[[@B14]\] noted that although the timeliness of the AIDS passive and active surveillance systems were comparable, the completeness of the active AIDS reporting system far exceeded the reporting completeness for the passive system. This highlights the importance of evaluating completeness and timeliness and other surveillance system attributes concurrently, before contemplating any changes to a surveillance system based on the assessment of a single attribute.
To improve public health surveillance infrastructure and performance in the United States, CDC and local and state health agencies are integrating a number of public health surveillance systems monitoring infectious diseases in the United States, including the NNDSS, into the National Electronic Disease Surveillance System (NEDSS) \[[@B19],[@B20]\]. NEDSS outlines a standards-based approach to disease surveillance and intends to connect public health surveillance to the clinical information systems infrastructure. As a result, NEDSS promises to improve the accuracy, completeness, and timeliness of disease reporting to state and local health departments and CDC.
Conclusions
===========
To facilitate comparisons of surveillance system timeliness studies across jurisdictions or health conditions, a more standardized approach to describing timeliness studies is warranted. Public health surveillance systems should ensure that timeliness can be measured for specific surveillance system processes and in the context of the goals of surveillance. In addition, when timeliness is being measured, it is important to be explicit about how it is being measured. Our analysis of NNDSS reporting timeliness suggests that current acute hepatitis A infection reporting timeliness may be sufficient to support a timely public health response in the event of a multistate outbreak. However, for the other conditions evaluated, the long reporting lag and the variability across states limits the usefulness of NNDSS data and aberration detection analysis of those data for identification of and response to multistate outbreaks. The NNDSS timeliness data presented in this paper represents a baseline against which timeliness can be measured in the future. Further study is needed to identify the major sources of reporting delay and to assess how NNDSS reporting timeliness may be improved for the timely detection of cases and disease clusters.
Competing interests
===================
None declared.
Author\'s contributions
=======================
Both authors contributed equally to project conception and write-up of the manuscript. RAJ was responsible for data analysis. Both authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2458/4/29/prepub>
Supplementary Material
======================
::: {.caption}
###### Additional File 1
Published reports quantitatively measuring timeliness of reporting infectious disease surveillance data. Table summarizes the findings of the review of published literature about quantitative measurements of infectious disease surveillance system timeliness
:::
::: {.caption}
######
Click here for file
:::
Acknowledgements
================
We wish to acknowledge the health departments from the 50 U.S. States, New York City, and Washington DC that collect nationally notifiable infectious disease data from various case ascertainment sources and voluntarily report these data to CDC.
| PubMed Central | 2024-06-05T03:55:46.680758 | 2004-7-26 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509250/",
"journal": "BMC Public Health. 2004 Jul 26; 4:29",
"authors": [
{
"first": "Ruth Ann",
"last": "Jajosky"
},
{
"first": "Samuel L",
"last": "Groseclose"
}
]
} |
PMC509251 | Background
==========
The Mini-Mental State Examination (MMSE) is a brief screening test that quantitatively assesses the cognitive status of elderly people \[[@B1],[@B2]\]. It is easy to administer and has shown good reliability. Although its validity as a screening test is acceptable for clinical samples, it has been shown to have difficulty in discriminating between demented and non-demented individuals in community-based samples \[[@B2]\]. The MMSE has been found to be influenced largely by pre-morbid ability and is less sensitive to focal brain dysfunction \[[@B3]\] or mild dementia \[[@B4]\].
These limitations led to the development of the Modified Mini-Mental State Examination (3MS) in 1987 \[[@B5]\], which expanded the MMSE from 30 to 100 points to provide finer discrimination. In addition, the 3MS added four items: personal information, including date and place of birth; verbal fluency; abstract reasoning; and a second delayed recall trial. The 3MS also graded temporal orientation and broadened the delayed recall measures, which included cued and recognition formats. While retaining the brevity and ease of administration of the MMSE, the 3MS improved the validity and reliability of identifying individuals with dementia \[[@B6],[@B7]\], and in predicting functional outcomes in patients with stroke \[[@B8]\].
In 2002, a group from the Cache County Study modified the 3MS for use as a cognitive screen in an epidemiologic study of dementia \[[@B9]\]. These modifications substituted the recall of personal demographic information with the recall of current and past prominent politicians. The main reason for this modification was the difficulty the researchers had in verifying personal demographic information. In addition, the scaling of the items in the time orientation and writing parts of the test was changed, and the time allotted for animal naming was shortened. This revised form of the 3MS (3MS-R) demonstrated good sensitivity in detecting dementia in a general population and providing age- and education-specific normative data and cut-off values at the 7^th^and 10^th^percentiles \[[@B9]\].
In Korea, the MMSE was translated into two versions, the Korean version of the Mini-Mental State Examination (MMSE-K) and the Korean Mini-Mental State Examination (K-MMSE) \[[@B10],[@B11]\]. Both Korean versions of the MMSE were tested for validity and efficacy in clinical settings \[[@B11],[@B12]\] and partly in epidemiologic research \[[@B13]\]. Both were somewhat modified to adjust better to the cultural background in Korea, but both shared all the limitations of the original MMSE. Recently the Korean Modified Mini-Mental State Examination (K-3MS) was introduced and validated in a clinical setting \[[@B14]\]. Although the K-3MS was found to be a reliable cognitive screening measure, there was no significant difference between the K-3MS and extracted MMSE for detecting individuals with dementia. In addition, components of the K-3MS could not be compared with items extracted from the K-MMSE and MMSE-K. We have therefore introduced the Korean version of modified Mini-Mental State Examination (K-mMMSE), and we have determined whether its validity is superior to that of the K-MMSE as a screen for cognitive impairment or dementia in a community setting.
Methods
=======
Subjects
--------
Potential participants for this study were recruited from all inhabitants of Noam-dong, Namwon-city, Jeonbuk-province, South Korea, aged 65 and over in 2003, as recorded in national residents registration lists. The area surveyed covered 8.93 km^2^and had an estimated population of 6,883, of whom about 7% were aged 65 or over. All participants gave informed consent, and the study was conducted in accordance with the guidelines in The Declaration of Helsinki and approved by the appropriate research ethics committee.
Among the 522 eligible subjects aged 65 and over identified from registration lists, 235 (45%) completed clinical examinations after the interview and formed the study sample for principal analysis. Of the remaining subjects, we were unable to establish contact with 162 (31%), 75 (14%) refused to participate, 18 (3%) did not complete the survey, 7 (1%) had severe pre-morbid illness including blindness and deafness, 4 (0.8%) had changed address, and 3 (0.6%) had died before the visit. The principal apparent reason for the difficulties in establishing contact was that the person was in a regular daily activity or away from home, visiting family members living elsewhere. The 18 individuals (3%) who did not complete the survey questionnaire or were not examined clinically had a mean age of 74.9 ± 10.3 years; 13 (72%) were females, and 4 (22%) were educated. Of all the eligible subjects, participants and non-participants did not differ in age (73.5 ± 6.8 y vs. 74.6 ± 7.8 y, respectively) or gender (66% and 62%, all *P*values \> 0.1).
Assessment and measurements
---------------------------
Interviewers received a seven-day training session on administering the screening instruments and were supervised throughout by the project neurologist. Cognitive status was classified in two stages. In the first stage, interviewers carried out home-based interviews for data on cognitive function, past medical history, and demographic characteristics. All participants were contacted for cognitive screening using a formulated battery, from which the K-mMMSE and K-MMSE were extracted. And they were rated by a knowledgeable informant using the Short form of Samsung Dementia Questionnaire (S-SDQ) and Korean Instrumental Activities of Daily Living (K-IADL) \[[@B15],[@B16]\]. The S-SDQ is a Korean version of the Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE), with 15 items and scores ranging from 0 to 30 \[[@B15]\]. The K-IADL is composed of 11 items that grade functional abilities, with scores calculated as the sum of points over the number of applicable questions and ranging from 0.0 to 3.0 \[[@B16]\]. High scores on the S-SDQ and K-IADL indicate poor performance. Both tests were found to be uncontaminated by pre-morbid ability, including education or age \[[@B15],[@B16]\].
At the second interview, physicians who were blinded to the cognitive scores performed a clinical examination and neuropsychiatric inventory on participants who completed the first survey questionnaire. The clinical examination verified the presence of cognitive impairment. The diagnostic criteria for dementia were based on those of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) \[[@B17]\] and were subdivided according to the guidelines of the National Institutes of Neurological and Communicative Disorders and Stroke and the Alzheimer\'s disease and Related Disorders Association (NINCDS-ADRDA) \[[@B18]\]. Severity of dementia was staged using the Korean version of the Expanded Clinical Dementia Rating (CDR) scale \[[@B19]\]. The physician and neurologist made independent diagnoses and CDR scoring and subsequently held a case conference to reach a consensus diagnosis, classifying the person as either cognitively normal, cognitively impaired with no dementia (CIND), or having dementia. A diagnosis of CIND represents an attempt to classify people with recognizable cognitive decline who did not meet the criteria for dementia. This group included people who complained of cognitive decline and showed impaired memory function, but did not have any non-cognitive alterations, including intact activities of daily living. At both stages, home visits were repeated on at least two occasions if no contact was made.
Instruments
-----------
We translated the original 3MS-R into Korean according to the guidelines recommended by the modifiers \[[@B9]\]. The item regarding political figures, which asked the participants to name the current president, vice president, and state governors, was replaced with questions about current and previous presidents, because there is no vice president in the government of South Korea. We assessed temporal orientation according to three methods used to calculate year and time in Korea: the solar, lunar and Tangun era. We replaced the words \"shirt,\" \"nickel\" and \"honesty\" in the memory task with the words \"airplane,\" \"pine tree\" and \"sincerity.\" In Korean, the first two words coincided with the K-MMSE items and ended with vowel sounds (\"bee-haeng-gi,\" and \"so-na-mu,\" respectively); while \"seong-sil\" in Korean, which means sincerity, was substituted for \"jeong-jik,\" which is equivalent to the English word honesty, inasmuch as a pilot study found that \"jeong-jik\" was more difficult to hear or perceive than \"seong-sil\", possibly because the latter ended with a voiced sound and could be heard more comfortably.
In explaining the appropriate questions and answers, we presented a simple example prior to asking the first question, specifically, \"the eyes and nose are different, but they are similar in being part of our face.\" In a pilot study, most elderly subjects could not understand the concept of similarity without this example, and they became intolerant to our interview unless an example was provided. After providing the example, however, most subjects were more cooperative and tried to answer properly. We asked subjects to write a spontaneous sentence and scored whether it was legible and correct, with or without prompting. The total possible score was 100, and a K-MMSE score could be generated from it.
Statistical analysis
--------------------
For demographic factors, mean or median values and proportions were calculated according to the cognitive impairment strata. Cronbach\'s alpha was used to quantify internal consistencies of the K-mMMSE and K-MMSE \[[@B20]\]. We assessed the stability and test-retest reliability with 30 subjects who took the K-mMMSE twice. Receiver operating characteristic (ROC) curves were used to determine the validity of the two screening tests graphically and statistically. The areas under the ROC curves (AUC) were calculated and described with standard errors (SE) using the trapezoidal rule \[[@B21]\], and comparison between AUCs was made by the algorithm with an estimated covariance matrix \[[@B22]\]. Cut-off points were chosen to optimize the trade-off between false-negative and false-positive rates. The choice of whether to judge the screening tests by their ability to identify CIND or dementia was addressed practically by assessing both. The validity of the K-mMMSE and K-MMSE were compared, first between those with CIND or dementia and those who were normal, and then between those with dementia and those with CIND or normal. All statistical calculations were performed using Stata 8.2 software (Stata, College Station, TX).
Results
=======
Descriptive statistics
----------------------
Descriptive statistics are displayed in Table [1](#T1){ref-type="table"} for the total sample and according to the cognitive impairment strata. Of the 235 participants, 46 (19.6%) were classified as having dementia and 54 (22.9%) as having CIND. Overall levels of education were very low, in that 118 participants (50.2%) had no formal education, of whom 83 (70.3%) were illiterate.
Severity of cognitive impairment was directly related to mean age and inversely related to number of years of education. Women had higher rates of being cognitively impaired or demented than men. The median scores on the K-mMMSE and K-MMSE decreased with severity of cognitive impairment. Median scores on the informant questionnaires of the S-SDQ and K-IADL increased with poorer cognitive status. The inter-quartile ranges (IQR) also steadily increased, reflecting increasing variability in cognitive and functional status. Four respondents scored perfectly on the K-MMSE, whereas none scored perfectly on the K-mMMSE, suggesting that the K-mMMSE might be less prone to the ceiling effect in this population.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Subject characteristics
:::
All subjects (n = 235) Normal (n = 135) CIND (n = 54) Dementia (n = 46)
--------------------------------------------- ------------------------ ------------------- ------------------- -------------------
**Demographic characteristics**
Age, mean ± SD, y 73.5 ± 6.7 71.9 ± 5.3 73.8 ± 7.4 77.8 ± 7.8
Women, % 66.4 57.8 74.1 82.6
Education, median (IQR), y 1 (0--6) 4 (0--6) 0 (0--5) 0 (0--2)
**Cognitive measures, median (IQR), score**
K-mMMSE 64 (48--80) 78 (66--85) 54 (44--63) 38 (29--47)
K-MMSE 20 (14--25) 24 (20--27) 16 (13--21) 12 (9--15)
S-SDQ 9 (5--13) 7 (3--10) 10 (5--14) 14 (10--21)
K-IADL 0.22 (0.09--0.50) 0.11 (0.00--0.29) 0.27 (0.11--0.60) 1.10 (0.40--1.67)
CIND; Cognitive Impairment No Dementia, SD; standard deviation, IQR; Inter-quartile range, K-mMMSE; Korean version of modified Mini-Mental State Examination, K-MMSE; Korean Mini-Mental State Examination, S-SDQ; Short form of the Samsung Dementia Questionnaire, K-IADL; Korean Instrumental Activities of Daily Living.
:::
Reliability
-----------
The estimated Cronbach\'s alpha was 0.91 for the K-mMMSE and 0.84 for the K-MMSE. Relative to each cognitive impairment stratum (normal, CIND, and dementia), the alphas were 0.84, 0.81 and 0.81, respectively, on the K-mMMSE and 0.74, 0.72, and 0.63, respectively, on the K-MMSE. Neither age nor gender had any substantial impact on internal consistency. Stratum-specific alphas of the K-mMMSE for the different subgroups ranged from 0.86 to 0.91 in men and 0.89 to 0.90 in women.
The retest of the K-mMMSE was assessed in 30 subjects (mean interval, 26 days; range, 19--32 days). The correlation coefficients for the total scores were 0.89 on the K-mMMSE and 0.85 on the K-MMSE. The coefficients of the 15 items of the K-mMMSE were all significant, ranging from 0.37 for similarities to 0.83 for time orientation. The re-tested subjects were representative of the entire study population, in that the sociodemographic characteristics of the 30 retested subjects were similar to the other participants in mean age (74.0 ± 7.4 y vs. 73.5 ± 6.7 y), educational years (3.5 ± 3.2 y vs. 3.4 ± 3.9 y), K-mMMSE scores (63.8 ± 21.0 vs. 63.1 ± 20.4), and proportion of women (80.0% vs. 66.4%; *P*= 0.132).
Validity of the K-mMMSE and K-MMSE
----------------------------------
### Construct validity
Construct validity data between the K-mMMSE and other cognitive or functional measures are shown in Table [2](#T2){ref-type="table"}. K-mMMSE was found to be significantly correlated with all measures, including CDR, Sum of Boxes of CDR (CDR-SB), and informant questionnaires such as the S-SDQ and K-IADL. The correlation coefficient between K-mMMSE and K-MMSE scores was 0.94. According to the CDR scores, the median values of the K-mMMSE and K-MMSE changed significantly (Table [3](#T3){ref-type="table"}).
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Correlations between K-mMMSE, K-MMSE and cognitive or functional measures (CDR, KIADL, and S-SDQ)
:::
K-mMMSE K-MMSE CDR CDR-SB KIADL S-SDQ
--------- ---------- ---------- --------- --------- --------- -------
K-mMMSE 1.000
K-MMSE 0.945\* 1.000
CDR -0.755\* -0.710\* 1.000
CDR-SB -0.750\* -0.702\* 0.966\* 1.000
KIADL -0.648\* -0.614\* 0.740\* 0.793\* 1.000
S-SDQ -0.489\* -0.454\* 0.529\* 0.555\* 0.617\* 1.000
\* *P*\< 0.001 by Pearson\'s correlation analyses. K-mMMSE; Korean version of modified Mini-Mental State Examination, K-MMSE; Korean Mini-Mental State Examination, CDR; Clinical Dementia Rating, CDR-SB; Sum of Boxes of CDR, K-IADL; Korean Instrumental Activities of Daily Living, S-SDQ; Short form of the Samsung Dementia Questionnaire
:::
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
K-mMMSE and K-MMSE scores for each CDR group
:::
CDR
------------------------- ------------- ------------- ------------- -------------
K-mMMSE, Median (IQR)\* 78 (66--85) 54 (44--63) 44 (35--50) 29 (12--31)
K-MMSE, Median (IQR)\* 24 (20--27) 16 (13--21) 12 (9--16) 10 (5--11)
\* *P*\< 0.001 by Kruskal-Wallis test. K-mMMSE; Korean version of modified Mini-Mental State Examination, K-MMSE; Korean Mini-Mental State Examination, CDR; Clinical Dementia Rating, IQR; Inter-quartile ranges.
:::
### Identification of combined CIND and dementia
The performance of the K-mMMSE (AUC ± SE, 0.91 ± 0.02) was significantly superior to that of the K-MMSE (0.89 ± 0.02; *P*= 0.041). The ROC curves plotted in the same graph suggested that the performance of the K-mMMSE was superior to that of the K-MMSE at almost all cut-off points (Figure [1](#F1){ref-type="fig"}). At a cut-off of 69/70 for CIND, the K-mMMSE had a sensitivity of 0.86 (95% Confidence Intervals, 0.78--0.92), a specificity of 0.79 (0.71--0.86), a positive likelihood ratio (LR) of 4.15, and a negative LR of 0.18. In comparison, at a cut-off of 20/21 for CIND, the K-MMSE had a sensitivity of 0.82 (0.73--0.89), a specificity of 0.79 (0.71--0.86), a positive LR of 3.95 and a negative LR of 0.23.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Receiver operating characteristic (ROC) curves of the K-mMMSE and the K-MMSE for combined CIND and dementia.**K-mMMSE (light blue), K-MMSE (brown), and diagonal line. Area under ROC Curves (AUC): K-mMMSE = 0.91, K-MMSE = 0.89.
:::
:::
### Identification of dementia
Both the K-mMMSE and the K-MMSE could properly discriminate demented from normal individuals, but there was no significant difference between them (AUC, 0.92 vs 0.91; *P*= NS). At the cut-off of 59/60, the K-mMMSE had a sensitivity of 0.91 (0.79--0.98), a specificity of 0.78 (0.72--0.84), a positive LR of 4.21 and a negative LR of 0.11. At a cut-off point of 18/19, the K-MMSE had a sensitivity of 0.91 (0.79--0.98), a specificity of 0.76 (0.69--0.82), a positive LR of 3.82 and a negative LR of 0.11.
Discussion
==========
We have shown here that the K-mMMSE is a valid, reliable, and stable cognitive screening instrument, as well as being more sensitive to all levels of CIND and dementia, compared to the K-MMSE. The K-mMMSE has been shown to have a broader spectrum of cognitive domains, including political figures, word fluency, similarities, and delayed recall. Furthermore, the expanded 100 point scoring allows finer discrimination of cognitive impairment. Thus, the K-mMMSE represents a summary form of administration and scoring.
Internal consistency results of the K-mMMSE and K-MMSE were comparable to those observed in previous community studies. Cronbach\'s alpha (á) for the 3MS has been reported to be 0.91 in a community study, a value identical to that found here \[[@B23]\]. Another population study has reported alphas for the 3MS and MMSE of 0.87 and 0.78, respectively, which were slightly lower than our findings of 0.91 and 0.84 \[[@B6]\]. Cronbach\'s alpha has been reported to be influenced by educational status or variability of response, in that it was higher in groups having fewer years of education \[[@B24]\] and in clinical populations having greater variability \[[@B25]\]. Our population consisted of a high percentage with no formal education (50.2%), and their scores were very variable (inter-quartile ranges for the K-mMMSE and the K-MMSE of 48--80 and 14--25, respectively).
Test-retest reliability results of the K-mMMSE were also comparable to those in previous studies. Correlation coefficients of 0.91 to 0.93 have been reported for small samples of community dwelling residents and dementia patients, which are slightly higher than our value of 0.89 \[[@B26]\]. The Stirling County Study found a coefficient of 0.78, but items requiring less judgment exhibited lower reliability than items requiring more judgment \[[@B23]\]. In contrast, we found markedly lower reliability in items requiring more judgment, i.e., similarities (*r*= 0.37), compared with simple items, i.e., temporal orientation (*r*= 0.81). The discrepancy might be due to a difference of time lag, in that the Stirling County Study retest was performed over a 3 year interval, with individual retests ranging from 0.9 to 4.0 years. Furthermore their retested subjects were not representative of all participants. We observed a correlation coefficient of 0.85 for the K-MMSE over all levels of cognitive status, which is in line with generally acceptable findings \[[@B2]\]. Scores on the K-mMMSE and K-MMSE increased after retest, with differences in mean values of 4.4 and 2.6 points, respectively, presumably due to a practice or studying effect after a short interval \[[@B1],[@B27],[@B28]\].
The K-mMMSE was superior to the K-MMSE for diagnosis of all levels of CIND or dementia, as well as being slightly superior at almost all cut-off points. Since dementia is usually preceded by CIND or mild cognitive impairment (MCI), the definition of both requires explication \[[@B29],[@B30]\]. Subjects with CIND or MCI have been found to be at increased risk for developing dementia or, more specifically, Alzheimer\'s disease and some vascular subtypes of dementia \[[@B30],[@B31]\]. The difference between the K-mMMSE and the K-MMSE in diagnosing this condition might mean that the former was more sensitive to the mild stage or pre-dementia than the latter. In this respect, the K-mMMSE seemed to partially overcome a weakness of the K-MMSE, that is, insensitivity to mild dementia \[[@B4]\]. The present findings suggested that the K-MMSE was actually a fairly reasonable instrument as well. Given the faster administration of the K-MMSE, it would be a choice of clinicians to use optimally, recognizing that the K-MMSE was slightly inferior in terms of its test characteristics.
The two cognitive screening measures did not differ significantly, however, in the detection of dementia. These results are comparable to the findings of McDowell et al. \[[@B6]\]; however, their validity results differed between the two language groups studied, namely French and English speakers. The 3MS was superior only in the diagnosis of combined CIND or dementia in French, but not English, speaking participants. There were fewer French than English participants (434 vs. 1166), and they had fewer years of education (6.8 vs. 9.2 years). These differences were also observed in our study samples, with the most important being the smaller sample size, inasmuch as statistical significance was directly influenced by the total number of participants \[[@B22]\].
Even Cache County modifications to the 3MS showed a good sensitivity and specificity, 3MS-R was also dependent on their cultural and social factors which might limit general use in non-US population \[[@B9]\]. For this reason, cross validation was a very important step for a cultural validation of the instrument. The K-mMMSE was shown to be more significantly correlated with other tests for cognitive status or functional abilities, such as the CDR, S-SDQ, and K-IADL, than was the K-MMSE. The correlation coefficients of the CDR were higher than the informant questionnaires, which might be due to the characteristics of the questions. That is, the K-mMMSE and K-MMSE are cognitive screening measures, and the CDR includes items about the cognitive aspects for scoring, whereas the informant questionnaires (S-SDQ and K-IADL) are comprised only of questions related to functional abilities. To the best of our knowledge, this is the first report showing concurrent validity of the modified MMSE series.
There are important limitations to our findings. First, the subjects who participated in this study showed very low levels of educational background, perhaps limiting its general usefulness, especially regarding the cut-off points for a diagnosis of CIND or dementia. The low educational attainment, however, has been one of important characteristics of our elder population, because they were largely deprived of education due to Korean War and Japanese colonial dominion over the country \[[@B32]\]. And the study design, which showed the validities of and comparison between the two cognitive screening measures, would be appropriate for selected community samples, because all participants have a two-stage interview and a clinical examination, thus reducing verification bias. Second, although the trapezoidal rule provide a more accurate method of estimating the \"true\" AUC, an AUC derived from the parameters of a straight-line fit to the ROC plot tends to slightly underestimate the AUC of a Gaussian-based ROC. Finally, although we observed no significant differences between participants and non-participants, the rate of participation in our study was somewhat low. The majority of non-participants were those with whom we could not meet on two separate visits, suggesting that individuals who refused to participate may be more intelligent or active than the participants. If this were true, however, our results would not change, and additional statistical power may be added to our analysis.
Conclusions
===========
We conclude that the K-mMMSE is a valid, stable, and reliable cognitive screen in an epidemiologic study. The K-mMMSE is more sensitive to all levels of CIND and dementia than the K-MMSE. Future investigations with the K-mMMSE will examine age-, sex-, and education-specific reference values to determine how performance patterns on individual items may discriminate between those with or without cognitive impairment and dementia subtypes.
Competing interests
===================
None declared.
Authors\' contributions
=======================
SKJ performed physical measurements, collected data, and drafted the manuscript. KHC participated in data collection and reviewed the manuscript. JMK conceived of the study and participated in its design, and also performed physical measurements. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2458/4/31/prepub>
Acknowledgements
================
We thank Professors Seol-Heui Han, Jin-Sang Yoon, and Young-Hyun Kim for their valuable assistance in preparing the manuscript.
| PubMed Central | 2024-06-05T03:55:46.684210 | 2004-7-30 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509251/",
"journal": "BMC Public Health. 2004 Jul 30; 4:31",
"authors": [
{
"first": "Seul-Ki",
"last": "Jeong"
},
{
"first": "Ki-Hyun",
"last": "Cho"
},
{
"first": "Jae-Min",
"last": "Kim"
}
]
} |
PMC509252 | Background
==========
Medical ethics has increasingly become a common component of the undergraduate curriculum at many medical schools, often within a defined humanities program \[[@B1]-[@B3]\]. In 1999, the European Federation of Internal Medicine, the American College of Physicians and the American Board of Internal Medicine launched the Medical Professionalism Project, which placed medical ethics at the centre of a charter of behaviour and attitudes for all physicians \[[@B4]\]. In 2003, the Liaison Committee on Medical Education in the USA identified the teaching of medical ethics as a core curriculum component of modern medical school education \[[@B5]\].
Medical ethics teaching can occur within the traditional model of medical education, where a separate program of clinical instruction follows several years of medical sciences \[[@B6]\]. However, there is some evidence to suggest that a vertically integrated learning model where the study of clinical subjects runs parallels to and is integrated with basic sciences may be more effective \[[@B7],[@B8]\]. This has resulted in a trend towards including clinical experience in the early years of the medical school curriculum. There is increasing evidence that this enhances student attitudes towards patients \[[@B9]\], provides a structure for teaching integrated clinical medicine \[[@B10]\] and better prepares students for the clinical years \[[@B11]\].
Although Islam has a long tradition of ethical reasoning, there had been little impetus to establish courses in ethics within medical school curricula in the Arabian Gulf. The Faculty of Medicine and Health Sciences (FMHS) was almost alone in having done so, an initiative introduced by expatriate family physicians from New Zealand who had undertaken training in Western medical ethics. Ethics had been taught at the FMHS since 1997 in a vertically integrated program that spanned all levels of the curriculum, based on the four-principle approach developed by Beauchamp and Childress \[[@B12],[@B13]\] as modified by Herbert; autonomy, beneficence, non-malfeasance and justice \[[@B14],[@B15]\] (See Table [1](#T1){ref-type="table"}). Although it was less than ideal to focus the medical ethics curriculum on an imported model, there was little support within the UAE University for a course centred on local culture and traditions.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Ethical Principles utilized in the teaching program
:::
*Primary Principle* *Subsection principles*
--------------------- -------------------------
1\. Autonomy
a\) Disclosure
b\) Truth telling
c\) Informed consent
d\) Competence
e\) Paternalism
f\) Confidentiality
g\) Decision-making
2\. Beneficence
3\. Non-malfeasance
4\. Justice
:::
English has become the lingua franca of medicine, with most international medical societies, publications and meetings conducted in English \[[@B16],[@B17]\]. This has encouraged the development of English language medical schools in countries where English is a second language, including the Arabian Gulf.
There is a significant body of literature describing how people from different cultures utilise different approaches to the clinical application of medical ethics \[[@B18],[@B19]\]. However, there is little information about teaching medical ethics across cultural and linguistic boundaries. Social anthropology has demonstrated that there is a strategic mixing of language with culture and that both culture and social systems are conceptualised in language \[[@B20],[@B21]\]. This would suggest that students learning medicine in English, their second language, could experience dissonance between the content of their courses and the context of their everyday lives, especially in socially formulated issues such as medical ethics.
The aim of this study was to investigate whether Arabic speaking students studying medicine in an Arabic country, albeit with the language of instruction being English, would be able to correctly identify some of the principles of Western medical ethical reasoning, a discipline that challenged significant cultural and linguistic boundaries.
Methods
=======
Setting
-------
The FMHS at the United Arab Emirates (UAE) University offers a six-year undergraduate program in medicine. Students are admitted after a one-year post high school tertiary orientation year, which includes additional English language instruction. Although all students are citizens of the UAE, speaking Arabic as their first language, the course is conducted in English therefore students are required to meet an English language proficiency level in order to be accepted into the medical school. Faculty members come from across the globe, with approximately 1/3 with English as their first language, 1/3 with Arabic as their first language and 1/3 with a different primary language.
All medical students participated in an introductory course in medical ethics, conducted in the first three weeks of first year and taught by a non-Muslim native English speaker from New Zealand with no knowledge of the Arabic language. Apart from his qualifications in Family Medicine, he had also undergone training in Western medical ethics. Although the course was respectful of Arabic culture and tradition, the content of the introductory course focused on Western medical ethical reasoning and excluded an analysis of Islamic medical ethics.
Based on the principles of adult learning, the educational strategy focused on the use of real clinical encounters after minimal orientation, followed by an interactive debriefing session. Rather than provide a sound basis for ethical reasoning, the aim of the introductory course was to introduce the students to the central importance of ethical principles in medical practice, knowing that a more complete and integrated understanding would develop over the ensuing six years of the course. Following two 45-minute interactive seminars, students in groups of 3 or 4 visited a primary health care centre for one morning, sitting in with an attending physician seeing his or her patients. In the absence of any clinical understanding at this early point in their training, the students were encouraged to focus all their energies on medical ethics.
Following the visit to the primary health care centre each student submitted a personal report for summative assessment in which they described the ethical situations they had observed and then provided an appropriate label according to the four-principal approach. Although the seminars were in held in English, the clinical sessions were conducted in Arabic. When questioned by the investigator (SM), the clinical staff stated they had never received formal instruction in medical ethics. The clinical staff were discouraged from indicating ethical issues to the students; the educational process was for the students to identify these on their own. Following submission of their personal report, a third seminar of the introductory course was held with the same teacher where students were invited to share and discuss their experiences.
Sample
------
This study examined two cohorts of first year students, 33 students from the 2002 -- 2003 academic year and 29 students from the 2003 -- 2004 academic year (i.e. all students enrolled in these courses). Each cohort experienced the same admission criteria, underwent the same curriculum, was taught ethics by the same native English-speaking teacher and utilised the same group of primary health care physicians.
Analysis methodology
--------------------
Although both experienced educators and educational researchers, the investigators (VY and SM) did not teach medical ethics at this level of the curriculum. Each investigator coded all personal reports independently and any discrepancies were resolved by mutual agreement. A positive score for labelling any of the four principles and the subsets of autonomy was only allocated when the accompanying description corresponded with the label given. Merely listing a label was not coded as positive.
As an indicator of general academic ability, the students\' score on a multidisciplinary unit test held at the end of the fifth week of year one was also considered. The results of this test have been considered as a variable in the study to determine if there was a correlation between general academic ability and the students\' competency in identifying ethical principals and to avoid associated bias. This multidisciplinary unit test covered all topics taught in the first 5 weeks including anatomy, chemistry, medical physics and medical ethics. The pass mark was set at 75%. The medical ethics component contributed 5% of the total mark.
As not all students had the same clinical experience, each individual student may not have had the opportunity to observe the same range of ethical issues during their clinical experience. For example, some students may not have seen an example of competence to detail in their report. Hence, as this manuscript concerns each individual student\'s ability to report what he or she as an individual had observed in an experience not shared across the whole group, only numbers of ethical issues identified by individual students are reported rather than the total numbers of students per ethical issue.
A third variable included was their score in a standardised test of English, the Test of English as a Foreign Language (TOEFL) \[[@B22]\], used in the selection process to enter medical school. Although initially designed as a measure of the English language proficiency of international students wishing to study at colleges and universities in the North America, this test was widely used in the Arabian Gulf. The TOEFL is a multiple-choice examination that measured listening, comprehension, vocabulary and reading \[[@B23]\]. This test measures the English language proficiency of non-native speakers of English and as such has relevance in the FMHS as the medical course there is taught in English. The nominal requirement at the FMHS is a minimum score of 500. As this test has a non-linear scoring system, this is substantially lower than the 550 -- 580 required in Western universities in North America, Australasia and the UK.
The Statistical Package for the Social Sciences was used for analysing the results \[[@B24]\]. Simple frequency analysis was used to describe demographics, TOEFL scores, unit test results and quantification of ethics issues. The correlation between the TOEFL score and Multidisciplinary Unit Test Score was assessed by Pearson\'s correlation coefficient as both variables were normally distributed and held a linear relationship. As the number of ethical issues identified was not distributed in a Gaussian fashion, frequency distributions were reported by median and range from minimum to maximum, while the appropriate non-parametric statistics were used: Mann-Whitney U test for comparisons between variables and Kendall\'s tau-b test for bivariate correlations. The level of statistical significance was defined as p \< 0.05.
The Research Ethics Committee of the United Arab Emirates University Faculty of Medicine and Health Sciences, which complies with the ethical rules for human experimentation that are stated in the Declaration of Helsinki, approved the project.
Results
=======
All members of each class participated in this study. Participant demographics are detailed in Table [2](#T2){ref-type="table"}. The age range was from 19--21 years; all were practicing Muslims and had Arabic as their first language.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Participant demographics, TOEFL score and test results
:::
Class
-------------------------------------------------- ------------------- ------------------- ---------------- ----------------
Cohort 2002--2003 Cohort 2003--2004
Male Female Male Female
n 13 20 11 18
TOEFL\* score prior to entry to medical school 498 +/- 27.8 518 +/- 58.2 514.9 +/- 40.9 505.6 +/- 29.0
Multidisciplinary Unit Test Score \[pass = 75%\] 82.8 +/- 5.1 82.7 +/- 6.6 81.9 +/- 6.8 83.9 +/- 3.6
\[All enrolled students in this course participated\] \* The Test of English as a Foreign Language
:::
The similarities of issues identified and described across the group of students who attended the same clinic were highly suggestive that the events described actually occurred. However, there were sufficient differences in identified principles, evidence provided and labels given, to indicate that reports were written independently.
Table [3](#T3){ref-type="table"} details the ethical issues identified. Although the initial expectation was that there were only a limited number of ethical issues that could be identified in a single clinical session, each student acting independently was able to correctly identify a median number of 4 different medical ethical issues (range 2--9) and correctly identify and label accurately a median of 2 different medical ethical issues (range 2--7) There was no significant difference in results between males and females except for \'autonomy mislabelled as a different ethical issue\' (male: median = 0, range= 0--2; female median = 0, range = 0--3; p = 0.002) and \'autonomy / confidentiality mislabelled as a different ethical issue\' (male: all results = 0; female median = 0, range = 0 -- 2; p = 0.03).
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Analysis of Ethics assignments: the median and range from minimum to maximum of the number of issues identified by individual students as detailed in their personal report
:::
Median Range
---------------------------------------------------------------------------- -------- --------
Total number of ethical issues identified:
Labelled correctly 2 2 -- 7
Mislabelled as a different ethical issue or subset of Autonomy not defined 2 3 -- 9
\[Labelled correctly\] + \[mislabelled\] 4 2 -- 9
Total number of issues identified as ethical which were not ethical issues 0 0--4
Total number of autonomy issues identified
Labelled correctly 1 0--4
Labelled as Autonomy, no subsection specified 0 0--3
Mis-labelled as a different ethical issue 0 0--3
Total number of autonomy: disclosure issues identified
Labelled correctly 0 0--1
Labelled as Autonomy, no subsection specified 0 0--1
Mis-labelled as a different ethical issue 0 0--1
Total number of autonomy: truth telling issues identified
Labelled correctly 0 0--1
Labelled as Autonomy, no subsection specified 0 0--1
Mis-labelled as a different ethical issue 0 0 -- 0
Total number of autonomy: informed consent issues identified
Labelled correctly 0 0 -- 2
Labelled as Autonomy, no subsection specified 0 0 -- 1
Mis-labelled as a different ethical issue 0 0 -- 1
Total number of autonomy: competence issues identified
Labelled correctly 0 0 -- 0
Labelled as Autonomy, no subsection specified 0 0 -- 1
Mis-labelled as a different ethical issue 0 0 -- 0
Total number of autonomy: paternalism issues identified
Labelled correctly 0 0 -- 1
Labelled as Autonomy, no subsection specified 0 0 -- 1
Mis-labelled as a different ethical issue 0 0 -- 1
Total number of autonomy: confidentiality issues identified
Labelled correctly 0 0 -- 1
Labelled as Autonomy, no subsection specified 0 0 -- 1
Mis-labelled as a different ethical issue 0 0 -- 2
Total number of autonomy: decision -- making issues identified
Labelled correctly 0 0 -- 2
Labelled as Autonomy, no subsection specified 0 0 -- 2
Mis-labelled as a different ethical issue 0 0 -- 2
Total number of beneficence issues identified
Labelled correctly 0 0 -- 2
Mis-labelled as a different ethical issue 0 0 -- 1
Total number of non-malfeasance issues identified
Labelled correctly 0 0 -- 2
Mis-labelled as a different ethical issue 0 0 -- 3
Total number of justice issues identified
Labelled correctly 0 0 -- 1
Mis-labelled as a different ethical issue 0 0 -- 2
:::
The four cohorts of students displayed a wide range of TOEFL scores ranging from the low 400 s through the mid 600 s. There was a moderate correlation between the TOEFL score and the multidisciplinary unit test score (r = 0.616, p \< 0.001). There was no statistically significant correlation between the TOEFL score and the number of ethical issues identified. There were weak statistically significant correlations between the multidisciplinary unit test scores and ethical issues identified for \'total number of issues correctly identified\' (r = 0.26, p = 0.008) and \'number of autonomy issues correctly identified\' (r = 0.21, p = 0.04). There were no other statistically significant correlations between test scores and ethical issues identified.
Discussion
==========
This study has demonstrated that these first year students were able to identify medical ethical issues in a clinical setting after minimal instruction. Although introductory courses by their very nature aim to build on the students\' previously accumulated knowledge, understanding and experience, especially the unstructured kind based on life experience, the students in this study were able to develop this skill with very minimal instruction, when compared to the normal length of introductory medical ethics courses \[[@B25]\].
Feldman et al found important differences in the ethical practices and beliefs of Internists in the USA and China, even amongst those practicing Western medicine and suggested that some basic bioethical principles may be culturally based rather than universal \[[@B26]\]. One important feature in this regard is the concept of autonomy, where more traditional or tribal based societies, such as is seen in the Arabian Gulf States, often view autonomy as relating to the group rather than the individual. Hence, students learning ethics that incorporate Western constructs and are delivered in a Western language may fail to see the relevance or appropriateness to their understanding of their culture and society. However, this study has demonstrated that the students were able to correctly identify the concept of autonomy and some of its sub groupings.
Although the students in this study demonstrated that they were able to use an imported model of ethical principles, this study did not directly address whether the students embraced the underlying conceptual framework, as the educational objectives of the introductory course were primarily to arouse interest and discussion amongst the students. However, informal review of the performance in examination settings of earlier cohorts of students who underwent similar educational programs suggests that by the end of their pre-registration medical training students do internalise the concepts and appear to understand their clinical applications. Further studies could investigate the impact of ethical training on their eventual behaviour as independent clinicians.
This study had an underlying a priori assumption that the students would not be successful in identifying ethical issues due to a combination of the brief nature of the introductory seminars, their relative youth and inexperience with the adult learning model and being at the beginning of their medical training. Hence, the relatively high number of issues identified and the acceptable accuracy in labelling suggests that these students did demonstrate a reasonable level of skill even in the absence of a control group to help validate this conclusion. With little research available on the impact of early education in medical ethics, further studies utilising a control group would prove beneficial to determine what issues students would have been able to identify with no training.
With ethics and professionalism being globally recognised as an integral component of primary medical degree curriculum, the results of the project suggest that early exposure may be beneficial in that it would provide a solid base in ethics, an essential requirement for their later clinical training. If ethical principles are clearly established early in the students\' training, all future clinical training can be considered from an ethical standpoint.
Jackson found that corporate managers\' ethical decision-making was more influenced by their home country than by the country in which they resided \[[@B27]\]. This suggests the overriding importance culture and tradition has on one\'s values and beliefs systems. Hence the absence of a correlation between English language ability, accuracy in identification of ethical issues and number of issues identified suggests that in this case the basic conceptualization of medical ethics transcended the possible values and beliefs systems implicit in teaching a Western ethical curriculum in the English language in this environment.
A number of researchers have suggested that the four principles of Western medical ethics have always existed in Islam \[[@B28]-[@B30]\]. Islam has an ethical and moral tradition which is intimately linked to Qur\'anic teachings \[[@B31]\]. While the students would be familiar with ethical principles as enshrined in their religious beliefs, the linguistic constructs and labels of Western medical ethics as outlined in the four principle approach would be unfamiliar to these students. Perhaps the ethical principles embedded in their religion enabled the students to juxtapose Western linguistic constructs onto the ethical events observed in the clinical situation. They demonstrated an ability to correctly identify, describe and label ethical events despite using new language constructs, given that English is their second language. The relative importance of the intervention compared to their background knowledge could be further addressed in an expanded study using a control group.
This study found at best a weak correlation between general academic ability and the number and accuracy of ethics issues identified. This suggests that even academically weak students were able to grasp the concepts being taught. Perhaps this outcome was enhanced by two factors; utilizing an adult learning model where students worked together in groups (although assignments were individually prepared) undergoing real rather than simulated experiences and the incorporation of their Islamic religious beliefs and self developed ethical reasoning.
The students in this study had a broad range of English language skills as suggested by the large range in TOEFL results. Although there is no clear evidence that TOEFL correlates with the ability to conceptualise theoretical constructs presented in English, there is strong evidence that TOEFL correlates with grade point average at a North American Universities \[[@B32],[@B33]\], and some evidence it correlates with the degree of participation of UAE students in problem based learning sessions \[[@B34]\].
This study only addressed the ability of medical students at the onset of their medical training to assimilate and conceptualise the basic principles of ethical reasoning. In particular, no assessment was made of the long-term impact of this course on \'moral enculturation\' as this study has only described one component of a longitudinal six-year course in medical ethics.
Conclusions
===========
This study has demonstrated that these students could identify medical ethical issues based on Western constructs, despite learning in English, their second language, being in the third week of their medical school experience and with minimal instruction. This result was independent of their academic and English language skills suggesting that ethical principles as espoused in the four principal approach may be common to the students\' Islamic religious beliefs, allowing them to access complex medical ethical reasoning skills at an early stage in the medical curriculum.
Competing interests
===================
None declared.
Authors\' contributions
=======================
SM conceived the study, prepared the ethics application, coded the student\'s material, analysed the results, prepared and reviewed the manuscript. VY prepared the ethics application, prepared the coding sheet, coded the student\'s material, analysed the results, prepared and reviewed the manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1472-6939/5/4/prepub>
Acknowledgements
================
Nil
| PubMed Central | 2024-06-05T03:55:46.686936 | 2004-7-30 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509252/",
"journal": "BMC Med Ethics. 2004 Jul 30; 5:4",
"authors": [
{
"first": "Valmae A",
"last": "Ypinazar"
},
{
"first": "Stephen A",
"last": "Margolis"
}
]
} |
PMC509278 | Background
==========
Histones H3 and H4 are among the most evolutionarily conserved proteins (\>90% identity from yeast→humans) \[[@B1]\]. Octamers composed of one histone H3/H4 tetramer and two histone H2A/H2B dimers package 146 bp of DNA into the basic repeating subunit of chromatin, the nucleosome \[[@B1]\]. Hence, as fundamental components of chromatin, these proteins are an integral part of all cellular processes involving chromosomal DNA.
The physical characteristics of the histones are precisely regulated in the cell by an elaborate network of post-translational modifications that include acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation \[[@B2]-[@B4]\]. These modifications are found primarily on the NH~2~-terminal tails of the histones. These domains, which protrude from the core of the nucleosome, are free to interact with, and be acted upon by, the nuclear environment. The past several years has seen the identification of numerous enzymes that are capable of modifying the histones. These enzymes are generally found in large, multi-subunit complexes and have activities that are not only specific for a given histone but are specific for particular amino acid residues within the histone \[[@B5],[@B6]\].
The most well characterized histone modifying enzymes are the histone acetyltransferases (HATs). HATs catalyze the transfer of an acetyl moiety from acetyl-coenzyme A to the ε-amino group of lysine residues in the histone NH~2~-terminal tails. Historically, these enzymes have been classified as either type A or type B, based upon substrate specificity and cellular localization \[[@B7]\]. Found in the nucleus, type A HATs utilize nucleosomal histones as substrates. A number of Type A HATs have been identified in yeast. These include Gcn5p (SAGA, ADA, SLIK, SALSA and HAT-A2 complexes), Sas2p (SAS complex), Sas3p (NuA3 complex), Esa1p (NuA4 and picNuA4 complexes) and Elp3 (Elongator complex) \[[@B8]-[@B22]\]. These enzymes have been characterized primarily in the context of transcriptional activation but are likely to be involved in other chromatin mediated events as well \[[@B23],[@B24]\].
Type B HATs were initially described as cytoplasmic enzymes that acetylate free histones in conjunction with chromatin assembly \[[@B7]\]. The *de novo*assembly of chromatin is a complex, multi-step process that occurs most prominently during DNA replication (but also accompanies other cellular processes involving DNA synthesis) \[[@B25],[@B26]\]. Following induction of histone mRNA synthesis, histone proteins are translated in the cytoplasm. For histones H3 and H4, synthesis is rapidly followed by the acetylation of specific lysine residues in their NH~2~-terminal tail domains \[[@B27]\]. For newly synthesized histone H4, this acetylation occurs on lysine residues at positions 5 and 12 in all eukaryotic organisms examined to date \[[@B28],[@B29]\]. For newly synthesized histone H3, acetylation appears to occur in distinct patterns that can differ from organism to organism \[[@B28],[@B30],[@B31]\]. The acetylated H3 and H4 form tetramers that are translocated into the nucleus and loaded onto DNA \[[@B32]\]. Following completion of the histone octamer by histone H2A/H2B addition, mature chromatin is formed following the deacetylation of histones H3 and H4 \[[@B33],[@B34]\].
In contrast to the type A HATs, only one type B HAT has been characterized to date, Hat1p. Hat1p is an evolutionarily conserved enzyme that specifically acetylates free histone H4 \[[@B35]-[@B38]\]. Consistent with its identification as a type B HAT, recombinant yeast Hat1p, as well the Xenopus and Human Hat1p homologs, acetylates both lysine 5 and lysine 12 \[[@B35]-[@B39]\]. Hat1p was originally purified from yeast cytoplasmic extracts in a complex with Hat2p, a yeast homolog of the mammalian Rbap46/48 proteins \[[@B36],[@B40],[@B41]\]. Subsequent studies have shown that yeast Hat1p, as well as its higher eukaryotic counterparts, can also localize to the nucleus \[[@B37],[@B38],[@B42]\]. These results suggest that, while specificity for free histones is a bona fide characteristic, cytoplasmic localization may not be a strict criterion for classification as a type B HAT.
Evidence has accumulated indicating that the acetylation of newly synthesized histones H3 and H4 play over-lapping roles in chromatin assembly. While yeast strains carrying a deletion of either the H3 or H4 NH~2~-terminal tail are viable, concomitant deletion of both NH~2~-termini (or combining tail deletions with alterations in specific sites of acetylation) results in a defect in nucleosome assembly and cell death \[[@B43],[@B44]\]. In addition, while deletion of the *HAT1*gene produces no observable phenotype, combining a deletion of *HAT1*with specific lys→arg mutations in the NH~2~-terminus of histone H3 generates defects in both telomeric silencing and DNA damage repair \[[@B45],[@B46]\]. However, despite the importance of the acetylation of newly synthesized histone H3 in chromatin assembly, there have been no type B histone acetyltransferases described that specifically target histone H3.
To identify potential histone H3-specific type B HATs, we have systematically surveyed yeast extracts for candidate activities. Here we detail one such activity, termed HatB3.1. We provide evidence that this is a novel complex that utilizes Gcn5p as its catalytic subunit. Intriguingly, unlike previously identified Gcn5p-containing HAT complexes, HatB3.1 contains Ada3p, but not Ada2p.
Results
=======
Identification of histone H3-specific type B histone acetyltransferase activities in yeast
------------------------------------------------------------------------------------------
The highly selective activity of the native Hat1p/Hat2p complex for free versus nucleosomal histone H4 is the primary characteristic that distinguishes this enzyme from the type A histone acetyltransferases \[[@B35],[@B36]\]. Therefore, to identify putative histone H3-specific type B HAT complexes, we systematically surveyed yeast extracts for activities that acetylated free histone H3 but not histone H3 packaged into chromatin. Extracts were prepared from cell cultures grown to mid-log phase to enrich for actively dividing cells, as the most robust period of chromatin assembly occurs during DNA replication. Yeast cell walls were digested with zymolyase and cytosolic extracts were produced by the lysis of the cells in low salt buffer followed by centrifugation to remove nuclei and large cell debris. Hence, this extract contained soluble cytoplasmic proteins as well as proteins loosely associated with the nucleus. The nuclear extract was obtained by incubating the nuclear pellet in buffer containing 1.0 M NaCl to extract proteins that are more tightly associated with the nucleus.
It is difficult to reliably detect histone acetyltransferase activities in the relatively crude cytosolic and nuclear extracts. Therefore, to evaluate the intrinsic HAT activities present in each of the extracts, they were fractionated by anion and cation exchange chromatography. Fractions were assayed for HAT activity using ^3^H-acetyl Coenzyme A and equivalent amounts of either free histones or chromatin as substrate. Histones were then resolved by SDS-PAGE and acetylated species visualized by fluorography.
Fractionation of the cytosolic extract on a DEAE column is shown Figure [1A](#F1){ref-type="fig"}. As expected, the predominant type B activity present in these preparations was attributable to Hat1p, as indicated by robust, free histone H4 acetylation (Fig. [1A](#F1){ref-type="fig"}, lanes 28--34). The identity of the Hat1p/Hat2p complex was confirmed by western blot analysis using polyclonal antibodies against both Hat1p and Hat2p (data not shown).
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Identification of putative histone H3-specific type B histone acetyltransferases.**Cytosolic and nuclear extracts were prepared and fractionated as outlined in the flow chart. Inherent HAT activities were identified by assaying column fractions with ^3^H-Acetyl-Coenzyme A and equivalent amounts of either free histones or chromatin (as indicated). Reaction products were resolved by 18% SDS-PAGE and visualized by fluorography. The relative migrations of the core histones, as determined from coomassie blue staining, are denoted at the side of each fluorogram. The positions of Hat1p and putative H3-specific type B HATs are indicated by brackets.
:::
:::
The cytosolic extract also contained at least two additional HAT activities. The first showed a clear peak that was centered on fraction 14 and acetylated free histones H3, H2B and H4. The activity of this HAT on chromatin was more difficult to determine as the H3 and H4 labeling seen in these fractions does not show a marked peak in fraction 14 and may be due to the leading edge of a HAT activity eluting at higher salt. Therefore, this activity may be a candidate type B HAT. There was also a distinct peak of HAT activity at fractions 18--20. With free histone substrates, this activity primarily acetylated histone H3. However, there was also a coincident peak of chromatin H3 and H4 acetylating activity in these fractions suggesting this activity is likely to be a type A HAT.
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**HatB3.1 is a chromatographically distinct activity.**DEAE fractions encompassing HatB3.1 activity were pooled and subjected to further fractionation. Fluorograms of liquid HAT assays, using free histones or chromatin as substrates, representative of gradient eluted Mono Q column fractions, showed that HatB3.1 activity can be separated from the overlapping activities present in the initial DEAE fractionation. Migration of the core histones is indicated. Migration of free histone H3 activity attributable to HatB3.1 is marked with an arrow.
:::
:::
DEAE fractionation of the nuclear extract also revealed several distinct HAT activities (Figure [1B](#F1){ref-type="fig"}). There were two H4-specific type A HAT activities that peaked at fractions 14 and 18, as indicated by activity on both free and nucleosomal histones. There was also a significant peak of activity that acetylated free histone H3 (there was also slight acetylation of histone H2B that is more easily seen in Figure [3](#F3){ref-type="fig"}) that was coincident with a minor nucleosomal H3 HAT activity (Fig. [1B](#F1){ref-type="fig"} right panel, lanes 16--34). This activity also partly overlapped the nucleosomal H4 activities. This peak of activity was rather broad and most probably results from the partial overlap of at least two distinct activities. In fact, the separation of these activities was readily apparent in Figures [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}. The strong overall preference of these activities for free histone H3 makes them good candidates for H3-specific type B HATs. As these are chromatographically distinct activities we have termed them HatB3.1 and HatB3.2 as indicated (Figure [1B](#F1){ref-type="fig"}, right panel).
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**HatB3.1 activity is dependent upon GCN5.**Nuclear extracts, generated as depicted in Figure 1 from the indicated isogenic deletion strains, were fractionated via DEAE anion exchange chromatography. Fluorograms of HAT assays resolved by 18% SDS-PAGE are shown with the migration of the core histones as indicated. Fractions of equivalent conductance are aligned for each strain. Regions containing HatB3.1, HatB3.2 and Hat1p are identified by brackets.
:::
:::
::: {#F4 .fig}
Figure 4
::: {.caption}
######
**Highly purified HatB3.1 contains Gcn5p and Ada3p in a high molecular weight complex.**A) Flowchart outlining the partial purification of HatB3.1. B) Fluorograms of liquid HAT assays of the Superose 6 column fractionation of HatB3.1 activity (top 2 panels). Assays used either free histones or chromatin as substrate (as indicated). The relative elution of molecular weight standards is shown along the top, while the migration of histones H3 and H4 is indicated at the right. Column fraction aliquots (15 μL) were also resolved by SDS-PAGE and visualized by silver staining (bottom panel, protein ladder mobility is represented at right). Corresponding fraction numbers for both the fluorograms and silver stained gel are indicated along the bottom. C) Peak HatB3.1 containing Superose 6 fractions, as indicated at top of blots, were resolved by three identical 10% SDS gels, transferred to nitrocellulose and probed with the indicated antibodies (left of blots). Presence of Gcn5p, Ada2p and Ada3p in nuclear extract and/or column fractions was visualized via chemifluoresence. Relative migration of protein standards is shown on the right.
:::
:::
Unbound material from the initial DEAE fractionation of the cytosolic and nuclear extracts was analyzed by cation exchange chromatography (carboxymethyl sepharose (CM)). While this fraction from the cytosolic extract appeared inactive, there were several additional HAT activities resolved from the nuclear extract (Figure [1C](#F1){ref-type="fig"}, data not shown). The presence of these activities in the DEAE flowthrough fraction is not simply due to column overloading as recycling the flowthrough fraction over the DEAE column a second time did not result in significant protein retention. Hence, these activities are chromatographically distinct from those that bind the DEAE resin. Two activities, centered on fractions 22 and 30, acetylated primarily histone H4. These appeared to be typical type A HAT\'s as they were active on both free histones and chromatin. A broad peak of histone H3-specific activity eluted from the CM column from fraction 10 through fraction 24 (with activity trailing through the remainder of the gradient). Comparison of the free histone and chromatin activities in these fractions suggested that this region of the gradient actually contained overlapping type A and type B activities. There was a distinct peak of free histone H3 acetylating activity centered on fractions 12 -- 14 while acetylation of chromatin associated H3 peaked in fraction 16. Hence, the activity in fractions 12--14 is another candidate H3-specific type B HAT (labeled HatB3.3).
HatB3.1 is specific for free histone H3
---------------------------------------
The fractions from the DEAE column that contained the activity that we have termed HatB3.1 modified not only free histone H3 but also free H4. In addition, a low level of nucleosomal H3 activity could also be seen in these fractions. To determine whether these activities were the result of a single enzyme complex or were due to multiple, overlapping complexes, these fractions were pooled, dialyzed and fractionated over a Mono-Q column (Figure [2](#F2){ref-type="fig"}). Inspection of the HAT activity profile of the fractions eluting from the Mono-Q column clearly demonstrated that multiple HAT activities overlapped with HatB3.1 during the initial fractionation of the nuclear extract. The HatB3.1 activity eluted from the Mono-Q column very early in the gradient and appeared to be highly specific for free histone H3. The second activity to elute from the Mono-Q column was specific for chromatin-associated histone H4. The third activity acetylated both free and nucleosomal histones H3, H2B and H4. These results indicated that the acetylation of multiple histones in the DEAE elution profile was the result of at least three overlapping activities and confirmed that HatB3.1 is a chromatographically distinct free histone H3-specific activity. Therefore, HatB3.1 was a good candidate for further characterization.
HatB3.1 activity is dependent on GCN5
-------------------------------------
To gain insight into the identity of the catalytic subunit of HatB3.1, we constructed null mutants for each of the yeast HAT\'s that have demonstrated histone H3 activity as well as the known type B HAT, *HAT1*. Isogenic deletion strains (Δ*gcn5*, Δ*sas2*, Δ*sas3*and Δ*hat1*) were grown and protein extracts prepared exactly as for the wild type strain. Nuclear extracts were again fractionated via DEAE column chromatography and fractions of equivalent conductivity assayed for HAT activity as described above.
Parallel comparison of the HAT activity profiles from each strain provided biochemical evidence for the dependency of specific histone acetyltransferase activities on the presence of a particular HAT catalytic subunit (compare Figure [3](#F3){ref-type="fig"} with Figure [1B](#F1){ref-type="fig"}). While subtle variations in observed specificity and intensity of HAT activity were seen throughout the profiles of the Δ*sas2*and Δ*sas3*strains, the robust H3 acetylation attributed to the HatB3.1 activity appeared unaffected by deletion of these enzymes (Figure [3](#F3){ref-type="fig"}, lanes 26--34). Conversely, HatB3.1 activity was abolished in a Δ*gcn5*strain (Figure [3](#F3){ref-type="fig"}, lanes 26--34). In addition, the HatB3.2 activity also appeared to be absent in extracts from a gcn5 strain indicating that both of these putative type B HAT activities are dependent on Gcn5p. Additionally, the integrity, in a Δ*gcn5*strain, of the overlapping free histone and chromatin (data not shown) activities in this region of the gradient confirmed that HatB3.1 was a chromatographically distinct HAT activity exhibiting specificity for free histone H3.
Analysis of the activity profile from nuclear extracts derived from a Δ*hat1*strain identified a broad peak of Hat1p dependent activity that spanned fractions \~22--34. Western blot analysis using antibodies against Hat1p and Hat2p confirmed the presence of these proteins in fractions from this region of the gradient from the wild type extract (data not shown). As with the Hat1p-dependent activity in cytosolic extracts, this activity also appeared to be specific for free histone H4. This result confirmed previous observations indicating that Hat1p is localized to both the cytoplasm and the nucleus \[[@B37],[@B42]\]. In addition, the presence of an authentic type B HAT activity in our nuclear extracts validated our use of these extracts for the identification of putative histone H3-specific type B HAT activities.
HatB3.1 activity is dependent on ADA3 but not ADA2
--------------------------------------------------
There are two proteins, Ada2p and Ada3p, that are components of all known Gcn5p-containing HAT complexes and that are required for the activity of these complexes \[[@B9]-[@B11],[@B13],[@B14]\]. To determine whether the HatB3.1 activity was also dependent on these proteins, nuclear extracts were prepared from isogenic Δ*ada2*and Δ*ada3*strains and the status of the HatB3.1 activity determined by DEAE chromatography. As shown in Figure [4](#F4){ref-type="fig"}, the loss of *ADA2*did not affect either the HatB3.1 or HatB3.2 activity but did cause a substantial increase in the free histone H4 specific activity that eluted late in the DEAE gradient. However, the HAT activity profile of the Δada3 extracts was strikingly similar to that seen for the gcn5 extracts with both the HatB3.1 and HatB3.2 activities absent. These results indicated that the HatB3.1 activity was dependent on *ADA3*and that Ada2p is either not a component of the HatB3.1 activity or is not required for its stability.
Partial purification of HatB3.1
-------------------------------
To further characterize HatB3.1, this activity was purified through several chromatographic steps. The purification scheme is diagramed in Figure [5A](#F5){ref-type="fig"}. HatB3.1 containing fractions from the DEAE column were pooled, dialyzed to a conductivity similar to that of the loading buffer (DN(50)) and the dialysate applied to a cation exchange column (CM sepharose). HAT activity assays indicated that the HatB3.1 activity flowed through the CM sepharose column while bound proteins, resolved by a linear salt gradient, contained co-purifying HAT activities that acetylated both free and nucleosomal, H3 and H4 (data not shown). The presence of HatB3.1 in the CM sepharose flow through also confirmed that HatB3.1 and HatB3.3 were distinct activities.
::: {#F5 .fig}
Figure 5
::: {.caption}
######
**ADA3, but not ADA2, is essential for HatB3.1 activity.**Nuclear extracts from isogenic Δada2 and Δada3 strains were prepared and fractionated as previously described for wild type and HAT deletion strains (Figures 1 and 3). Fluorograms reflecting HAT activity assays from fractions of equivalent conductivity from each strain, using free histones, are shown (fraction numbers are displayed at bottom \[compare lanes to those in figure 3 as well\]). Regions of HatB3.1 and HatB3.2 are highlighted by brackets.
:::
:::
The proteins that flowed through the CM sepharose column were applied to a Mono-Q column and then eluted with a linear salt gradient. Fractions containing free histone H3 activity were pooled and concentrated by precipitation with 75% ammonium sulfate. The sample was then fractionated by size exclusion chromatography using a Superose 6 column. As seen in Figure [5B](#F5){ref-type="fig"}, the HatB3.1 activity peaked at fractions 48--50, indicating that a high molecular weight complex of \~500 kDa was responsible for this activity. The size of HatB3.1 remained stable throughout the course of purification as Superose 6 fractionation of the pooled HatB3.1 activity from the initial DEAE column displayed an identical mass (data not shown). The highly purified HatB3.1 retained its high degree of specificity for free histone versus chromatin substrates. There were also two peaks of free histone H4 specific activity seen in the Superose 6 elution profile. Western blot analysis indicated that Hat1p co-eluted with the low molecular weight species. The second peak of H4 activity co-purified with HatB3.1. Whether this acetylation of histone H4 was the result of a weak specificity of HatB3.1 for H4 or due to a second, co-eluting, HAT activity has not been resolved.
Gcn5p and Ada3p, but not Ada2p, co-purified with the HatB3.1 activity
---------------------------------------------------------------------
The absence of HatB3.1 activity in extracts from a Δgcn5 strain indicated that HatB3.1 was dependent on Gcn5p, either indirectly via Gcn5p-mediated transcriptional regulation, or directly, as its catalytic subunit. While the HatB3.1 activity was highly purified relative to the initial nuclear extract, the peak Superose 6 fractions were still too complex to allow the definitive identification of specific bands that co-purified with the activity (Figure [5B](#F5){ref-type="fig"}). Extensive efforts to purify HatB3.1 to homogeneity have been unsuccessful. To determine whether Gcn5p was likely to be functioning as the catalytic subunit of HatB3.1, fractions across the peak of HatB3.1 activity from the Superose 6 column were probed with anti-Gcn5p antibodies. As seen in Figure [5C](#F5){ref-type="fig"}, Gcn5p was present in the fractions containing the peak of HatB3.1 activity from the Superose 6 column. This result is consistent with direct association of Gcn5p with the HatB3.1 complex.
Duplicate blots were probed with anti-Ada2p and anti-Ada3p antibodies to determine whether these proteins also co-fractionated with the HatB3.1 complex. As expected, both Ada2p and Ada3p are present in the nuclear extracts (Figure [5C](#F5){ref-type="fig"}). However, while Ada3p precisely co-purified with Gcn5p and the peak of HatB3.1 activity, Ada2p did not appear to be associated with this complex. The absence of the Ada2p from the peak of HatB3.1 activity is consistent with the observation that HatB3.1 activity is independent of the ADA2 gene and suggests that Ada2p is not a component of the HatB3.1 complex. The absence of an Ada2p signal on the Western blot was not due to problems with sensitivity as comparison of the relative signals of Gcn5p, Ada2p and Ada3p in the nuclear extracts and Superose 6 fractions demonstrated that the presence of Ada2p in the Superose 6 fractions would have been readily apparent. While the HatB3.1 activity is enriched in the Superose 6 peak fractions relative to the original nuclear extract, the amount of Gcn5p and Ada3p present in these fractions is not enriched relative to the nuclear extract due to the fact that these proteins are components of at least five other histone acetyltransferase complexes. Hence, only a fraction of the Gcn5p and Ada3p present in the cell extracts was associated with HatB3.1.
Discussion
==========
Considerable genetic and biochemical evidence indicates that, in most organisms, newly synthesized histone H3 is acetylated and that this acetylation plays a role in the *de novo*assembly of chromatin \[[@B28],[@B30],[@B31],[@B43]-[@B48]\]. However, the enzymes responsible for this modification have remained elusive. In the present study we have comprehensively surveyed yeast extracts for putative, histone H3-specific, type B histone acetyltransferase activities. At least three candidate activities were identified, HatB3.1, HatB3.2 and HatB3.3. Further characterization of HatB3.1 indicated that this activity is a novel \~500 kDa HAT complex. In addition, our results suggest that Gcn5p and Ada3p are components of this complex but that, contrary to all previously isolated Gcn5p complexes, HatB3.1 is not associated with Ada2p. It does not appear that the HatB3.1 complex is merely an unstable form of one of the previously characterized Gcn5p-containing complexes as the apparent molecular weight of HatB3.1 did not vary during the course of its purification.
There have been at least a dozen distinct HAT complexes identified in yeast \[[@B8]-[@B22],[@B36],[@B42]\]. Conservative analysis of our systematic fractionation of yeast cytosolic and nuclear extracts resolved 12 chromatographically separable activities. However, many of these activities were represented by rather broad peaks, likely to be composed of partially overlapping activities that may differentiate upon further purification (as seen in Figure [2](#F2){ref-type="fig"}). While many of the activities identified here may correspond to previously characterized complexes, it is difficult to determine these relationships, as our initial purification steps differ from those typically used for the isolation of other yeast HAT complexes. In particular, the purification of the SAGA, ADA, SLIK, SALSA, NuA3 and NuA4 complexes start from Ni^2+^-NTA agarose fractionated whole cell extracts, as these enzymes fortuitously bind to this resin \[[@B9],[@B10],[@B13],[@B14],[@B17],[@B22]\].
Most histone acetyltransferases have substrate specificities that direct the acetylation of specific residues within one or more of the core histones \[[@B5]\]. However, these substrate specificities are not fixed and can be altered by the association of the catalytic subunits with different protein complexes \[[@B19],[@B49]\]. The presence of numerous HAT complexes expands the repertoire of modification states that can be generated on the chromatin template. Therefore, as growing evidence indicates that specific cellular processes are associated with precise patterns of histone modification, the presence of multiple HAT complexes in cells is likely to be a reflection of the myriad events that must take place in the context of chromatin \[[@B50]\].
Despite the importance of histone acetylation in regulating chromatin structure, with the exception of Esa1p, none of the yeast histone acetyltransferases are essential for viability \[[@B51],[@B52]\]. Also, the deletion of most HAT genes results in only relatively mild phenotypes \[[@B35],[@B36],[@B53]-[@B57]\]. One explanation for this observation is that some HATs perform functionally redundant roles in the cell \[[@B58],[@B59]\]. Alternatively, examination of the HAT activity profiles of fractionated extracts derived from HAT deletion strains presented here suggests that there may be mechanisms that can compensate for the lack of one histone acetyltransferase by increasing the activity of other HAT complexes. For example, in a Δ*sas2*strain, there is a dramatic increase in an activity present in nuclear extracts that acetylates free histone H4 and which elutes from a DEAE column at a salt concentration similar to that of the nuclear form of Hat1p (Figure [3](#F3){ref-type="fig"}). In addition, deletion of the *HAT1*gene causes a large increase in an activity that is coincident with the HatB3.1 activity. These results suggest the possibility that cells may monitor levels of histone modification and adjust specific HAT activities accordingly.
HatB3.1 is the third native HAT complex identified from yeast that is only capable of acetylating free histones \[[@B15],[@B36]\]. In addition to the histone H4 specific Hat1p/Hat2p complex, the SAS complex, composed of Sas2p, Sas4p and Sas5p, was recently shown to acetylate free histones H3 and H4. The potential classification of the SAS complex as a type B HAT is supported by the fact that the SAS complex has also been shown to be physically associated with the histone deposition proteins Cac1p and Asf1p \[[@B16],[@B60],[@B61]\]. However, the specific target of SAS complex acetylation, histone H4 lysine 16, has not been found to be acetylated in the pool of newly synthesized histones in any organism \[[@B15],[@B30]\]. Therefore, it remains to be determined whether the SAS complex participates in the acetylation of newly synthesized histones H3 and H4 prior to histone deposition or whether it is involved in the post-assembly modification of histones.
Gcn5p is the prototypical type A histone acetyltransferase. While rGcn5p is only capable of acetylating free histones under most experimental conditions, it has been identified as the catalytic subunit of five native HAT complexes that acetylate nucleosomal substrates (SAGA, ADA, A2, SLIK and SALSA) \[[@B9]-[@B11],[@B13],[@B14],[@B31],[@B62]\]. The most straightforward interpretation of the dependence of the HatB3.1 activity on a functional *GCN5*gene and the co-elution of Gcn5p with highly purified HatB3.1 is that Gcn5p is also the catalytic subunit of HatB3.1. In the context of the type A HAT complexes, the Ada2p, Ada3p and TAF~II~68 proteins have been shown to be important for expanding the substrate specificity of Gcn5p to allow for the acetylation of nucleosomal histones \[[@B49],[@B63]-[@B68]\]. Hence, the ability of Gcn5p to acetylate histones in chromatin is a property that must be conferred upon it by association with other proteins. The identification of Gcn5p as a component of a type B histone acetyltransferase activity suggests that classification as either type A or type B may not be an inherent property of an enzyme but, rather, may be a function of the association of the enzyme with specific accessory factors.
Several properties of HatB3.1 indicate that it is distinct from previously identified Gcn5p-containing complexes. First, HatB3.1 is the only native Gcn5p-containing complex that does not have detectable activity on nucleosomal substrates. Second, the apparent molecular weight of HatB3.1 (\~500 kDa), as determined by size exclusion chromatography, is much lower than that of the SAGA, ADA, SALSA and SLIK complexes but is similar to that reported for the A2 complex \[[@B9],[@B13],[@B14],[@B64]\]. However, unlike HatB3.1, the A2 complex is both dependent upon, and co-purifies with, Ada2p. These results clearly distinguish HatB3.1 as a novel Gcn5p-containing HAT complex \[[@B64]\].
Ada2p, Ada3p and Gcn5p form a module that provides the catalytic activity to their associated type A HAT complexes \[[@B5]\]. In these complexes, there does not appear to be any direct physical interaction between Ada3p and Gcn5p but, rather, their association is mediated through Ada2p \[[@B55],[@B67],[@B69],[@B70]\]. The absence of Ada2p from the HatB3.1 activity suggests that Ada3p and Gcn5p can directly associate under certain circumstances or that another subunit(s) of the HatB3.1 complex can replace the function of Ada2p in bridging the interaction of Ada3p and Gcn5p. The identification of a Gcn5p-containing complex that is independent of Ada2p also suggests that there are cellular processes, such as histone deposition, that are influenced by Gcn5p (and Ada3p) but that do not require Ada2p. However, with the exception of the specific synthetic lethality seen with Δ*gcn5*Δ*sas3*mutants, deletions of the *GCN5*, *ADA2*and *ADA3*genes have similar *in vivo*consequences \[[@B22],[@B59],[@B71],[@B72]\]. The absence of phenotypes unique to Δ*gcn5*and Δ*ada3*mutants may be the result of the complex functional redundancies observed in the assembly of chromatin. For example, Δ*hat1*and Δ*hat2*mutants only display phenotypes when combined with mutations in multiple lysine residues in the histone H3 NH~2~-terminal tail \[[@B45],[@B46]\]. Uncovering these redundancies and deciphering the potential role of Gcn5p in the acetylation of newly synthesized histones is likely to require the characterization of the complete set of complexes that display type B histone acetyltransferase activity.
Conclusions
===========
In conclusion, we have fractionated yeast cytoplasmic and nuclear extracts and resolved several putative histone H3-specific type B histone acetyltransferase activities. One of these activities, HatB3.1, is highly specific for histone H3 that is free in solution. A combination of genetic and biochemical evidence indicates that HatB3.1 is a novel complex that depends on *GCN5*and *ADA3*but that is independent of *ADA2*.
Methods
=======
Yeast strains
-------------
UCC1111 was used as the wild type yeast strain that serves as the genetic background for all deletion strains \[[@B45]\]. Null mutants for *GCN5*, *SAS3*, *SAS2*, *ADA2*, *ADA3*and *HAT1*were constructed using PCR-mediated gene disruption with the *HIS3*reporter gene \[[@B73]\].
Extract preparation
-------------------
Cells were grown to mid-log phase in 1% yeast extract, 2% peptone, 2% glucose and 50 μg/mL ampicillin at 30°C. Cells were harvested at 4000 × g, 10\', 4°C and total grams of cells recorded. All buffers contain 1.0 mM PMSF. Spheroplasts were prepared essentially as described previously using 0.25 mg of Zymolyase (U.S. Biologicals) per gram of cells for spheroplasting \[[@B74]\]. Spheroplasts were burst in 0.5 mL/g cells Lysis Buffer (18% Ficoll 400, 10 mM HEPES \[pH 6.0\]) followed by dilution in 1.0 mL/g cells Buffer A (50 mM NaCl, 1.0 mM MgCl~2~, 10 mM HEPES \[pH 6.0\]). Supernatant from a 1500 × g, 15\' spin at 4°C was retained as a cytosolic extract. Pelleted material was washed once with Buffer A then resuspended in DN(1000) (DN buffers contain 25 mM Tris \[pH 7.5\], 10% glycerol, 0.1 mM EDTA and mM \[NaCl\] listed in parentheses). Supernatant from another 1500 × g spin as above yielded the nuclear extract. This extract was dialyzed O/N at 4°C into DN(0) to a conductivity similar to that of the cytosolic extract. Extracts were cleared by high speed centrifugation (\~30,000 × g) prior to their chromatographic fractionation.
Extract fractionation
---------------------
All columns were equilibrated with and run using DN Buffers. HPLC (ÄKTA *purifier*-- Pharmacia) was employed for all column runs.
### Anion and cation exchange chromatography
DEAE -- Cleared extracts were loaded onto a HiPrep 16/10 DEAE FF column (Pharmacia). Following a 5 C.V. wash with DN(50), proteins were eluted with a linear, 20 C.V., salt gradient from 50 mM to 1.0 M NaCl. A flow rate of 1.0 mL/min. was used and 3.0 mL fractions were collected.
CM -- Either pooled peak fractions, dialyzed into DN(0) until at similar conductivity as DN(50) start buffer, or Flowthrough from the DEAE were loaded onto a HiPrep 16/10 CM FF column (Pharmacia). The column was washed and proteins eluted as described above.
Mono Q -- The flowthrough fraction from the CM column was loaded onto a Mono Q HR 5/5 column (Pharmacia). Following a 5 C.V. wash with DN(50), a 20 C.V., linear, salt gradient was employed as above and 0.5 mL fractions were collected.
### Ammonium sulfate precipitation
Peak fractions of HatB3.1 activity from the Mono Q column were pooled and brought to 75% (NH~4~)~2~SO~4~(0.516 g/mL) over 30\' at 4°C. Following an additional 30\' equilibration period at 4°C, precipitated protein was pelleted (10,000 × g, 10\', 4°C) and resuspended in 300 μL cold, DN(0).
### Gel filtration chromatography
A 250 μL aliquot of resuspended ammonium sulfate precipitate was loaded onto a Superose 6 HR 10/30 column (Pharmacia). The column was equilibrated with and run in DN(350) at a flow rate of 0.3 mL/min. and 0.25 mL fractions were collected. Molecular weight standards (Sigma, MW-GF-1000) were run using the same parameters and 24 μL aliquots of every other fraction run on a 10% SDS-polyacrylamide gel. The elution profile of the MW standards was determined by protein visualization via Coomassie blue staining.
Liquid HAT assays
-----------------
Chicken erythrocyte core histones and chromatin were isolated as previously described \[[@B75],[@B76]\]. Typically 10 μL aliquots of column fractions were incubated with 0.1 μM ^3^H-Acetyl Coenzyme A (5.50 Ci/mmol, Pharmacia) and \~1.0 mg/mL core histones or chromatin in a final volume of 100 μL at 1X \[DN(75)\]. 50 μL of each reaction was analyzed for HAT activity via liquid scintillation counting. The remaining assay mixture was brought to 1X \[SDS Load Dye\] to stop the reaction. In general, aliquots (24 μL) of these remaining assay mixtures were run on 18% SDS-polyacrylamide gels to resolve the histones. Gels were incubated in Autofluor (National Diagnostics), dried down and acetylated histone species visualized via fluorography.
Western blot and gel analysis
-----------------------------
Superose 6 fractions exhibiting HAT B3 activity, as determined above, were run on 10% SDS-polyacrylamide gels and proteins were either visualized by silver staining or transferred to nitrocellulose using a semi-dry transfer apparatus (Biorad). Blots were processed following standard procedures. Goat, polyclonal antibodies against Gcn5p, Ada2p and Ada3p (Santa Cruz Biotechnology, Inc.) were used at 1:100 dilutions in 5% Milk/TBS-T. Donkey, HRP-labeled Anti-Goat IgG secondary antibody (Santa Cruz Biotechnology, Inc.) was used at 1:2500 dilution followed by detection with ECL+Plus (Pharmacia) and visualization via phosphoimager (STORM 860, Pharmacia).
Authors\' contributions
=======================
A.R.S. performed all of the experiments presented here and drafted the manuscript. M.R.P. directed the project and edited the manuscript.
Acknowledgements
================
This work was supported by grants from the American Cancer Society (RPG-00-340-01-CSM) and the National Institutes of Health (1 R01 GM62970) to M.R.P..
| PubMed Central | 2024-06-05T03:55:46.689324 | 2004-7-26 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509278/",
"journal": "BMC Biochem. 2004 Jul 26; 5:11",
"authors": [
{
"first": "Amy R",
"last": "Sklenar"
},
{
"first": "Mark R",
"last": "Parthun"
}
]
} |
PMC509279 | Background
==========
Malaria threatens public health in regions of the world where more than a third of the human population lives \[[@B1],[@B2]\]. It has been shown that immunization with radiation-attenuated *Plasmodium*sporozoites, the infective stage of the malaria parasite, confers protective immunity \[[@B3],[@B4]\]. The role of specific antibody in conferring protection was demonstrated with passive administration of murine mAbs directed against the major repeat epitope of the circumsporozoite (CS) protein \[[@B5]\] in a rodent model. The corresponding epitope of the human malaria parasite *Plasmodium falciparum*is contained within the repeat tetramer peptide (Asn-Pro-Asn-Ala)~n~, (NPNA)~n~\[[@B6]\]. In some studies of volunteers protected against malaria by immunization with radiation attenuated *P. falciparum*sporozoites, protected individuals had significant elevations of anti-repeat antibodies (\>19 μg/ml) \[[@B7]\].
With the advent of recombinant combinatorial antibody technology \[[@B8],[@B9]\] and phage display \[[@B10]-[@B13]\] it is possible to attempt to dissect the human antibody response against a wide range of pathogens. In order to further investigate the role of the human antibody response in *P. falciparum*sporozoite induced protection, a phage display library of antibody gene fragments isolated from the peripheral blood lymphocytes of such a protected donor (WR5) \[[@B7]\] was assembled. Recombinant antibodies against the PfCSP structural repeat (NPNA)~3~epitope were selected. Recognition was restricted to a single antibody designated PfNPNA-1, encoded by V~H~3 and V~κ~I families. This restricted humoral response has implications for rational vaccine design and the potential use of this human monoclonal antibody to prevent *P. falciparum*infection.
Methods
=======
RT-PCR of Immunoglobulin genes
------------------------------
A human volunteer (WR5), who was previously exposed to the bites of γ-irradiated *P. falciparum*infected *Anopheles*mosquito\'s and subsequently shown to be protected against a non-irradiated parasite challenge, donated lymphocytes by leukophoresis five days after a booster challenge (appropriate informed consent was obtained) for details see Egan et al., \[[@B7]\]. The irradiated sporozoite immunization protocol was approved by the Naval Medical Research Institute\'s Committee for the Protection of Human Subjects in accordance with the US Navy regulation (SECNAVINST3900.39B) governing the use of human participants in medical research. Total RNA was extracted from 2 ml of packed cells using an RNA isolation kit (Stratagene, La Jolla, CA) with a modified protocol \[[@B9]\]. The equivalent of 2.5 μg total RNA template were used in each cDNA synthesis reaction using reverse transcriptase (Invitrogen, CA) with oligonucleotide oligo dT or 3 \'HuVH (5\'GCCCCCAGAGGTGCTCTTGGA-3\', anneals in CH1 domain) following the instructions provided by the supplier.
The genes encoding variable heavy (V~H~) and the kappa chain (κ) were accessed by RT-PCR and combined by overlap extension PCR, resulting in shuffling of the V~H~and the V~L~domains. The V~H~PCR amplification was carried out with the cDNA template generated using the 3\'HuVH primer. The V~H~domains were amplified using 5\'HuVHA and 3\'HuVH-Link 3\' designed to anneal with the sequence corresponding to the first β-strand of the CH1 domain and overlap with the 5\'HuVk primer. The κ chains were amplified using 5\'HuVk and the 3\'Hukappa primers. The V~H~and the κ chain PCR products were combined by overlap extension PCR using a V~H~flanking primer 5\'HuVHB (to introduce a *NheI*site) and the 3\'HuKappa primer.
Oligonucleotide primer sequences
--------------------------------
### 5\'HuVk
5\'-TATTAGCGGCCGCCCAACCAGCCATGGCCGAEFIJLOPETGACBCAGTCTCC-3\' (where B=G+C+T, S=G+C, E = 50%A+33%C+17%T, F = 83%A = 17%G, I = 83%T+17%C, J = 50%T+33%C+17%G, L = 67%G+17%T+17%C, O = 67%T+17%A+17%C, and P = 83%G+17%C)
### 3\'HuKappa
5\'-TCCTGAAGCTTGACGACCTTCGATCTCTCCCCTGTTGAAGCTCTT-3\'
### 5\'HuVHA
5\'-SAGGTGCAGCTGSTGSAGTCTGG-3\'
### 5\'HuVHlink3\'
5\'-GGCTGGTTGGGCGGCCGCTAATATGGAGGAGGGTGCCAGGGGGAAGAC-3\'
### 3\'HuVHB
5\'-GTTTCGCTAGCGTAGCTCAGGCTSAGGTGCAGCTGSTGSAGTCTGG-3\'
The procedural steps are illustrated in Figure [1](#F1){ref-type="fig"}.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**V~H~/κ library construction.**A schematic diagram of the steps involved in constructing a V~H~/κ library from mRNA isolated from PBL.
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Cloning PCR fragments into pORFES and JC-M13-88
-----------------------------------------------
The PCR amplified V~H~/κ products were digested with restriction enzymes *NheI*and *HindIII,*and ligated into pORFES \[[@B14]\]. An aliquot of *E. coli*transformed with the ligation mixture was plated with and without carbenicillin selection, to determine the number of functional inserts. The V~H~/κ coding sequences are directionally inserted for expression between an OmpA leader peptide (to direct the polypeptide into the periplasm), and the β-lactamase. Functional full-length V~H~/κ β-lactamase fusion polypeptide is secreted into the periplasm. Bacteria harbouring plasmids conferring antibiotic resistance may be positively selected. The V~H~/κ coding insert may be readily transferred as a *XbaI*-*HindIII*fragment into the JC-M13-88 phage vector to display the insert polypeptide as a gpVIII fusion. The selected \"functional\" library of V~H~/κ inserts were excised from pORFES using *XbaI*and *HindIII*, ligated into pre-digested JC-M13-88 \[[@B4]\], and transformed into *E. coli*(XL1-Blue: Stratagene). Phage was produced overnight at 37°C in the presence of 1 mM IPTG, unless otherwise stated. A schematic outline of the vectors is shown in Figure [2](#F2){ref-type="fig"}.
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Illustration of vectors pORFES, JC-M13-88 and pAbHIS.
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Phage panning
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The peptide (NPNA)~3~C (Chiron Mimotopes Peptide Systems, San Diego, CA.) was conjugated to BSA using Imject Activated Immunogen kit (Pierce, Rockford, IL) according to the manufacturers guidelines. ELISA plates (Dynatech Immunlon I, Alexandria, VA) were coated with BSA or (NPNA)~3~C-BSA and used in phage panning experiments essentially as described elsewhere \[[@B5]\]. To blocked antigen coated wells a total of 4 × 10^10^plaque forming units (pfu) of the phage library in dilution buffer (PBS pH 7.2, Tween-20 0.05%, BSA 0.1%, NaN~3~0.02%) was added (1 × 10^10^plaque forming units (pfu) per well). After 4 h the wells were washed and the bound phage were eluted by applying either 0.1 M glycine-HCl, pH2.2 or a solution of the free peptide (NPNA)~3~(\~8 μM) dissolved in dilution buffer, for 15 min at ambient temperature. An aliquot of the phage elute was titered, and the remainder was used to propagate phage for further rounds of panning. The three-domain single chain antibody retains the kappa constant domain thus permits plaques filter lifts to be probed with anti-human kappa chain antibodies for immunodetection. V~H~and V~L~coding sequences were determined by sequencing of replicative form (rf) phage DNA prepared from κ-positive plaques, using the oligonucleotides primers:
3\'Seq VH-JC130 (5\'-CGGCCATGGCTGGTTGGGCGGCC-3\') and
3\'Seq VL-JC128 (5\'TTCAACTGCTCATCAGATGGCGG-3\').
Expression of PfNPNA-1 V~H~/*k*in *E. coli*
-------------------------------------------
The expression vector pAbHIS, was constructed by modification of pUC18. The β-galactosidase coding region was removed and *XbaI*-*HindIII*sites introduced upstream of a sequence encoding a six histidine tail. Insertion of V~H~/κ coding sequence selected by phage display as *XbaI*-*HindIII*fragment would result in the expressed polypeptide being secreted into the periplasmic space with a hexa-histidine tag. The plasmid pAbHIS was constructed by PCR modification of pUC18 using the primers PUCSpe-JC127(5\'-TCATCATACTAGTAACGACACCCGCCAACACCC-3\') and M13-JC118 (5\'-AAGCTTATGATGTCTAGAGCTGTTTCCTGTGTGAA-3\'). A pair of annealed oligonucleotides designed to encode a 6×His tag were ligated into the *HindIII*digested plasmid to complete pAbHIS. The selected PfNPNA-1 V~H~/κ gene was excised from the rf JC-M13-88 DNA by digestion with *XbaI*and *HindIII*and ligated into similarly digested pAbHIS. An additional 6×His-coding pair of oligonucleotides was ligated into the PfNPNA-1 V~H~/κ linker sequence as NotI-NcoI insert. The expression of PfNPNA-1 V~H~/κ in *E. coli*D29A1 cells at 25°C, and the isolation of bacterial periplasmic material was performed as described \[[@B16]\] with modifications; Dnase I n(1 μg/ml) and MgCl~2~(20 mM) were added, the bacterial suspension was incubated on ice for a further 20 min before final centrifugation step. The periplasmic extract was passed over Ni-NTA resin (Qiagen), washed and the PfNPNA-1 V~H~/κ was eluted with 300 mM imidazole. SDS PAGE and western blotting were used to asses purity and integrity of the expressed V~H~/κ polypeptide during the purification procedure (data not shown). Purified PfNPNA-1 V~H~/κ was quantified spectrophotometrically assuming an OD at 280 nm of 1 = 0.72 mg/ml protein.
ELISA affinity and specificity determination
--------------------------------------------
ELISA Plates (Dynatech Immunlon I) were coated with (NPNA)~3~C-BSA (10 μg/ml). Dilutions of the peptide (NPNA)~3~were made in dimethyl formamide (DMF) before mixing with the PfNPNA-1 V~H~/κ diluted in PBST. Aliquots of 0.1 ml were added to duplicate wells, incubated for 2 h at 37°C. In all wells the final concentration of DMF was 1% (v/v). After washing 4 times with PBST, anti-human kappa chain alkaline phosphatase conjugate diluted 1:1000 in PBST was added and incubated as before. The wells were washed 4 × with PBST and rinsed 1× with PBS and substrate *p*-nitrophenyl phosphate was added, the absorbance was determined at 405 nm
The binding of immune serum (WR5), non-immune serum and PfNPNA-1 V~H~/κ to R32tet32, recombinant hepatitis core containing (NANP)~4~peptide sequence and (NPNA)3C-BSA conjugate coated microtiter plate well was determined by ELISA essentially as described above. The serum(s) and the recombinant PfNPNA-1 V~H~/κ were diluted 1/16 and 1/10 respectively.
Phage ELISA
-----------
Phage at 1 × 10^12^pfu/ml in dilution buffer were applied (0.1 ml/well) to duplicate wells coated with (NPNA)~3~-C-BSA or BSA (10 μg/ml). After incubation at ambient temperature for 4 h, plates were washed with PBST. The bound phage was detected with sheep anti-M13 antibodies (5\'-prime 3\'-prime), followed by rabbit anti-sheep alkaline phosphatase antibodies in PBST added sequentially for 1 h at 37°C. Plates were washed and developed as described above.
Indirect immunofluorescence assay (IFA) on *P. falciparum*sporozoites
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The PfNPNA-1 V~H~/κ was compared with a well-characterized murine monoclonal anti-*Pf*repeat antibody 2A10 \[[@B17],[@B18]\] in IFA. All incubations were at 37°C in a humid container. Printed multiwell slides coated with *Plasmodium falciparum*NF54 strain sporozoites were either fixed in ice cold acetone for 10 min or used unfixed. Slides were first blocked with 4%BSA in PBS for 1 h. Antibodies diluted in PBST were applied for 2 h, then slides were washed 4× with PBS and fluoroscein-conjugated anti-human kappa chain or anti-mouse immunoglobulin (Sigma) was applied, diluted 1:25 in PBST. After 2 h slides were washed as above and mounted in SlowFade anti-fade reagent (Molecular Probes, Eugene, OR) and viewed by fluorescence microscopy.
Other antibodies
----------------
The murine mAb 2A10 \[[@B17],[@B18]\] (IgG2b, κ), which recognizes the (NANP)~3~sequence of the *P. falciparum*CSP was provide as whole ascitic fluid (a kind gift from Dr P. Sinnis New York University). Concentration of the whole IgG was estimated using a standard antibody capture ELISA. Immune IgG (denoted (Vol-IgG) was purified from serum of the immune volunteer (WR5), donated at the time of lymphophoresis using Protein A Sepharose (Pharmacia) and quantified assuming OD at 280 nm of 1.0 represents 0.8 mg/ml IgG. Within the Vol-IgG, the proportion of (NPNA)~3~specific IgG with κ or λ light chains were determined by ELISA (data not shown).
Results
=======
Library construction
--------------------
Sera from the protected individual (WR5) \[[@B7]\] contained antibodies against the PfCSP, which were predominantly IgG/κ and against the structural repeat peptide as determined by ELISA. Gene fragments encoding V~H~/κ single chain antibodies were amplified and assembled by PCR from cDNA derived from the peripheral blood lymphocytes of the immune donor WR5 (as outlined in Figure [1](#F1){ref-type="fig"}). The library of PCR amplified V~H~/κ sequences were inserted into pORFES \[[@B14]\] and an aliquot compared for number of functional inserts by selecting in the presence of either chloramphenicol (total transformation events) or chloramphenicol and carbenicillin (functional inserts). Approximately half of the initial library contained non-functional domains (data not shown). The remainder of the library was selected on 100 μg/ml carbenicillin, yielding a primary library of 1.3 × 10^6^members, these V~H~/κ sequences were transferred to the phage display vector JC-M13-88 \[[@B15]\] with ten fold over representation of the primary library.
Panning
-------
Samples of the V~H~/κ-phage library were subjected to four rounds of panning on (NPNA)~3~C-BSA coated wells. Both the acid and peptide elution strategies yielded significantly greater numbers of phage after four cycles of panning on (NPNA)~3~C-BSA when compared to panning on BSA alone (Table [1](#T1){ref-type="table"}). Analysis of fifteen individual phage after the fourth round of panning on (NPNA)~3~C-BSA eluted with free peptide revealed, twelve kappa positive phage, of these three clones (NP 04, 12, 13) were positive in the phage ELISA for binding to (NPNA)~3~C-BSA and were encoded by an identical sequence, henceforth denoted PfNPNA-1. Prior to panning ten kappa positive clones were randomly selected for sequencing (R 01-10; Table [2](#T2){ref-type="table"}). The PfNPNA-1 V~H~and V~L~sequences were members of the V~H~3 and V~κ~I families respectively and were not found amongst the random sampling of phage prior to panning. In an independent experiment with phage propagated at 30°C, but otherwise an identical panning procedure 12 out of 12 selected phage clones were identical to PfNPNA-1. Likewise, phage selected by acid elution and evaluated by ELISA for binding to (NPNA)~3~C-BSA were all identical to PfNPNA-1. Despite extensive sampling of phage that were positive in the phage ELISA for binding to (NPNA)~3~C-BSA (n = 25), only the PfNPNA-1 sequence was observed.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Phage panning experiments ELISA plates (Dynatech Immulon I) were coated with BSA or (NPNA)~3~C-BSA and used in phage panning experiments. To the blocked antigen coated wells a total of 4 × 10^10^pfu of the phage library in dilution buffer were added 1 × l0^10^pfu per well. After 4 h the wells were washed and phage eluted by applying either 0.1 M glycine-HCl pH 2.2 or a solution of the free peptide (\~8 μM) (NPNA)~3~dissolved in dilution buffer for 15 min at ambient temperature. An aliquot of the phage eluate was titered and the output determined.
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**Eluate after** **Coating antigen / Elution method (×l0^5^pfu)\***
-------------------- ---------------------------------------------------- -------------------- -------------------------- -------------------------------
**panning rounds** **BSA / acid** **BSA /(NPNA)~3~** **(NPNA)~3~C BSA /acid** **(NPNA)~3~C BSA /(NPNA)~3~**
1 2.9 (0.38) 0.82 (0.032) 3.0 (0.34) 0.51 (0.024)
2 1.4(0.03) 0.24 (0.020) 3.5 (0.24) 1.5 (0.028)
3 1.2(0.06) 0.47 (0.020) 4.3 (0.024) 12 (0.68)
4 13 (0.70) 1.0(0.032) 170 (30) 370 (20)
\* Figures represent the mean of the total plaque forming units eluted by either acid or excess free peptide, after repeated panning against BSA or (NPNA)~3~C-BSA. Values for the standard deviation are shown in brackets ().
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::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
V~H~and V~L~assignments and alignment of CDR 3 sequences The selected (NP 04, 12, 13 designated Pf NPNA-1 bind to the repeat epitope), all other NP clones were randomly picked after the panning procedure and were subsequently shown not to be reactive with the repeat epitope. Non-selected (R01-10) were randomly picked from the library prior to initiating panning. The peptide sequence of the heavy and light chain complementarity-determining region 3 (CDR3) is shown below. V~H~/V~L~families, segments and the number of differences from germline segments were determined by using the V BASE sequence directory (Tomlinson, I. M., Williams, S. C., Corbett, S. J., Cox, J. P. L. & Winter, G., MRC Centre for Protein Engineering, Cambridge, UK) and the DNAPLOT alignment package (Müller, W. & Althaus, H.-H., Köln University)
:::
**clone code\*** **V~H~family** **V~H~Segment** **Differences from germline** V~H~CDR3 **V~L~family** **V~L~Segment** **Differences from germline** V~L~CDR3
------------------ ---------------- ----------------- ------------------------------- ------------------- ---------------- ----------------- ------------------------------- -------------
PfNPNAl VH3 DP46 10 DRDSSSYFDS VkI L12a 15 QQYNSYSGLT
NP04, NP12, NP13 VH3 DP46 10 DRDSSSYFDS VkI L12a QQYNSYSGLT
R01 VH1 4M28^†‡^ 28(+6)\* ^§^- DSESVAQWRY VkIV DPK24 43 QQSLSPVWT
R02 VH3 COS-3^‡^ 27 (+3)\_ GVNWCSDY VkI DPK9 10 QQSYSTSWT
R03 VH5 DP73 35 LYTSIYYFDS VkIV DPK24 7 QQYYSTPLT
R04 VH3 DP46 8 DRVTNFWSGYFDY VkIII DPK22 13 QQYGSSPGFT
R05 VH3 DP58 23 DSTVKTVTKMRYGLD V VkIII DPK22 8 QQYGSSPFT
R06 VH1 4M28^†^ 12 DNYGDPGGGFDI VkIII DPK22 11 QQYGNSPRT
R07 VH5 DP73 9 RFWFGELYDAFDI VkIV DPK24 16 HQYYSTPQT
R08 VH5 DP73 34 LYTSIYYFDS VkIII DPK22 14 QQYGRSPWT
R09 VH3 V3-21^†^ 34 DQGGGWSSEVDS VkIII Vg 5 QQRSNWPLT
R10 VH1 DP7^‡^ 21 (+9)\*\* ALYGHDAFDI VkI DPK4 12 PKYNSALHT
NP02 VH3 DP47 36 ERPYDAFDS VkIII DPK22 23 QQYSTSPPMYN
NP03 VH5 DP73 40 LYTSIYYFDS VkIII Vg 17 KQRSKWPPIT
NP05 VH3 V3-48 14 EPRGAGTTLYFDY VkIII DPK22 22 QQYGGSPGYN
NP08 VH4 4.30^†^ 18 DRGVSSGWTFDC VkII DPK16 32 MQLTAFPWT
NP09 VH4 DP71 17 FRGGVAAGYDY VkIII DPK22 24 QHYRESCS
NP10 VH4 DP78 29 DRVRVPYYYIDV VkIII DPK22 15 QQYGTSPYS
NP11 VH3 VH3-8^†^ 12 DTTVTHYFDY VkI DPK9 21 QQSFSSPRT
NP14 VH1 DP88 20 GPGATIHYYYMDV VkI DPK8 18 QQLDNYPLT
NP15 VH5 DP73 36 LYTSIYYFDS VkIII DPK22 28 QQYGNSPPT
\*Phage clones were either selected from the library at random (prefix R) or after four rounds of panning against (NPNA)~3~C-BSA, eluting with free (NPNA)~3~peptide (prefix NP). ^†^The segment given the best DNAPLOT match, although the segment sequence has not been verified by duplication. ^‡^Aligned after removal of the unusual sequence additions (see^§,^-, ^‡,^-, \*\*). ^§^Figure in brackets indicates an unusual sequence addition. - Sequence has two additional codons in CDR1. - Sequence has one additional codon in CDR1. \*\* Sequence has three additional codons in CDR2.
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Expression and evaluation of the recombinant antibody fragment
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The PfNPNA-1 sequence was transferred to the expression vector pAbHIS (as outlined in Figure [2](#F2){ref-type="fig"}. Purification of the V~H~/κ polypeptide was carried out on Ni-NTA agarose beads, yielding 0.5 mg of the 38 kDa V~H~/κ polypeptide/L bacterial culture.
Fine specificity and affinity determination
-------------------------------------------
Anti-sporozoite activity of the PfNPNA-1 V~H~/κ molecule was clearly evident in an immunofluorescence assay (IFA) with *P. falciparum*sporozoites (Figure [3](#F3){ref-type="fig"}). The human single chain monoavalent antibody (panel A) was compared with a known *in vitro*protective whole murine antibody 2A10 (panel B). The murine antibody and the recombinant PfNPNA-1 V~H~/κ molecule both labelled the parasites.
::: {#F3 .fig}
Figure 3
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######
**Indirect immunofluorescence assay (IFA) on *Plasmodium falciparum*sporozoites.**Panel (A) PfNPNA-1 V~H~/κ, (B) 2A10 MAb.
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Competitive ELISA was carried out and the IC~50~value used to approximate the affinity of binding. Binding affinity of the monovalent PfNPNA-1 for (NPNA)~3~compared favourably with values previously reported for a panel of conventional murine monoclonal antibodies directed against the repeat epitope \[[@B18]\], which also have affinities in the μM range (Figure [4](#F4){ref-type="fig"}).
::: {#F4 .fig}
Figure 4
::: {.caption}
######
Competition ELISA.
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Analysis of the fine specificity of the antibody PfNPNA-1 revealed weak binding to the repeat based \[NVDP(NANP)~15~\]~2~, R32tet32 \[[@B19]\], whilst binding to the (NANP)~4~epitope contained within the hepatitis B virus nucleocapsid (C75CS2) \[[@B20]\] was strong. This activity profile pattern was mirrored in the protected donor serum (Figure [5](#F5){ref-type="fig"}). The very high binding observed with WR5 immune serum with the (NPNA)~3~C-BSA conjugate is probably due to the multivalent array of the capture ligand (i.e. multiple peptides coupled per BSA molecule), favouring more efficient retention of the antibody.
::: {#F5 .fig}
Figure 5
::: {.caption}
######
**Determination of specificity of PfNPNA-1.**The binding of immune serum (WR5), non-immune serum and PfNPNA-1 V~H~/κ to R32tet32, recombinant hepatitis core containing (NANP)~4~peptide sequence and (NPNA)3C-BSA conjugate coated microtiter plate well was determined by ELISA essentially as described in Figure 4. The serum(s) and the recombinant PfNPNA-1 V~H~/κ were diluted 1/16 and 1/10 respectively.
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Discussion
==========
The recombinant antibody library construction differed from conventional antibody phage display library assembly \[[@B10]-[@B13]\], a pre-selection step was introduced to remove antibody inserts that were either; prematurely terminated, intact but did not translate well or were intact, translated well but failed to translocate into the bacterial periplasmic space, a prerequisite for functional display. Previously an approach towards developing a vector to select for fully intact functional sequences for antibody or peptide display had shown promise with model sequences \[[@B21]\], but had not been applied for large-scale random antibody library assembly. A \"clean-up\" vector, plasmid open reading frames expression secretion (pORFES) \[[@B14]\] was developed and used to remove these non-functional sequences. Up to 50% of the clones from the initial transformed library were non-functional. Some of the non-functional antibody fragments could in part be due to errors introduced during PCR amplification resulting in frame shifts. However it may be that some sequences either did not express well or did not translocate into the periplasmic space. Irrespective of the explanation, the size of the functional library was half of the total transformation events. An initial enhancement of the initial library by removing most non-functional inserts may at first appear to be a minor improvement. However, in conventional phage display the initial expansion of the library prior to panning results in a preferential growth of phage that do not make and display encoded inserts, moreover phage that lack an insert have a greater growth advantage. This results in a phage population that is greatly biased towards non-productive elements, which impacts directly on the panning efficiency. Incorporation of the pORFES step assured that only the functional (1.3 × l0^6^) sequences were subsequently transferred to the phage display vector. Panning with a functionally enhanced library resulted in very efficient enrichment and recovery. Previously it had been demonstrated that manipulating the conditions of phage production results in modulation of the density of antibody display on phage \[[@B15]\]. The phage library was expanded using parameters that would result in either monovalent display (0-1 antibody/phage) or multivalent display (0--5 antibodies/phage) \[[@B15]\] prior to initiating panning. It was anticipated that a range of antibodies with varying affinities would be present in the library, and modulating antibody display on phage would permit capture antibodies with a range of affinities and sequence diversity.
Induction of protective immunity against sporozoite challenge by exposure to radiation attenuated malaria sporozoite has been demonstrated in humans \[[@B4],[@B7],[@B22]\]. Protection is thought by most investigators to be primarily cellular in nature \[[@B23]\], but there is no question that antibodies with significant sporozoite neutralizing activity are elicited \[[@B22]\] and may play a role in protection. The antibody response is primarily directed against the repeat region of the PfCSP. Studies of subunit vaccines which induce antibodies only against the repeat region demonstrate that protective immunity can be induced in some individuals \[[@B24],[@B25]\]. At the onset of this study it was proposed that the dissection of the anti-*P*. *falciparum*sporozoite antibody response by combinatorial antibody library phage display would permit individual selected antibodies to be evaluated for protective potential and the information generated could be used in vaccine design. In particular, attention was focused on antibodies against the structural motif (NPNA)~n~. Despite using two different strategies for the elution of repeat region peptide specific antibodies (acid and peptide specific) it would appear that the anti-structural repeat response by this protected individual is restricted to a single V~H~/V~L~combination observed in the panel of selected phage (n = 25). Sequencing of randomly picked phage prior to panning revealed that a diverse range of V~H~and V~L~families were represented in the library as shown in Table [2](#T2){ref-type="table"}. Moreover the PfNPNA-1 V~H~/V~L~was not represented in the sampling and was only detected after enrichment.
Comparison of the monovalent PfNPNA-1 molecule with the conventional bivalent murine mAb, such as the in vitro inhibitory 2A10 against *P. falciparum*sporozoites indicates that they recognize the repeat epitope(s) with equivalent affinities \[[@B18]\]. The sequence revealed extensive somatic hyper mutations in both the V~H~and V~L~genes suggesting antigen driven affinity maturation. Based on these observations, PfNPNA-1 may be a good candidate to develop and evaluate as a protective antibody.
Analysis of field samples in rural Gambia \[[@B26]\], Thailand \[[@B27]\] Indonesia \[[@B28]\] and Kenya \[[@B29]\], suggest that anti-sporozoite antibody is poorly developed under natural conditions of exposure and does not protect against clinical malaria. In contrast to exposure to *P. falciparum*sporozoites under natural conditions in the field, immunization with irradiated *P. falciparum*sporozoites induces in general higher levels of antibodies against the PfCSP repeats, and does induce sterile protective immunity \[[@B4],[@B7],[@B30]-[@B38]\]. In the study by Egan et al., 3 of the 4 volunteers were protected against challenge with *P. falciparum*sporozoites. The generally accepted explanation for the lack of protection in the one volunteer is that the volunteer did not receive an adequate immunizing dose of irradiated sporozoites (less than 1000 infective bites \[[@B4],[@B7]\]). However, it is of interest that this non-protected volunteer (WR1, \[[@B22]\])had significantly lower levels of antibodies against the PfCSP repeat than did the protected volunteer who donated cells for this study (WR5, \[[@B22]\]) (2.4 μg/ml vs 50 μg/ml of specific antibody). This raises the question as to whether the antibodies are markers for adequate immunization or are actually major mediators of protection. Regardless, this anti-repeat response in this protected individual appeared to be restricted to a single antibody. This does not preclude that antibodies directed against non-repeat epitopes on PfCSP and other sporozoite proteins \[[@B39]\] play a role in protection. It is not possible to conclude that the response against the structural repeat epitope is restricted to a single antibody of moderate affinity, since only a single protected donor has been used in this study. One may speculate that in concordance with the argument put forward by Saul \[[@B40]\] that the inability to recover high affinity antibody, may reflect that high affinity antibodies may not be required for protection. Due to the repetitive nature of the antigen one can further speculate that only limited affinity maturation is required to obtain physiologically relevant efficacy. The restricted recovery of antibodies is unlikely to be a technical limitation on the phage technology since others have generated panels of very high affinity human antibodies against a range of antigens \[[@B13]\]. Very few examples of different approaches of generating human antibodies from immune donors are described in the literature, in particular when attempting to make antibodies against the same antigen. Currently it is not possible to fully understand the limitations of a technology. Using an alternative technology of engrafting immune human PBL\'s directly into SCID mice from donors vaccinated against anthrax vaccine adsorbed, boosting with protective antigen (PA), recovering immortalizing antibody-producing cells via conventional hybridoma technology \[[@B41]\] resulted in a panel of very high affinity potent neutralizing antibodies against anthrax toxin. Independently, an antibody phage display library from a similar (not identical) immune donor PBL\'s was constructed and panned against PA \[[@B42]\] also resulted in a panel of high affinity anti-anthrax PA antibodies. This would suggest that the methodology is not limiting. However in this example, unlike CSP, the PA antigen does not contain repeating epitopes.
Further it is speculated that antibodies directed against the structural (NPNA)~n~repeat play a role in conferring protection against *P. falciparum*sporozoites in some of the protected volunteers and this protection may be associated with circulating levels of this specific antibody against the structural repeat.
Efforts are being directed towards producing a fully human IgG based on the PfNPNA-1 V~H~and V~L~domains for further in vitro and in vivo evaluation. The use of a human monoclonal antibody as a preventive measure against *P. falciparum*malaria, would be independent of factors which hinder active vaccination, such as adjuvant effects, the requirement to be effectively presented in a diverse range of human leukocyte class I and II molecules, and immunlogical antagonism \[[@B43],[@B44]\]. In practice, the utility of monoclonal antibodies as anti-infectious agents is often negated by the presence and or the inevitable emergence of variants with altered surface epitopes (in particular with viral targets). Fortunately, there has never been a *P. falciparum*isolate that does not contain the (NPNA)~n~repeats on the PfCSP \[[@B45]\], and the number of tandem array of repeats on the PfCSP reduces the likelihood of variants arising which evade antibody recognition. This would suggest that an effective antibody directed against the repeats would be effective against all *P. falciparum.*If this restricted antibody response to the repeat epitope plays a role in preventing *P. falciparum*infection, PfNPNA-1 may be a useful prophylactic agent. Moreover, if PfNPNA-1 is shown to be protective in passive immunization in humans or monkeys as previously demonstrated for anti-*P. vivax*CSP murine mAb, NVS3 \[[@B46]\], it would provide a template that could be used in defining the precise conformation of the structural repeat required for the induction of desired antibodies that can neutralize parasites.
Conclusions
===========
Over the past 25 years the antibody response against the PfCSP repeat epitope has been pursued as a target for active vaccination, with encouraging results \[[@B47]\]. Our attempt to dissect the protective antibody response against the structural PfCSP repeat revealed that the response was restricted to a single V~H~/V~L~pairing, designated PfNPNA-1 encoded by V~H~3 and V~κ~I families (with evidence of somatic mutations). The affinity for the ligand was in the μM range, which in the context of a whole antibody may be more than sufficient for retention on a polyvalent surface such as the *P. falciparum*CSP. It is speculated that the induction and the maintenance of high circulating levels of antibodies against the structural PfCSP repeat may be more important than intrinsic high affinity for the ligand for protection against *P. falciparum*infection. The absence of high affinity anti-repeat antibodies is in concordance with the expected response against a multivalent antigen (i.e. sporozoite surface). Under physiological conditions a whole IgG antibody and a multimeric ligand result in bivalent binding. Such complexes can have avidities estimated to be approaching the product of two independent monomeric interactions. In this case, the 1 × 10^-6^M monovalent affinity of PfNPNA-1 may approach a theoretical higher avidity (1 × 10^-12^M) in the context of a whole antibody. This implies that further affinity maturation either in vivo or in vitro may not necessarily increase physiological effectiveness of the whole IgG antibody. Public health officials have acknowledged the urgency for development of an effective anti-*P*. *falciparum*malaria vaccine. One of the key criteria of such a putative vaccine may be the induction and maintenance of high levels of anti-(NPNA)n antibodies. The fully human PfNPNA-1 IgG could be used as a positive control in evaluating sera from immunized donors, or possibly be developed as a prophylactic agent that could be used alone or in combination with various vaccination strategies. One immediate hurdle for the development of such an antibody as a prophylactic would be the anticipated high cost of commercial manufacture in mammalian cells. However, advances in alternative antibody production technology may one day provide some more cost effective solutions \[[@B48],[@B49]\].
With the availability of an antibody phage display library constructed from a protected individual immunized via bites of irradiated *P. falciparum*infected *Anopheles*mosquitoes, it should be possible to further dissect the antibody response against \"other\" sporozoite antigens \[[@B39]\].
Authors\' contributions
=======================
JAC was the postdoctoral researcher on this project. WOR and SLH co-investigators. ASK was the PI and recipient of the Department of Army award. All authors read and approved the final manuscript
Disclaimer
==========
The views and opinions expressed herein are those of the author and do not purport to reflect those of the U.S. Navy or the Department of Defense, Sanaria Inc or Avanir Pharmaceuticals Inc.
Acknowledgements
================
Ms. Kiyoko Shimizu and Ms. Yan Su are thanked for administrative and technical assistance respectively and Dr Ritsuko-Sawada-Hirai for comments on the manuscript. The staff at Walter Reed Army Research Institute (WRAIR) and the Naval Medical Research Center (NMRC) (in particular donor WR5), Dr. Dan Gordon and his colleagues at WRAIR and NMRC carried out the immunizations of the volunteer, Dr. Robert Wirtz provided *P. falciparum*sporozoite slides and Dr. Ripley Ballou supplied R32tet32 and continued support, advice and guidance. Dr. David Milich (TSRI) provided the recombinant hepatitis nucleocapsid protein C75CS2. ASK was a recipient of an Investigators Award from the Cancer Research Institute/Partridge Foundation and this work was supported by the Department of the Army ARL No DAAL03-92-G-0215 and the Naval Medical Research and Development Command Work Unit 61102A3M161102BK13AK111. This article has been assigned manuscript number 9934a-MB from The Scripps Research Institute.
| PubMed Central | 2024-06-05T03:55:46.692869 | 2004-7-29 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509279/",
"journal": "Malar J. 2004 Jul 29; 3:28",
"authors": [
{
"first": "Jonathan A",
"last": "Chappel"
},
{
"first": "William O",
"last": "Rogers"
},
{
"first": "Stephen L",
"last": "Hoffman"
},
{
"first": "Angray S",
"last": "Kang"
}
]
} |
PMC509280 | Background
==========
Antimicrobial resistance has emerged as an important factor in predicting outcomes and overall resource use after infections in intensive care units (ICU) \[[@B1]\]. Globally ICUs are encountering emergence and spread of antibiotic-resistant pathogens. For some pathogens there are few therapeutic options available, e.g., vancomycin-resistant *Enterococcus faecium*. Awareness of these problems has been underscored with data from a number of surveillance studies aimed at improving the use of empiric therapy. In the United States there have been several national programs, which have focused on both the etiology of infections and resistance patterns of nosocomial or ICU infections including the National Nosocomial Infections Surveillance (NNIS) \[[@B2]\] and more recently an ICU-specific study examining the epidemiology of antimicrobial resistance, Project ICARE \[[@B3],[@B4]\]. Stephen et al. collected strains from 28 ICUs from across the United States as part of the SENTRY Antimicrobial Surveillance Program in 2001 \[[@B5]\].
European data on the antimicrobial resistance of ICU pathogens has also been collected in several recent surveillance studies. A large prevalence survey of nosocomial infections in ICUs in 17 countries was published in 1995 \[[@B6]\], and more recently a number of nation-specific surveys were reported \[[@B7]-[@B9]\]. Several key points emerge: first, antimicrobial resistance among ICU pathogens is generally increasing, but variations do exist among different countries, probably due to individual antimicrobial use patterns; second, when new medical practices and alternative antimicrobials are introduced changes in the dominant microbial etiologies may emerge prompting novel empiric selections; and third, the standards of hygiene and infection control also vary across countries. Finally, appropriate therapy of ICU infections directed by local resistance data can have significant consequences for both patient and the healthcare system. It is against this background that local resistance surveillance programs are of most value in developing appropriate therapeutic guidelines for specific infections and patient types. For example, the recent modification to the American Thoracic Society guidelines for the treatment of hospital-acquired pneumonia \[[@B10]\] considered contemporary resistance data. Local surveillance data can be applied to other infections to assist in local formulary policy such as those governing treatment of nosocomial urinary tract infections \[[@B11]\].
This study using TSN program reports the antimicrobial resistance profiles of bacterial isolates from ICU patients in five countries during the period 2000--2002. The relevance of these recent nation-specific data will be discussed on a country-by-country basis, as part of improving and updating empiric therapeutic approaches to specific pathogens causing infections in the ICU setting according to each country. These surveillance programs help to maintain current knowledge of susceptibilities and relevant treatment options.
Methods
=======
TSN Database -- United States and Europe
----------------------------------------
TSN is a queriable, real-time database that electronically assimilates daily antimicrobial susceptibility testing and patient demographic data from a network of geographically dispersed laboratories in the United States (283 hospital sites), France (63 hospital sites), Germany (169 hospital sites), Italy (48 hospital sites) and Canada (87 hospital sites) \[[@B12]\].
Laboratories included in TSN include those servicing university, community, and private hospitals with bed sizes ranging from 100 to \>1000 beds. Routine diagnostic susceptibility testing results are collected daily from each participating laboratory. The methods used by these laboratories include VITEK (bioMérieux, St. Louis, MO), MicroScan (Dade-Microscan, Sacramento, CA), Sceptor and Pasco MIC/ID (Becton Dickinson, Sparks, MD) and Etest (AB Biodisk, Solna, Sweden), as well as manual broth microdilution MIC, disk diffusion and agar dilution. TSN reflects current testing in participant laboratories and represents the data reported to physicians from the respective laboratories \[[@B13]\].
Although some European countries have alternate breakpoints, all data forwarded to TSN Databases are derived from hospitals that utilized NCCLS standards and definitions (United States, Canada, Italy, and Germany) \[[@B14]\] or the Comité de L\'Antibiogramme de La Societé Français de Microbiologie (France) \[[@B15]\] thus standardizing datasets. Results were interpreted as susceptible, intermediate (if available), or resistant in TSN, based upon the NCCLS interpretative guidelines in place during 2001 \[[@B16]\]. In addition, a series of quality-control filters (i.e., critical rule sets) were used in TSN to screen susceptibility test results for patterns indicative of testing error and suspect results were removed from analysis for laboratory confirmation. In TSN, any result from the same patient with the same organism identification and the same susceptibility pattern received within five days was considered a repeat culture and was counted only once in the database.
Bacterial species and antimicrobials tested
-------------------------------------------
For this study, data from TSN results for each individual database from January 1, 2000 through to December 31, 2002 were included in the analysis to determine the proportion of species and their susceptibility to antimicrobial agents commonly tested in clinical laboratories throughout the participating regions. Only isolates derived from patients located in hospital ICUs were considered in the analysis. Gram-positive species included in the analysis were comprised of *S. aureus*, coagulase negative staphylococci, *Enterococcus faecalis*,*Enterococcus faecium*, *Streptococcus pyogenes*, *Streptococcus pneumoniae* and viridans group streptococci. Gram-negative species studied comprised the predominantly encountered enteric species (*Escherichia coli*, *Klebsiella oxytoca, Klebsiella pneumoniae, Proteus mirabilis, Morganella morganii* and *Serratia marcescens*), and *Pseudomonas aeruginosa* and *Acinetobacter* spp.
The antibiotics studied are listed in Tables [2](#T2){ref-type="table"},[3](#T3){ref-type="table"},[4](#T4){ref-type="table"},[5](#T5){ref-type="table"}. Among *E. coli*, putative ESBL production was defined as those isolates that were intermediate or resistant (non-susceptible) to ceftazidime \[[@B17]\]. Given the large number of isolate results included in the majority of analyses in this study, statistical analysis was not performed, as even subtle differences in percent resistance (\<1%) to an antimicrobial agent for any time period or demographic parameters would be reported as highly significant (*P* \<0.001).
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
*S. aureus*, Coagulase-negative staphylococci, *E. faecalis*, and *E. faecium* isolated from ICU patients during 2000--2002
:::
United States Canada Italy Germany France^a^
--------------------------------------------- ------------------------- --------------- -------- ------- --------- ----------- ------ ------- ------ ------ ------- ------ ------ -------- --------- ------
*Staphylococcus aureus* Ampicillin 19,703 6.7 93.3 3,792 12.6 87.4 1,665 5.6 94.4 2,867 16.2 83.8 15 6.7 93.3
Cefepime 1,260 52.9 46.9 NT^b^ NT NT 304 15.8 84.2 483 80.5 17.0 \<10 NA^c\>^ NA
Cefotaxime 6,898 50.2 49.7 220 55.5 44.5 671 36.4 63.6 729 92.0 8.0 490 63.9 36.1
Ceftriaxone 5,914 45.6 54.3 153 69.3 30.7 1,048 28.1 71.8 220 88.6 11.4 23 73.9 26.1
Ciprofloxacin 24,350 47.4 51.0 5,958 74.5 24.1 4,600 39.7 58.6 5,243 73.4 26.1 316 57.0 40.5
Gentamicin 35,034 85.6 13.7 6,641 89.4 10.3 5,531 40.9 58.0 5,735 90.0 9.7 10,100 90.4 9.4
Oxacillin 44,939 47.7 52.3 10,105 80.3 19.7 6,147 40.6 59.4 6,475 79.0 21.0 10,512 59.4 40.6
Teicoplanin NT NT NT NT NT NT 5,868 100 0 4,632 99.8 0.2 8,232 100 0
Vancomycin 43,245 100 0 7,882 100 0 5,937 100 0 5,276 100 0 9,453 100 0
*Staphylococcus aureus*
OSSA Ampicillin 9,047 14.5 85.5 3,055 15.7 84.3 741 12.6 87.4 2,414 19.3 80.7 10 0 100
Cefepime 672 99.1 0.4 NT NT NT 49 98.0 2.0 387 99.5 0.3 NT NT NT
Cefotaxime 3,451 99.7 0.2 122 100 0 244 100 0 653 100 0 312 100 0
Ceftriaxone 2,707 99.5 0.2 106 100 0 295 99.0 0.3 194 100 0 16 100 0
Ciprofloxacin 11,827 91.2 6.5 4,692 93.5 4.8 1,902 91.4 4.9 4,171 91.4 8.0 188 90.4 6.4
Gentamicin 16,951 98.3 1.4 5,384 98.1 1.8 2,223 95.1 4.5 4,527 98.4 1.5 5,958 99.4 0.5
Oxacillin 21,416 100 0 8,110 100 0 2,495 100 0 5,115 100 0 6,244 100 0
Teicoplanin NT NT NT NT NT NT 2,402 100 0 3,593 99.9 0.1 5,018 100 0
Vancomycin 20,110 100 0 6,046 100 0 2,430 100 0 4,002 100 0 5,580 100 0
*Staphylococcus aureus*
ORSA Ampicillin 10,656 0 100 737 0 100 924 0 100 453 0 100 \<10 NA NA
Cefepime 588 0 100 NT NT NT 255 0 100 96 4.2 84.4 \<10 NA NA
Cefotaxime 3,447 0.6 99.3 98 0 100 427 0 100 76 23.7 76.3 178 0.6 99.4
Ceftriaxone 3,207 0 100 47 0 100 753 0.3 99.7 26 3.8 96.2 \<10 NA NA
Ciprofloxacin 12,523 6.1 93.1 1,266 3.9 95.5 2,698 3.3 96.4 1,072 3.3 96.6 128 7.8 90.6
Gentamicin 18,083 73.7 25.2 1,257 52.0 46.8 3,308 4.5 94.0 1,208 58.7 40.5 4,142 77.5 22.2
Oxacillin 23,523 0 100 1,995 0.2 99.8 3,652 0 100 1,360 0 100 4,268 0 100
Teicoplanin NT NT NT NT NT NT 3,466 100 0 1,039 99.7 0.3 3,214 100 0
Vancomycin 23,135 100 0 1,836 100 0 3,507 100 0 1,274 100 0 3,873 100 0
*Staphylcoccus* species, coagulase-negative
Ampicillin 16,288 5.7 94.3 3,533 6.3 93.7 2,142 10.6 89.4 4,075 8.1 91.9 \<10 NA NA
Cefepime 991 11.8 88.1 \<10 NA NA 116 0 100 625 11.0 73.1 \<10 NA NA
Cefotaxime 5,538 17.7 82.3 240 17.9 82.1 335 16.7 83.3 625 37.4 62.4 174 28.7 69.0
Ceftriaxone 3,471 14.8 84.8 116 22.4 77.6 512 11.7 88.3 412 25.0 74.8 \<10 NA NA
Ciprofloxacin 18,829 40.2 58.3 5,366 44.4 54.7 5,102 42.7 54.0 6,197 29.5 67.6 198 44.4 53.0
Gentamicin 27,248 51.5 38.1 5,571 40.6 47.3 5,241 33.8 60.7 6,848 41.5 51.7 9,422 46.8 51.5
Oxacillin 35,135 15.8 84.2 9,172 20.6 79.4 5,961 15.2 84.8 7,442 18.6 81.4 9,884 30.1 69.9
Teicoplanin NT NT NT NT NT NT 5,797 93.7 2.4 5,096 95.6 0.7 7,670 84.6 3.1
Vancomycin 34,424 100 0 8,239 100 0 5,937 100 0 6,953 100 0 8,300 100 0
*Staphylcoccus* species, coagulase-negative
Oxacillin susceptible Ampicillin 2,582 35.7 64.3 638 34.6 65.4 437 51.7 48.3 824 39.6 60.4 \<10 NA NA
Cefepime 117 100 0 NT NT NT NT NT NT \<10 NA NA NT NT NT
Cefotaxime 978 99.5 0.2 42 100 0 56 100 0 128 100 0 54 92.6 0
Ceftriaxone 523 98.3 0.4 26 100 0 59 100 0 103 100 0 \<10 NA NA
Ciprofloxacin 2,844 82.4 16.6 988 91.8 7.6 779 87.7 10.1 1,103 89.5 9.2 78 83.3 14.1
Gentamicin 4,424 93.5 4.2 1,068 91.9 5.3 698 94.3 5.3 1,263 96.5 2.7 2,822 93.9 5.4
Oxacillin 5,565 100 0 1,886 99.9 0.1 904 100 0 1,383 100 0 2,980 100 0
Teicoplanin NT NT NT NT NT NT 890 99.1 0.3 691 98.4 0.3 2,454 95.8 0.2
Vancomycin 5,240 100 0 1,587 100 0 897 100 0 981 100 0 2,467 100 0
*Staphylcoccus* species, coagulase-negative
Oxacillin resistant Ampicillin 13,706 0.1 99.9 2,895 0 100 1,705 0 100 3,251 0.2 99.8 \<10 NA NA
Cefepime 874 0 99.9 \<10 NA NA 116 0 100 624 10.9 73.2 \<10 NA NA
Cefotaxime 4,560 0.1 99.9 198 0.5 99.5 279 0 100 497 21.3 78.5 120 0 100
Ceftriaxone 2,948 0 99.8 90 0 100 453 0.2 99.8 309 0.0 99.7 \<10 NA NA
Ciprofloxacin 15,985 32.7 65.8 4,378 33.7 65.3 4,323 34.7 61.9 5,094 16.5 80.2 120 19.2 78.3
Gentamicin 22,824 43.3 44.7 4,503 28.5 57.3 4,543 24.5 69.2 5,585 29.1 62.8 6,600 26.6 71.3
Oxacillin 29,570 0 100 7,286 0 100 5,057 0 100 6,059 0 100 6,904 0 100
Teicoplanin NT NT NT NT NT NT 4,907 92.7 2.8 4,405 95.1 0.8 5,216 79.3 4.5
Vancomycin 29,184 100 0 6,652 100 0 5,040 100 0 5,972 100 0 5,833 100 0
*Enterococcus faecalis*
Ampicillin 7,865 98.8 1.2 1,000 99.4 0.6 1,289 95.3 4.7 1,902 99.6 0.4 1,183 99.5 0.2
Ciprofloxacin 3,311 56.9 38.7 625 45.3 50.4 1,159 64.0 31.1 2,012 39.7 39.5 559 78.5 17.0
Gentamicin (HL Testing) 5,503 65.1 34.8 706 63.0 36.8 1,156 62.9 37.1 965 64.8 35.2 1,563 63.6 13.4
Teicoplanin NT NT NT \<10 NA NA 1,248 97.1 2.4 1,294 99.7 0.2 1,747 99.9 0.1
Vancomycin 7,656 95.1 4.5 1,005 98.3 0.9 1,303 96.7 2.8 1,636 99.4 0.3 1,811 99.7 0.2
*Enterococcus faecium*
Ampicillin 3,896 9.7 90.3 383 17.2 82.8 260 21.5 78.5 481 12.3 87.7 151 41.7 49.7
Ciprofloxacin 1,846 5.3 92.5 221 10.9 85.5 234 10.3 77.4 591 6.9 73.9 66 21.2 39.4
Gentamicin (HL Testing) 2,512 57.5 42.5 291 59.5 40.5 223 67.7 32.3 349 60.2 39.8 263 65.4 12.2
Teicoplanin 23 8.7 87.0 \<10 NA NA 234 86.3 13.7 517 97.9 2.1 266 99.6 0.4
Vancomycin 4,066 23.2 76.3 415 85.1 14.5 264 75.4 24.2 628 93.9 4.8 247 98.4 0.8
^a^NCCLS breakpoints were used for all countries, except (CA-SFM) ^b^Not tested ^c^Not applicable if \<10 isolates were tested
:::
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
*S. pneumoniae*, *S. pyogenes*, *S. agalactiae*, and Viridans group streptococci isolated from ICU patients during 2000--2002
:::
United States Canada Italy Germany France^a^
-------------------------------- ---------------- --------------- -------- ------- --------- ----------- ------ ------ ------ ------ ------- ------ ------ ------- ------- ------
*Streptococcus pneumoniae* Amoxicillin 120 91.7 2.5 31 100 0 60 93.3 6.7 17 100 0 1,328 71.2 2.3
Cefepime 22 90.9 4.5 25 60.0 12.0 66 90.9 7.6 NT^b^ NT NT \<10 NA^c^ NA
Cefotaxime 1,571 82.2 6.3 145 93.8 0.7 108 93.5 4.6 63 100 0 1,181 77.1 0.8
Ceftriaxone 2,373 88.3 3.2 145 91.7 0.7 145 91.7 3.4 29 100 0 544 80.1 0.6
Clarithromycin 184 71.7 25.5 56 69.6 30.4 90 64.4 31.1 \<10 NA NA NT NT NT
Erythromycin 3,029 67.9 30.5 539 78.5 20.8 313 69.6 28.1 405 88.6 9.4 1,567 59.0 38.8
Levofloxacin 2,133 99.1 0.4 356 98.6 1.1 174 98.3 0.6 340 99.4 0.3 62 98.4 1.6
Penicillin 3,096 51.5 20.2 325 59.1 7.1 198 77.3 7.6 102 96.1 2.0 1,387 45.5 17.9
Vancomycin 2,865 100 -^c^ 271 100 \- 231 100 \- 190 100 \- 1,479 100 \-
*Streptococcus pyogenes*
Amoxicillin NT NT NT NT NT NT NT NT NT NT NT NT 58 100 0
Cefepime \<10 NA NA NT NT NT NT NT NT NT NT NT NT NT NT
Cefotaxime 32 100 \- 29 100 \- \<10 NA NA 11 100 \- 30 100 \-
Ceftriaxone 75 100 \- \<10 NA NA \<10 NA NA \<10 NA NA \<10 NA NA
Clarithromycin 19 84.2 5.3 \<10 NA NA 17 88.2 11.8 NT NT NT NT NT NT
Erythromycin 118 92.4 6.8 102 81.4 11.8 59 74.6 23.7 63 84.1 11.1 170 82.9 14.7
Levofloxacin 71 97.2 1.4 \<10 NA NA \<10 NA NA 61 77.0 4.9 NT NT NT
Penicillin 140 100 \- 97 100 \- 58 100 \- 64 100 \- 139 100 \-
Vancomycin 121 100 \- 42 100 \- 12 100 \- 34 100 \- 162 100 \-
*Streptococcus agalactiae*
Amoxicillin NT NT NT NT NT NT NT NT NT NT NT NT 165 100 0
Cefepime 28 100 \- NT NT NT \<10 NA NA NT NT NT NT NT NT
Cefotaxime 71 100 \- 17 100 \- 24 100 \- 50 100 \- 50 100 \-
Ceftriaxone 184 100 \- \<10 NA NA 38 100 \- 37 100 \- \<10 NA NA
Clarithromycin 21 81.0 9.5 \<10 NA NA 21 71.4 28.6 NT NT NT \<10 NA NA
Erythromycin 489 76.3 21.7 222 82.9 14.9 121 77.7 18.2 192 83.9 10.9 588 79.9 16.2
Levofloxacin 333 97.9 1.2 \<10 NA NA 51 98.0 0 180 91.1 1.7 173 99.4 0
Penicillin 518 100 \- 226 100 \- 145 100 \- 184 100 \- 369 100 \-
Vancomycin 463 100 \- 179 100 \- 143 100 \- 65 100 \- 526 100 \-
*Streptococcus* viridans group
Amoxicillin NT NT NT NT NT NT NT NT NT NT NT NT 268 92.9 0.7
Cefepime 23 95.7 4.3 NT NT NT 12 66.7 33.3 NT NT NT NT NT NT
Cefotaxime 434 83.6 11.1 101 92.1 4.0 31 90.3 9.7 75 97.3 2.7 56 94.6 0
Ceftriaxone 678 87.3 7.7 130 89.2 3.8 99 81.8 18.2 40 97.5 2.5 \<10 NA NA
Clarithromycin 34 52.9 38.2 21 76.2 19.0 21 71.4 23.8 \<10 NA NA NT NT NT
Erythromycin 959 57.2 37.7 289 71.6 23.2 192 64.6 32.8 796 88.1 9.2 626 59.9 31.6
Levofloxacin 331 96.1 2.7 \<10 NA NA 16 87.5 0 93 89.2 4.3 \<10 NA NA
Penicillin 1,047 63.7 6.2 303 79.2 0 61 78.7 8.2 \<10 NA NA 452 69.0 3.1
Vancomycin 1,095 100 \- 276 100 \- 180 100 \- 277 100 \- 580 100 \-
^a^NCCLS breakpoints were used for all countries, except France (CA-SFM) ^b^Not tested ^c^Breakpoints do not currently exist to interpret as S (susceptible) or R (resistant)
:::
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
*Enterobacteriaceae* isolated from ICU patients during 2000--2002
:::
United States Canada Italy Germany France^a^
------------------------------- ------------------------------- --------------- -------- ------- --------- ----------- ------ ------- ------ ------ ------- ------ ------ ------- ------- ------
*Escherichia coli* Cefepime 10,356 98.1 1.5 207 98.1 1.9 1,426 98.1 1.4 2,830 98.6 1.2 4,358 98.9 0.6
Cefotaxime 9,086 96.5 2.2 3,231 96.3 2.5 1,748 94.5 3.8 5,828 97.8 1.8 9,362 98.8 0.6
Ceftazidime 14,574 95.3 3.0 4,438 97.7 1.6 2,548 94.7 3.7 3,924 97.9 1.6 9,164 97.8 1.2
Ceftriaxone 15,897 97.4 1.7 3,829 96.8 2.2 1,423 94.4 4.2 534 99.8 0.2 834 98.6 1.0
Ciprofloxacin 17,294 89.0 10.7 5,028 90.3 9.5 2,616 87.0 12.7 4,615 86.7 12.4 8,577 93.1 6.5
Gentamicin 20,581 92.4 6.5 6,654 92.8 5.3 2,650 92.2 6.6 4,825 94.3 5.2 9,442 95.4 4.2
Imipenem 15,353 100 0 3,386 100 0 2,254 100 0 5,172 100 0 8,994 100 0
Levofloxacin 14,920 88.2 11.6 776 85.1 13.9 496 86.5 13.3 3,137 88.2 11.0 NT^b^ NT NT
Piperacillin-tazobactam 13,573 93.1 3.6 4,305 95.1 2.4 1,879 95.8 2.4 5,637 93.6 3.4 7,255 95.4 1.1
Trimethoprim-sulfamethoxazole 20,296 79.2 20.7 6,737 84.6 15.3 2,440 75.0 24.9 5,598 73.1 26.6 9,028 78.2 21.1
*Klebsiella oxytoca*
Cefepime 1,476 96.2 3.3 19 100 0 255 99.6 0 566 96.8 2.7 478 97.1 0.4
Cefotaxime 1,324 92.7 4.7 486 94.2 4.5 230 96.5 1.7 1,117 93.8 4.4 865 96.3 0.8
Ceftazidime 1,909 91.7 7.0 661 94.9 4.1 361 83.4 15.2 749 95.3 4.5 870 98.3 0.5
Ceftriaxone 2,035 89.9 6.6 536 93.8 2.8 197 81.7 2.0 83 97.6 0 79 87.3 2.5
Ciprofloxacin 2,226 92.5 5.9 745 96.0 3.0 368 96.7 3.0 905 90.1 7.8 815 94.5 4.8
Gentamicin 2,569 89.9 8.3 857 95.0 4.9 366 89.6 3.0 1,016 98.2 1.2 865 97.1 2.4
Imipenem 2,061 100 0 516 100 0 337 100 0 1,062 100 0 845 100 0
Levofloxacin 1,754 93.3 3.4 159 96.9 1.3 133 97.0 3.0 560 94.6 3.2 NT NT NT
Piperacillin-tazobactam 1,801 82.7 13.9 624 91.2 7.1 313 81.8 11.2 1,113 78.9 18.1 742 88.3 10.4
Trimethoprim-sulfamethoxazole 2,467 92.5 7.5 863 96.3 3.6 308 95.1 4.9 1,084 93.7 6.3 802 94.1 5.7
*Klebsiella pneumoniae*
Cefepime 7,276 95.8 3.4 98 100 0 552 93.5 5.6 1,068 95.7 3.5 840 95.6 3.0
Cefotaxime 6,243 91.0 6.1 1,411 97.9 1.5 850 76.7 16.4 2,414 93.1 6.0 1,553 94.4 1.9
Ceftazidime 9,597 88.5 10.1 2,238 97.5 2.2 1,142 69.8 28.5 1,665 90.0 8.2 1,591 92.5 5.2
Ceftriaxone 10,337 92.7 4.7 1,736 97.9 1.1 816 75.2 15.0 166 98.8 0.6 112 86.6 5.4
Ciprofloxacin 11,089 89.9 8.4 2,484 91.8 7.2 1,190 88.2 9.9 2,128 85.4 9.4 1,473 89.5 8.7
Gentamicin 13,012 91.6 7.0 2,906 96.7 2.9 1,211 81.4 14.5 2,065 91.6 6.1 1,553 97.1 2.7
Imipenem 10,263 100 0 1,766 100 0 1,066 100 0 2,351 100 0 1,567 100 0
Levofloxacin 9,626 91.0 6.4 485 93.4 3.7 287 78.4 21.3 1,228 92.6 4.4 NT NT NT
Piperacillin-tazobactam 9,359 85.9 7.4 2,160 91.5 2.7 746 82.2 14.6 2,408 84.9 8.3 1,286 89.4 5.1
Trimethoprim-sulfamethoxazole 12,641 88.6 11.1 2,924 92.8 7.1 1,103 82.0 18.0 2,324 82.2 17.2 1,443 88.2 10.9
*Morganella morganii*
Cefepime 566 95.9 2.3 \<10 NA NA 121 97.5 2.5 262 94.7 5.0 412 96.1 0.2
Cefotaxime 499 78.8 8.4 156 91.0 3.8 144 74.3 6.3 437 86.7 3.9 678 81.1 5.9
Ceftazidime 715 73.6 17.3 256 79.7 10.9 213 75.6 15.0 313 84.0 7.7 673 78.6 8.0
Ceftriaxone 806 91.1 2.2 219 96.3 1.4 125 91.2 3.2 22 86.4 0 57 84.2 5.3
Ciprofloxacin 841 78.1 20.7 292 94.2 4.5 220 87.3 9.5 344 97.7 2.0 634 88.6 8.5
Gentamicin 967 84.0 14.1 329 94.5 4.6 222 90.1 8.6 378 96.8 2.1 679 95.6 3.4
Imipenem 784 100 0 196 100 0 206 100 0 402 100 0 649 99.8 0
Levofloxacin 725 78.1 19.3 42 95.2 4.8 55 90.9 9.1 251 98.0 1.6 NT NT NT
Piperacillin-tazobactam 725 91.2 5.1 254 97.2 1.6 150 94.0 3.3 430 94.2 3.5 564 91.0 4.6
Trimethoprim-sulfamethoxazole 936 75.1 24.7 329 91.8 8.2 193 79.8 20.2 435 93.1 6.9 627 83.9 14.2
*Proteus mirabilis*
Cefepime 1,964 98.2 1.0 20 100 0 395 87.6 11.4 599 99.2 0.8 736 99.0 0.1
Cefotaxime 1,794 99.1 0.5 295 99.7 0 441 69.4 23.4 1,209 98.8 0.7 1,503 99.5 0.1
Ceftazidime 2,684 98.0 1.1 463 99.4 0.2 630 86.0 9.4 821 98.5 1.0 1,505 99.3 0.2
Ceftriaxone 3,034 99.4 0.3 392 99.5 0 385 80.5 13.8 77 98.7 0 72 100 0
Ciprofloxacin 3,169 85.2 12.7 504 95.2 4.6 657 70.6 22.7 980 92.9 5.1 1,424 90.9 6.8
Gentamicin 3,796 91.5 7.7 698 92.6 7.2 670 61.6 37.2 992 92.9 5.9 1,509 91.3 7.9
Imipenem 2,850 100 0 367 100 0 580 100 0 1,020 100 0 1,319 100 0
Levofloxacin 2,825 87.8 10.5 94 100 0 202 61.9 34.7 688 96.5 2.3 \<10 NA^c^ NA
Piperacillin-tazobactam 2,715 97.7 0.8 449 98.2 0.2 465 95.7 2.8 1,201 98.6 0.8 1,231 99.3 0.2
Trimethoprim-sulfamethoxazole 3,706 85.2 14.7 708 89.4 10.6 615 61.6 38.0 1,159 80.8 19.1 1,411 79.7 18.6
*Serratia marcescens*
Cefepime 3,653 96.7 2.3 52 96.2 1.9 497 96.8 2.2 546 94.1 3.5 509 98.6 0.2
Cefotaxime 3,134 87.0 5.7 670 92.8 2.7 470 79.6 9.8 951 84.0 7.5 809 81.5 3.3
Ceftazidime 4,718 89.7 7.9 1,113 95.2 3.0 738 81.4 13.3 851 89.7 7.5 812 94.7 3.0
Ceftriaxone 4,710 90.5 4.6 846 95.4 1.7 444 86.7 6.3 160 45.6 0 115 77.4 4.3
Ciprofloxacin 5,006 91.0 6.7 1,292 85.0 11.7 757 83.5 4.5 978 72.6 12.4 787 78.9 10.5
Gentamicin 5,905 92.9 5.9 1,313 94.6 5.2 758 97.4 2.1 665 92.9 6.3 808 91.6 6.6
Imipenem 4,960 100 0 880 100 0 727 100 0 1,018 100 0 805 100 0
Levofloxacin 4,356 94.3 4.2 264 92.4 4.2 266 95.5 1.5 595 87.6 6.6 \<10 NA NA
Piperacillin-tazobactam 4,337 88.1 5.1 1,155 91.6 3.3 547 92.7 3.8 1,053 77.6 3.1 749 82.6 2.4
Trimethoprim-sulfamethoxazole 5,697 95.9 3.9 1,325 94.9 5.1 646 81.4 18.6 908 88.1 10.9 699 84.1 13.6
^a^NCCLS breakpoints were used for all countries, except France (CA-SFM) ^b^Not tested ^c^Not applicable if \<10 isolates were tested
:::
::: {#T5 .table-wrap}
Table 5
::: {.caption}
######
*P. aeruginosa* and *Acinetobacter* spp isolated from ICU patients during 2000--2002
:::
United States Canada Italy Germany France
------------------------------- ------------------------------- --------------- -------- ------- --------- -------- ------- ------- ------ ------- ------- ------ ------ ------- ------ ------
*Acinetobacter species* Cefepime 5,162 43.8 40.2 97 67.0 23.7 475 17.9 73.7 623 74.2 10.8 857 28.0 40.3
Cefotaxime 3,830 23.3 49.9 705 36.7 34.9 555 11.0 78.7 1,254 34.9 24.6 671 15.4 38.7
Ceftazidime 5,954 42.2 40.8 1,162 70.8 22.9 692 25.6 68.5 988 66.7 14.5 1,106 34.9 35.5
Ceftriaxone 4,709 16.3 55.9 874 32.4 28.7 452 8.8 72.6 104 42.3 11.5 81 9.9 51.9
Ciprofloxacin 5,808 39.7 58.0 1,156 72.1 25.9 686 21.1 76.7 1,126 74.8 22.9 1,038 37.7 61.2
Gentamicin 6,618 47.2 47.2 1,185 72.8 22.8 768 23.3 72.4 979 82.0 14.1 936 49.3 43.5
Imipenem 6,006 87.0 7.5 918 95.8 1.9 569 77.9 19.0 1,253 96.2 3.4 1,088 93.8 3.8
Levofloxacin 5,099 43.8 52.2 489 61.1 25.6 295 13.9 75.3 840 82.0 10.5 NT^b^ NT NT
Meropenem 2,154 66.3 26.5 348 93.7 4.9 455 74.5 13.6 1,024 96.0 3.4 147 68.0 28.6
Piperacillin 4,658 35.4 45.9 959 66.5 19.5 635 19.5 69.9 1,171 59.7 12.9 805 35.0 50.3
Piperacillin-tazobactam 3,429 53.6 28.5 903 70.7 23.1 425 35.1 46.4 1,225 81.8 7.5 878 74.5 10.5
Trimethoprim-sulfamethoxazole 5,697 51.4 48.4 1,155 74.8 25.2 750 44.1 55.7 1,234 83.6 15.6 93 45.2 52.7
*Pseudomonas aeruginosa*
Cefepime 20,220 72.5 12.4 371 73.3 12.4 5,056 58.9 28.9 3,483 80.3 7.8 7,967 52.6 16.2
Cefotaxime 11,283 9.2 50.4 1,836 13.3 47.5 4,181 6.0 70.7 2,689 7.7 52.2 NT NT NT
Ceftazidime 26,353 71.2 17.4 6,036 73.7 13.4 7,640 56.7 31.3 5,141 76.2 14.9 8,547 70.2 14.9
Ceftriaxone 14,066 12.1 56.4 2,847 11.3 59.7 3,383 8.4 70.4 154 26.6 7.8 NT NT NT
Ciprofloxacin 26,700 62.8 33.1 5,924 67.2 30.2 7,388 58.4 38.8 4,746 68.6 24.4 8,560 55.3 40.6
Gentamicin 29,268 69.4 21.5 5,951 72.2 15.9 7,522 52.2 41.7 3,913 74.0 14.3 7,327 44.0 46.1
Imipenem 26,076 73.5 22.1 3,775 77.9 18.2 7,057 59.7 27.8 4,412 70.5 19.0 8,575 69.5 21.4
Levofloxacin 21,059 62.7 31.7 713 56.8 33.5 2,427 44.9 51.0 2,953 68.0 23.9 NT NT NT
Meropenem 7,540 76.0 18.2 1,266 80.3 14.5 4,082 57.3 32.7 4,351 77.8 13.8 1,818 81.1 6.4
Piperacillin 22,855 77.7 22.2 5,520 80.9 18.8 7,004 63.1 36.7 4,554 81.7 14.1 8,454 64.1 24.1
Piperacillin-tazobactam 21,848 85.5 14.4 4,190 91.0 9.0 5,252 77.7 22.0 4,746 85.8 10.7 8,256 69.6 15.9
Trimethoprim-sulfamethoxazole 15,618 3.6 96.4 4,283 4.0 96.0 7,054 4.1 95.8 3,375 4.2 95.8 NT NT NT
^a^NCCLS breakpoints were used for all countries, except France (CA-SFM) ^b^NT = not tested
:::
Results
=======
In vitro susceptibility data from over 220,000 isolates from ICUs in five countries over the period 2000--2002 were assimilated. The most frequent species isolated from infections in the ICU was *S. aureus*, being most common in three of the five countries (Table [1](#T1){ref-type="table"}). The oxacillin resistance rates among *S. aureus* varied markedly across countries from 19.7% in Canada to 59.5% in Italy. *E. coli* (7.7%--15.5%) and *P. aeruginosa* (10.8%--22.3%) were the most frequent Gram-negative organisms encountered. The Gram-positive genus *Enterococcus*, either as *E. faecalis, E. faecium* or non-speciated isolates accounted for \<10% of isolates in most countries with *E. faecalis* being the most common species \<4.3%. Community-acquired respiratory pathogens such as *Streptococcus pneumoniae* and *Haemophilus influenzae* were relatively uncommon in all five countries.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Incidence of pathogens isolated from ICU patients by country (%)
:::
United States Canada Italy Germany France
------------------------------------------------ -------- --------------------------------------------- --------- --------------------------------------------- -------- --------------------------------------------- -------- --------------------------------------------- --------
*S. aureus*^a^ 20.2 *S. aureus*^a^ 17.4 *P. aeruginosa* 22.3 CNS 16.4 *S. aureus*^1^ 17.2
CNS^b^ 15.9 CNS 16.1 CNS 18.7 *S. aureus*^a^ 13.6 CNS 16.7
*P. aeruginosa* 13.1 *E. coli* 12.6 *S. aureus*^a^ 18.1 *E. coli* 12.3 *E. coli* 15.5
*E. coli* 9.2 *P. aeruginosa* 11.3 *E. coli* 7.7 *P. aeruginosa* 10.8 *P. aeruginosa* 13.8
*K. pneumoniae* 5.8 *Enterococcus* spp 7.6 *E. faecalis* 3.9 *Enterococcus* spp 7.4 S. pneumoniae 3.3
*Enterococcus* spp 5.4 *K. pneumoniae* 5.5 *K. pneumoniae* 3.5 *K. pneumoniae* 5.4 *E. cloacae* 3.3
*E. cloacae* 4.3 *E. cloacae* 4.2 *Enterococcus* spp 3.3 *E. cloacae* 4.7 *E. faecalis* 3.0
*E. faecalis* 3.7 *S. marcenscens* 2.5 *E. cloacae* 2.6 E. faecalis 4.3 *K. pneumoniae* 2.7
*S. marcescens* 2.7 *H. influenzae* 2.1 *S. marcescens* 2.2 *P. mirabilis* 2.6 *P. mirabilis* 2.5
*A. baumanii* 2.6 *E. faecalis* 2.1 *P. mirabilis* 1.9 *K. oxytoca* 2.4 *Enterococcus* spp 2.3
*Enterobacteriaceae*^c^ (all species combined) 29.5 *Enterobacteriaceae* (all species combined) 33.0 *Enterobacteriaceae* (all species combined) 30.2 *Enterobacteriaceae* (all species combined) 36.0 *Enterobacteriaceae* (all species combined) 32.1
Total (n) 26,624 Total (n) 54,445 Total (n) 34,609 Total (n) 48,385 Total (n) 62,459
^a^Proportion of *S. aureus* testing as MRSA was USA (52.3%), Canada (19.7%), Italy (59.4%), Germany (21.0%), and France (40.6%) ^b^CNS = Coagulase-negative staphylococci ^c^*Enterobacteriaceae* includes all species of genera occurring at \>0.1%
:::
Tables [2](#T2){ref-type="table"},[3](#T3){ref-type="table"},[4](#T4){ref-type="table"},[5](#T5){ref-type="table"} show the antimicrobial susceptibility profiles of various Gram-positive and Gram-negative pathogens isolated from ICU patients against a range of relevant antimicrobials.
Specifically notable susceptibility patterns include the vancomycin susceptibility of all strains of staphylococci. Generally, there was a low proportion of vancomycin resistant *E. faecalis* \<5%, whereas vancomycin-resistant *E. faecium* was more prevalent ranging from 0.8% in France to 76.3% in the United States, with a wide inter-country variation (Table [2](#T2){ref-type="table"}). Penicillin resistance rates varied among *S. pneumoniae*, from 2.0% in Germany to 20.2% in the US with concurrent ceftriaxone resistance rates of 0% in Germany to 3.4% in Italy (Table [3](#T3){ref-type="table"}).
β-lactam activity was assessed by comparing four different cephalosporins and a β-lactam/β-lactamase inhibitor combination, piperacillin-tazobactam. Overall, the putative production of ESBLs among *E. coli* was low, \<6%, but ceftazidime resistance was reported at higher rates in *K. pneumoniae* and *S. marcescens*, with the highest rates seen in *M. morganii*, from 16.0% in Germany to 26.4% in the United States (Table [4](#T4){ref-type="table"}). Among the gram-negative organisms tested, ceftriaxone resistance rates were usually lower than ceftazidime, with the exception among *P. aeruginosa* and *Acinetobacter* spp. Cefepime, a fourth generation cephalosporin with anti-pseudomonal activity was also more active than ceftazidime (Table [5](#T5){ref-type="table"}). Against the *Enterobacteriaceae*, the β-lactam combination agent piperacillin-tazobactam was generally less active than ceftriaxone. These species showed a wide variation in fluoroquinolone susceptibility among both species and countries. Gentamicin resistance rates among the *Enterobacteriaceae* varied from 1.2% among *K. oxytoca* from Germany to 37.2% in *P. mirabilis* from Italy. Ciprofloxacin resistance rates among *E. coli* ranged from 6.5% in France to 12.7% in Italy. Variable fluoroquinolone resistance rates among *S. marcescens* were also demonstrated, with a range of resistance from 4.5% in Italy to 12.4% in Germany.
Discussion
==========
Data derived from international surveillance studies, such as those presented here, can provide a unique contemporary perspective on the susceptibility of commonly encountered organisms to commonly used antibiotics. Such surveillance systems play a crucial role in detecting emerging trends in resistance. Comparisons of these with data of other recent surveillance programs show the wide variations in susceptibility profiles and the need for ongoing unit-specific surveys.
In Germany the prevalence of resistance among gram-positive organisms remained comparatively low with an incidence of 21% MRSA. In 2000, Frank et al. reported that 96% of German isolates of *S. marcescens* and *M. morganii* were susceptible to ceftazidime, yet in this study we found 89.7% and 84.0%, respectively \[[@B9]\]. A similar decrease in activity was noted with *E. coli* and ciprofloxacin between the two studies, 91% in 1996--1997 compared with 86.7% in this study. Marked decreases in susceptibility of *P. aeruginosa* in Germany were also evident, with no agent showing \>85.8% susceptibility (piperacillin-tazobactam) compared with most agents having 85%--94% susceptibility in 1996--1997. Changes of 15--20% have been reported with ceftazidime, imipenem, ciprofloxacin and meropenem, while piperacillin-tazobactam has shown the smallest decrease in susceptibility with \<6% over the 4-year period. Piperacillin plus or minus tazobactam and cefepime were the most active agents, based on susceptibility, against *P. aeruginosa* in Germany. Conversely, ceftriaxone and imipenem were the most active agents, based on susceptibility, against *Klebsiella* spp., which account for almost 8% of ICU isolates.
Staphylococcal species from French ICU isolates showed a high proportion of oxacillin resistance, 40.6% and 69. 9% of *S. aureus* and coagulase-negative staphylococci spp., respectively. *S. pneumoniae* showed penicillin resistance of 17.9%, higher than the other four countries, although the activity of third-generation cephalosporins, ceftriaxone and cefotaxime, showed only 0.6% and 0.8% resistance, respectively. Despite a lower ceftazidime susceptibility breakpoint compared to NCCLS standards (MIC 4 μg/ml instead of 8 μg/ml) putative ESBL expression were slightly lower in France than in Germany in 2000--2002. Ceftazidime non-susceptibility rates among *E. coli*, *K. oxytoca*, and *P. mirabilis* were ≤ 2.2%; however, ceftazidime non-susceptibility rates among *K. pneumoniae*, *M. morganii* and *S. marcescens* were 7.5%, 21.4%, and 5.3%, respectively. Imipenem was active against all *Enterobacteriaceae*. Against *P. aeruginosa* and *Acinetobacter* spp., imipenem resistance rates were 21.4% and 3.8%, respectively. Previously, a lower imipenem resistance of 24% among French isolates of *P. aeruginosa* was reported \[[@B7]\].
Among the Italian isolates of staphylococci, oxacillin resistance occurred in 59.4% of *S. aureus* and 84.8% of coagulase-negative isolates. This MRSA rate was similar to that reported by Frank et al. from bacteremic isolates in Italy; however, they reported an increase in MRSA from 25% to 55% over the period 1997 to 2001 \[[@B18]\]. Vancomycin resistance rates of 2.8% for *E. faecalis* and 24.2% for *E. faecium* are some of the highest rates recorded in Europe, although still modest compared to rates experienced in the United States; however, teicoplanin was more active with 2.4% and 13.7% of strains being resistant, respectively. Pneumococcal resistance to penicillin and erythromycin was 7.6% and 28.1%, respectively. The impact of alterations in penicillin-binding protein that reduce penicillin susceptibility have less effect on the activity of third-generation cephalosporins such as ceftriaxone with 3.4% and cefotaxime with 4.6% resistance, respectively. *S. pyogenes* was fully susceptible to penicillin; however, 11.8% of isolates were resistant to clarithromycin and 23.7% were resistant to erythromycin.
The proportion of ESBLs was slightly higher in Italy with *E. coli* showing ceftazidime non-susceptibility of 5.3%, whereas *K. pneumoniae* and *K. oxytoca* demonstrated 30.2% and 16.6% ceftazidime non-susceptibility, respectively. Fluoroquinolone resistance rates among the *Enterobacteriaceae*, using ciprofloxacin as a marker, varied from 3.0% for *K. oxytoca* to 22.7% for *P. mirabilis*, and 12.7% for *E. coli*. Thus, among *Enterobacteriaceae*, ciprofloxacin was generally less active than the third-generation cephalosporin, ceftriaxone. *P. aeruginosa* and *Acinetobacter* spp. strains from Italian ICUs demonstrated significant resistance rates. Isolates of *P. aeruginosa* showed resistance rates of \>28% for all agents tested except piperacillin-tazobactam. Thus empiric therapy for possible pseudomonal infections will require combination therapy. *Acinetobacter* spp. showed a similar lack of susceptibility except to imipenem and meropenem (19.0% and 13.6% resistant). An increase in fluoroquinolone resistance in *E. coli* and *K. pneumoniae* in bacteremic isolates from Italy was observed during 1997--2001, with rates of 26.7% and 24%, respectively \[[@B9]\]. An increase in ureidopenicillin resistance was noted in *P. aeruginosa* isolates in Italy from 30% to 37% in a 4-year period \[[@B9]\]. This study showed 22.0% piperacillin-tazobactam and 36.7% piperacillin resistance among ICU *P. aeruginosa* isolates.
In Canada oxacillin-resistance among *S. aureus* was noted in 19.7% and coagulase-negative staphylococci in 79.4%. Vancomycin resistance was reported among 0.9% and 14.5% of *E. faecalis* and *E. faecium*, respectively. The lowest rate of penicillin resistance in *S. pneumoniae* in this study was noted from Canada at 7.1%; however, clarithromycin resistance was 30.4%. Ceftriaxone showed 0.7% resistance whereas cefepime exhibited 12.0% resistance among pneumococci from the ICU.
Overall the susceptibility rates for Gram-negative isolates from Canadian ICUs were higher than those in the other four countries examined. A low rate of ESBLs was reported, but there was variable activity of piperacillin-tazobactam which showed \>9% resistance among *Klebsiella* spp. and *S. marcescens* tested. The rate of fluoroquinolone resistance was similar to those of other countries with *E. coli* showing 13.9% levofloxacin resistance. Among *Enterobacteriaceae*, \<10% of most species were resistant to third-generation cephalosporins tested with the exception of ceftazidime and *M. morganii*. Resistance among *P. aeruginosa* and *Acinetobacter* spp. was generally lower than in other countries apart from Germany. Only piperacillin-tazobactam showed reliable activity against *P. aeruginosa* (9% resistant), while resistance to all other agents was \>19%. *Acinetobacter* spp. remained susceptible to only the carbapenems, imipenem and meropenem.
Comparison of the data from Canadian isolates with those from the United States shows some significant differences. This demonstrates the limitations of pooling Canadian and United States data since the differences between the two regions, such as the rate of MRSA, may have some impact on empiric therapy. Data from the NNIS system has previously reported an increasing trend towards resistance within ICUs in the United States \[[@B19]\]. Oxacillin resistance among staphylococci from ICUs in the United States was 52.3% and 84.2% for *S. aureus* and coagulase-negative species, respectively.
This value is identical to that of *S. aureus* and very similar to the CNS data reported by the 1999 NNIS system. The NNIS highlighted a 37% increase in MRSA over the period 1994--98 to 1999, but only a 2% increase among CNS strains \[[@B4]\]. Vancomycin resistance in the United States was observed in 4.5% of *E. faecalis*; however, over 76% *E. faecium* were vancomycin non-susceptible.
Although streptococci are uncommon ICU pathogens they can be rapidly invasive and possibly fatal unless adequate therapeutic approaches are adopted. *S. pneumoniae* in the United States has acquired a range of resistance mechanisms with resistance to penicillin and the macrolides, clarithromycin and erythromycin, being common, 20.2% and 25.5%--30.5% respectively. The newer generation cephalosporins, ceftriaxone, cefotaxime and cefepime showed good activity against pneumococci, 3.2%, 6.3% and 4.5% resistant, respectively. Less than 1.0% of isolates were resistant to levofloxacin. These data are similar to other recent reports \[[@B20]\].
For *Enterobacteriaceae* which account for approximately 30% of all isolates from ICU infections, the incidence of putative ESBLs was low in *E. coli*, 4.7% but ceftazidime non-susceptibility was higher in *K. oxytoca* 8.3%,*K. pneumoniae* 11.5%,*S. marcescens* 10.3% and *M. morganii* 26.4%. These data are consistent with other recent reports \[[@B21]\]. Fluoroquinolone resistance was observed in all *Enterobacteriaceae* tested, in the US for example, resistance rates were as follows, using ciprofloxacin as a marker: *E. coli* 10.7%, *K. oxytoca* 5.9%, *K. pneumoniae* 8.4%, *M. morganii* 20.7%, *P. mirabilis* 12.7% and *S. marcescens* 6.7%. These data show increased fluoroquinolone resistance compared with recent reports \[[@B21]\]. Jones et al. previously reported susceptibility data on ICU pathogens isolated over the period 1998--2001 \[[@B22]\].
Specifically, enteric bacteria showed changes over this time. Fluoroquinolone resistance doubled among *E. coli* isolates from 3.3--5.5% to 10.8--11.4% \[[@B22]\]. This study showed a generally higher level of activity among third-generation cephalosporins than other reports \[[@B23]\], with ceftriaxone showing \<10% resistance rates against most species tested. Piperacillin-tazobactam showed less consistent activity with some species being \>14% resistant, e.g. *Klebsiella* spp.,*P. aeruginosa*, and *Acinetobacter* spp. present significant therapeutic challenges in ICUs in the United States. With the exception of cefepime, all other tested antimicrobials demonstrated \>12% resistance to *P. aeruginosa*, many considerably higher. Piperacillin-tazobactam showed the next lowest resistance rate, 14.4%, with all other agents having rates of 17% or higher. Non-susceptibility to ciprofloxacin among *P. aeruginosa* was 37.2%, higher than in the Neuberger report. Sahm et al. reported a 10% increase in fluoroquinolone resistance among *P. aeruginosa* in the United States, whereas resistance emerged more slowly with the other classes of antimicrobials tested \[[@B12]\]. *Acinetobacter* infections continue to present significant therapeutic challenges due to the extensive resistance mechanisms demonstrated by the \>25% resistance shown in Table [5](#T5){ref-type="table"}. Only imipenem has any reliable activity against *Acinetobacter* spp. with an 87% susceptibility rate.
There are several implications of these data. It is essential that local surveillance programs be maintained in each country\'s ICU setting. The local data are vital to the formulary committees as they select appropriate agents to treat infections. There are clear differences among the five countries studied in this report. Although the predominant pathogens are similar, ongoing surveillance is essential to detect the emergence of resistant species. It is clear that certain classes of compounds are losing activity against the ICU pathogens tested. For example, the fluoroquinolones have reduced susceptibility among many Gram-negative species as well as staphylococci; however, the newer class members have enhanced activity against pneumococci. Advanced-generation cephalosporins have variable activity, with ceftriaxone showing consistently good activity against the *Enterobacteriaceae* and some staphylococci. Ceftazidime has lost potency due to the emergence of ESBL enzymes and also has diminished activity against *P. aeruginosa*. Piperacillin-tazobactam is generally active against *P. aeruginosa* in ICUs. The aminoglycoside, gentamicin has shown continued activity against most *Enterobacteriaceae* in all five countries, and modest activity against *S. aureus* but not against CNS strains. The gentamicin susceptibility of *P. aeruginosa* ranged from 44.0% in France to 74.0% in Germany, whereas *Acinetobacter* spp *.* showed more variable gentamicin susceptibility varying from 23.3% in Italy to 82.0% in Germany. These local data should be considered when treating infections in the ICU.
Use of agents with anti-pseudomonal activity such as cefepime, piperacillin-tazobactam or the carbapenems should preferably be reserved for patient types or infections where this pathogen is present or risk factors exist, as per the ATS Community acquired-pneumonia guidelines \[[@B24]\]. A combination of a third-generation cephalosporin such as ceftriaxone with vancomycin may be appropriate for bloodstream infections based upon the NNIS etiology data from 1992--1999.
Conclusions
===========
The current study confirmed the emergence of fluoroquinolone resistance among various Gram-negative species and staphylococci, which may be increasing due to the heightened use of these drugs; however the reported ESBL rates among *Enterobacteriaceae* was lower than noted in other studies and appeared to be stable. The prevalence of MRSA, perhaps the most significant resistant hospital pathogen, varied among the five countries and appeared to be increasing. Parenteral cephalosporins such as ceftriaxone and cefotaxime remained quite active against *Enterobacteriaceae*. Up-to-date susceptibility data should be made available as rapidly as possible to physicians so that appropriate targeted empirical therapy can be instituted, this approach can assist in maintaining the activity of the current antimicrobials. While local surveillance studies remain crucial, national surveillance studies such as this can provide an invaluable data source to provide guidance in formulary decision-making.
Authors Contributions
=====================
MJ conceived the study, provided data interpretation and drafted the manuscript. DD analyzed the study data; JK and DS provided expert microbiological analysis and interpretation of study data; RW provided clinical expertise in interpretation of data and drafting manuscript. All authors read and approved the final manuscript.
Acknowledgments
===============
We thank F. Hoffmann-La Roche Ltd., Basel, Switzerland for financial support of this study. Additionally, we thank the many clinical microbiology laboratories around the world that contribute data to TSN Databases, without whom such studies would not be possible.
| PubMed Central | 2024-06-05T03:55:46.696697 | 2004-7-29 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509280/",
"journal": "Ann Clin Microbiol Antimicrob. 2004 Jul 29; 3:14",
"authors": [
{
"first": "Mark E",
"last": "Jones"
},
{
"first": "Deborah C",
"last": "Draghi"
},
{
"first": "Clyde",
"last": "Thornsberry"
},
{
"first": "James A",
"last": "Karlowsky"
},
{
"first": "Daniel F",
"last": "Sahm"
},
{
"first": "Richard P",
"last": "Wenzel"
}
]
} |
PMC509281 | Introduction
============
The incidences of both the acute and chronic forms of sinusitis have been increasing, and between 10 and 15% of the population of central Europe are affected annually \[[@B1]\]. There are an estimated 30 million cases of ABS in the USA each year \[[@B2]-[@B4]\]. Acute sinusitis accounts for 0.5--2.0% of all upper respiratory tract infections in adults and between 5--10% in children and therefore is a common reason for visits to primary care physicians \[[@B5]\]. Although usually mild in severity, complications can be life threatening, including meningitis, brain abscess, orbital cellulitis and abscess, subempyema, osteomyelitis, and nasal polyposis \[[@B6]-[@B9]\].
*S. pneumoniae*is the most common pathogenic bacterium responsible for ABS, isolated in 30--50% of cases, followed by *H. influenzae*, isolated in 20--40% of cases. *Moraxella catarrhalis*is isolated in 5--10% of cases, beta haemolytic streptococci in less than 5%, and *Staphylococcus aureus*in less than 10% although it is often found co-infecting with other bacteria \[[@B10]\].
Treatment options for ABS are controversial as up to 40% of patients recover spontaneously, however, studies have shown that treatment with an antibacterial reduces the time to recovery from sinusitis, improves symptoms, and helps to prevent complications \[[@B11],[@B12]\]. Guidelines on antibacterial use for ABS vary, possibly because of different regulations, antibacterial resistance patterns, and etiology in different countries, however, the choice of first-line antibacterial is similar across treatment guidelines \[[@B3],[@B13]-[@B17]\]. Nearly all recommend amoxicillin, as it is active against the major causative pathogens of AMS and is generally well tolerated. For patients with penicillin allergy, the recommended first-line agents vary in different countries. Trimethoprim or trimethoprim-sulfamethoxazole is commonly recommended \[[@B3],[@B14]\]. In addition, the French guidelines have recently been reviewed and telithromycin has been included as an alternative first-line agent \[[@B15]\]. Macrolides are not included in the French guidelines due to the high macrolide resistance prevalence in France \[[@B15],[@B18]\].
Second line, or alternative antibacterial agents of choice, are clarithromycin or second-generation cephalosporins such as cefuroxime and cefpodoxime, third-generation cephalosporins such as cefdinir, and trimethoprim-sulphamethoxazole \[[@B3],[@B5],[@B16],[@B17]\]. Telithromycin has been recommended as an alternative agent in Germany \[[@B13],[@B16]\]. High-dose amoxicillin-clavulanate should be used if the patient does not improve \[[@B3],[@B16],[@B17]\]. In France, anti-pneumococcal fluoroquinolones are recommended after bacterial confirmation or if the patient is at high risk of complications \[[@B15]\]. A single dose of ceftriaxone can be used in a child who cannot be treated orally, i.e. vomiting \[[@B17]\].
However, due to increasing levels of resistance in bacterial respiratory tract pathogens to these commonly used antibacterials (particularly the rapid emergence of penicillin-and macrolide-resistant strains of pneumococci), new agents are required that have high *in vitro*activity and demonstrated clinical efficacy against bacterial pathogens causing community-acquired respiratory tract infections (RTI\'s) \[[@B19]-[@B22]\]. Telithromycin is the first ketolide approved for clinical use. The ketolides are semisynthetic derivatives of the 14-membered ring macrolide erythromycin and have high *in vitro*activity against the common community-acquired RTI pathogens \[[@B23]\]. Clinical trials have demonstrated the efficacy and tolerability of telithromycin therapy in ABS \[[@B24]-[@B26]\].
The PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study is a longitudinal, global multicentre surveillance study designed in part to determine the activity of telithromycin against community-acquired RTI isolates, in relation to the frequency of prescribing, in the regions where the study is conducted \[[@B27]\]. The aim of this paper is to focus on the data gathered in the PROTEKT surveillance study to determine the in vitro efficacy of the new ketolide telithromycin and comparator agents against bacterial pathogens isolated from the subset of patients with clinically diagnosed ABS collected in PROTEKT (2000--2001, and 2001--2002).
Materials and Methods
=====================
Patients and bacterial isolates
-------------------------------
Detailed study design, including patient selection and methodology for isolate identification and storage in the PROTEKT study has been described previously \[[@B27]\]. The isolates in this sub-study of PROTEKT were selected from those patients presenting with clinically diagnosed ABS in which the isolates were determined clinically to be the pathogenic organism and the specimen type was sinus aspirate or nasopharyngeal swab/aspirate only. Methodology for sinus aspiration was that used routinely by the investigator.
Antimicrobial testing
---------------------
MIC susceptibility status was determined, using the National Committee of Clinical and Laboratory Standards (NCCLS) breakpoints, at a central laboratory (GR Micro Ltd, London, UK) from a panel of existing and new antibacterials, using the NCCLS broth microdilution method and lyophilised microtitre plates (Sensititre, Trek Diagnostics) \[[@B28]\]. NCCLS breakpoints were used for interpretation of MIC\'s \[[@B29]\]. Tentative NCCLS breakpoints for telithromycin are: *S. pneumoniae*and *S. aureus*, ≤ 1 μg/ml is susceptible, 2 μg/ml is intermediate, and ≥ 4 μg/ml is resistant; for *Haemophilus influenzae*, ≤ 4 μg/ml is susceptible, 8 μg/ml is intermediate, and ≥ 16 μg/ml is resistant \[[@B29]\].
Statistical analysis
--------------------
Statistical analysis was performed using a χ^2^test.
Results
=======
A total of 1,336 bacterial pathogens in all were collected from 25 countries within Western Europe (*n*= 652), North America (*n*= 14), Latin America (*n*= 207), Asia (*n*= 464), Eastern Europe (*n*= 68), Australia (*n*= 2), and South Africa (*n*= 126) in the PROTEKT study from years 2000--2001 and 2001--2002 for analysis of the susceptibility of bacterial pathogens isolated from patients with acute sinusitis. Gender distribution was 52.2% male (695 patients), 46.7% female (624 patients); gender was not provided for 1.3% of patients. Almost two thirds (66.3%) of patients were in the 0--12 year age group, one third (29.7%) in the 13--65 year age group, 2.7% in the over 65 year age group and age was not specified in 1.3% of patients. *S. pneumoniae*was the pathogen most frequently isolated (47.9% of isolates) followed by *H. influenzae*(24.6% of isolates) (Table [1](#T1){ref-type="table"}).
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Distribution of species by specimen type for the 1336 bacterial pathogens causing acute sinusitis \[n (%)\]
:::
**Specimen** ***S. pneumoniae*** ***H. influenzae*** ***M. catarrhalis*** ***S. aureus*** ***S. pyogenes*** **Total**
---------------- --------------------- --------------------- ---------------------- ----------------- ------------------- ------------
Sinus 272 (47.5) 148 (25.9) 67 (11.7) 64 (11.2) 21 (3.7) 572 (42.8)
Nasopharynx^1^ 368 (48.1) 181 (23.7) 145 (19.0) 52 (6.8) 18 (2.4) 764 (57.2)
Total 640 (47.9) 329 (24.6) 212 (15.9) 116 (8.7) 39 (2.9) 1336 (100)
^1^Aspirate or swab
:::
MIC data for isolates from patients with ABS demonstrated that the *in vitro*activity of telithromycin against gram-positive cocci was similar to amoxicillin-clavulanate and was higher and more potent than clarithromycin and beta-lactams tested such as cefuroxime (Table [2](#T2){ref-type="table"}). In total, 99.5 % of streptococcal isolates were susceptible to telithromycin. With the exception of *S. aureus*isolates more than 90% of gram-positive cocci were inhibited at a telithromycin MIC of 0.25 mg/L (Table [2](#T2){ref-type="table"}).
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
*In vitro*activity of antibacterial agents and percent susceptible against 1336 bacterial pathogens isolated from patients with clinically diagnosed acute sinusitis
:::
---------------------- ----------------------------- -------------------------------- ---------------- ------------------------------------------ -------- --------------------
**Organism** **N (total, SA^1^, NP^2^)** **Antibiotic** **MIC (mg/L)** **Percent susceptible**(Total, *SA, NP*)
**Range** **50** **90**
***S. pneumoniae*** 640, 272, 368 Penicillin 0.008 -- 8 0.06 2 58.0, *64.7, 53.0*
Amoxicillin-clavulanate 0.015 -- 8 0.03 2 95.5, *95.2, 95.7*
Cefuroxime 0.015 -- 16 0.12 8 66.1, *73.2, 60.9*
Cefpodoxime 0.12 -- 32 0.12 2 65.0, *71.7, 60.1*
Trimethoprim-sulphamethoxazole 0.12 -- 32 0.5 8 56.3, *58.1, 54.9*
Erythromycin 0.03 - \>64 0.06 \>64 55.9, *60.7, 52.5*
Clarithromycin 0.015 - \>32 0.06 \>32 55.8, *60.7, 52.2*
Azithromycin 0.03 - \>64 0.12 \>64 55.8, *60.7, 52.2*
Telithromycin 0.008 -- 8 0.015 0.12 99.5, *98.9, 100*
***H. influenzae*** 329, 148, 181 Ampicillin 0.12 -- 32 0.25 8 88.2, *91.2, 85.6*
Amoxicillin-clavulanate 0.12 -- 4 0.5 1 100, 100,100
Cefuroxime 0.12 -- 16 1 2 99.4, *99.3, 99.5*
Cefpodoxime 0.015 -- 4 0.06 0.25 99.4, *100, 98.9*
Cefdinir 0.06 -- 4 0.25 0.5 97.3, 96.0, 98.3
Trimethoprim-sulphamethoxazole 0.03 -- 16 0.06 4 84.5, *82.4, 86.2*
Erythromycin 0.25 -- 16 4 8 -^3^
Clarithromycin 0.25 -- 32 8 16 87.5, *87.2, 87.9*
Azithromycin 0.06 -- 4 1 2 100, 100,100
Telithromycin 0.06 -- 4 1 2 100, 100,100
***M. catarrhalis*** 212, 67, 145 Ampicillin 0.12 -- 32 4 16 \-
Amoxicillin-clavulanate 0.12 -- 0.5 0.12 0.25 \-
Cefuroxime 0.12 -- 16 1 4 \-
Cefpodoxime 0.06 -- 4 0.5 1 \-
Cefdinir 0.06 -- 1 0.12 0.25 \-
Trimethoprim-sulphamethoxazole 0.03 -- 2 0.12 0.25 \-
Erythromycin 0.25 -- 1 0.25 0.25 \-
Clarithromycin 0.25 -- 0.5 0.25 0.25 \-
Azithromycin 0.06 -- 0.25 0.06 0.06 \-
Telithromycin 0.004 -- 0.5 0.06 0.12 \-
***S. aureus*** 116, 64, 52 Methicillin \- \- \- 90.5, *92.2, 88.5*
Amoxicillin-clavulanate 0.06 -- 8 0.5 4 90.5, *92.2, 88.5*
Cefuroxime 0.5 -- 16 1 2 90.5, *92.2, 88.5*
Cefpodoxime 1 -- 32 2 4 88.8, *92.2, 84.6*
Trimethoprim-sulphamethoxazole 0.12 -- 32 0.12 0.12 96.6, *95.3, 98.1*
Erythromycin 0.03 - \>64 0.25 \>64 69.0, *73.4, 63.5*
Clarithromycin 0.015 - \>32 0.25 \>32 69.8, *75.0, 63.5*
Azithromycin 0.03 - \>64 0.5 \>64 69.0, *75.0, 61.5*
Telithromycin 0.015 - \>32 0.06 2 89.7, *89.1, 90.4*
***S. pyogenes*** 39, 21, 18 Penicillin 0.008 -- 0.008 0.008 0.008 100, *100,100*
Amoxicillin-clavulanate 0.008 -- 0.03 0.015 0.015 100, *100,100*
Cefuroxime 0.015 -- 0.015 0.015 0.015 100, *100,100*
Cefpodoxime 0.12 -- 0.12 0.12 0.12 100, *100,100*
Trimethoprim-sulphamethoxazole 0.12 -- 0.5 0.12 0.25 \-
Erythromycin 0.03 -- 4 0.06 0.25 92.3, *100, 83.3*
Clarithromycin 0.015 -- 2 0.03 0.25 92.3, *100, 83.3*
Azithromycin 0.03 -- 16 0.12 0.25 92.3, *100, 83.3*
Telithromycin 0.008 -- 0.12 0.015 0.015 \-
---------------------- ----------------------------- -------------------------------- ---------------- ------------------------------------------ -------- --------------------
^1^Sinus aspirate ^2^Nasopharyngeal aspirate or swab ^3^No NCCLS interpretive guidelines available or pending
:::
Resistance to most antibiotics was slightly greater in nasopharyngeal specimens than sinus aspirates (Table [2](#T2){ref-type="table"}). Considerable variation in *in vitro*antibiotic activity was apparent between geographical regions as observed in the key examples shown in Table [3](#T3){ref-type="table"}. Insufficient data were available for analysis by country.
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Key example of regional variation in *in vitro*antibiotic activity
:::
***Streptococcus pneumoniae*** ***Haemophilus influenzae***
---------------- -------------------------------- ------------------------------ -------------------------- ----- -------------------------
REGION^1^ N Penicillin susceptible Erythromycin susceptible N Beta-lactamase positive
Eastern Europe 40 70.0% 82.5% 13 0.0%
Far East 185 24.9% 15.7% 96 13.5%
Latin America 108 63.0% 73.1% 31 6.5%
South Africa 61 37.7% 59.0% 34 2.9%
Western Europe 246 83.7% 73.6% 153 11.8%
Grand Total 640 58.0% 55.9% 329 10.3%
^1^Australasia and North America not included due to insufficient data
:::
Combined macrolide resistance and reduced susceptibility to penicillin was present in 200/640 (31.3 %) of *S. pneumoniae*isolates (128 isolates were resistant to penicillin \[MIC \>= 2 mg/L\], 72 intermediate \[MIC 0.12--1 mg/L\]). Of note, 3 isolates of *S. pneumoniae*were non-susceptible to telithromycin (2 isolates intermediate with an MIC of 2 mg/L, 1 isolate resistant with an MIC of 8 mg/L). This represented 0.5% of isolates, a value that is significantly (p \< 0.001) lower than those obtained by erythromycin (44.1%), clarithromycin (44.2%) and cefuroxime (33.9%).
Of the 329 *H. influenzae*isolates, 34 (10.3 %) were positive for β-lactamase production. All isolates of *H. influenzae*were susceptible to amoxycillin-clavulanate and telithromycin with an MIC~90~of 1 and 2 mg/L, respectively. Amoxycillin-clavulanate and telithromycin were more potent and had greater activity than clarithromycin (MIC~90~= 16 mg/L, 87.5% susceptible). This activity was comparable to azithromycin (MIC~90~= 2 mg/l, 100% susceptible).
Although the number of *S. aureus*isolated from the total number of specimens was small (116/1366 isolates), telithromycin was as efficacious as comparators. Of the 116 isolates, 11 were resistant to methicillin (MRSA) and 105 were methicillin susceptible (MSSA). Ninety-nine (94.3%) of the MSSA isolates and 5 of the 11 MRSA isolates were susceptible to telithromycin. Of note, all of the *S. pyogenes*isolates were inhibited by ≤ 1 mg/L telithromycin, despite 17.7% resistance to erythromycin and clarithromycin.
Telithromycin was the most potent antimicrobial against *M. catarrhalis*with an MIC~50~of 0.06 mg/L and MIC~90~of 0.12 mg/L. β-lactamase production was detected in 97.6% of these isolates.
Discussion
==========
The data in this analysis demonstrates that telithromycin has high *in vitro*activity against bacterial pathogens isolated from a large, globally distributed population of patients diagnosed with ABS. Telithromycin was the most active and potent agent against all isolates of the pathogens isolated from patients with ABS with 99.4% of isolates susceptible. Not surprisingly, high levels of penicillin resistance, macrolide resistance, and combined penicillin and macrolides resistance were prevalent in *S. pneumoniae*although prevalence varied widely between geographical regions.
Amoxicillin has been the treatment of choice in ABS because of its general effectiveness, safety, tolerability, low cost and narrow spectrum \[[@B17]\]. The high prevalence of beta-lactamase in *H. influenzae*and *M. catarrhalis*found in the present study demonstrate compromised *in vitro*efficacy of amoxicillin against these isolates. Although the cephalosporins (cefuroxime, cefpodoxime and cefdinir) showed high activity against *H. influenzae*(including beta-lactamase positive strains), resistance to these agents was high in *S. pneumoniae*: \>30% for cefuroxime and cefpodoxime -- cefdinir was not tested against pneumococci in PROTEKT, however susceptibility is usually similar to the other cephalosporins reported here \[[@B30]\].
Similarly, macrolides are prescribed in various countries for ABS and an overall resistance rate for *S. pneumoniae*of 44.1% to erythromycin, azithromycin, and clarithromycin was found. Trimethoprim-sulfamethoxazole activity was low for *S. pneumoniae*(56.3% susceptible) and decreased for *H. influenzae*(84.5% susceptible).
Respiratory fluoroquinolones are recommended second-line treatment options in some countries (references needed to support this statement). However, recent evidence suggests that resistance to fluoroquinolones is rapidly developing in pneumococci and other pathogens (including gram-positive and gram-negative \[[@B31]-[@B34]\]. To preserve the long-term utility of fluoroquinolones, including their use in the treatment of serious non-respiratory infections, it has been recommended that respiratory fluoroquinolones be reserved for treating severe (e.g. hospitalized) community-acquired RTIs only \[[@B35],[@B36]\].
The high prevalence of beta-lactam, macrolide, TMP-SMX resistance demonstrated in the large number of isolates from patients with clinically diagnosed sinusitis in our study demonstrates the need to be exploring new therapeutic options, especially in geographical regions of high prevalence such as the Far East.
The high *in vitro*activity of telithromycin against ABS pathogens reported in this study, regardeless of geographical region, also demonstrates its potential as an empiric therapeutic option for ABS. There are several other reasons to consider this option -- 1) High rates of clinical cure and bacteriological eradication have been demonstrated using telithromycin against sinus isolates of *S. pneumoniae*, *H. influenzae*, *M. catarrhalis*and *S. aureus*\[[@B24]-[@B26]\]. 2) Telithromycin has been shown to have a targeted spectrum of activity against the major bacterial respiratory tract pathogens and has less effect on normal bacterial ecology \[[@B37]-[@B39]\]. 3) The pharmacokinetic profile of oral telithromycin allows it to be prescribed with a dosing regime of 800 mg once daily for 5 days \[[@B40],[@B41]\]. This contrasts favourably with its comparators, where a 10 -- 14 day course with administration either 2 or 3 times daily, depending on the chosen antibacterial, is generally prescribed. Studies have shown that the once daily dosing regime affords greater patient treatment compliance, thereby avoiding clinical failure and the ensuing development of antibacterial resistance \[[@B41]-[@B43]\]. 5) Telithromycin has been shown to have high penetration levels in paranasal sinuses, and it is preferentially absorbed by polymorphonuclear neutrophils (PMNs) within the azurophil granules allowing effective delivery to phagocytized intracellular bacteria \[[@B44],[@B45]\].
Although this study provides valuable information on the overall antimicrobial profile of bacteria causing ABS care should be taken when interpreting data related to specific demographics. A major limitation of this study, inherent to most surveillance studies, is the requirement for collecting centers to fulfill a specified quota of isolates over a defined time period (1 year). If, for instance, 1 center manages to fulfill their quota for *S. pneumoniae*isolates from patients with community-acquired pneumonia, they may then only send *H. influenzae*from patients with ABS to fulfill their quota for this organism. Thus, the potential exists to over or under estimate the prevalence of a species in a particular disease. A further limitation of this study is it is restricted to the major bacterial pathogens causing sinusitis and does not therefore assess anaerobic bacteria, which are also known to be involved in this disease. However, a recent study of sinus puncture specimens demonstrated that telithromycin had good *in vitro*activity against anaerobes involved in sinusitis \[[@B46]\].
The inclusion of nasopharyngeal specimens is a potential limitation of this study and the higher rate of resistance compared to sinus aspirates may indicate some isolates were nasopharyngeal flora rather than pathogens. However, the difference in resistance prevalence between nasopharyngeal specimens and sinus aspirates was not great for any species/antibiotic combination, and assuming the majority of isolates were the responsible pathogen, significant bias of resistance patterns is unlikely. The treatment of ABS is complicated by a difficulty in establishing the causative pathogen(s). Sampling of infected fluid using sinus puncture is a painful and rare procedure \[[@B47]\]. Nasopharyngeal culture is a painless and reliable method that can help identify patients that may benefit from antibacterial therapy \[[@B48]\] and hence, could be useful in determining antibiotic resistance implicated in sinusitis -- particularly *Streptococcus pneumoniae*and *Haemophilus influenzae*. Additionally, there are regional differences in the susceptibility patterns observed and, as therapy is usually empirical, choosing an effective therapy can be challenging \[[@B18],[@B20],[@B49]\].
In summary, the data presented here demonstrates that telithromycin has good *in vitro*activity against *S. pneumoniae*, *H. influenzae, M. catarrhalis*and *S. aureus*respiratory pathogens commonly isolated in ABS. It is as active as or more active than antibacterial agents that are currently used in this clinical setting. The development of resistance will always be a threat to the usefulness of antibacterial compounds, however surveillance studies such as PROTEKT allow the rapid detection and characterization of resistance mechanisms and highlight the need for and examine the *in vitro*efficacy of newer antibacterial agents. Providing careful surveillance for the development of resistance is maintained telithromycin currently offers a useful therapeutic option in the treatment of AS.
Authors\' contributions
=======================
JD, RCa and RCo reviewed the data and provided clinical and microbiological interpretation and discussion. DF and DJF participated in the design of the study, supervised the scientific testing, and provided data analysis, microbiological interpretation and discussion. All authors drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgements
================
We are grateful to our colleagues worldwide for the supply of bacterial isolates as part of the PROTEKT study and the GR Micro PROTEKT team who performed the testing. Aventis is acknowledged for their financial support of the PROTEKT study.
| PubMed Central | 2024-06-05T03:55:46.711463 | 2004-8-2 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509281/",
"journal": "Ann Clin Microbiol Antimicrob. 2004 Aug 2; 3:15",
"authors": [
{
"first": "Joseph",
"last": "Dohar"
},
{
"first": "Rafael",
"last": "Cantón"
},
{
"first": "Robert",
"last": "Cohen"
},
{
"first": "David John",
"last": "Farrell"
},
{
"first": "David",
"last": "Felmingham"
}
]
} |
PMC509282 | Background
==========
Syndrome X comprises a plethora of conditions such as obesity, dyslipidemia, impaired glucose tolerance, insulin resistance and hypertension \[[@B1]\]. It places stress on multiple organ systems and plays a significant role in the development of other related cardiovascular disorders. Western style diet, which contains high levels of fat, is also considered to be one of the main factors in the development of obesity and insulin resistance. The most common reason for the development of hyperinsulinemia (decreased hepatic insulin clearance and/or increased insulin secretion) from insulin resistance is obesity. Excess fat deposits in the white adipose tissue affects insulin mediated glucose metabolism in non adipose tissues, causes disordered insulin response and increases lipid deposition \[[@B2]\]. However, pathophysiologies of vascular complications in syndrome X have not been fully understood. 5-hydroxytryptamine (5-HT) is shown to be related to pathogenesis of vasculopathy in diabetes. Various studies in humans and rabbits have reported increased plasma 5-HT levels and enhanced contraction to 5-HT in diabetes mellitus \[[@B3]-[@B5]\]. Another vasopressor peptide, Angiotensin II (Ang II) is involved in cardiovascular complications of diabetes mellitus. Many have reported that the vascular Renin angiotensin system is one of the key systems in the etiology of vascular alterations in early stages of diabetes. It is already established that 5-HT and Ang II responses are altered in the aortic rings of streptozotocin (STZ) induced diabetic rats \[[@B6],[@B7]\] and high fructose diet fed rats \[[@B8]\]. However, very little information is available in the literature regarding the vascular contractile responses to vasopressor agents such as 5-HT and Ang II in HFD fed rats.
The aim of this study was to elucidate the contractile responses to 5-HT and Ang II in HFD fed rat thoracic aorta which will provide an avenue for further exploratory studies. We have selected HFD fed rat model for our study because it is a useful model of the putative effects of excess fat intake in humans and it represents the major sub type of diabetes mellitus, non insulin dependent diabetes mellitus (NIDDM).
Results
=======
Biochemical measurements
------------------------
HFD fed rats showed significant increase in body weight as compared to NPD fed rats (Table [2](#T2){ref-type="table"}). In addition, the rats fed HFD showed significant elevation in the basal plasma glucose, triglyceride, total cholesterol and insulin levels at the end of four weeks of dietary manipulation as compared to NPD fed control groups (Table [2](#T2){ref-type="table"}). The HFD fed rats exhibited significant elevation in basal fasting glucose and showed significant impairment in glucose tolerance to exogenously administered glucose (Fig [1](#F1){ref-type="fig"}). Estimation of AUC values indicated 28.4 % increase in plasma glucose of HFD fed when compared to NPD fed rats.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Various parameters of NPD and HFD fed rats
:::
**Parameter** **NPD fed** **HFD fed**
---------------------------------- ------------- -------------------
**Body weight (gm)** 259.2 ± 5.3 315.0 ± 6.5\*\*\*
**plasma glucose (mg/dl)** 87.5 ± 2.5 111.0 ± 0.9\*\*\*
**Plasma triglycerides (mg/dl)** 41.3 ± 1.7 89.7 ± 7.2\*\*\*
**Plasma cholesterol (mg/dl)** 48.9 ± 3.7 87.5 ± 2.6\*\*\*
**Plasma Insulin (ng/ml)** 1.90 ± 0.15 3.05 ± 0.21\*
**pD~2~of 5-HT** 5.74 ± 0.09 6.00 ± 0.06
**E~max~of 5-HT** 30.0 ± 1.8 49.0 ± 2.0\*\*
**pD~2~of Ang II** 7.69 ± 0.08 7.31 ± 0.18
**E~max~of Ang II** 25.0 ± 3.4 34.0 ± 3.0\*
Each point is represented as mean ± SEM \*p \< 0.05, \*\*p \< 0.01 and \*\*\*p \< 0.001 Vs NPD fed group
:::
::: {#F1 .fig}
Figure 1
::: {.caption}
######
Effect of HFD on intra peritoneal glucose tolerance test (IPGTT) in rats, as compared to NPD fed group. All values are expressed as mean ± SEM (n = 8) \*p \< 0.05, \*\*p \< 0.01, \*\*\*p \< 0.001 Vs NPD fed group
:::
:::
Vascular Responses
------------------
Cumulative concentration response curves of 5-HT and Ang II for both HFD fed rat thoracic aortae showed an increase in E~max~(Fig [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}) with out any change in pD~2~values when compared to NPD fed rats (Table [2](#T2){ref-type="table"}). There was no significant change in both E~max~and pD~2~values with KCl in both groups (data not shown). The order of potency of agonists in both groups was Ang II\>5-HT\>KCl.
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Cumulative concentration response curve to 5-HT in helically cut aortic strip preparations obtained from NPD fed and HFD fed rats. Each point is represented as mean ± SEM (n = 5) \*p \< 0.05, \*\*p \< 0.01 Vs NPD fed group
:::
:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
Cumulative concentration response curve to Ang II in helically cut aortic strip preparations obtained from NPD fed and HFD fed rats. Each point is represented as mean ± SEM (n = 5) \*p \< 0.05, \*\*p \< 0.01 Vs NPD fed group
:::
:::
Discussion
==========
Obesity is a major risk factor for several metabolic diseases, frequently clustering to form the metabolic syndrome or syndrome X \[[@B9]\]. Obese people have increased incidence of NIDDM with high percentage of mortality and morbidity \[[@B10]\]. Western style diet, which is abundant with calorically dense and saturated fatty foods, is considered to be the main factor in the development of obesity and insulin resistance. Our studies have shown that HFD causes increase in bodyweight when compared to NPD after four weeks of dietary manipulation in rats.
Hyperglycemia is observed in insulin resistance where glucose utilization is reduced. We saw significant elevations in blood glucose levels. Intraperitoneal glucose tolerance tests confirm severe glucose intolerance. Oversupply of dietary lipids causes insulin resistance in rats \[[@B11]\]. Randle glucose fatty acid cycle suggested that the body prefers excess lipid stores to glucose for metabolic oxidation in insulin resistance \[[@B12]\]. Our HFD model also exhibited high plasma triglyceride levels. Hence studies on our experimental model in compliance with Randle et al findings suggested that HFD feeding causes insulin resistance \[[@B13]\].
Insulin resistance with compensatory hyperinsulinemia is a prominent feature of metabolic syndrome. The most common reason for the development of hyperinsulinemia in insulin resistance is obesity. It stands as one of the major cardiovascular risk factors in patients with obesity. The present study on HFD rats demonstrated higher plasma insulin levels than control values. This marked hyperinsulinemia could be due to a combination of increased β-cell mass and decreased insulin clearance, as well as failure of insulin to suppress hepatic gluconeogenesis \[[@B14]\].
Elevated cholesterol is also observed in insulin resistant individuals. For this reason we measured plasma cholesterol levels which were found to be more than normal values. Previous studies have reported a down regulation of LDL receptors and associated decrease in LDL clearance, increased total cholesterol levels \[[@B15]\].
According to National Cholesterol Education Program\'s Adult Treatment Panel III (Third report) easily measured clinical findings for syndrome X includes increased abdominal circumference, elevated triglycerides, low high-density lipoprotein-cholesterol, and elevated fasting blood glucose and/or elevated blood pressure. Three of these five are required for diagnosis. Our study demonstrated three of the clinical parameters indicating conditions of syndrome X in HFD fed rats \[[@B16]\]. Insulin resistance along with other conditions of syndrome X might induce hypertension by a host of mechanisms involving insulin itself, increased sodium reabsorption and/or enhanced intra cellular concentration of free calcium in vascular smooth muscle \[[@B17]\]. Although the etiology of vascular disorders in metabolic syndrome has not completely been revealed, it is suggested that alterations in the reactivity of blood vessels to neurotransmitters and circulating hormones are responsible for the functional abnormalities of blood vessels.
In order to elaborate the pathways that connect syndrome X to hypertension we have studied the contractile responses to 5-HT and Ang II in both HFD and NPD fed rat thoracic aortae. Previously we have demonstrated increased contractile responses with synthetic alpha adrenoceptor agonist, phenylephrine, in HFD fed rat thoracic aorta \[[@B18]\]. This enabled us to explore the role of these endogenous mediators in the same animal model.
The present vascular studies demonstrated that the magnitude of responses to 5-HT and Ang II was significantly enhanced in HFD fed animals without change in pD~2~value. Endothelial denudation obviates any related mechanisms such as impairment of NO release, increased destruction of EDRF and substrate availability for the production of EDRF. Hence, the probable reasons for these enhanced 5-HT and Ang II responses may be due to receptor mediated or non-receptor mediated pathways.
The role of non-receptor mediated contraction can be ruled out for there was no change in contractile response to KCl. Vascular studies have also shown functional evidence that hypertension developed in HFD fed rats may be associated with enhanced vasoreactivity to various vasoconstrictor agents. Further the enhanced responses to 5-HT in HFD fed rats could be due to increased PKC as previously reported with STZ and alloxan (ALL) induced diabetic animals \[[@B19]\]. Increased contractions to 5-HT can also be related to 5-HT~2A~upregulation as observed in spontaneously hypertensive rats or due to serotonin acting through alpha adrenoceptors \[[@B20]\].
Increased Ang II responses in HFD fed rats are may be due to upregulation of Ang II receptors as observed in hyperinsulinemia or via amplified secondary messenger systems \[[@B21]\]. A proposed scheme of events is given in Fig [4](#F4){ref-type="fig"}.
::: {#F4 .fig}
Figure 4
::: {.caption}
######
Proposed events underlying syndrome X in HFD rats. HFD leads to condition, in rats, similar to syndrome X. This includes obesity, insulin resistance, hyperglycemia and dyslipidemia which all are interrelated. These events are the initial steps in the cascade towards hypertension. This could be mediated via increased contractile responses to various endogenous mediators such as 5-HT and Ang II.
:::
:::
In summary, the present study has shown that HFD feeding in rats produces conditions similar to syndrome X. Increased vasocontractile responses observed in the model are not only mediated via alpha adrenoceptors but also due to 5-HT and Ang II (see Figure [4](#F4){ref-type="fig"}).
More robust studies on the secondary messenger systems of 5-HT and Ang II will provide valuable insights into the mechanisms underlying increased vascular contractility in insulin resistance.
Materials and Methods
=====================
Tissue Preparation
------------------
Male Sprague-Dawley rats (central animal facility, National Institute of Pharmaceutical Education and Research (NIPER), India), 160--200 g, were kept in controlled environmental conditions with room temperature 22 ± 2°C, humidity 55 ± 5% and 12-h light/dark cycles. All the animals had free access to food and water. The rats were divided into two dietary groups and fed with standard rat normal pellet diet (NPD) (3.8 kcal/g, carbohydrate 67%, protein 21%, fat 12% kcal) and HFD (5.3 kcal/g, carbohydrate 17%, protein 25%, fat 58% kcal). Composition of HFD is described in Table [1](#T1){ref-type="table"}.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Composition of HFD
:::
(g)
-------------------------------- -----
Powdered pellet diet 364
Lard 310
Casein 250
Cholesterol 10
DL-Methionine 3
Yee-sac powder 1
Vitamin and mineral mix powder 60
Sodium chloride 2
:::
### Biochemical measurements
Blood samples from the retro orbital plexus of anaesthetized rats (Pentobarbitone 45 mg/kg, i.p.,) were collected into the heparinized tubes and immediately centrifuged at 5000 rpm for the separation of plasma. Plasma was stored at -20°C until assayed. The plasma was used for the estimation of glucose (Qualigens, Mumbai, India), triglycerides, and cholesterol (Chema diagnostica, Jesi, Italy) by commercial kits. Plasma insulin was determined by radioimmuno assay using rat insulin as standard (Linco research, St. Charles, MO, USA)
### Intra peritoneal glucose tolerance test (IPGTT)
Glucose tolerance tests were carried out after four weeks of feeding of both NPD and HFD. After an overnight fast, blood samples were collected from the retro orbital plexus. Glucose levels were measured at time zero (0 min) and glucose was injected into the rats (2 g/Kg/4 ml, i.p.,). Additional blood samples were taken at 15, 30, 60 and 120 min. following the glucose load. Plasma glucose levels were measured by the glucose oxidase reaction (GOD/POD) using commercial kit (Qualigens, Mumbai, India). Area under the curve (AUC) was calculated for both NPD and HFD fed rats.
### Vascular Studies
After 4 weeks of feeding, rats were sacrificed by cervical dislocation. The section of the aorta from between the aortic arch and the diaphragm was removed from the euthanized rats and placed in oxygenated, modified Krebs-Henseleit solution (KHS; in mM: 118 NaCl, 4.7 KCl, 25 NaHCO~3~, 2.6 CaCl~2~2H~2~O 1.2 NaH~2~PO~4~, 1.2 MgCl~2~6H~2~O, 5.5 glucose). With the help of a steel rod, aortic endothelium was deliberately denuded. The aorta was cut into helical strips 3 mm wide, 20 mm long and then placed in a well-oxygenated (95% O~2~-5% CO~2~) bath of 10-ml KHS with one end connected to a tissue holder and the other to an isotonic transducer (Bio Devices, Ambala, India). The tissue was equilibrated for 60 min under a resting tension of 1.0 g. At the beginning of each experiment, aortic strips were primed with depolarizing concentration (90 mM) potassium chloride (KCl). After the equilibration period, contractile responses to various concentrations of 5-HT (10 nM-30 μM) and Ang II (1 nM-300 nM) were recorded.
Data analysis
-------------
Contraction responses are expressed as percentage. For each contractile agent, both the maximal contraction (E~max~) and the concentration necessary to produce 50% of its maximal response (EC~50~) were determined. The EC~50~values were converted to the negative logarithms and expressed as pD~2~. Results were shown as mean ± SEM; n refers to the number of animals from which vessels were taken. Agonist potencies and maximal effects were compared by student\'s *t*test by using statistical software (GraphPad Prism 3.01). Values were considered significantly different at p \< 0.05.
Drugs and solutions
-------------------
The following drugs were used in this study: 5-HT (RBI, USA), Ang II (Bachem, Switzerland), All drugs were dissolved in KHS. Drugs were added to the organ chambers in volumes not greater than 0.2 ml.
List of abbreviations
=====================
HFD -- High fat diet
NPD -- Normal pellet diet
NIDDM -- Non insulin dependent diabetes mellitus
STZ -- Streptozotocin
5-HT -- 5-Hydroxytryptamine
Ang II -- Angiotensin II
KHS -- Krebs-Henseleit solution
GOD/POD -- Glucose oxidase/peroxidase
AUC -- area under the curve
E~max~-- Maximal response
EC~50~-- Concentration required producing 50% of maximal response
pD~2~-- -log EC~50~
Authors\' contributions
=======================
SG carried out the all experimentations. PR has conceived the study and participated in its design and coordination. Authors read and approved the final manuscript.
Acknowledgements
================
This work was supported by a grant from NIPER. The authors thank Dr. Srinivasan for help in Radioimmunoassay of insulin. We thank Ms. Deepthi Nimmagadda for help in preparation of the manuscript.
| PubMed Central | 2024-06-05T03:55:46.714270 | 2004-8-2 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509282/",
"journal": "Lipids Health Dis. 2004 Aug 2; 3:19",
"authors": [
{
"first": "Srinivas",
"last": "Ghatta"
},
{
"first": "Poduri",
"last": "Ramarao"
}
]
} |
PMC509283 | Introduction
============
While many women complain of worsening acne and water retention during their menstrual cycle, there exist a small number in whom the menstrual cycle is associated with a variety of other skin manifestations such as urticaria, eczema, folliculitis, and angioedema. This condition is known as autoimmune progesterone dermatitis (APD) due to the fact that progesterone is most frequently identified as the etiologic agent. In women with irregular menses, the diagnosis may remain elusive for years. We present a case of APD, and review the current literature in regards to clinical features, pathogenesis, diagnosis, and treatment options.
Case
====
A 33y/o woman with a history of endometriosis presented with complaints of chronic urticaria. The patient noted that the urticaria began at the age of 12, and did not seem to have any obvious trigger. Each individual lesion would last from 12--24 hours, and the entire episode would last 5--10 days. Lesions would usually start on the chest and then spread over the entire body. She had seen multiple physicians, including allergists and dermatologists, and had been treated with a variety of medications including certirizine, desloratadine, hydroxyzine, montelukast, ranitidine, and diphenhydramine without relief. Prednisone at high doses would provide temporary relief, and she had required multiple courses of prednisone over the past 20 years. In addition, she complained of occasional angioedema, usually at the same time as the hives but occasionally occurring when hives were not present. The patient also had acne that had been very difficult to control since her teenage years, and she noted that the acne would also respond to prednisone.
Multiple lab tests over the years had been unremarkable. These included SSA/SSB, anti-Smith, ACE level, C3/C4, hepatitis B, ANA, anti double-stranded DNA, immunoglobulins, SPEP, C1 esterase inhibitor level and function, chemistry panel, liver tests, TSH, T4, thyroid antibodies, rheumatoid factor, ESR, and CBC. Skin biopsy of a lesion had been read as \"chronic urticaria\".
Upon further questioning, it was learned that due to the patient\'s endometriosis, she had very irregular menstrual cycles in terms of length and timing. It was determined that the hives and/or angioedema would begin approximately 4 days prior to the onset of menses, and would last about 2 days into menses. The symptoms would not occur with every episode of menses. The patient\'s acne would often occur on her face during the urticarial episodes. Of note, the patient had 2 children, and during each pregnancy her hives, acne, and angioedema had been markedly improved. Because of her endometriosis, she had been started on Depo-Provera (medroxyprogesterone acetate) in her twenties. After 1 injection, she developed severe hives that lasted over 2 months and required multiple courses of prednisone. Due to the urticaria, Depo-Provera was discontinued after one injection. As the patient complained of acne, Ortho Tri-Cyclen (norgestimate/ethinyl estradiol) was initiated by her dermatologist. This treatment modality did not have any effect on the urticaria, angioedema, or acne.
The patient was evaluated in our clinic. Physical examination was essentially normal, and no hives were noted. Allergy skin testing was performed with progesterone 50 mg/mL in normal saline. Prick test was normal, but a full strength intradermal test revealed a 7 mm wheal with erythema. The histamine control showed a 9 mm wheal with erythema, and saline control was negative for wheal and erythema. Two healthy controls also underwent intradermal testing to exclude irritant reaction, and were found to be negative
Based on the above results, the patient was diagnosed with autoimmune progesterone dermatitis. The patient was started on a GnRH agonist (nafarelin acetate nasal spray, 200 mcg twice a day). Within one month, she noted dramatic improvement in her urticaria and angioedema. Acne was still occasionally present, but much improved. She did complain of mild hot flashes, but felt these were tolerable.
Discussion
==========
In a small group of women, the menstrual cycle has been associated with a spectrum of dermatologic diseases including eczema, erythema multiforme, stomatitis, papulopustular lesions, folliculitis, angioedema, urticaria, and others (Table [1](#T1){ref-type="table"}) \[[@B1]-[@B8]\]. As progesterone sensitivity has been the most commonly identified cause, dermatologic diseases associated with the menstrual cycle have been labeled autoimmune progesterone dermatitis (APD) \[[@B4]\]. The first documented case of APD was in 1921, in which a patient\'s premenstrual serum caused acute urticarial lesions. In addition, it was shown that the patient\'s premenstrual serum could be used to desensitize and improve her symptoms \[[@B9]\]. Since 1921, approximately 50 cases of APD have been published in the medical literature.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Dermatologic manifestations of autoimmune progesterone dermatitis
:::
-------------------------------------------
\- Urticaria
\- Angioedema
\- Eczema
\- Erythema multiforme
\- Stomatitis
\- Folliculitis
\- Papulopustular/papulovesicular lesions
\- Stephens-Johnson syndrome
\- Vesiculobullous reactions
\- Dermatitis herpetiformis-like rash
\- Mucosal lesions
-------------------------------------------
:::
Clinical Features
-----------------
The clinical symptoms of APD (eczema, urticaria, angioedema, etc.) usually begin 3--10 days prior to the onset of menstrual flow, and end 1--2 days into menses. Severity of symptoms can vary from nearly undetectable to anaphylactic in nature, and symptoms can be progressive \[[@B10],[@B11]\]. There are no specific histological features on biopsy in APD \[[@B12]\]. The age of onset is variable, with the earliest age reported at menarche \[[@B13]\]. Some studies have noted that a majority of patients had taken an oral contraceptive (OCP) prior to the onset of APD \[[@B14]\], but multiple cases exist in which women have never been exposed to exogenous progesterone \[[@B15]-[@B17]\].
The symptoms of APD correlate with progesterone levels during the luteal phase of the menstrual cycle. Progesterone begins to rise 14 days prior to the onset of menses, peaks 7 days prior to menses, and returns to a low baseline level 1--2 days after menses begins. In studies where an etiologic agent has been sought, progesterone has been found most frequently. However, estrogen, prostacyclin, and gonadotropin levels have correlated with symptoms in some cases \[[@B18]-[@B21]\].
Symptoms may first appear, improve, or worsen during pregnancy and the peripartum period \[[@B2],[@B22]-[@B24]\]. In addition, APD during pregnancy has been associated with spontaneous abortions \[[@B2],[@B25]\]. Pregnancy is associated with an increase of maternal progesterone levels, which may explain the initiation or worsening of symptoms. In regards to an improvement of symptoms during pregnancy, a number of theories have emerged. Explanations include a slow rise of progesterone during pregnancy that acts as a method of desensitization, a decrease in maternal immune response during pregnancy, or an increased production of anti-inflammatory glucocorticoids \[[@B13],[@B25],[@B26]\].
Pathogenesis
------------
The exact pathogenesis of APD is unknown. If exogenous progesterones (i.e. OCPs) are initially used, it is conceivable that uptake by antigen presenting cells and presentation to T~H~2 cells could result in subsequent IgE synthesis; however this mechanism would not explain the pathogenesis in patients such as ours who have the onset of APD prior to exogenous progesterone exposure. Some authors have suggested that hydrocortisone or 17-α-hydroxyprogesterone have cross-sensitivity with progesterone and may cause initial sensitization, but this has not been observed in all studies \[[@B27],[@B28]\].
To further delineate the pathogenesis, antibodies against progesterone have been investigated. Using immunofluorescent techniques and basophil degranulation tests, studies have found that such antibodies do exist in certain patients with APD \[[@B1],[@B13],[@B29]\]. However, negative results looking for antibodies have also been reported \[[@B24]\]. In addition, skin test results with progesterone have shown immediate reactions (within 30 minutes), delayed reactions (24--48 hours later), and reactions with features of both immediate and delayed features \[[@B13],[@B14],[@B30],[@B31]\]. This presumably indicates both type I and type IV hypersensitivity reactions. Progesterone has also been reported to have a direct histamine releasing effect on mast cells, yet very little research has been done to support this hypothesis \[[@B32]\]. Additionally, one study found an in vitro increase of an interferon-γ release assay, possibly implying a role for T~H~1-type cytokines in APD \[[@B33]\].
Eosinophils may also be involved in the pathogenesis of APD. Eosinophilia has been correlated with cutaneous symptoms in some cases, and studies have found a decrease in total eosinophil count after therapy \[[@B13],[@B29],[@B34]\]. Whether increased eosinophils are a response to cytokines from lymphocytes or play a primary mechanistic role in APD remains to be determined.
Diagnosis
---------
The diagnosis of APD requires an appropriate clinical history accompanied by an intradermal injection test with progesterone. An aqueous suspension or aqueous alcohol solution of progesterone is the preferable vehicle of testing as progesterone in oil can cause an irritant reaction \[[@B35]\], though many published case reports have used progesterone in oil for testing. Various authors have advocated different amounts of progesterone or medroxyprogesterone to be used for testing \[[@B12],[@B33],[@B36]\]. As had been done in some prior studies, the patient presented here was tested with progesterone in aqueous solution at a concentration of 50 mg/mL.
As mentioned above, APD may be due to an immediate or delayed hypersensitivity reaction. Therefore, intradermal testing may not become positive until 24--48 hours later \[[@B14],[@B24]\]. In addition, some authors have advocated patch testing with progesterone to further evaluate for a hypersensitivity reaction \[[@B33]\]. Of note, intradermal testing has been negative in some patients with typical clinical symptoms of APD and who improved after APD treatment \[[@B2],[@B3],[@B24]\].
Some authors have recommended further tests to evaluate the immunologic evidence in APD. These include circulating antibodies to progesterone, basophil granulation tests, direct and indirect immunofluorescence to luteinizing cells of the corpus luteum, in vitro interferon-γ release, and circulating antibodies to 17-α-hydroxyprogesterone \[[@B1],[@B7],[@B13],[@B29],[@B33],[@B36]\]. However, most case reports in the medical literature do not routinely check for serologic evidence of APD, and when checked these markers have not always been found to be reliable. This is most likely due to the fact that, as mentioned above, the pathogenesis of APD is incompletely understood.
Treatment
---------
Autoimmune progesterone dermatitis is usually resistant to conventional therapy such as antihistamines. The use of systemic glucocorticoids, usually in high doses, has been reported to control the cutaneous lesions of APD is some studies, but not in others \[[@B3],[@B10],[@B37]\]. Early reports of APD describe attempts of progesterone desensitization, and some authors even attempted injections derived from the corpus luteum \[[@B18],[@B24],[@B38]\]. However, results were usually temporary, and such methods of treatment have now fallen out of favor.
Current therapeutic modalities often attempt to inhibit the secretion of endogenous progesterone by the suppression of ovulation. Table [2](#T2){ref-type="table"} lists some of the pharmacologic strategies used in APD. Oral contraceptives (OCPs) are often tried as initial therapy, but have had limited success, possibly due to the fact that virtually all OCPs have a progesterone component. Conjugated estrogens have also been used in the treatment of APD. These did show improvement in many of the patients, but often required high doses \[[@B2],[@B16],[@B22]\]. However, due to the increased risk of endometrial carcinoma with unopposed conjugated estrogens, this treatment is not commonly used today \[[@B39]\].
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Treatment options used in autoimmune progesterone dermatitis
:::
**Treatment Option** **Advantages** **Disadvantages**
---------------------------- ------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------
Oral Contraceptives (OCPs) \- Usually tried as initial therapy \- Limited success due to the progesterone component of OCPs
\- Fewer side effects than other most other therapies
Antihistamines \- Well tolerated, few side effects \- Rarely effective as monotherapy
\- Does not address underlying mechanism
Conjugated Estrogens \- Avoids progesterone component of OCPs \- Increased risk of endometrial cancer, not commonly used today
\- Often require high doses
Glucocorticoids \- Able to suppress multiple components of the immune system \- Usually not effective alone
\- Can be combined with other therapies \- Often require high doses
GnRH Agonists \- Often used if OCPs and glucocorticoids are not effective \- Can cause symptoms of estrogen deficiency (hot flashes, decreased bone mineral density)
Alkaylated Steroids \- Can be combined with low dose steroids \- Can cause symptoms of excess androgens (facial hair, hepatic dysfunction, mood disorders)
\- Interferes with gonadal hormone receptors
Tamoxifen \- Has been used successfully in patients unresponsive to conjugated estrogen \- Can cause symptoms of estrogen deficiency
\- Increased risk of venous thrombosis and cataract formation
Bilateral oopherectomy \- Definitive treatment, used if medical options unsuccessful \- Surgical procedure, associated morbidity
\- Symptoms of estrogen deficiency
:::
Various other therapy modalities are currently used in APD, and there is no clear treatment of choice. GnRH agonists, such as buserelin and triptorelin, have been used to induce remission of symptoms by causing ovarian suppression \[[@B7],[@B11],[@B15]\]. However, side effects include symptoms of estrogen deficiency (hot flashes, vaginal dryness, decreased bone mineral density), and estrogen supplementation may be needed \[[@B40]\]. Alkaylated steroids such as stanozol have been used to successfully suppress ovulation, sometimes in combination with chronic low doses of corticosteroids \[[@B37]\]. Side effects of alkaylated steroids include abnormal facial or body hair growth, hepatic dysfunction, and mood disorders, any of which may limit their use. To decrease the risk of side effects, some authors have recommended using the alkaylated steroid only in the perimenstrual period \[[@B37]\]. Another therapeutic option used in APD has been the antiestrogen tamoxifen, which also can suppress ovulation \[[@B3],[@B5]\]. As with GnRH agonists, patients on tamoxifen may experience symptoms of estrogen deficiency. In addition, tamoxifen has been associated with an increased risk of venous thrombosis and cataract formation. In some patients with unremitting symptoms of APD, bilateral oopherectomy has been required \[[@B10],[@B15],[@B24]\]. While this definitive treatment has been successful in controlling symptoms, today it is rarely used before all medical options have been exhausted.
Conclusion
==========
Autoimmune progesterone dermatitis is a condition seen in a small number of women who present with eczema, erythema multiforme, stomatitis, papulopustular lesions, folliculitis, angioedema, urticaria, and other skin manifestations in relation to the menstrual cycle. It is usually seen 3--10 days prior to the onset of menstrual flow, but may be difficult to recognize in women with irregular menses. The exact pathogenesis is unknown, and is thought to involve a hypersensitivity reaction to progesterone. The diagnosis of APD is made by an appropriate clinical history accompanied by an intradermal injection test with progesterone. Current treatment modalities often attempt to inhibit the secretion of endogenous progesterone, but may be unsuccessful. More research is needed into the pathogenesis of APD to most appropriately care for these patients.
| PubMed Central | 2024-06-05T03:55:46.715602 | 2004-8-2 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509283/",
"journal": "Clin Mol Allergy. 2004 Aug 2; 2:10",
"authors": [
{
"first": "Alan P",
"last": "Baptist"
},
{
"first": "James L",
"last": "Baldwin"
}
]
} |
PMC509284 | Background
==========
Acute chest pain is a common reason for emergency hospital attendance and admission. Patients with chest pain that remains undiagnosed after clinical assessment, ECG and chest radiograph pose a particular problem. They carry a low, but important risk of an acute coronary syndrome \[[@B1]\]. The potentially life-threatening nature of this diagnosis means that a cautious approach is often taken, with many patients being admitted to hospital for observation and investigation \[[@B2]\]. Yet most patients with undifferentiated chest pain do not have a coronary syndrome, whereas anxiety and psychological morbidity are common \[[@B3]-[@B5]\] and appear to be associated with impaired quality of life \[[@B6]\]. It is possible that anxiety could be influenced by the investigation and management of chest pain. If this is so, then decision analysis modelling suggests that the potential health gains that could be achieved by reducing anxiety and improving quality of life among the majority of patients who do not have an acute coronary syndrome substantially outweigh the potential health gains from detecting and treating acute coronary syndromes \[[@B7]\].
The chest pain unit (CPU) was developed to provide rapid and accurate diagnosis for patients presenting with acute undifferentiated chest pain \[[@B8]\]. Patients receive up to six hours of observation and biochemical testing followed by an exercise treadmill test. If these tests are positive then they are admitted to hospital with a clear diagnosis, if negative they are discharged home. Evaluation of CPU care has focussed upon cardiac events, process measures and economic measures \[[@B9]\]. There is some evidence that CPU care is associated with improved diagnostic certainty \[[@B10]\] and patient satisfaction \[[@B11]\], but no data to compare psychological morbidity and quality of life after CPU and routine care, despite substantial data to suggest that this is an important problem for patients \[[@B3]-[@B5],[@B12]-[@B14]\].
The ESCAPE (effectiveness and safety of chest pain evaluation to prevent emergency admission) trial was a randomised controlled trial and economic evaluation of CPU versus routine care that showed that CPU care was associated with reduced hospital admission \[[@B15]\], improved health utility \[[@B15]\] and improved patient satisfaction \[[@B16]\], and was likely to be considered cost-effective \[[@B15]\]. This paper reports quality of life and psychological measures from the ESCAPE trial. We aimed to measure the effect of CPU care upon anxiety, depression, and health related quality of life, and to determine whether CPU care reduced subsequent symptoms of chest pain.
Methods
=======
The Northern General Hospital Emergency Department provides adult emergency care to the 530,000 population of Sheffield, United Kingdom. In 1999 a CPU was established in the emergency department, staffed by three specialist chest pain nurses, and able to accommodate up to six patients with acute undifferentiated pain. Patients were selected using validated clinical predictors and received two to six hours of observation and biochemical cardiac testing, followed by, where appropriate, an exercise treadmill test. Full details of the CPU protocol have been published \[[@B17]\]. Routine care, prior to development of the CPU, consisted of assessment by a doctor who had access to biochemical cardiac tests, but not observation facilities or exercise treadmill testing.
From 5^th^February 2001 to 5^th^May 2002 the CPU was subject to a cluster randomised controlled trial. Days of the week (N = 442) were randomised to CPU or routine care in equal numbers. All patients attending with acute chest pain were screened for eligibility in the trial. Patients were excluded if they had ECG changes diagnostic for an acute coronary syndrome, clinically obvious unstable angina, co-morbidity or alternative pathology requiring hospital admission (e.g. suspected pulmonary embolus), negligible risk of acute coronary syndrome (e.g. age less than 25 years), or if they were unable to consent to participation. Written, informed consent was requested and patients who agreed to participate were followed up in a review clinic at two days, and by postal questionnaire at one month. The study protocol was approved by the North Sheffield Research Ethics Committee. Full details of the ESCAPE trial have been published \[[@B15]\].
Health related quality of life was measured using the SF-36 questionnaire \[[@B18]\]. Anxiety and depression were measured using the Hospital Anxiety Depression Scale (HADS) \[[@B19]\]. Both are widely used, validated, self-completed questionnaires. Both were administered at two days and one month. At two days patients were handed the questionnaires in the review clinic and asked to complete it in their own time and return it to the Medical Care Research Unit. No reminder was sent to non-responders to this questionnaire. Further questionnaires were mailed at one month with one re-mailing for non-responders.
A brief additional questionnaire was sent at one month that was designed specifically for the study. This predominantly asked questions about health service use for the economic evaluation, but also asked participants whether they had suffered any further chest pain. If they responded that they had, they were asked to score the severity of the chest pain on a 100 mm visual analogue scale. A further question asked whether the patient had taken time off work since their hospital attendance.
The sample size estimate of 988 was based upon the primary outcome measure, the proportion of patients admitted to hospital. Assuming a response rate of 65% to the questionnaires, this sample size would provide 80% power to detect an effect size of 0.25 for these outcomes (alpha = 0.05). Using standard deviations derived from a two-week pilot study, this effect size equates to 1.1 points on the HADS anxiety or depression scores, 11.5 points of the SF-36 physical or emotional role dimensions, and 6 points on the other SF-36 dimensions. Data was analysed using Stata statistical software (version 8.0). Multi-level random effects modelling was used with day of week as a random effect to adjust for clustering by day of week. For the principal analysis no adjustment for confounding was made. For secondary analysis age, gender and past history of coronary heart disease were included as covariates (determined a priori to be important potential confounders), along with any variable that showed significant (p \< 0.05) baseline imbalance between the study groups.
Results
=======
During the 442-day study period there were 6957 attendances with chest pain or a related complaint. Of these, 764 (11.0%) had ECG changes diagnostic for an acute coronary syndrome, 2402 (34.5%) had clinically obvious unstable angina, 869 (12.5%) had co-morbidity or alternative pathology requiring hospital admission, 1291 (18.6%) had negligible risk of acute coronary syndrome, and 513 patients (7.4%) were unable to participate in the trial or provide consent. The remaining 1118 patients (16.1%) were asked to participate in the trial and 972 agreed (86.9%). Response rates were: 717 (73.8%) to the initial questionnaire and 679 (69.9%) to the one-month questionnaire. The CONSORT diagram and full details of exclusions have been published elsewhere \[[@B15]\]. Baseline characteristics of the study groups are shown in Table [1](#T1){ref-type="table"}. Source of referral, smoking status, and ECG at presentation showed significant baseline imbalance. Hence secondary analyses adjusted for these covariates, along with age, gender and past history of coronary heart disease.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Baseline characteristics of the study groups
:::
**CPU care** **Routine care**
------------------------- -------------- ------------------
Age (years) 49.4 49.6
Male sex (%) 304 (63.5%) 318 (64.5%)
Known CHD (%) 16 (3.3%) 27 (5.5%)
Hypertension (%) 127 (26.5%) 120 (24.3%)
Diabetes (%) 17 (3.5%) 29 (5.9%)
Hyperlipidaemia (%) 58 (12.1%) 70 (14.2%)
Smoker (%) 169 (35.3%) 143 (29.0%)
Family history (%) 189 (39.5%) 200 (40.6%)
**Pain nature**
Indigestion / burning 60 (12.5%) 56 (11.4%)
Stabbing / sharp 116 (24.2%) 113 (22.9%)
Aching / dull / heavy 175 (36.5%) 181 (36.7%)
Gripping / crushing 66 (13.8%) 59 (12.0%)
Other 57 (11.9%) 71 (14.4%)
**Pain site**
Central 317 (66.2%) 335 (68.0%)
Left chest 129 (26.9%) 125 (25.4%)
Right chest 19 (4.0%) 16 (3.2%)
Other 8 (1.7%) 8 (1.6%)
**Pain radiation**
None 183 (38.2%) 189 (38.3%)
Left arm 118 (24.6%) 142 (28.8%)
Right arm 31 (6.5%) 26 (5.3%)
Neck 22 (4.6%) 22 (4.5%)
Jaw 15 (3.1%) 13 (2.6%)
Back 70 (14.6%) 53 (10.8%)
Other 27 (5.6%) 30 (6.1%)
**Pain duration**
Continuous pain 312 (65.1%) 341 (69.2%)
Intermittent pain 93 (19.4%) 95 (19.3%)
**Other symptoms**
Nausea 129 (26.9%) 161 (32.7%)
Vomiting 25 (5.2%) 31 (6.3%)
Dyspnoea 185 (38.6%) 202 (41.0%)
Sweating 192 (40.1%) 210 (42.6%)
**ECG at presentation**
ECG normal (%) 412 (89.0%) 382 (82.2%)
ECG non-specific (%) 38 (8.2%) 64 (13.8%)
ECG old change (%) 13 (2.8%) 19 (4.1%)
**Source of referral**
GP referral 138 (28.8%) 116 (23.5%)
Self referred 173 (36.1%) 155 (31.4%)
999 145 (30.3%) 189 (38.3%)
Other 23 (4.8%) 33 (6.7%)
:::
Table [2](#T2){ref-type="table"} shows the final diagnosis recorded in the case notes, after hospital attendance and admission, of the most senior clinician to care for the patient. Those receiving routine care were more likely to have received a diagnosis of angina, whereas those receiving CPU care were more likely to have received a non-specific or non-cardiac diagnosis.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Diagnostic impression after initial hospital attendance
:::
**Diagnosis** **CPU care** **Routine care**
------------------------- -------------- ------------------
Non-specific chest pain 144 (30.1%) 125 (25.4%)
Anxiety 13 (2.7%) 21 (4.3%)
Angina 63 (13.2%) 123 (24.9%)
Myocardial infarction 28 (5.8%) 27 (5.5%)
Gastro-oesophageal pain 74 (15.4%) 60 (12.2%)
Musculo-skeletal pain 122 (25.5%) 106 (21.5%)
Other diagnosis 26 (5.4%) 18 (3.7%)
Not recorded 9 (1.9%) 13 (2.6%)
P \< 0.0001 for the difference in distribution across the categories
:::
Table [3](#T3){ref-type="table"} shows the mean SF-36 scores for both groups at two days, with the adjusted difference, 95% confidence interval, p-value and intraclass correlation coefficient. Table [4](#T4){ref-type="table"} shows these estimates at one month. At two days, CPU care was associated with significant improvements in physical functioning, vitality and general health. At one month, CPU care was associated with significant improvements in all dimensions of quality of life, except the emotional role dimension.
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Mean SF-36 scores at two days
:::
**N (% completed)** **CPU care** **Routine care** **Difference** **95% CI** **P-value** ***ρ***
---------------------- --------------------- -------------- ------------------ ---------------- -------------- ------------- ---------
Physical functioning 694 (96.7%) 74.8 69.7 5.1 1.1 to 9.0 0.012 0.002
4.2 0.4 to 7.9 0.029
Social functioning 703 (98.0%) 72.2 69.8 2.4 -1.7 to 6.6 0.252 0
1.5 -2.7 to 5.6 0.49
Role-physical 684 (95.4%) 50.4 46.0 4.4 -2.2 to 11.0 0.191 0.028
3.3 -3.3 to 10.0 0.326
Role-emotional 685 (95.5%) 64.7 59.5 5.2 -1.2 to 11.6 0.113 0
5.1 -1.2 to 11.4 0.111
Mental health 700 (97.6%) 66.9 64.7 2.2 -0.9 to 5.3 0.158 0
2.3 -0.7 to 5.4 0.132
Vitality 697 (97.2%) 52.3 47.6 4.6 1.3 to 8.0 0.007 0
4.6 1.3 to 8.0 0.007
Pain index 701 (97.7%) 50.8 49.0 1.8 -1.9 to 5.5 0.351 0
2.0 -1.7 to 5.7 0.284
General health 688 (96.0%) 60.3 54.5 5.7 2.3 to 9.2 0.001 0
5.4 2.0 to 8.8 0.002
Upper row shows unadjusted analysis (primary analysis) Lower row shows adjusted analysis (secondary analysis) *ρ*= Intraclass correlation coefficient. This provides a measure of the amount of clustering of each outcome by the unit of randomisation (day).
:::
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
Mean SF-36 scores at one month
:::
**N (% completed)** **CPU care** **Routine care** **Difference** **95% CI** **P-value** ***ρ***
---------------------- --------------------- -------------- ------------------ ---------------- -------------- ------------- ---------
Physical functioning 654 (96.3%) 74.1 66.2 7.8 3.8 to 11.9 \<0.001 0.025
7.6 3.6 to 11.5 \<0.001
Social functioning 654 (96.3%) 74.6 67.0 7.6 3.2 to 12.0 0.001 0
6.8 2.4 to 11.2 0.002
Role-physical 638 (94.0%) 54.1 46.0 8.2 1.3 to 15.0 0.02 0
7.0 0.4 to 13.6 0.039
Role-emotional 630 (92.8%) 63.9 60.2 3.7 -3.0 to 10.5 0.281 0
3.9 -2.8 to 10.5 0.256
Mental health 653 (96.2%) 69.1 64.4 4.7 1.3 to 8.2 0.007 0
5.2 1.9 to 8.6 0.002
Vitality 649 (95.6%) 52.6 47.1 5.5 1.8 to 9.2 0.003 0
5.8 2.2 to 9.3 0.002
Pain index 655 (96.5%) 66.4 62.0 4.4 0.2 to 8.5 0.04 0
4.3 0.2 to 8.3 0.041
General health 651 (95.9%) 59.7 51.7 8.0 4.6 to 11.5 \<0.001 0
8.1 4.6 to 11.5 \<0.001
Upper row shows unadjusted analysis (primary analysis) Lower row shows adjusted analysis (secondary analysis) *ρ*= Intraclass correlation coefficient
:::
Table [5](#T5){ref-type="table"} shows the summary HADS data at two days and one month. CPU care was associated with lower depression scores at both two days and one month. An early significant reduction in anxiety associated with CPU care was no longer significant at one month. HADS data is also summarised in the Figure [1](#F1){ref-type="fig"}, categorised according to severity of anxiety and depression. Scores of zero to seven are normal, eight to ten are mild, eleven to fourteen are moderate, and fifteen to twenty-one are severe. Most participants had normal levels of depression, but only half reported normal levels of anxiety. CPU care was associated with increased prevalence of normal levels of anxiety at two days (53.4% vs 45.1%; p = 0.028) but not at one month (56.7% vs 50.8%; p = 0.129), and increased prevalence of normal levels of depression at two days (81.8% vs 72.9%; p = 0.005) and one month (80.4% vs 73.2%; p = 0.029).
::: {#T5 .table-wrap}
Table 5
::: {.caption}
######
Mean HADS scores at two days and one month
:::
**N (% completed)** **CPU care** **Routine care** **Difference** **95% CI** **P-value** ***ρ***
---------------------- --------------------- -------------- ------------------ ---------------- --------------- ------------- ---------
Anxiety- two days 702 (97.9%) 7.73 8.62 0.89 0.21 to 1.56 0.01 0
0.75 0.09 to 1.41 0.027
Depression-two days 701 (97.8%) 4.30 5.23 0.93 0.34 to 1.51 0.002 0
0.84 0.26 to 1.42 0.005
Anxiety-one month 645 (95.0%) 7.29 7.77 0.48 -0.26 to 1.23 0.203 0
0.58 -0.15 to 1.31 0.117
Depression-one month 644 (94.8%) 4.42 5.43 1.00 0.36 to 1.66 0.002 0
1.02 0.37 to 1.66 0.002
Upper row shows unadjusted analysis (primary analysis) Lower row shows adjusted analysis (secondary analysis) *ρ*= Intraclass correlation coefficient
:::
::: {#F1 .fig}
Figure 1
::: {.caption}
######
HADS scores categorised according to severity
:::
:::
At one-month follow-up, 143 out of 318 participants (45.0%) receiving CPU care reported having further pain, compared to 168 out of 314 (53.5%) receiving routine care (unadjusted OR for further pain if receiving CPU care = 0.71, 95% CI 0.52 to 0.97, p = 0.032; adjusted OR = 0.65, 95% CI 0.10 to 0.76, p = 0.010). For those reporting further pain, the mean score on a 100 mm visual analogue pain score was 36.5 mm among those receiving CPU care and 43.0 mm among those receiving routine care (unadjusted difference = 6.5 mm, 95% CI 2.2 to 10.8, p = 0.003; adjusted difference= 6.8 mm, 95% CI 2.2 to 11.5, p = 0.004). Thus, at one month, CPU care was associated with a reduction in the incidence and severity of subsequent chest pain.
One month after hospital attendance, 49 out of 315 participants receiving CPU care (15.6%) reported that they had taken time off work, compared to 58 out of 316 receiving routine care (18.4%). The unadjusted odds ratio for taking time off work after receiving CPU care was 0.82 (95% CI 0.54 to 1.24, p = 0.35; adjusted OR 0.79 (95% CI 0.59 to 1.22, p = 0.287).
Discussion
==========
Main findings
-------------
Patients with acute, undifferentiated chest pain who received CPU care had improved quality of life and reduced psychological symptoms. All dimensions of quality of life were improved at one month apart from the emotional role dimension. Anxiety was reduced two days after assessment, but there was no significant difference by one month, whereas reduced symptoms of depression at one month were still significant at one month. Patients receiving CPU care reported that subsequent symptoms of chest pain were less frequent and (if present) less severe. However, these reported differences in symptoms and quality of life were not associated with any significant difference in the need to take time off work.
Comparison to other studies
---------------------------
Previous studies of CPU care have focussed on cardiac events, process measures and economic measures \[[@B9]\]. One previous randomised trial found that CPU care was associated with greater diagnostic certainty \[[@B10]\] and improved patient satisfaction \[[@B11]\]. Our study suggests a more complicated picture, since more patients in the CPU group received a diagnosis of non-specific chest pain. CPU assessment may allow cardiac disease to be ruled out, but if an alternative diagnosis is not offered then this can hardly be said to increase diagnostic certainty, except in the somewhat convoluted sense that we may be more certain of what we know the cause is not.
Nevertheless, CPU assessment was associated with reduced anxiety and improved quality of life. This is consistent with a previous study of diagnostic testing by Sox et al \[[@B20]\] that showed reduced anxiety among patients who were randomised to a more thorough outpatient diagnostic work-up for non-specific chest pain, but inconsistent with the findings of a study of exercise testing by Channer et al \[[@B21]\] that found no evidence of reassurance.
Limitations of this study
-------------------------
The main limitation of this study relates to our inability to blind participants to the intervention they received and to fact that they were involved in a trial of CPU care. Participants may have been influenced by this knowledge and improvements in psychological symptoms and quality of life may represent a positive response to receiving a novel form of care, rather than improvements specifically related to CPU care.
The use of cluster randomisation has substantial advantages for pragmatic evaluation of changes in organisation, particularly if economic evaluation is undertaken \[[@B22]\]. However, the fact that randomisation occurs before recruitment means that there is the potential for selection bias. We attempted to reduce this risk by applying rigorous selection criteria and to address any potential bias by undertaking a secondary, adjusted analysis. Nevertheless it is possible that selection bias may have influenced the results.
Although the measures used have been validated, they have not been widely used in the emergency setting. Changes in health status after an episode of chest pain may be very rapid, hence our need to measure outcomes only two days after intervention. Yet the HADS measures anxiety and depression over the previous week, while some SF-36 questions refer to the previous month. A recent episode of chest pain is likely to be an important determinant of reported health, but it may be that, if participants interpreted the questionnaires strictly, the initial questionnaire was recording health status before the intervention. Also, there may be doubts regarding what some of the outcomes are actually measuring. For example, some of the questions in the HADS measure symptoms that are useful markers for depression, such as levels of activity, which may also be changed by other health or social processes. Thus it may be that the reduced scores associated with CPU care measured on the depression scale relate to increased activity in response to the CPU exercise treadmill test, rather than reduced depression.
Implications for practice and future research
---------------------------------------------
This study suggests that the assessment that patients receive when they present with acute chest pain can have an impact upon their subsequent health, even if this assessment does not, in most cases, provide a definitive diagnosis. It supports the findings of decision analysis modelling \[[@B7]\] that the potential health impact of chest pain assessment lies as much in addressing quality of life and psychological symptoms as in detecting and treating cardiac disease. The CPU assessment simply provides a rigorous and structured evaluation, yet this appears to have a significant effect upon anxiety (although this is not maintained), depression and quality of life.
Yet it is not clear how this effect is achieved. It is possible that early, rigorous testing, particularly the exercise treadmill test, has a valuable effect in reassuring the patient that they are healthy and capable of normal physical functioning. Alternatively, it could be that consistent, reliable advice and attention from specialist chest pain nurses, rather than a variety of different doctors, is the key element. A third possibility, as previously discussed, is that bias plays an important role.
Future research needs to determine which of these possibilities is the key factor. This is important for the specific issue of determining whether and how CPU care is effective, and thus what elements of CPU care are essential, and for the more general issue of exploring how diagnostic assessment effects subsequent well being.
Conclusions
===========
CPU care for patients attending hospital with acute, undifferentiated chest pain is associated with reduced initial anxiety, reduced depression over the following month, and improvements in most dimensions of quality of life. Further research is required to establish how this effect is achieved.
List of abbreviations
=====================
OR: odds ratio
CI: confidence interval
ECG: electrocardiograph
CPU: chest pain unit
ESCAPE: effectiveness and safety of chest pain assessment to prevent emergency admission
HADS: hospital anxiety depression scale
Authors\' contributions
=======================
SG conceived and designed the study, analysed the data, drafted the paper, and participated in writing the final paper. JN assisted with study conception and design, supervised data analysis, and participated in writing the final paper.
Acknowledgements
================
We would like to thank all the members of the ESCAPE Research Team: Liz Cross, Simon Dixon, Simon Capewell, Deborah Quinney, Jane Arnold, Karen Angelini, Sue Revill, Tom Locker, Stephen Campbell and Francis Morris.
| PubMed Central | 2024-06-05T03:55:46.717349 | 2004-7-29 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509284/",
"journal": "Health Qual Life Outcomes. 2004 Jul 29; 2:39",
"authors": [
{
"first": "Steve",
"last": "Goodacre"
},
{
"first": "Jon",
"last": "Nicholl"
}
]
} |
PMC509285 | Background
==========
Adenotonsillar enlargement is the main cause of obstructive sleep apnea (OSA) in the pediatric population. However, this prevalent syndrome is more complicated in adults \[[@B1]\]. OSA has also been described in cases of benign lymphoid hyperplasia, plasmacytoma, amyloidosis, pharyngeal tumors and diseases that involve the nasopharyngeal structures. A series of careful examinations of the upper airway should be performed in every adult patient to check for anatomic causes related to upper airway obstruction \[[@B2]\]. We report here a patient with severe obstructive sleep apnea treated by uvulopalatopharyngoplasty (UPPP).
Case presentation
=================
A 41-year-old man presented with complaints of snoring, excessive daytime sleepiness, and pavor nocturnes for more than 10 years. Systemic diseases were denied. Physical examination revealed bilateral symmetric and enlarged palatine tonsils without abnormal surface change. There were no palpable cervical lymph nodes or B symptoms (fever, body weight loss and cold sweats). White and red blood cell counts, biochemistry and chest radiographs were within normal limits. The results of an overnight polysomnography (PSG) showed mean SaO~2~, 91%, minimal SaO~2~, 62%, and a desaturation index (≥ 4%) of 61.8/h. The arousal index was 64.8/h and the respiratory disturbance index (RDI) was 66.0/h. Believing that the patient was suffering from severe OSA and hyperplastic palatine tonsils, he received UPPP.
The postoperative course was uneventful and sleep apnea improved. PSG performed 4 months after surgery demonstrated that the RDI had reduced to 23.9/h. Pathology indicated small B cell lymphocytic lymphoma (Figure [1](#F1){ref-type="fig"},[2](#F2){ref-type="fig"}) with bone marrow involvement. During the whole course, the patient was free from B symptoms and no further abnormal lymphadenopathy was detected even after head and neck computed tomography (CT) and thallium scan (figure [3](#F3){ref-type="fig"}). Chemotherapy was started after evaluation at the oncology clinic. The patient is doing well and is on regular follow-up in the ENT and oncology clinics.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
a\) surgical specimen of palatine tonsils; b) picture of oropharynx post UPPP 3 months later
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Photomicrograph a) effacement of normal architecture and infiltration of monotonous small lymphoid cells is visible (Hematoxylin and Eosin 100X); b) Bone marrow showing monotonous small lymphoid cells infiltration (Hematoxylin and Eosin 100X).
:::
:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
a):Mild lymphadenopathy over bilateral posterior neck area; b:Gallium scan: gallium-avid lymphoma in bilateral submandibular regions and suspected lesions in the mid-abdomen
:::
:::
Discussion
==========
Adenotonsillar enlargement is the leading cause of OSA in the pediatric population \[[@B1]\] though it is not so rare disorder in adults as well. The morbidity of OSA includes hypertension, arrhythmia, heart disease, erythrocytosis, and hyperlipidemia. Malignancy should be considered a potential contributing factor that rarely contributes to OSA and has never been shown to be related to it\[[@B2]\].
Small lymphocytic lymphoma (SLL) is an indolent but relentless malignancy, with a median survival of about 10 years. Because It usually presents as neck lymphadenopathy in the later stages, SLL is not easy to diagnose in the early stage. The effectiveness of chemotherapy for treating SLL is controversial. Most studies have found no benefit in treating patients until they develop symptoms \[[@B3]\]. Lymphoma presenting as OSA is extremely rare, but this case report illustrates that malignancy should be considered a potential contributing factor of OSA; a careful oropharyngeal examination in patients with OSA is necessary. Both tonsillectomy and UPPP can improve the patency of upper airway in OSA patients presenting with abnormally enlarged palatine tonsils. However, pathology of unsuspicious tissues can reveal malignancy with specific staining, and structural abnormalities secondary to a hidden malignancy might present initially as OSA. Therefore, a thorough physical examination should be performed and the pathological results should be closely traced.
Nolan described a case of adenotonsillar enlargement due to chronic lymphatic leukemia which caused severe OSA \[[@B4]\]. His report highlights the need to consider OSA as a cause of constitutional symptoms in adults with lymphoreticular disease, especially when there is involvement of the Waldeyer\'s ring. Zorick *et al*., \[[@B5]\] reported that upper airway sleep apnea was exacerbated by lymphocytic lymphoma but that chemotherapy led to complete remission of well differentiated lymphocytic lymphoma and subsidence of OSA \[[@B5]\]. Abe *et al*., \[[@B6]\] described a patient with Non-Hodgkin\'s lymphoma who was successfully treated by tonsillar surgery and chemotherapy. In one published case, complete remission of centrocytic-centroblastic diffuse B cell lymphoma was found after tonsillectomy with UPPP, as in our case \[[@B7]\].
Conclusions
===========
Tonsillar surgery should be performed even on patients highly suspected of having lymphoma to improve OSA \[[@B8]-[@B10]\]. Neck CT is also suggested as a preoperative examination for patients with OSA and neck lymphadenopathy. Whether the prognosis or the outcome of chemotherapy or radiation therapy will be affected by tonsillar surgery is controversial. We conclude that SLL might be a contributing factor of OSA. Therefore careful neck examination should also be performed on patients complaining of snoring or sleep disturbances.
Competing interest
==================
None declared.
Authors\' contributions
=======================
YT, YC, CL, WC and MT made substantial contributions to the intellectual content of the paper, in the interpretation of results and in drafting the manuscript. All authors read and approved the manuscript
Acknowledgement
===============
Patient consent was obtained for publication of his case record, scan and specimen photograph.
| PubMed Central | 2024-06-05T03:55:46.721162 | 2004-7-29 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509285/",
"journal": "World J Surg Oncol. 2004 Jul 29; 2:26",
"authors": [
{
"first": "Yung-An",
"last": "Tsou"
},
{
"first": "Yuan-Kai",
"last": "Cheng"
},
{
"first": "Chia-Der",
"last": "Lin"
},
{
"first": "Weng-Cheng",
"last": "Chang"
},
{
"first": "Ming-Hsui",
"last": "Tsai"
}
]
} |
PMC509286 | Background
==========
The author has been conducting research on behavioral treatment of obesity for about 25 years. During that time, the dominant conceptual models guiding intervention development have been cognitive behavior models that have their origin in psychological theory. Those most often cited include the Health Belief Model \[[@B1]\], Protection Motivation Theory \[[@B2]\], Subjective Expected Utility Theory \[[@B3]\], the Theory of Reasoned Action \[[@B4]\], Social Cognitive Theory \[[@B5]\], and the Transtheoretical Model \[[@B6]\]. All of these theories are concerned with how people make behavioral choices and the general idea is that people decide what to do based on the extent to which they expect that their choices will produce results that they value. Much of the content of the theories is concerned with factors that may affect value/expectancy calculations. As summarized by Weinstein in a comparative review of four social psychological theories \[[@B7]\], variables thought to influence value/expectancy judgments include such factors as perceived rewards of current behavior, self-efficacy, normative beliefs, motivation, and the perceived consequences of not changing behavior.
Weinstein\'s summary is illustrative of the fact that Health Behavior Theory has tended to be particularly interested in understanding people\'s motivation to change behavior rather than ability to change. Moreover, motivation is thought to be the result of a relatively complex, but logical, interpretation of large quantities of information about self and environment. The theories that Weinstein reviewed deal almost exclusively with behavioral decision processes in people\'s minds. They have few if any terms relating to how information gets into peoples minds or how subsets of it receive more or less attention. Broader health behavior theories such as Social Cognitive Theory or the Transtheoretical model have addressed issues and variables outside the person to a greater extent, but the fundamental interest in and belief in psychological variables as the key force in determining health behavior remains.
The implications of the focus of health behavior theory on psychological determinants of behavioral decision-making for my own research area of interest, obesity treatment, are several. One is the inclusion of measures of psychological characteristics in most research protocols (e.g., assessment of behavioral intentions, self-efficacy, perception of barriers to change, perception of social support, and outcome expectations). A second is the inclusion of treatment elements that specifically target psychological perceptions and processes independent of the diet and physical activity behaviors that actually produce weight change (e.g., how to deal with emotional eating, how to deal with the frustration of lapses and relapses, and how to talk to yourself to increase self-motivation). A third is the belief that psychological reactions to treatment experiences themselves are very important and deserve independent attention. Common behavioral prescriptions for weight-loss goals and frequency of self-weighing are exemplary (i.e., recommending infrequent weighing to prevent discouraging feedback about progress and encouraging smaller and thus \"more attainable\" behavior and weight-loss goals in the belief that they will be more motivating).
The problem with the emphasis on cognitive variables in weight-control research is that they have so far failed to meet fundamental scientific criteria for empirical verification. Thus, they also have not led to a better understanding of the weight-loss process, have not improved our ability to predict weight-loss outcomes, and have not led to improvement in treatment methods. In some cases it is even arguable that they have made treatment worse. I will illustrate these problems with results from my own research.
Discussion
==========
Like most behavioral researchers in the obesity area, I have attempted to measure elements of health behavior theory in every obesity intervention project I have ever conducted. I have assessed weight-loss goals, behavioral and weight-loss self-efficacy, psychological well-being, perceived barriers to diet and physical activity change, stages-of-change, and perceived social support. How well have empirical examinations of these factors fared as predictors of success in weight control?
Self-efficacy
-------------
We have examined the predictive value of self-efficacy assessments in several of our studies and describe the results from three of these here in more detail \[[@B8]-[@B10]\]. In the first study, self-efficacy was assessed at baseline, posttreatment, and one year later in 85 men participating in a 15-week weight-loss program \[[@B8]\]. The self-efficacy instrument had subscales for emotional states (e.g., anxiety) and situations (e.g., eating away from home). Higher baseline self-efficacy on both subscales was associated with greater weight loss in treatment and at 1- and 2-year follow-up. Emotional self-efficacy at posttreatment did not predict weight loss at 1- or 2-year follow-up. Situational self-efficacy at posttreatment predicted weight loss at 1-year but not 2-year follow-up.
The second study examined mood and situational self-efficacy in 55 men and 58 women before and after a 16-week weight-loss treatment with a 1-year follow-up \[[@B9]\]. Women had lower pretreatment self-efficacy than men. Self-efficacy was predictive of weight loss and maintenance in men but not in women. Change in self-efficacy over time was positively related to weight change in women but not in men.
The third study examined predictors of weight change over a 2-year period in 460 men and 1172 women who received a low-intensity weight-loss intervention delivered through their HMO \[[@B10]\]. The self-efficacy measure was the WEL questionnaire. Men again were found to have higher baseline self-efficacy than women. Self-efficacy did not predict weight change in men but was positively, though weakly, related to weight change at 6 months only in women.
Our overall conclusion from the analyses described above, as well as others not pursued in as great detail, is that self-efficacy is a weak predictor of weight loss and is inconsistent across study populations and gender. It tends to increase with weight loss. However, treatment-induced increases in efficacy are not predictive of longer-term weight-loss success.
Barriers to Adherence
---------------------
We have also attempted to measure barriers to adherence to weight-control behaviors in many of our studies \[[@B11]-[@B14]\]. The instruments used for this have typically been formatted similarly to efficacy questionnaires in that people are asked to indicate how difficult they find situational, knowledge, and motivational challenges to achieving diet and exercise changes. The findings in these studies have been quite consistent. Baseline assessments of perceived barriers to behavior change are not predictive of weight change. Weight loss is associated with reported decreases in perceived barriers. Treatment-induced change in perceived barriers are not predictive of future weight change. In other words, barrier perceptions as we have measured them do not appear to have pragmatic significance.
Weight Goals
------------
Goal-setting has long been of interest to health behavior theory and in recent years has attracted attention in weight-loss research when it was realized that most people who enter weight-loss treatments want to lose a lot more weight than is realistic given the potency of current weight-loss methodologies \[[@B15]\]. When asked to describe weight losses they deem to represent \"dream, happy, acceptable, and disappointing,\" many individuals in treatment fail to reach even \"disappointing\" weight losses even though in objective medical terms the results are positive. Based on the argument that failure to reach gratifying weight-loss goals leads to psychological distress that lowers weight self-efficacy and undermines weight-loss efforts, it has become popular to recommend counseling in weight-loss treatments specifically targeting the lowering of weight-loss goals. The theoretical argument is that excessive outcome expectations undermine behavioral efforts. We have now completed three sets of formal analyses examining whether weight goals are predictive of weight-loss success. In one of these analyses the relationship between weight-loss goals, weight-loss goal attainment, and long-term (30 months) weight-loss attainment and psychological well-being were assessed in 69 men and 61 women participating in an intensive behavioral treatment program \[[@B16]\]. Results indicated that weight-loss goals were unrealistically high on average and that lower goals were more likely to be reached. Nevertheless, weight-loss goals did not predict either short- or long-term weight losses and were not associated with elevated psychological distress. Two more recent analyses we have conducted looking at weight-loss goals as predictors of success have produced similar results \[Linde JA, Jeffery RW, Levy RL, Pronk NP and Boyle RG, unpublished data \[[@B17]\]\]. Weight-loss goals either did not predict weight loss at all or were slightly positively related to weight-loss success.
Perceived Social Support
------------------------
Perceived social support is another psychological factor thought to influence health behavior decision-making. We have measured social support in a variety of ways in our studies, ranging from single-item questions to multipaged assessments attempting to differentiate among informational, instrumental, and emotional support. The results, unfortunately, have closely paralleled those we have seen with other assessments of barriers to adherence. Assessments of social support prior to treatment do not predict weight loss. Average reports of social support tend to parallel weight loss itself. When people lose weight they report more social support. When they regain, they report less. In other words, perceptions of social support are not predictive of success in weight-loss treatments.
Frequency Weight Self-monitoring
--------------------------------
Self-monitoring of health behavior is incorporated into many health behavior theories, usually as part of a person\'s assessment of achieved outcomes. Although self-monitoring is usually considered a positive element in the adoption of health behavior, in obesity treatment frequent self-monitoring of weight has tended to be down-played or even discouraged on the grounds that disappointing results (i.e., less than desired weight change) may undermine motivation. This is another example in which health behavior theory may have indirectly led to incorrect treatment recommendations. In weight-loss treatments, active discouragement of frequent self-observation of weight has become popular based on the premise that more frequent weighting will cause psychological stress and lower self-efficacy. Recently, we have examined the relationship between frequency of self-weighing and body weight in both clinical and population samples and have found, somewhat to our surprise, that frequency of self-weighing is one of the strongest single predictors of body weight cross-sectionally, and change in the frequency of self-weighing is one of the strongest predictors of weight change \[Linde JA, Jeffery RW and French SA, unpublished data\]. The direction of predictions, however, is opposite that derived from theory. People who weigh themselves more weigh less and are more successful in losing weight.
Stage-of-Change
---------------
A final failure of current health behavior theory to prove useful in weight-control research is a recent examination of the relationship between a stage-of-change measure adopted from Prochaska and short- and long-term weight loss \[[@B18]\]. Categories of precontemplation, contemplation, preparation, and action were defined based on questions about weight-loss intentions and recent weight-loss attempts. Despite a large sample size, excellent follow-up rates, and well-measured objective outcomes, we were unable to demonstrate that staging algorithms recommended by proponents of the Transtheoretical Model could predict weight-loss outcomes.
Experimental Modification of Expectations
-----------------------------------------
Our most recent effort to utilize health behavior theory in obesity intervention research is a study that attempted to examine the effectiveness of experimentally-induced outcome expectancies on weight loss \[Finch EA, Linde JA, Jeffery RW, Rothman AJ and King CM, unpublished data\]. Obese men and women participated in an 8-week weight-loss program with 18-month follow-up in which they were assigned to one of two expectancy groups. The optimistic group was told that focusing exclusively on the positive benefits of weight loss would be valuable in ensuring that they remained motivated in their weight-loss efforts and was given assignments during weekly group sessions and homework between sessions to reinforce this optimistic mindset. A \"balanced\" expectancy group received the instructions that focusing on both the positive and negative aspects of weight loss, a balanced approach, would be most conducive to maintaining weight-loss motivation. This group also received assignments to reinforce their message. Results of this study indicated that the expectation induction was successful initially but difficult to maintain in the face of real weight-loss experience. We were also unable to show that experimentally-induced expectations influenced weight-loss success.
Summary and Conclusion
======================
To summarize the findings described above, I have had considerable difficulty over the last 25 years in confirming that the psychosocial variables favored by health behavior theory are of much value for obesity intervention research. They do not predict weight loss well, either as mediators or moderators. There is little evidence to support the idea that targeting them for intervention improves weight-loss outcomes. It is, of course, arguable that the weak findings relating to health behavior theory variables are due in large part to methodological weaknesses, either in measurement tools and/or their frequency of measurement. I would argue, however, that 25 years is long enough to wait for improved methods and that it is time to look elsewhere for variables that better predict weight-change outcomes and that, therefore, may form a better basis for improving future treatments.
Implication for Weight-Loss Treatment
-------------------------------------
Given the lack of success finding support for cognitive mediators of behavior change in weight loss, one might surmise that progress in improving weight-loss interventions over the last 20 years must have been dreary indeed. Somewhat surprisingly, however, that is not the case. In fact, the short-term (6 to 12 months) success of weight-loss treatments has approximately doubled over that time and several variables have been identified that reliably enhance treatment outcomes. It has been clearly shown experimentally that increasing treatment length \[[@B19]\], prescribing low-energy intakes \[[@B20]\], prescribing high-energy expenditure \[[@B21]\], using a deposit contract and group-based reward systems \[[@B22]\], and simplifying adherence to diet through meal substitutes \[[@B23]\] and exercise by providing exercise equipment \[[@B24]\] all improve initial weight loss. From a theoretical perspective, however, one thing is noteworthy about these successful innovations. Although not incompatible with health behavior theory, none of them are specifically derived from cognitive decision-making models. Indeed, health behavior theory does not include variables like these in its models.
Where Do We Go From Here?
-------------------------
The argument above about the practical limitations of many popular theories of health behavior is not meant to be a call to abandon theory. Behavior scientists have amassed much useful information about the principles underlying human behavior that should be valuable for health behavior interventions. Much is known about human perception, learning, motivation, and responsiveness to environmental opportunities and contingencies. Health behavior intervention lies at the interface between people and their environment. Interventionists change aspects of the environment (cues, information, behavioral contingencies) with the intention of producing changes in how people behave. What is needed to advance health behavior intervention is theory that addresses relationships between modifiable aspects of the environment and behavior. There is no doubt that cognitive processes are involved in these relationships. However, the extent to which current theories capture this is questionable. Data now available suggest that easily obtainable information about people\'s cognitive processes adds little to our ability to predict the results of interventions. Thus, it may be wise to pay more attention to applied theories like classical behavior theory \[[@B25]\], communications theory \[[@B26]\], and learning theory \[[@B27]\] than to those coming out of the social cognitive traditions.
Competing interests
===================
None declared.
Acknowledgements
================
This research was supported by grant DK50456 from the National Institute of Diabetes and Digestive and Kidney Diseases.
| PubMed Central | 2024-06-05T03:55:46.721964 | 2004-7-23 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509286/",
"journal": "Int J Behav Nutr Phys Act. 2004 Jul 23; 1:10",
"authors": [
{
"first": "Robert W",
"last": "Jeffery"
}
]
} |
PMC509287 | Introduction
============
Although considerable advances have been made in terms of our molecular and cellular knowledge, for most human disease states a fundamental understanding of causal disease onset, disease mechanism and progression, and optimal treatment is still significantly limited.
In part, this advancement has been hampered by our inability to fully and rapidly delineate complex cellular metabolic processes and molecular pathways.
Organisms are complex self-organizing entities made up of such parts: organs, tissues, cells, organelles and ultimately molecules and atoms. One question that arises, concerns the relationship between the whole and its component parts. The issue at stake is sometimes called \"the question of reduction\" or \"the problem of reductionism\" \[[@B1]\].
The inefficacy of contemporary science to describe biological systems, consisting of non-identical parts that have different and non-local interactions has tended to limit progress in the human healthcare. Many biological systems remain incomprehensible because their multifarious nature has been combined with a reductionist approach based on the linear conception of *cause*and *effect*.
The use, however, of a more *holistic*multidimensional system level-based approach may provide new insights into the understanding of disease processes and mechanisms of action of therapeutical agents \[[@B2]\].
Herein we aim to introduce a system level-based approach that includes morphological and molecular techniques for validating the appropriateness of using novel tumor-associated antigens (TAA) for clinical purposes. This approach might be easily implemented for identifying prognostic, diagnostic and alternative biomarkers.
Finally, this type of analysis of appropriately designed cohorts might also provide a key to understanding the differences in patients who do or do not respond to any particular therapy. This information may be helpful for a more effective (and therefore more *cost-effective*) design of clinical trials \[[@B2]\].
Immunotherapy and the human complexity
======================================
The recognition and characterization of novel TAA is fundamental to the advance of cancer immunotherapy. The original hypothesis of Boon \[[@B3]\] and Rosenberg \[[@B4]\] that human cancers express antigens that can be specifically targeted by cell mediated immunity has become a scientifically justifiable rationale for the design and clinical testing of novel TAA based immunotherapies and therapeutic vaccines \[[@B5]-[@B7]\].
However, although a number of TAA have been discovered and it has been suggested that they could be useful in the immunological treatment of cancer, the *complexity*of human beings leads us to reflect on the need to establish compelling new criteria for validating their real applicability. *Biological complexity*can be intuitively appreciated -- at least in terms of morphological or behavioral complexity, or the variety of cell types in an organism -- but the term itself is notoriously difficult to define \[[@B8]\]. Human beings are *complex hierarchical system*s consisting of a number of *levels of anatomical organization*(genes, cells, tissues, organs, apparatuses, and organism) that interrelate differently with each other to form networks of growing complexity. The concept of anatomical entities as hierarchy of graduated forms, and the increasing number of known structural variables, have highlighted new properties of organized biological matter and raised a series of intriguing questions. In order to understand biology at the system *level*, we need to examine the structure and dynamics of the functions of organisms rather than the characteristics of their constitutive isolated parts \[[@B8]-[@B13]\].
The expression of TAA in biological materials has mainly been studied at the level of gene expression and gene level measurement by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis and the Quantitative real-time PCR (qrt-PCR) technology \[[@B14]-[@B17]\]. However, the *information*provided by these approaches is limited by the fact that the phenomena observed at each level of anatomical organization have properties that do not exist at a lower or higher level: RT-PCR and qrt-PCR may offer a satisfactory *qualitative/quantitative*description of *small-scale structures*, but this is likely to be irrelevant when it comes to *large-scale features*. The above considerations, in conjunction with the *complexity of tumor-host interactions*within the tumor microenvironment caused by temporal changes in tumor phenotypes and an array of immune mediators expressed in the tumor microenvironment \[[@B18]\] might clarify the limited reliability and applicability of current immunotherapeutic approaches.
Here, we suggest a *system level-based*approach (Figure [1](#F1){ref-type="fig"}) for validating the appropriateness of using TAA for clinical purposes, which includes the following never defined before key points:
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Figure 1
::: {.caption}
######
System level-based approach for validating candidate TAA for clinical application. Aside from the well defined experimental procedure, the method presented here is based on the *complex hierarchical nature*of the human beings. The analysis begins at level of gene expression and then continues to higher levels of anatomical organization, (cell, tissue, organ, apparatuses and organism). This approach includes both morphological and molecular techniques. It also introduces the concept of dynamics of TAA expression at the level of the *cell cycle*, the *physiological status of the organism*and the *process of aging*.
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• *Discriminating*the *cell types*expressing the candidate antigen on the basis of the morphological visualization of all of the parts making up the *organ*under investigation.
• *Discriminating*the candidate antigen\'s sub-cellular localization (at the level of cell *nucleus*, *cytoplasm*and/or *plasma membrane*) by ultra-structural morphological visualizations.
• *Mapping*candidate antigen expression in all of the *organs*making up the *apparatuses*.
• *Mapping*candidate antigen expression in all of the *apparatuses*making up the living *organism*.
• *Estimating*the percentage of normal cells and their neoplastic counterparts expressing the candidate antigen.
• *Evaluating*the *dynamics*of candidate antigen expression at the level of the *cell cycle*, the *physiological status of the organism*(*i.e.*the woman\'s menstrual cycle) and the *process of aging*.
In order to advance our knowledge in a currently widely debated field of investigation, a clearer distinction must be made between *in vitro*laboratory results (the discovery and validation of target antigens) and their *in vivo*application (*in vivo*validation), and it is necessary to adopt a more complete experimental approach that forcefully includes both morphological and molecular techniques \[[@B19]\].
Conclusions
===========
Translational science which is aimed to test, in humans, novel therapeutic strategies developed through experimentation \[[@B20]\] should begin to consider the role of *emergence*in other words the appearance of *unexpected structures*and/or the occurrence of *surprising behaviors*in large systems composed from microscopic parts, whether physical or biological. By unexpected and surprising we mean structures and behaviors which are not *intuitive*and are not simply *predictable*.
Since our understanding of complex human disease such as cancer, is still limited and pre-clinical models have shown a discouraging propensity \[[@B2],[@B6]\] to fail when applied to humans, a new way of thinking is strongly needed that unites physicians, biologists, mathematicians and epidemiologists, in order to develop a better theoretical framework of tumor development, progression and tumor-host interactions.
Although the model presented here is based on a multidisciplinary system-level approach probably within the reach of only very large and multi-talented laboratories, it is aimed to introduce a different way of investigating *human cancer,*which takes into account the complexity of the human being as a system.
The use of a holistic approach, which enables a more accurate selection of immunotherapeutic target antigens in the first phase of the experimental research, will reduce the notable fragmentation of the biological information in the post-genomic era, and will facilitate a more accurate transfer of the acquired knowledge to the bedside.
Further, this new multidisciplinary approach is specifically required to improve the capacity to produce desired results with a minimum expenditure of *energy*, *time*, or *resources*for immunotherapeutic treatments and to allow cancer immunotherapy to re-emerge from the mist in which it is currently shrouded.
| PubMed Central | 2024-06-05T03:55:46.723355 | 2004-7-29 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509287/",
"journal": "J Transl Med. 2004 Jul 29; 2:26",
"authors": [
{
"first": "Maurizio",
"last": "Chiriva-Internati"
},
{
"first": "Fabio",
"last": "Grizzi"
},
{
"first": "Robert K",
"last": "Bright"
},
{
"first": "W",
"last": "Martin Kast"
}
]
} |
PMC509288 | Background
==========
In eukaryotes, transcription by RNA polymerase II requires the orderly recruitment of basal transcription factors and activators to the core promoter and enhancers, respectively \[[@B1],[@B2]\]. The core promoter contains the transcription initiation site, and it provides the docking sites for the basal transcription factors that nucleate the assembly of a functional preinitiation complex (PIC). The TATA box is one of four major core promoter elements, and it is specifically recognized by the TATA-binding protein (TBP), a subunit of the basal transcription factor TFIID which also contains at least 14 TBP-associated factors (TAFs). On the other hand, enhancers are bound by sequence-specific transcriptional activators that are thought to promote PIC assembly through interactions with components of the basal transcription machinery.
Human T-cell leukemia virus type I (HTLV-I) Tax protein is a unique transcriptional regulator \[[@B3]\]. Tax can modulate the HTLV-I long terminal repeats (LTR), heterologous viral promoters, and a variety of cellular genes. In most context, Tax acts as a potent transcriptional activator through Tax-responsive DNA elements that are recognized by cellular transcription factors CREB, NFκB and serum response factor (SRF) \[[@B4]-[@B6]\]. For activation of the HTLV-I LTR, Tax targets three imperfectly conserved 21-bp direct repeats flanked by GC-rich sequences. In this scenario, Tax forms a ternary complex with CREB and the 21-bp repeat through physical interaction with CREB and direct contact with the flanking GC-rich sequences \[[@B7]-[@B9]\]. Tax-induced activation of other promoters is thought to be mediated through protein-protein interactions. Thus, Tax is a pleiotropic transcriptional activator that targets multiple enhancer elements through multiple cellular transcription factors.
To date, the molecular mechanisms for Tax trans-activation have been well studied. Due to its pleiotropic activities, there are likely nuances to Tax\'s activity which remain unrevealed. Currently, we understand Tax to harbor a minimal activation domain \[[@B10]\], to interact with basal transcription factors such as TBP \[[@B11]\], to form a homo-dimer \[[@B12]-[@B14]\], and to stimulate the dimerization of cellular regulatory factors such as CREB \[[@B15],[@B16]\] and IKK-γ \[[@B17]\]. Moreover, we also know that Tax can directly engage transcriptional coactivators such as CREB-binding protein, p300 and P/CAF \[[@B18]-[@B20]\]. However, it remains unclear what is Tax\'s optimal preference for an enhancer -- TATAA configuration. It has also been unaddressed whether Tax has a transcriptional activity after the formation of an initiation complex at the TATAA-box.
In mammalian cells, the artificial recruitment of TBP sufficiently activates transcription from some promoters \[[@B21]-[@B24]\]. It is understood that the structure of core promoter is one important determinant for this activation \[[@B23]\]. On the other hand, DNA-tethered TBP can also work synergistically with selective natural activators such as human immunodeficiency virus type 1 (HIV-1) Tat protein \[[@B21]-[@B23]\] and cytomegalovirus IE2 protein \[[@B25]\]. In this regard, it is not known whether TBP recruitment suffices for activation of HTLV-I minimal promoter. Nor is it clear whether Tax can cooperate with promoter-tethered TBP.
Here, we have constructed a series of chimeric enhancer-TATAA reporters to analyze the functional roles of these transcription elements in Tax-mediated activation. We observed that Tax activates the HTLV-I 21-bp repeats more potently than other enhancer elements. Analysis of ten mutants of Tax revealed that Tax utilizes different domains to target different cellular factors. We also found that multiple bZIP transcription factors including the newly-identified LZIP are involved in Tax activation of HTLV-I LTR. Finally, two other salient findings are that optimal Tax-responsiveness is specified by the HTLV-I-specific TATAA element, and that Tax synergizes with artificially recruited, DNA-tethered, TBP in a phase of transcription after the assembly of an initiation complex at the promoter.
Results
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Specific preference by Tax for only one enhancer element
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Tax can activate transcription through 21-bp repeats, CRE, κB site or SRE \[[@B4]-[@B9]\]. However, a direct head-to-head comparison between the relative preferences of Tax for each of these elements is complicated by the context of additional DNA elements in the various promoters tested to date (i.e. the HTLV-I LTR versus the HIV-1 LTR versus the interleukin-2 promoter). To directly compare enhancer motifs, they should be placed in identical TATAA-context and tested in identical experimental settings. Towards this end, we constructed a series of six reporters to dissect the ordered preference of Tax for various enhancers.
Each reporter contains two copies of enhancer motifs (21-bp repeats, CRE, AP1, Sp1, κB or SRE) and a minimal HTLV-I TATAA promoter (Fig. [1A](#F1){ref-type="fig"}). Because all reporters have the same HTLV-1 minimal promoter and are otherwise devoid of any known enhancer elements, side-by-side comparisons would reflect directly the contribution of the variously added cis-enhancer. We observed that the κB- and CRE- motifs had the highest basal activities in HeLa cells in the absence of Tax (Fig. [1B](#F1){ref-type="fig"}, lanes 3, 4, 9 and 10; and Fig. [1C](#F1){ref-type="fig"}, columns 3 and 6 compared to column 1). Of significant interest, in stark contrast to the cellular CRE elements, the reiterated HTLV-I 21-bp repeats (normally considered as viral CRE elements) and the SRE exerted little or no basal activity (Fig. [1B](#F1){ref-type="fig"}, lanes 1, 2, 11 and 12; and Fig. [1C](#F1){ref-type="fig"}, lanes 2 and 7 compared to lane 1). The AP1 and Sp1 sites were moderately active (Fig. [1B](#F1){ref-type="fig"}, lanes 5--8 and Fig. [1C](#F1){ref-type="fig"}, lanes 4 and 5). Hence for basal expression in the context of the HTLV-I TATAA promoter, κB, CRE \> AP1, Sp1 \>\> 21 bp, SRE.
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Figure 1
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######
Relative responsiveness of enhancers to Tax in HeLa cells. (**A**) CAT reporter plasmid. Each plasmid contains two copies of enhancer elements (21-bp repeats, CRE, AP1, Sp1, κB and SRE) and one copy of HTLV-I minimal promoter (HTLV TATAA). The enhancer (Enh.) sequences are shown in green. (**B**) A representative example of CAT assay. Increasing amounts (5 to 10 μg) of p21-HTLV-CAT (lanes 1 and 2), pCRE-HTLV-CAT (lanes 3 and 4), pAP1-HTLV-CAT (lanes 5 and 6), pSP1-HTLV-CAT (lanes 7 and 8), pKB-HTLV-CAT (lanes 9 and 10) and pSRE-HTLV-CAT (lanes 11 and 12) were transfected into HeLa cells. CAT assays were performed 48 h after transfection. AcCM: acetyl chloramphenicol. CM: chloramphenicol. (**C**) Basal transcriptional activities of enhancer elements. Five microgram of plasmids containing the HTLV TATAA alone (pHTLV-CAT; column 1) or the indicated enhancer elements (columns 2 to 7) were transfected into HeLa cells and the relative CAT activities were compared. CAT activity from pKB-HTLV-CAT-transfected HeLa cells was taken as 100% (lane 6). (**D**) Tax-dependent transcriptional activities of enhancer elements. The same plasmids as in C plus 1 μg of Tax-expressing plasmid pIEX were co-transfected into HeLa cells and the CAT assays were performed. Fold activation in the presence of Tax versus in the absence of Tax was calculated and compared. All CAT results are representative of three independent experiments.
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When the reporters were tested in the presence of Tax, a different pattern emerged. Transcription from the 21-bp repeats was stimulated approximately 70-fold (Fig. [1D](#F1){ref-type="fig"}, lane 2 compared to lane 1) while that from the Sp1 site, not prototypically known to be responsive to Tax, was not activated significantly over the activity of the HTLV-I minimal promoter (Fig. [1D](#F1){ref-type="fig"}, lane 5 compared to lane 1). All other responses to Tax were markedly weaker than that seen from the 21-bp repeats. Hence, for all practical purposes, only a duplicated 21-bp repeat in the context of isolated placement upstream of an authentic HTLV-I minimal TATAA box could be regarded as significantly Tax-responsive in HeLa cells.
We repeated the experiments in Jurkat T lymphocytes and obtained similar results (Fig. [2](#F2){ref-type="fig"}). Thus, while the κB and CRE enhancers displayed the highest activities in the absence of Tax (Fig. [2B](#F2){ref-type="fig"}, lanes 3 and 6 compared to lanes 5, 4, 2, 1, and 7), only the 21-bp repeats were highly responsive to Tax (Fig. [2A](#F2){ref-type="fig"}, lanes 1 and 2; Fig. [2C](#F2){ref-type="fig"}, lane 2). Our results from HeLa and Jurkat cells consistently support the preferential activation of the 21-bp repeats by Tax.
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Figure 2
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Relative responsiveness of enhancers to Tax in JPX9 cells. (**A**) A representative example of CAT assay. Tax-expressing plasmid pIEX (1 μg) and increasing amounts (0.5 to 1 μg) of p21-HTLV-CAT (lanes 1 and 2), pCRE-HTLV-CAT (lanes 3 and 4), pAP1-HTLV-CAT (lanes 5 and 6), pSP1-HTLV-CAT (lanes 7 and 8), pKB-HTLV-CAT (lanes 9 and 10) and pSRE-HTLV-CAT (lanes 11 and 12) were transfected into Jurkat cells. CAT assays were performed 48 h after transfection. AcCM: acetyl chloramphenicol. CM: chloramphenicol. (**B**) Basal transcriptional activities of enhancer elements. One microgram of plasmids containing the HTLV TATAA alone (pHTLV-CAT; column 1) or the indicated enhancer elements (columns 2 to 7) were transfected into Jurkat cells and the relative CAT activities were compared. CAT activity from pKB-HTLV-CAT-transfected Jurkat cells was taken as 100% (lane 6). (**D**) Tax-dependent transcriptional activities of enhancer elements. The same plasmids as in C plus 1 μg of Tax-expressing plasmid pIEX were co-transfected into Jurkat cells and the CAT assays were performed. Fold activation in the presence of Tax versus in the absence of Tax was calculated and compared. All CAT results are representative of three independent experiments.
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Multiple activation surfaces are configured in Tax
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In Fig. [1D](#F1){ref-type="fig"}, the 21-bp repeats were activated by Tax \>75 fold, while κB and SRE motifs were activated five and three fold, respectively. The low activation of the latter motifs, although comparatively less significant than that from the 21 bp elements, was real and reproducible. To further understand how Tax works, we wondered whether the different magnitudes of activation were due to quantitative or qualitative differences in protein-protein interaction. To address this question, we examined the separate responses of the three motifs to a battery of Tax mutants.
Previously we had characterized 47 mutations in Tax that affect transcriptional activity \[[@B26]\]. Here we selected 10 of these Tax mutants to shed light on the discrete surfaces used by Tax to mediate effects on 21-bp repeats, κB and SRE. All mutants were expressed to comparable levels in HeLa cells (data not shown). Their relative activities on 21-bp repeats, κB and SRE were assessed (Fig. [3](#F3){ref-type="fig"}).
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Figure 3
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Differential activities of Tax mutants on 21-bp repeats (**A**), κB (**B**), and SRE (**C**) motifs. One microgram of plasmid expressing the indicated Tax mutants plus 5 μg of p21-HTLV-CAT, pKB-HTLV-CAT or pSRE-HTLV-CAT was individually transfected into HeLa cells. CAT activity from wild type Tax-transfected cells (lane 1) was taken as 100%.
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Based on percentage of activation relative to wild type Tax, we saw three patterns of mutant activity for 21 bp, κB and SRE (Fig. [3](#F3){ref-type="fig"}). Hence, the activation domain mutant Tax L320G \[[@B10]\] and the zinc finger mutant Tax H52Q \[[@B26]\] were defective in activating either 21-bp repeats or SRE, but were fully competent for κB (Fig. [3](#F3){ref-type="fig"}, lanes 4 and 10). By contrast, the N-terminal mutant Tax Δ3--6 and the point mutant Tax S258A activated 21-bp repeats and SRE well, but did not activate κB (Fig. [3](#F3){ref-type="fig"}, lanes 2 and 7). Additionally, mutants Tax Δ94--114, Tax S150A and Tax Δ337--353 were active on all three motifs (Fig. [3](#F3){ref-type="fig"}, lanes 5, 6 and 11), while neither Tax Δ2--58, Tax Δ 284--353 nor Tax L296G (Fig. [3](#F3){ref-type="fig"}, lanes 3, 8 and 9) activated any of the motifs. These non-identical patterns suggest that Tax may use different contact surfaces to target factors docked at the 21-bp repeats, κB or SRE. We note some similarity in the Tax mutant activity profiles for the 21-bp repeats and SRE suggesting that overlapping surfaces may be utilized.
Amongst bZIP factors, CREB is specifically preferred by Tax
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Tax activates the HTLV-I LTR through the viral 21-bp repeats \[[@B7]-[@B9]\]. When compared to κB and SRE, the activation of 21-bp repeats by Tax is particularly effective (Fig. [1](#F1){ref-type="fig"} and Fig. [2](#F2){ref-type="fig"}) and, based on mutant profiles (Fig. [3A](#F3){ref-type="fig"}), relies upon unique structural surfaces. Previously, it has been proposed that bZIP cellular transcription factors including CREB \[[@B9],[@B27],[@B28]\], ATF4 \[[@B29],[@B30]\] and c-Jun \[[@B31]\] play roles in Tax activation of 21-bp repeats. However, the relative contribution of these bZIP factors has not been compared directly in the same experimental setting. Furthermore, it remains undetermined whether additional newly identified bZIP proteins may also participate in Tax activation of 21-bp repeats.
We next used dominant-negative proteins to assess the contributory roles of different bZIP transcription factors on Tax-dependent activation. We employed several well-documented dominant-negative inhibitors of CREB and Jun proteins including KCREB \[[@B32]\], A-CREB \[[@B33]\], A-Fos \[[@B34]\] and TAM67 \[[@B35]\]. In addition, we constructed dominant-negative versions of ATF4 and LZIP \[[@B36]\] using the strategies suggested by Vinson et al. \[[@B37]\]. The dominant-inhibitory activities of the latter two proteins A-ATF4 and A-LZIP were verified using electrophoretic mobility shift assay and CAT reporter assay (data not shown). We interrogated these dominant negative bZIP proteins for inhibition of Tax activation of HTLV-I LTR (Fig. [4A](#F4){ref-type="fig"}). All, KCREB, A-CREB, A-ATF4 and TAM67, suppressed Tax activation in a dose-dependent manner (Fig. [4A](#F4){ref-type="fig"}, lanes 3--10 compared to lane 2). However, different dominant negative inhibitors constructed to the same protein using different strategies might have different potencies. For example, KCREB contains a mutation of a single amino acid in the CREB DNA-binding domain \[[@B32]\], whereas A-CREB was constructed by fusing a designed acidic amphipathic extension onto the N terminus of the CREB leucine zipper region \[[@B33]\]. Differential inhibitory effects of KCREB and A-CREB were observed (Fig. [4A](#F4){ref-type="fig"}, lanes 3--6). In light of this, we quantitated and compared the inhibitory activities of dominant negative proteins all constructed using the same strategy (Fig. [4B](#F4){ref-type="fig"}). Since NFκB is not involved in Tax activation of HTLV-I LTR, we included a dominant negative form of IKKβ, IKKβ DN, as a neutral control (Fig. [4B](#F4){ref-type="fig"}, group 7). When we compared four dominant negative bZIP proteins, A-CREB, A-LZIP, A-Fos and A-ATF4, constructed using the identical molecular strategy, we observed the most dramatic suppression of Tax activation of HTLV-I LTR with A-CREB (Fig. [4B](#F4){ref-type="fig"}, group 3, red column). The second most significant reduction in activity was seen with A-LZIP \[[@B36]\] (Fig. [4B](#F4){ref-type="fig"}, group 6, red column). Thus, although several bZIP proteins can redundantly serve to mediate Tax-activation of the LTR, a clear preference for CREB is revealed by our assay.
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Figure 4
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Specific preference for CREB by Tax. (**A**) An example of CAT assay. HeLa cells were transfected with pU3RCAT alone (lane 1), pU3RCAT plus Tax expression plasmid pIEX (lane 2) or pU3RCAT plus pIEX plus increasing amounts (5 to 10 μg) of plasmids expressing the indicated dominant-negative proteins (lanes 3--10). D-Threo-\[dichloroacetyl-1-^14^C\]-chloramphenicol was as used as substrate in the CAT assay. (**B**) Influence of dominant-negative proteins on Tax activation. The cells received pU3RCAT (red) or pKB-SV40-CAT (blue) only (group 1), pU3RCAT/pKB-SV40-CAT plus Tax-expressing plasmid pIEX (group 2) or pU3RCAT/pKB-SV40-CAT plus pIEX plus plasmids expressing the indicated dominant-negative proteins. The empty vector was used to normalize the amount of plasmids given to each group of cells. DN: dominant-negative.
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To verify the specificity of dominant negative effects, we also tested the activities of dominant negative proteins on an NFκB-dependent reporter (Fig. [4B](#F4){ref-type="fig"}, blue columns). Noticeably, none of the dominant negative bZIP proteins had an effect on Tax activation of NFκB (Fig. [4B](#F4){ref-type="fig"}, groups 3--6 compared to group 2, blue columns). In contrast, the expression of IKKβ DN led to more than 50% suppression of NFκB activity (Fig. [4B](#F4){ref-type="fig"}, group 7, blue column). These results ruled out the possibility that A-CREB, A-ATF4, A-Fos and A-LZIP might non-specifically inhibit transcription.
Functional significance of the HTLV-I TATAA element to transcriptional activation by Tax
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In the course of our analyses, we noted that Tax can activate the HTLV-I minimal TATAA-promoter without any known enhancer element by approximately 4-fold (Fig. [1D](#F1){ref-type="fig"}, lane 1). This responsiveness of the HTLV-I minimal promoter is compatible with the concept that the core promoter can also be an important determinant of transcriptional specificity \[[@B2]\]. We next asked whether all TATAA-elements are recognized by Tax in the same way for purposes of activated transcription. Hence, we constructed reporter plasmids that contain two 21-bp repeats and a minimal TATAA promoter from HTLV-I, HIV-1 or SV40 (Fig. [5A](#F5){ref-type="fig"}). Since the TATAA promoters were all placed within the same context, we consider this a valid comparison of their relative responsiveness to Tax activation.
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Figure 5
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Tax preferentially activates the HTLV-I minimal TATAA promoter. (**A**) CAT reporter plasmid. Each plasmid contains two 21-bp repeats and one copy of minimal promoter (TATAA) from HTLV-I, HIV-1 and SV40. The minimal promoter sequences are shown in blue. (**B**) A representative example of CAT assay. The cells received 0, 0.5 and 1 μg of Tax-expressing plasmid pIEX and 5 μg of the indicated CAT reporter constructs (p21-HTLV-CAT, p21-HIV-CAT and p21-SV40-CAT). (**C, D**) Basal and Tax-induced transcriptional activities. HeLa cells were co-transfected with 5 μg of the indicated CAT reporter plasmids (p21-HTLV-CAT, p21-HIV-CAT and p21-SV40-CAT) plus 0.5 μg of pCMV empty vector (w/o Tax) or pIEX (w/ Tax). Basal CAT activity from p21-SV40-CAT-transfected cells was taken as 100% (**C**, column 3).
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While the basal activities of HIV-1 and SV40 minimal promoters were measurably greater than that from HTLV-I (Fig. [5C](#F5){ref-type="fig"}), replacement of the HTLV-I TATAA with the counterpart element from either HIV-1 or SV40 led to a significant reduction in Tax responsiveness (Fig. [5B](#F5){ref-type="fig"}, lanes 4--9; and Fig. [5D](#F5){ref-type="fig"}). To further verify the importance of the TATAA-promoter, we asked the same question using a different approach. Above, Tax was recruited presumably to the downstream TATAA-box via factors bound to the HTLV-1 21bp repeats (see Fig. [5A](#F5){ref-type="fig"}). We next investigated whether the same conclusion could be established if a Gal4 DNA-binding domain-Tax fusion protein (Gal4-Tax) was delivered to downstream TATAA element by tethering to upstream Gal4-binding sites (see Fig. [6A](#F6){ref-type="fig"} for reporter schematic). For this assay, we tested the HTLV-I, the HIV-1, and the E1b TATAA-elements. Consistent with the results from the 21 bp-TATAA experiments (Fig. [5](#F5){ref-type="fig"}), Gal4-Tax activated most strongly the HTLV-I TATAA element (Fig. [6B](#F6){ref-type="fig"}, lane 9 and Fig. [6D](#F6){ref-type="fig"}, group 3) and was minimally potent for the adenoviral E1b promoter (Fig. [6B](#F6){ref-type="fig"}, lane 7 and Fig. [6D](#F6){ref-type="fig"}, group 1). As a control for Gal4-Tax, we checked in parallel the activity of the artificial Gal4-VP16 activator. In contrast with Gal4-Tax, Gal4-VP16 showed no preference for the various TATAA elements (Fig. [6B](#F6){ref-type="fig"}, lanes 4--6 and Fig. [6D](#F6){ref-type="fig"}). Thus, two lines of evidence here support that the HTLV-I TATAA promoter is an additional Tax-specific responsive element.
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Figure 6
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DNA-tethered Tax is specifically active on the HTLV-I minimal promoter. (**A**) CAT reporter plasmid. Each plasmid contains five tandem copies of Gal4-binding sites and one copy of minimal promoter (TATAA) from adenovirus E1b, HIV-1 and HTLV-I. The minimal promoter sequences are shown in blue. (**B**) A representative example of CAT assay. The cells were co-transfected with 2 μg of a Gal4DB plasmid (pM vector alone for lanes 1--3, pGal4-VP16 for lanes 4--6, and pGal4-Tax for lanes 7--9) and 5 μg of a CAT reporter construct (pG5-E1B-CAT for lanes 1, 4 and 7; pG5-HIV-CAT for lanes 2, 5 and 8; and pG5-HTLV-CAT for lanes 3, 6 and 9). (**C, D**) Basal and activated transcriptional activities. HeLa cells were co-transfected with 5 μg of the indicated CAT reporter plasmids (pG5-E1B-CAT, pG5-HIV-CAT and pG5-HTLV-CAT) plus 2 μg of pM empty vector (**C**), pGal4-VP16 (**D**, blue) or pGal4-Tax (**D**, yellow). Basal CAT activity from pG5-HIV-CAT-transfected cells was taken as 100% (**C**, column 2).
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Evidence for Tax activity after assembly of an initiation complex
-----------------------------------------------------------------
Artificial recruitment of TBP to some higher eukaryotic promoters bypasses transcriptional activation by a DNA-tethered activator \[[@B21]-[@B24]\]. When observed at such promoters, this finding is evident that those activators act mechanistically to enhance TBP recruitment to the TATAA box. For general transcriptional activation, additional events subsequent to TBP recruitment are also known to be functionally critical \[[@B21]-[@B23],[@B25]\]. To date, it remains unclear whether Tax works transcriptionally through a mechanism solely to recruit TBP or whether there are additional mechanistic implications after TBP is recruited to the TATAA-element.
To investigate the mechanism(s) of Tax function with respect to TBP recruitment, we constructed a series of reporter plasmids (Fig. [7A](#F7){ref-type="fig"}) with two copies of 21-bp repeat, five copies of Gal4-binding sites and a minimal TATAA sequence from one of four viral promoters (HTLV-I, HIV-1, SV40 and E1b). We artificially delivered TBP to each promoter by provision of Gal4-TBP, and we asked whether Tax has an additional transcriptional effect which is independent of TBP-recruitment to the TATAA-element. If Tax were to serve only for TBP-recruitment, then when TBP is tethered to the TATAA via Gal4-TBP one should expect to see no transcriptional enhancement from Tax. Provocatively, for both the HTLV-I and HIV-1 TATAA elements, Tax stimulated reporter expression greatly over that already achieved with Gal4-TBP (Fig. [7](#F7){ref-type="fig"}, groups 1 and 2). Consistent with above findings, the SV40 and E1b TATAA elements appear to be transcriptionally rate-limited by TBP recruitment, and Tax has minimal activity on these promoters. However, the findings from the HTLV-I and the HIV-1 reporters provide evidence that more than simply accelerating TBP recruitment Tax can serve transcriptional function(s) subsequent to TBP (TFIID) assembly at the core promoter. This is the first time that Tax has been shown to have a role subsequent to transcriptional initiation complex formation at the promoter.
::: {#F7 .fig}
Figure 7
::: {.caption}
######
Tax further activates a promoter with DNA-tethered TBP. (**A**) CAT reporter plasmid. Each plasmid contains two copies of 21-bp repeat, five copies of Gal4-binding sites and one copy of minimal promoter (TATAA) from adenovirus HTLV-I, HIV-1, SV40 and adenovirus E1b. (**B**) CAT assay. HeLa cells were co-transfected with 5 μg of the indicated CAT reporter plasmids (p21-G5-HTLV-CAT, p21-G5-HIV-CAT, p21-G5-SV40-CAT and p21-G5-E1B-CAT) and 2 μg of pGal4-TBP (yellow) or 2 μg of pIEX (Tax; pink) or 2 μg of pGal4-TBP plus 2 μg of pIEX (Gal4-TBP + Tax; blue). Basal CAT activity from cells transfected with pGal4-TBP plus p21-G5-E1B-CAT was taken as 100% (group 4, yellow).
:::
:::
Discussion
==========
Here, we have delineated functional requirements for both the TATAA promoter and the 21-bp enhancer elements in HTLV-I Tax mediated activation of the viral LTR. To date Tax has been considered solely to initiate transcription. Our study shows for the first time that Tax has a transcriptional role after assembly of an initiation complex at the promoter.
Preferential requirements for 21-bp repeats, CREB, and the HTLV-I TATAA box
---------------------------------------------------------------------------
HTLV-I is etiologically associated with adult T-cell leukemia \[[@B38],[@B39]\]. Expression of Tax leads to immortalization of T lymphocytes \[[@B40]-[@B42]\] and transformation of rat fibroblasts \[[@B43],[@B44]\]. Tax is a transcriptional activator that can interact pleiotropically with several different enhancers. In addition to the HTLV-I 21-bp repeats, κB and SRE elements can also mediate Tax activation \[[@B4]-[@B6]\]. Amongst these three enhancers, it is clear that the viral 21-bp repeats are the most highly responsive to Tax-activation (Fig. [1D](#F1){ref-type="fig"}). However, data elsewhere have raised questions as to the identity of the 21-bp binding bZIP factor which is best used to mediate Tax activation \[[@B30]\]. In direct comparisons, we have used matched A-CREB, A-Jun, A-ATF4 and A-LZIP dominant negative mutants to ask which bZIP factor is most contributory to Tax activation. In our cell system, a novel bZIP factor called LZIP \[[@B36]\] can apparently participate in LTR transcription; however, for Tax activation CREB is preferred over ATF4 or c-Jun (Fig. [4](#F4){ref-type="fig"}).
Beyond the requirement for the 21-bp enhancer, our experiments revealed that the HTLV-I TATAA is also specifically preferred by Tax (Fig. [5](#F5){ref-type="fig"} and Fig. [6](#F6){ref-type="fig"}). This finding is consistent with the general notion that core promoters can contribute specificity to transcriptional regulation \[[@B2]\]. Indeed, core promoter preference by other cellular and viral activators such as Sp1, VP16 and Tat have been documented previously \[[@B45]-[@B47]\]. However, the reasons underlying core promoter preferences are poorly understood. TAFs have been suggested to be responsible for the core promoter selectivity of some activators \[[@B48]-[@B50]\]. In this vein, the interaction of Tax with TBP \[[@B11]\] and TBP-associated factors such as TAF~II~28 \[[@B51]\] might provide mechanistic explanations.
Roles of Tax subsequent to TBP recruitment
------------------------------------------
A provocative notion which emerges from our study is that Tax can further activate a promoter at which TBP has already been artificially tethered (Fig. [7](#F7){ref-type="fig"}). Experiments in yeast and mammalian cells indicate that many genes can be activated through artificial recruitment of TBP and other components of the basal transcription machinery to their promoters \[[@B52],[@B53]\]. In yeast, artificial recruitment of TBP bypasses the effect of DNA-tethered activators whereas the activators fail to activate transcription when physically fused to components of the basal transcription machinery \[[@B54]\]. This and other lines of evidence support the notion that activator-dependent recruitment of TBP and basal transcription machinery is a major mechanism for transcriptional activation in yeast cells \[[@B54],[@B55]\]. In contrast, artificial recruitment of TBP to mammalian promoters has not yet been extensively studied. Among the few promoters examined, some such as the ones from E1b and thymidine kinase genes can be fully activated by artificially recruited TBP, while others such as HIV-1 and c-fos promoters are stimulated weakly \[[@B21]-[@B25]\]. On the other hand, some activators such as VP16, E1A, Tat, E2F1 and IE2 work synergistically with artificially recruited TBP, while others such as Sp1 cannot further enhance the activity of DNA-tethered TBP \[[@B21],[@B22]\]. Thus, artificial recruitment of TBP might insufficiently activate transcription in mammalian cells and different activators might function at different steps with respect to TBP recruitment. Our results indicate that DNA-bound TBP can activate HTLV-I LTR only weakly, but its activity is further enhanced by Tax (Fig. [6](#F6){ref-type="fig"}). While such experimental results do not exclude that under physiological circumstances the primary function of Tax may be to enhance initiation complex formation (i.e. TBP-recruitment), they do indicate that Tax has an additional transcriptional activity that extends to phases after transcriptional initiation. Currently, we do not know whether this is at the step of promoter clearance, transcriptional elongation, or some other processes. However, we do believe that Tax should be added to the list of mammalian activators that can function at steps subsequent to TBP recruitment \[[@B21]-[@B25]\].
All the transcriptional assays in the present study were based on transiently transfected reporters. We noted that transiently transfected and stably integrated promoters might behave differently \[[@B24],[@B56]\]. Obviously, chromatin structure and copy numbers can account for significant differences \[[@B56],[@B57]\]. Future experiments are required to verify whether the observations established here also hold for stably integrated HTLV-I LTRs.
Methods
=======
Plasmids
--------
Chloramphenicol acetyltransferase (CAT) reporter plasmid pG5CAT was from Clontech. CAT plasmid pU3RCAT containing the HTLV-I LTR has been previously described \[[@B13]\]. Other CAT plasmids were derived from pCAT-basic (Promega). For each construct, one copy of a minimal promoter and two copies of an enhancer were chemically synthesized and cloned into pCAT-basic. For example, pCRE-HTLV-CAT contains two copies of canonical CRE motif plus one copy of HTLV-I minimal promoter (Fig. [1A](#F1){ref-type="fig"}). Five copies of Gal4-binding sites as in pG5CAT were also inserted in some reporters. All constructs have the same spacing between the TATAA box and the CAT open reading frame (44 bp) or between the enhancer and the TATAA box (23 bp).
Sequences of canonical CRE, Sp1, AP1 and κB motifs in the reporter plasmids have been described \[[@B36],[@B58],[@B59]\]. HTLV-I 21-bp repeats and serum response element (SRE) in the plasmids were derived from the following synthetic oligonucleotides: 21-bp repeats, 5\'-AGCTTAGGCC CTGACGTGTCCCCCTGGATCCTAGGCCCTGACGTGTCCCCCTA-3\' and 5\'-AGCTTAG GGGGACACGTCAGGGCCTAGGATCCAGGGGGACACGTCAGGGCCTA-3\'; SRE, 5\'-AGCTACCATATTAGGATCCATATTAGGT-3\' and 5\'-AGCTACCTAATATGGATCCTAATATGGT-3\'. Sequences of the minimal promoter elements from HTLV-I, HIV-1, SV40 and adenoviral E1b have been described \[[@B60]\]. The SV40 early promoter naturally used for expression of the viral T/t antigens was used.
Expression plasmids for wild type and mutant Tax have been described elsewhere \[[@B26],[@B61]\]. pIEX is a Tax expression vector driven by cytomegalovirus IE promoter. Tax mutants are indicated by the amino acid to be changed, the position of the residue, and the replacement amino acid (e.g. Tax S150A). Amino acids that were removed in mutants are indicated as in Tax Δ3--6. Expression vector pM for Gal4 DNA binding domain (Gal4DB; amino acids 1--147) was from Clontech. Tax, human TBP and the activation domain of VP16 fused to Gal4DB were designated Gal4-Tax, Gal4-TBP and Gal4-VP16, respectively. Expression plasmids for Gal4-Tax and Gal4-TBP have been described \[[@B10],[@B21]\]. Expression plasmid for Gal4-VP16 was from Clontech.
Expression plasmid pRSV-KCREB for the dominant-negative CREB protein KCREB \[[@B32]\] was kindly provided by Dr. Richard Goodman. Expression plasmids pCMV-ACREB and pCMV-AFOS for dominant-negative CREB and AP1 proteins A-CREB \[[@B33]\] and A-Fos \[[@B34]\] were gifts from Dr. Charles Vinson. Expression plasmid pCMV-TAM67 for dominant-negative c-Jun protein TAM67 \[[@B35]\] was from Dr. Michael Birrer. Expression plasmids pCMV-AATF4 and pCMV-ALZIP for dominant-negative ATF4 and LZIP proteins A-ATF4 and A-LZIP were derived from pCMV500 provided by Dr. Charles Vinson \[[@B33],[@B37]\]. A-ATF4 contains 304--352 amino acids of human ATF4 and A-LZIP contains 175--223 amino acids of human LZIP. A-ATF4 and A-LZIP can specifically and dominantly inhibit the CRE-binding and CRE-activating activities of ATF4 and LZIP, respectively, in electrophoretic mobility shift assay and CAT reporter assay (data not shown). Expression plasmid for dominant-negative IKKβ (IKKβ DN) was a gift from Dr. Michael Karin \[[@B62]\].
Reporter assay
--------------
HeLa cells were grown in Dulbecco\'s modified Eagle\'s medium supplemented with fetal calf serum and antibiotics, seeded at 5 × 10^5^cells/well into six-well culture plates and transfected using calcium phosphate method as described \[[@B13]\]. Jurkat cells were cultured in RPMI 1640 medium and transfected by FUGENE 6 reagents (Roche). CAT activity was assayed as previously described \[[@B63]\]. Briefly, transfected cells were harvested and lysed by freezing and thawing. Protein concentration of clarified lysates was determined by Bradford reagent (Bio-Rad). Equal amounts of lysates were mixed with ^14^C-labeled chloramphenicol (Amersham) and acetyl coenzyme A (Calbiochem) for CAT reaction. CAT activities were detected using thin-layer chromatography and quantified by phosphorimager (Molecular Dynamics). For transfection of cells, each well received the same dose of plasmids. The empty vector or pUC19 was added to compensate for the different amounts of plasmids when necessary.
Competing interests
===================
None declared.
Acknowledgements
================
We thank E.W.M. Cheng for technical assistance, R.H. Goodman, C. Vinson, M.J. Birrer and M. Karin for plasmids, and C.M. Wong and M.L. Yeung for critical reading of manuscript. D.-Y. J. is a Leukemia and Lymphoma Society Scholar. This work was supported by a Concern Foundation Research Grant, a Young Investigator Award from the National Natural Science Foundation of China (Project 30029001) and a matching grant from the University of Hong Kong.
| PubMed Central | 2024-06-05T03:55:46.724460 | 2004-7-30 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509288/",
"journal": "Retrovirology. 2004 Jul 30; 1:18",
"authors": [
{
"first": "Yick-Pang",
"last": "Ching"
},
{
"first": "Abel CS",
"last": "Chun"
},
{
"first": "King-Tung",
"last": "Chin"
},
{
"first": "Zhi-Qing",
"last": "Zhang"
},
{
"first": "Kuan-Teh",
"last": "Jeang"
},
{
"first": "Dong-Yan",
"last": "Jin"
}
]
} |
PMC509289 | Introduction {#s1}
============
Hematopoiesis in *Drosophila* shares several features with the analogous process in vertebrates. A first population of embryonic hemocyte precursors (prohemocytes) is specified from the head mesoderm very early during embryogenesis. At the end of larval stages and the onset of metamorphosis, a second population of hemocytes is released from a specialised hematopoietic organ, the larval lymph gland ([@pbio-0020196-Rizki1]; [@pbio-0020196-Tepass1]; [@pbio-0020196-Campos-Ortega1]; [@pbio-0020196-Evans1]; [@pbio-0020196-Holz1]). Both populations give rise to plasmatocytes, which are dedicated phagocytes, and crystal cells, which are responsible for melanisation of pathogens. Lymph glands contain precursors of a third type of hemocyte that is not generated in embryos, the lamellocyte. Lamellocytes are large, adhesive cells devoted to the encapsulation of foreign bodies too large to be phagocytosed; these cells differentiate only in response to specific conditions, such as parasitization of larvae by Hymenoptera ([@pbio-0020196-Lanot1]; [@pbio-0020196-Sorrentino1]). Striking similarities with vertebrate hematopoiesis were revealed when it was shown that Serpent (Srp), a GATA factor, and Lozenge (Lz), a transcription factor related to Runx1/AML1, are required for the development of hemocytes and of crystal cells, respectively ([@pbio-0020196-Rehorn1]; [@pbio-0020196-Lebestky1]; [@pbio-0020196-Orkin1]). However, except for the observation that gain-of-function mutations in the Janus kinase Hopscotch and in the Toll receptor lead to constitutive production of lamellocytes ([@pbio-0020196-Harrison1]; [@pbio-0020196-Luo1]; [@pbio-0020196-Qiu1]), the mechanisms and factors underlying the specification of this cell type remain unknown ([@pbio-0020196-Evans1]; [@pbio-0020196-Meister1]).
During our search for genes involved in specification of lamellocytes, we observed that *collier (col)* is expressed in the lymph glands at the end of embryogenesis ([@pbio-0020196-Kambris1]). The gene *col* encodes the *Drosophila* orthologue of mammalian early B-cell factor (EBF) ([@pbio-0020196-Hagman1]; [@pbio-0020196-Crozatier2]), a key factor controlling B-cell lymphopoiesis in mice ([@pbio-0020196-Lin1]; [@pbio-0020196-Maier1]). We show here that Col activity is required for specification of the lamellocyte lineage in *Drosophila.* On the basis of Col expression and *col* mutant phenotypes, we propose that this factor confers an instructive function on a discrete subpopulation of cells in the *Drosophila* definitive hematopoietic organ.
Results/Discussion {#s2}
==================
Col Expression Identifies Lymph Gland Precursors in Early Embryos {#s2a}
-----------------------------------------------------------------
We first observed that Col is expressed in *Drosophila* lymph glands at the end of embryogenesis ([Figure 1](#pbio-0020196-g001){ref-type="fig"}). In the absence of a specific molecular marker, the embryonic anlage of lymph glands has been mapped to the thoracic lateral mesoderm by lineage analysis of transplanted cells ([@pbio-0020196-Holz1]). By histochemical staining, we observed that Col is expressed in two discrete clusters of cells in the dorsal mesoderm of thoracic segments T2 and T3, starting at the germ-band extension, when lymph gland hemocyte precursors become specified (stage 11; [Figure 1](#pbio-0020196-g001){ref-type="fig"}A) ([@pbio-0020196-Holz1]). These clusters of Col-expressing cells grow closer during germ-band retraction before coalescing to form the paired lobes of the lymph glands (early stage 13; [Figure 1](#pbio-0020196-g001){ref-type="fig"}B and [1](#pbio-0020196-g001){ref-type="fig"}C). Double staining for Col and Odd-skipped, a lymph gland marker expressed from that stage onward ([@pbio-0020196-Ward1]), confirmed that Col-expressing cells are lymph gland precursors ([Figure 1](#pbio-0020196-g001){ref-type="fig"}E). Thereafter, only three to five cells located at the posterior tip of each lobe maintain high levels of Col expression, although low levels are still detected in the other cells of the lymph glands and in some pericardial cells ([Figure 1](#pbio-0020196-g001){ref-type="fig"}D, [1](#pbio-0020196-g001){ref-type="fig"}F, and [1](#pbio-0020196-g001){ref-type="fig"}G). Col expression thus identifies a few cells of the thoracic dorsal mesoderm as the lymph gland primordium and distinguishes a specific posterior region of this hematopoietic organ ([Figure 1](#pbio-0020196-g001){ref-type="fig"}H). The embryonic hematopoietic primordium has been defined as the cephalic domain of Srp expression at the blastoderm stage ([@pbio-0020196-Rehorn1]; [@pbio-0020196-Lebestky1]). Srp is not detected, however, in lymph gland precursors prior to stage 12 (Berkeley Drosophila Genome Project gene expression report \[<http://www.fruitfly.org/cgi-bin/ex/insitu.pl>\]**;** [@pbio-0020196-Lebestky2]). Consistent with this result, larval hematopoietic progenitors expressing Col are observed in *srp^6G^* (an amorphic allele; [@pbio-0020196-Rehorn1]) mutant embryos ([Figure 1](#pbio-0020196-g001){ref-type="fig"}I), indicating that the specification of the embryonic and larval lymph gland progenitors may involve different processes.
::: {#pbio-0020196-g001 .fig}
Figure 1
::: {.caption}
###### Col Expression during Lymph Gland Ontogeny
\(A) Col expression in lymph gland precursors is first observed in two separate clusters of cells (black arrows) in the dorsal-most mesoderm of thoracic segments T2 and T3 at stage 11 (stages according to [@pbio-0020196-Campos-Ortega1]). Col expression in the head region is ectodermal (parasegment 0) and related to its function in head segmentation ([@pbio-0020196-Crozatier3]).
(B and C) The clusters of Col-expressing cells get closer between stage 12 and early stage 13 (B) before coalescing (C).
(D and E) Col expression becomes progressively restricted to the posterior-most cells of the forming lymph glands (arrowhead) during stage 14, as shown by the partial overlap between Odd-skipped (Odd) and Col expression.
(F and G) Enlarged view of lymph glands after completion of embryogenesis, stage 16. Col expression marks the prospective PSC ([@pbio-0020196-Lebestky2]) in a dorsal-posterior position (arrowheads).
\(H) Schematic representation of Col expression in the lymph glands and pericardial cells in stage 16 embryos.
\(I) A *srp^6G^* mutant embryo arrested at stage 13. Col is expressed in the presumptive lymph gland primordium (black arrow), although it is not possible to distinguish between high and low levels of expression. All embryos are oriented anterior to the left. (A--C), (G), and (I) are lateral views; (D--F) are dorsal views. (B), (C), and (E--G) are higher magnifications of the dorsal thoracic region. White arrows in (A) and (I) indicate Col expression in a developing dorsal muscle ([@pbio-0020196-Crozatier1]).
:::
:::
Lamellocyte Differentiation in Response to Parasitization Requires Col Activity {#s2b}
-------------------------------------------------------------------------------
Expression of Col in the embryonic lymph gland prompted us to investigate its possible function during larval hematopoiesis. Loss-of-function mutations of *col* (e.g., *col^1^*) are lethal at the late embryonic stage ([@pbio-0020196-Crozatier3]), but lymph glands form normally, indicating that Col activity is not required for formation of the organ per se. Rescue of the embryonic lethality by expressing the *col* cDNA under the control of a truncated *col* promoter that is active in the head ectoderm but not in the lymph glands ([@pbio-0020196-Crozatier1]) thus allowed us to analyse hematopoiesis in *col^1^* larvae. The presence of plasmatocytes and crystal cells in the circulation of these mutants indicated that *col* is not required for specification of either of these lineages ([Table 1](#pbio-0020196-t001){ref-type="table"}). We then tested the competence of *col^1^* larvae to respond to wasp *(Leptopilina boulardi)* parasitization by producing lamellocytes. This dedicated cellular response is maximal in wild-type *(wt)* larvae 48 h after wasp egg-laying ([Figure 2](#pbio-0020196-g002){ref-type="fig"}A and [2](#pbio-0020196-g002){ref-type="fig"}B) ([@pbio-0020196-Lanot1]). No circulating lamellocytes were detected in the hemolymph of parasitized *col^1^* larvae; as a consequence, the wasp eggs were not encapsulated and they developed into parasitic larvae ([Figure 2](#pbio-0020196-g002){ref-type="fig"}C). That this phenotype completely lacked lamellocytes was confirmed by using a lamellocyte marker, *misshapen-lacZ,* provided by the enhancer trap line *l(3)06949* ([@pbio-0020196-Braun1]). Whereas in *wt* larvae, numerous *lacZ*-positive cells could be seen adhering to and surrounding wasp eggs, no such cells were detected in *col^1^* larvae ([Figure 2](#pbio-0020196-g002){ref-type="fig"}D and [2](#pbio-0020196-g002){ref-type="fig"}E). To ascertain that the absence of lamellocytes was the consequence solely of the *col* mutation, we tested *col^1^* in transheterozygous combinations with two other *col* loss-of-function alleles and over the deficiency *Df(2R)AN293* ([@pbio-0020196-Crozatier1]). In no case did we observe lamellocyte differentiation (we tested 10--20 larvae for each genotype) in response to parasitization by *L. boulardi,* thereby confirming the critical requirement for Col activity in rendering hematopoietic precursors competent to differentiate into lamellocytes. Although gain-of-function mutations that lead to constitutive activation of either the Janus kinase or the Toll signalling pathways result in hematopoietic defects, including differentiation of lamellocytes in the absence of infestation ([@pbio-0020196-Harrison1]; [@pbio-0020196-Luo1]; [@pbio-0020196-Qiu1]), *col^1^* is, to our knowledge, the first identified loss-of-function mutation that abolishes lamellocyte production upon parasitization.
::: {#pbio-0020196-g002 .fig}
Figure 2
::: {.caption}
###### *col* Requirement for Lamellocyte Differentiation
(A--C) 4′,6-diamidino-2-phenylindole (DAPI) staining of hemocytes from *wt* (A and B) and from *col^1^* (C) third instar larvae. (A) Uninfected larva; (B) and (C) infected larvae. Plasmatocytes (inset in \[A\]) are always present, whereas lamellocytes (inset in \[B\]) are detected in the hemolymph of *wt* (B) but not *col^1^* (C) larvae 48 h after infestation by *L. boulardi.* In *col^1^* mutants, the wasp eggs are not encapsulated (white arrows) and develop into larvae (bottom right organism in \[C\]).
(D--F) Lamellocytes expressing the P-*lacZ* marker *l(3)06949* ([@pbio-0020196-Braun1]) surround the wasp eggs in *wt* larvae (D), are completely absent in infected *col^1^* mutant larvae (E), and differentiate in the absence of wasp infection following enforced Col expression in hematopoietic cells (*srpD-Gal4/UAS-col* larvae) (F). (G) *srpD-Gal4/UAS-col* pupa showing the presence of melanotic tumors.
Bars: 50 μm.
:::
:::
::: {#pbio-0020196-t001 .table-wrap}
Table 1
::: {.caption}
###### Circulating Hemocytes in Third Instar Larvae
:::
Values are expressed as mean (SD). Hemocyte types were counted as described in [@pbio-0020196-Duvic1]
^a^Crystal cells were counted in the three posterior-most segments
^b^The strong adhesive properties of lamellocytes preclude an accurate counting of individual cells
^c^Observed in a fraction of the larvae
Bal, balancer chromosome; ND, not determined
:::
Enforced Col Expression Triggers Lamellocyte Differentiation in the Absence of Immune Challenge {#s2c}
-----------------------------------------------------------------------------------------------
We then asked whether forced expression of Col in hematopoietic cells could induce lamellocyte differentiation in the absence of infestation. Because the *e33C-Gal4* line, which drives expression in lymph glands ([@pbio-0020196-Harrison1]) but also epidermis and some other tissues, was lethal in combination with *UAS-col,* we designed a new Gal4 driver. The driver *srpD-Gal4* contains distal elements of the *srp* gene promoter and drives expression of a *UAS* reporter gene in prohemocytes and hemocytes (see below) ([@pbio-0020196-Waltzer1]), with a low level of expression in pericardial cells and the fat body (data not shown). Although embryonic-lethal at 25 °C, the *srpD-Gal4/UAS-col* combination was viable when embryos were allowed to develop to the second larval instar at 18 °C before shifting to 25 °C. Examination of hemolymph samples from late third instar larvae expressing Col under the control of the *srpD-Gal4* driver revealed the presence, in a fraction of the larvae, of numerous lamellocytes identified on the basis of both cell morphology and expression of *misshapen-lacZ* ([Figure 2](#pbio-0020196-g002){ref-type="fig"}F; [Table 1](#pbio-0020196-t001){ref-type="table"}). Around 5% of all larvae developed melanotic tumors ([Figure 2](#pbio-0020196-g002){ref-type="fig"}G), which have been previously observed in other genetic contexts that lead to overproduction of lamellocytes ([@pbio-0020196-Hou1]). This phenomenon is considered to be a consequence of an autoimmune reaction in which hemocytes encapsulate self-tissue ([@pbio-0020196-Sparrow1]). Thus, we conclude that enforced *col* expression in hematopoietic cells can induce differentiation of lamellocytes in the absence of immune challenge. We also observed a concomitant drop in the number of circulating crystal cells ([Table 1](#pbio-0020196-t001){ref-type="table"}), consistent with the hypothesis that lamellocytes and larval crystal cells could differentiate from a common precursor ([@pbio-0020196-Evans1]). No production of lamellocytes was observed, however, when *col* expression was targeted to already specified crystal cells or plasmatocytes by using the *lz-Gal4* ([@pbio-0020196-Lebestky1]) and *hml-Gal4* drivers ([@pbio-0020196-Goto1]), respectively. This indicates that lamellocytes differentiate only when *col* expression is forced in yet-uncommitted progenitors.
Col-Expressing Cells Play an Instructive Role {#s2d}
---------------------------------------------
At the end of larval stages, the lymph gland is composed of four to six paired lobes. The two anterior (primary) lobes that formed in the embryo ([Figure 1](#pbio-0020196-g001){ref-type="fig"}) contain prohemocytes, plasmatocytes, and crystal cells, whereas the posterior (secondary) lobes, which form during the third larval instar, contain predominantly prohemocytes, suggesting that they correspond to a more immature stage of development ([@pbio-0020196-Shrestha1]; [@pbio-0020196-Lanot1]). Col expression in the anterior primary lobes was found to be restricted to a posterior cluster of about 30--40 posterior cells ([Figure 3](#pbio-0020196-g003){ref-type="fig"}A--[3](#pbio-0020196-g003){ref-type="fig"}C). Consistent with, on average, three to four cell divisions between embryo hatching and the third larval instar---as observed both in circulating hemocytes and imaginal tissues ([@pbio-0020196-Schubiger1]; [@pbio-0020196-Qiu1])---these cells are likely to represent the entire progeny of the three to five cells that strongly express Col in the late embryo (see [Figure 1](#pbio-0020196-g001){ref-type="fig"}E and [1](#pbio-0020196-g001){ref-type="fig"}F). They remain clustered at the posterior end of the primary lobes throughout larval development. Col is expressed in a variable number of cells in secondary lobes ([Figure 3](#pbio-0020196-g003){ref-type="fig"}A and [3](#pbio-0020196-g003){ref-type="fig"}C) but is never observed in circulating hemocytes. Despite the dramatic burst of lamellocyte production that occurs in lymph glands when larvae are parasitized (see [Figure 2](#pbio-0020196-g002){ref-type="fig"}) ([@pbio-0020196-Lanot1]; [@pbio-0020196-Sorrentino1]), the number and posterior clustering of Col-expressing cells were unchanged ([Figure 3](#pbio-0020196-g003){ref-type="fig"}D and [3](#pbio-0020196-g003){ref-type="fig"}E). This indicates that the small group of Col-expressing cells are not likely to be the direct precursors of lamellocytes, but rather that they play an instructive role in orienting hematopoietic precursors present in the lymph glands towards the lamellocyte lineage.
::: {#pbio-0020196-g003 .fig}
Figure 3
::: {.caption}
###### Col Expression in Lymph Glands of Third Instar Larvae
(A and B) Col is expressed in the primary lobes, in a posterior cluster of cells (arrow), and in a variable number of secondary lobes. Low expression is also detected in some pericardial cells (asterisks), the significance of which remains unknown. PI, propidium iodide.
\(C) Schematic representation of the lymph glands and Col expression in late third instar larvae.
(D and E) Col expression 24 h (D) and 48 h (E) after wasp infection; despite strong cell proliferation, including in secondary lobes, Col expression remains unchanged (black arrow).
(F--H) Overlap between Ser-lacZ ([@pbio-0020196-Bachmann1]) and Col expression in PSC cells; note a few scattered Ser-expressing cells that do not stain for Col.
Bars: 50 μm (A, B, D, and E) ; 10 μm (F--H).
:::
:::
Col expression in a posterior cluster of cells of the primary lobes is reminiscent of that of Serrate (Ser), a Notch ligand ([@pbio-0020196-Lebestky2]). The Ser/Notch pathway has recently been shown to be essential for crystal cell development ([@pbio-0020196-Duvic1]; [@pbio-0020196-Lebestky2]). Analysis of clones of *Ser* mutant cells in the larval lymph glands further indicated that Ser-expressing cells are responsible for activation of Lz expression in surrounding cells and their commitment to a crystal cell fate ([@pbio-0020196-Lebestky2]). Together with the Ser expression pattern, this observation led the authors to propose that the posterior cluster of Ser-expressing cells could act as a signalling centre, which they termed the posterior signalling centre (PSC). Through double-labelling experiments, we confirmed the overlap between Col and Ser expression (as visualised by Ser-LacZ \[[@pbio-0020196-Bachmann1]\]) in the posterior cells of the primary lobe ([Figure 3](#pbio-0020196-g003){ref-type="fig"}F--[3](#pbio-0020196-g003){ref-type="fig"}H). However, Ser, but not Col, is expressed in scattered cells throughout the primary lymph gland lobes in addition to the PSC ([Figure 4](#pbio-0020196-g004){ref-type="fig"}) ([@pbio-0020196-Lebestky2]).
::: {#pbio-0020196-g004 .fig}
Figure 4
::: {.caption}
###### PSC-Specific Gene Expression Is Dependent upon Col Activity
PSC-specific expression of *col*, Ser-lacZ, and Ser (arrowhead in \[A\], \[C\], and \[E\]) is lost in *col^1^* mutant larvae (B, D, and F); only Ser expression in scattered cells is maintained (arrow in \[E\] and \[F\]). Bar: 50 μm.
:::
:::
PSC-Specific Gene Expression Is Dependent upon Col Activity {#s2e}
-----------------------------------------------------------
Because Col expression and function suggested that the PSC was playing an instructive role in orienting other lymph gland cells towards the lamellocyte fate, we asked whether Col was necessary for the PSC to form properly. We looked at *col* and *Ser* expression in *col^1^* mutant lymph glands, using in situ hybridisation for *col* because Col antibodies do not recognise the Col^1^ protein ([@pbio-0020196-Crozatier1]). In *wt* larvae, consistent with the results of immunostaining, *col* transcripts were restricted to the PSC ([Figure 4](#pbio-0020196-g004){ref-type="fig"}A). In contrast, we could not detect *col* expression in *col^1^* mutant lymph glands ([Figure 4](#pbio-0020196-g004){ref-type="fig"}B). Furthermore, expression both of Ser-lacZ and Ser in the PSC ([Figure 4](#pbio-0020196-g004){ref-type="fig"}C and [4](#pbio-0020196-g004){ref-type="fig"}E) was also abolished ([Figure 4](#pbio-0020196-g004){ref-type="fig"}D and [4](#pbio-0020196-g004){ref-type="fig"}F), indicating that proper specification of PSC identity is dependent upon Col activity. Although Ser expression was lost from the PSC region, it was still observed in scattered cells in the primary lobe ([Figure 4](#pbio-0020196-g004){ref-type="fig"}E and [4](#pbio-0020196-g004){ref-type="fig"}F, arrows), suggesting that Ser-lacZ expression reflected the presence of a PSC-specific transcriptional enhancer without reproducing the entire Ser expression pattern.
Evidence for a Bipotential Crystal Cell/Lamellocyte Precursor {#s2f}
-------------------------------------------------------------
Ser signalling through the Notch signalling pathway is critical for the specification of crystal cell precursors ([@pbio-0020196-Duvic1]; [@pbio-0020196-Lebestky2]). However, numerous crystal cells differentiate in *col* mutant lymph glands, including in secondary lobes, despite the loss of Ser expression in the PSC (see [Figures 4](#pbio-0020196-g004){ref-type="fig"}E, [4](#pbio-0020196-g004){ref-type="fig"}F, [5](#pbio-0020196-g005){ref-type="fig"}A, and [5](#pbio-0020196-g005){ref-type="fig"}B). These data, together with the clonal analysis of [@pbio-0020196-Lebestky2], lead us to conclude that crystal cell development is triggered by signalling from the scattered Ser-expressing lymph gland cells, rather than from the PSC itself. In contrast, no differentiating lamellocytes could be detected in *col* mutant lymph glands, even under conditions of wasp infestation that induced massive lamellocyte differentiation in *wt* glands ([Figure 5](#pbio-0020196-g005){ref-type="fig"}C--[5](#pbio-0020196-g005){ref-type="fig"}F), confirming the key role of the PSC in this process.
::: {#pbio-0020196-g005 .fig}
Figure 5
::: {.caption}
###### Col-Expressing Cells Play an Instructive Role in Lamellocyte Production
Expression of the crystal cell marker *doxA3* ([@pbio-0020196-Waltzer1]) (A, B, and G); of the lamellocyte markers *α-ps4* (M. Meister, unpublished data) (C--F and H) and L1 ([@pbio-0020196-Asha1]) (J); and of Col (I and J); in *wt* (A, C, and E), *col* loss-of-function mutant (B, D, and F), and *srp-Gal4/UAS-col* (G--J) larvae. In (E) and (F), larvae were taken 48 h after infestation. An increased number of *doxA3*-positive cells (B) parallels the absence of lamellocyte differentiation (F) in *col^1^* mutant lymph glands. Conversely, lamellocyte differentiation and a reduced number of *doxA3*-positive cells are observed upon enforced Col expression (G and H). Double staining for Col and L1 shows that Col-expressing cells and differentiating lamellocytes do not overlap in the lymph gland. (I) shows ectopic Col expression compared to expression in the PSC (arrowhead; not visible in \[J\]). Antibody and in situ probes are indicated on each panel. In all panels, larvae are oriented with the head to the left: a single primary lobe is shown, with sometimes a few secondary lobes. Bar: 50 μm.
:::
:::
We then looked at the production of crystal cells and lamellocytes in lymph glands with enforced Col expression (*srpD-Gal4/UAS-col*; [Figure 5](#pbio-0020196-g005){ref-type="fig"}G--[5](#pbio-0020196-g005){ref-type="fig"}J). Very few crystal cells and numerous lamellocytes were observed, consistent with the circulating hemocyte picture ([Figure 5](#pbio-0020196-g005){ref-type="fig"}G and [5](#pbio-0020196-g005){ref-type="fig"}H). The *srpD-Gal4*-driven Col expression in the lymph gland is not uniform. Some cells express high levels when compared to the PSC, whereas many others show no detectable expression. A similar pattern was also observed in combination with UAS-lacZ ([Figure 5](#pbio-0020196-g005){ref-type="fig"}I; data not shown). Double-labelling experiments showed that the lymph gland cells induced to differentiate into lamellocytes surround but do not overlap with the Col-expressing cells ([Figure 5](#pbio-0020196-g005){ref-type="fig"}J), confirming the instructive role of Col-expressing cells. In all genotypes that we tested, we found equally large numbers of plasmatocytes in the lymph glands (data not shown), which indicates that this cell type is not affected by *col* loss-of-function and gain-of-function mutations. Altogether, the absence of lamellocytes after parasitization that is associated with the increase in the number of crystal cells in *col* mutant lymph glands, and the opposite situation in *srpD-Gal4/UAS-col* lymph glands ([Figure 5](#pbio-0020196-g005){ref-type="fig"}; [Table 1](#pbio-0020196-t001){ref-type="table"}), support the existence of bipotential crystal cell/lamellocyte precursors.
A Model for Induction of Lamellocytes in Response to Parasitization {#s2g}
-------------------------------------------------------------------
In summary, our data show that (i) Col expression defines a specific group of cells within the lymph glands; (ii) lamellocyte differentiation, which is an exclusive feature of lymph gland hematopoiesis, depends upon Col activity; and (iii) the massive production of lamellocytes that follows parasitization does not involve changes in Col expression. We thus propose a two-step signalling model for induction of lamellocytes in response to wasp egg-laying ([Figure 5](#pbio-0020196-g005){ref-type="fig"}A). According to this scheme, Col endows PSC cells with the competence to respond to a primary signal emitted by plasmatocytes as these permanent immune supervisors form a first layer around the parasite egg ([@pbio-0020196-Russo1]). Subsequently, PSC cells send a secondary signal that orients prohemocytes towards the lamellocyte fate. The production of lamellocytes upon enforced *col* expression suggests that the need for the primary signal to activate the secondary signal can be bypassed in overexpression experiments. Although several aspects of this model remain to be translated into molecular terms, it certainly sheds a new light on the genetic control of hemocyte lineages in *Drosophila.*
Concluding Remarks {#s2h}
------------------
B- and T-lymphocytes mediate adaptive immunity, a phylogenetically recent component of the immune system as it is found only in gnathostomes ([@pbio-0020196-Kimbrell1]; [@pbio-0020196-Mayer1]). How adaptive immunity emerged during evolution, and was built on top of the innate immune system by which it is controlled and assisted, remains a fascinating question. The requirement for Col function in the *Drosophila* cellular immune response, and EBF function in B-cell development in vertebrates, suggests that Col/EBF function was co-opted early during the evolution of cellular immunity. A puzzling question remains, however, of how the cell-autonomous function of EBF in B-cell development, and the non--cell-autonomous function of Col in lamellocyte development, could relate to an ancestral Col/EBF function. We would like to propose that the ancestral expression of Col/EBF in a subset of hematopoietic cells conferred on these cells the ability to respond to signals from circulating immune supervisors (generically designated as macrophages in [Figure 6](#pbio-0020196-g006){ref-type="fig"}) and provide a secondary line of defence against specific intruders. This cell-specific property in turn laid the ground for the emergence of the vertebrate lymphoid cells on one side and the *Drosophila* PSC on the other. Although admittedly highly speculative, this proposal takes into account the following considerations. B-cell development represents the default fate of lymphoid progenitors ([@pbio-0020196-Schebesta1]; [@pbio-0020196-Warren1]). Although specification of B-cells critically depends on EBF (and the basic helix-loop-helix protein E2A), commitment depends on another gene, *Pax5.* The *Pax5^−/−^* pro--B-cells retain the ability to generate a whole range of both 'innate' myeloid and lymphoid cells ([@pbio-0020196-Nutt1]; [@pbio-0020196-Rolink1]; [@pbio-0020196-Mikkola1]). Thus, the ontogeny of the B-cell lineage from preexisting myeloid cell types has occurred through several steps, one key event being the co-opting of *Pax5,* acting downstream of EBF, for which there is no known counterpart in *Drosophila* hematopoiesis. Second, the co-opting of Col activity for lamellocyte differentiation in larval hematopoiesis most likely came on top of a preexisting hematopoietic system, such as that operating in *Drosophila* embryos ([@pbio-0020196-Evans1]; [@pbio-0020196-Meister1]). Further investigation of Col/EBF functions in intermediate phyla should provide more insight into the diversity of myeloid lineages and ontogeny of the lymphoid lineages during evolution.
::: {#pbio-0020196-g006 .fig}
Figure 6
::: {.caption}
###### A Model for Lamellocyte Specification
\(A) A model for the induction of lamellocyte differentiation in the *Drosophila* lymph glands in response to wasp parasitization. Col enables PSC cells to respond to a primary signal (S1) that is likely emitted by plasmatocytes upon their encounter with a parasite ([@pbio-0020196-Russo1]; [@pbio-0020196-Meister1]). As a result, the PSC cells send a secondary signal (S2) that causes prohemocytes to develop into lamellocytes. Notch (N) signalling instructs a fraction of prohemocytes to become crystal cells ([@pbio-0020196-Duvic1]; [@pbio-0020196-Lebestky2]). The circular arrow indicates that increased proliferation leading to increased numbers of crystal cells and lamellocytes follows parasitization ([@pbio-0020196-Sorrentino1]).
\(B) Schematic view of hematopoiesis in *Drosophila* and mouse*.* Left: Lymph gland cells contain two types of hematopoietic cells, PSC cells and uncommitted precursors. These precursors can give rise to either plasmatocytes or crystal cells. Crystal cell precursors can also give rise to lamellocytes upon receiving a signal from the PSC cells expressing Col (dotted arrows); this signalling is itself dependent upon a communication between circulating plasmatocytes and the PSC (A). Right: In mice, hematopoietic stem cells (HSC) give rise to common myeloid precursors (CMP) and common lymphoid precursors (CLP) (adapted from [@pbio-0020196-Orkin1] and [@pbio-0020196-Schebesta1]). Signalling between CMP- and CLP-derived cells is an essential component of adaptive immunity. Col and EBF functions, in *Drosophila* and vertebrate hematopoiesis, respectively, suggest an ancestral role in their conferring on a subset of hematopoietic cells the ability to respond to signals from circulating immune supervisors (generically designated here as macrophages) and to provide a secondary line of defence against specific intruders.
:::
:::
Materials and Methods {#s3}
=====================
{#s3a}
### Fly stocks and hemocyte counting {#s3a1}
Unless otherwise stated, all fly stocks were maintained at 25 °C on standard medium, and genotypes were verified with marked balancer chromosomes. For wasp infection, second instar larvae were submitted to egg-laying by L. boulardi for 2--4 h, then allowed to develop at the appropriate temperature and analysed 24 or 48 h later. Hemocyte observation and counting, and *lacZ* staining of lamellocytes, were as previously described ([@pbio-0020196-Braun1]; [@pbio-0020196-Duvic1]).
### Transgenic constructs and flies {#s3a2}
The *srpD-Gal4* transgene: A distal promoter fragment, between 8.8 and 6 kb upstream of the *srp* transcription start site and a 340-bp fragment overlapping this site were amplified by PCR using 5′-GCTAGCGACGCGTGATGCAACTTAATCAA-3′ and 5′-CTGCAGTTTATGAATGGAAGACGCGGACG-3′ primers, and 5′-CTGCAGACGGCCAAGTCCAACAACAACAA-3′ and 5′-GGATCCCTGTTGCTGCTGTAACTGTTGAT-3′ primers, respectively, then fused before subcloning upstream of the *Gal4* coding sequence in a pCaSpeR vector. Transgenic lines were obtained by standard procedures. Because they are embryonic-lethal at 25 °C, the *srpD-Gal4/UAS-col* animals were kept at 18 °C before shifting to 25 °C at the second larval instar.
### Immunostaining and in situ hybridisation. {#s3a3}
Immunostaining and in situ hybridisation of larval lymph glands and embryos were performed as in [@pbio-0020196-Crozatier1] using rabbit anti-Col (1:250), rat anti-Ser (gift from K. Irvine; 1:500), mouse anti-β-galactosidase (Promega, Madison, Wisconsin, United States; 1:1000), mouse lamellocyte-specific L1 (gift from I. Ando; 1:10), and guinea-pig anti-Odd-skipped (gift from J. B. Skeath; 1:100). Peroxidase and Alexa Fluor 546 or 488 labelled secondary antibodies (Molecular Probes, Eugene, Oregon, United States) were used at a 1:500 dilution. In some cases, lymph glands were incubated for 30 min at 37 °C in a propidium iodide solution in the presence of RNase. Mounting in Vectashield medium (Vector Laboratories, Burlingame, California, United States) preceded observation by confocal microscopy (Zeiss LSM 510 \[Zeiss, Oberkochen, Germany\] and Leica SP2 \[Leica, Wetzlar, Germany\]). Single-stranded digoxigenin-labelled RNA probes were synthesised from corresponding cDNAs cloned in pGEM (Promega).
We thank M. Lagueux for help with the *srpD-Gal4* transgene; E. Knust, U. Banerjee, R. Reuter, and the Bloomington Stock Center for fly stocks; J. B. Skeath, I. Ando, and K. Irvine for antibodies; and J. A. Hoffmann, J. Smith, L. Waltzer, and many colleagues for critical reading of the manuscript and discussion. We are grateful to J. Mutterer and B. Ronsin for assistance with confocal microscopy. This work was supported by Centre National de la Recherche Scientifique, EntoMed, Exelixis Inc., l\'Association de la Recherche contre le Cancer, and Ministère de la Recherche (ACI Biologie du Développement).
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** MC, AV, and MM conceived and designed the experiments. MC, J-MU, and MM performed the experiments. MC, AV, and MM analysed the data. AV and MM wrote the paper.
Academic Editor: Michael Levine, University of California, Berkeley
Citation: Crozatier M, Ubeda JM, Vincent A, Meister M (2004) Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier. PLoS Biol 2(8): e196.
Col
: Collier
EBF
: early B-cell factor
Lz
: Lozenge
PSC
: posterior signalling centre
Ser
: Serrate
Srp
: Serpent
*wt*
: wild-type
| PubMed Central | 2024-06-05T03:55:46.727383 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509289/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e196",
"authors": [
{
"first": "Michèle",
"last": "Crozatier"
},
{
"first": "Jean-Michel",
"last": "Ubeda"
},
{
"first": "Alain",
"last": "Vincent"
},
{
"first": "Marie",
"last": "Meister"
}
]
} |
PMC509290 | Introduction {#s1}
============
DNA base substitutions do not occur randomly ([@pbio-0020199-Graur1]). Instead, they may be clustered in hotspots, for example around methylated CG dinucleotides, or subject to more general biases such as the excess of transitions relative to transversions. In addition, local structural context may be important, with neighbouring bases interacting to favour some changes over others ([@pbio-0020199-Blake1]; [@pbio-0020199-Morton1]; [@pbio-0020199-Goodman1]; [@pbio-0020199-Zavolan1]). However, many nonrandom patterns of sequence evolution remain unexplained. Here we explore how an abundant class of repetitive sequences, microsatellites, may influence the pattern of mutations in sequences that surround them.
Microsatellites are sequences of repeated 1--6-bp motifs that mutate primarily through the gain and loss of repeat units, in a process thought to depend on DNA replication slippage ([@pbio-0020199-Levinson1]; [@pbio-0020199-Tautz1]). Previous studies indicate that their flanking sequences evolve unusually and often contain mutated versions of microsatellites ([@pbio-0020199-Matula1]). Estimates of flanking sequence mutation rates vary greatly. Very slow evolution is suggested by sequence comparisons between distantly related species, where divergence rates may be as low as 0.016% to 0.1% per million years ([@pbio-0020199-Schlotterer2]; [@pbio-0020199-Rico1]; [@pbio-0020199-Zardoya1]). Elsewhere, pedigree studies suggest much higher rates and even hypermutability ([@pbio-0020199-Stallings1]). There is also disagreement about trends in mutation rate, some studies indicating an increase towards the microsatellite ([@pbio-0020199-Blanquer-Maumont1]; [@pbio-0020199-Zardoya1]; [@pbio-0020199-Grimaldi1]; [@pbio-0020199-Brohede1]) while others claim a more even distribution ([@pbio-0020199-Karhu1]).
To our knowledge, no one has yet conducted a systematic study of mutational biases operating around microsatellites. The direct study of naturally occurring mutations in flanking sequences is virtually prohibited by their slow rate of accumulation, and inferences based on comparisons between homologous microsatellite loci rely on small numbers of sequences. However, an indirect approach is possible, based on comparisons among very large numbers of microsatellite flanking sequences from the finished human genome. If microsatellites have little or variable influence on their flanking regions, among-locus similarities will be minimal or absent. Conversely, if microsatellites generate similar local mutation biases, nonhomologous loci should betray evidence of convergent evolution. With the publication of large blocks of sequence from the chimpanzee genome, one can extend this approach to ask questions about rate of divergence between homologous flanking sequences.
Here we use a combination of these indirect approaches to show that microsatellites appear to create regions around them in which both the rate and spectrum of mutations are modified.
Results {#s2}
=======
We studied the most abundant class of human dinucleotide repeats, (AC)*~n~,* and for simplicity considered only 'isolated' repeats, defined as those at least 100 bp from the nearest AC repeat as small as two units in length. 47% of AC repeats on human Chromosome 1 match these criteria. From the human genomic sequence, maximum sample size was set at 5,000 randomly selected loci for length classes (AC)~2~ to (AC)~5~. For longer microsatellites, of which fewer than 5,000 could be found, all sequences encountered were included. [Figure 1](#pbio-0020199-g001){ref-type="fig"} displays the length frequency distribution and sample sizes. Additionally, a control set of 5,000 randomly selected, non-microsatellite-associated sequences, each 50 bases long and containing no (AC)~2+~ repeats, was generated from Chromosome 1.
::: {#pbio-0020199-g001 .fig}
Figure 1
::: {.caption}
###### Frequency Distribution of Isolated, Pure (AC)*~n~* Microsatellite Lengths (in Repeat Units) in the Human Genome
Black shading indicates numbers of microsatellites used for analyses in this study.
:::
:::
Distribution of Cassette Type {#s2a}
-----------------------------
To distinguish between (AC)*~n~* and (CA)*~n~* repeats, (AC)*~n~* microsatellites were divided into subclasses, termed 'cassettes', according to their immediate 5′ and 3′ flanking bases such that 5′-X(AC)*~n~*Y-3′ is referred to as cassette X/Y. Hence, a (CA)~3~ repeat would be classed as cassette C/A around (AC)~2~. Such a distinction may appear pedantic, but since both DNA replication fidelity and repair efficiency are known to be influenced by base order and local sequence context ([@pbio-0020199-Goodman1]; [@pbio-0020199-Marra1]), it seems by no means certain that (AC)*~n~* and (CA)*~n~* are equivalent. This nomenclature also helps to resolve the problem of defining microsatellite length because (CA)~3~ equals (AC)~2~. [Figure 2](#pbio-0020199-g002){ref-type="fig"} shows the frequency distribution of cassette types relative to expectations, calculated assuming that each cassette base forms a dinucleotide with the end base of the microsatellite it flanks. Thus, cassette X(AC)*~n~*Y is viewed as comprising two dinucleotides, XA and CY. The probability of observing XA and CY jointly is then calculated from the individual frequencies of XA and CY estimated using 1 Mb of randomly sampled sequence from Chromosome 1 containing no (AC)~2+~ repeats.
::: {#pbio-0020199-g002 .fig}
Figure 2
::: {.caption}
###### Frequency Distribution of the 16 Possible Microsatellite-Flanking 5′--3′ Base Combinations Relative to Random Expectation
\(A) Cassette frequencies around (AC)~2~ microsatellites: black bars, observed; white bars, expected. Error bars show 95% confidence intervals and asterisks indicate significant difference (*χ^2^* tests, 1 d.f. *p* \< 0.05 with sequential Bonferroni corrections).
\(B) Deviation of cassette frequencies from random expectations around (AC)~2~, (AC)~5~, and (AC)~10~ microsatellites: black, white, and hatched bars, respectively.
\(C) Sampled number (solid line) and proportion (dotted line) of microsatellites with cassette T/A as a function of microsatellite length.
:::
:::
Around (AC)~2~ microsatellites, the cassette frequencies are broadly similar to those expected from the frequencies of the component dinucleotides in unique sequence DNA, though eight cassettes show significant differences ([Figure 2](#pbio-0020199-g002){ref-type="fig"}A). However, as AC repeat number increases, the relative frequencies of several cassettes begin to deviate more and more from expectations, either decreasing or increasing in frequency, summarised in [Figure 2](#pbio-0020199-g002){ref-type="fig"}B. Specifically, cassettes of the kind X/A, and particularly T/A are overrepresented, while cassettes X/C and X/T tend to be underrepresented. The total proportion of (AC) repeats with cassette T/A are shown as a function of repeat length in [Figure 2](#pbio-0020199-g002){ref-type="fig"}C. The observed pattern could arise either if cassette type influences the rate at which microsatellites change length, or if mutation biases generated by the microsatellite cause interconversion between cassette types.
Flanking Sequence Base Composition {#s2b}
----------------------------------
We consider flanking sequences to extend 50 bases either side of a microsatellite. To compare flanking sequences, we first divided microsatellites according to (AC) repeat number and cassette type, and then calculated the frequency of each of the four nucleotides at each of the 100 possible positions. Any mutation biases present should be revealed by locally changed base composition, and this appears to be the case. For many microsatellite length--cassette combinations, the flanking sequences exhibit strong deviations from random. The observed patterns can be placed in six broad classes according to the strength of a two-base periodicity and the degree of 5′ to 3′ asymmetry. These patterns are illustrated in [Figure 3](#pbio-0020199-g003){ref-type="fig"} and summarised in [Table 1](#pbio-0020199-t001){ref-type="table"}. Three further classes of less regular patterning can also be defined ([Figure S1](#sg001){ref-type="supplementary-material"}). For illustration, we chose length (AC)~5~, since this exhibits the strong patterns while at the same time retaining sufficient sample sizes for analyses to be conducted on the rarer cassette types. Several features are apparent. First, levels of patterning can be remarkably strong, with the probability of observing a given base at a given site varying from less than half of that in unique sequence to more than double, often at adjacent sites. Second, many of the patterns show strong dinucleotide periodicities, presumably reflecting the dinucleotide structure of the microsatellite. Third, there are several examples of clear 5′ to 3′ asymmetry, indicating that mutational patterns on one side of a microsatellite may not be the same as those on the other.
::: {#pbio-0020199-g003 .fig}
Figure 3
::: {.caption}
###### Flanking Sequence Frequency Distributions for Six Representative Nucleotide--Cassette Combinations for (AC)~5~ Microsatellites
In each panel, the microsatellite is centrally placed, represented as a gap at position zero, and the cassette type, base, and number of sequences considered *(n)* are given. Frequency distributions are plotted with separate 95% confidence intervals for odd- and even-numbered positions (shading). Horizontal lines indicate mean frequencies for the 3′ and 5′ flanking regions, calculated separately. (A--F) illustrate the six main classes of patterning where either dinucleotide periodicity or 5′--3′ asymmetry are present, summarised for all cassette--base combinations in [Table 1](#pbio-0020199-t001){ref-type="table"}.
:::
:::
::: {#pbio-0020199-t001 .table-wrap}
Table 1
::: {.caption}
###### Summary of Patterns in Flanking Sequence Base Frequencies by Cassette
:::
:::
As well as showing variation between cassettes, flanking sequence patterning also changes with microsatellite length. For those cassettes where sample size is sufficient to show a trend, that is, T/A and to some extent C/A and A/A, both amplitude of deviation from random and the breadth of patterning tend to increase with increasing AC repeat number. We do not see dramatic changes such as a reversal in 5′ to 3′ asymmetry or the emergence of new patterns. For cassette T/A, pattern strength increases up to (AC)~9~ but then declines in longer microsatellites. [Table 1](#pbio-0020199-t001){ref-type="table"} reveals a dominant role for nucleotides A and T, with excesses of base A tending to be complemented by deficits at the same positions of base T. In some cases, the excesses of A are interleaved with only weak deviations from base frequency expectation. In other cases, excesses of A are interleaved with excesses of base T.
Dinucleotide Patterns {#s2c}
---------------------
In view of the strong two-base periodicities seen for some base--cassette combinations, we next examined the distribution of frequencies of all 16 possible dinucleotide motifs. As expected from the single nucleotide patterning, dinucleotides also tend to show periodic patterning ([Figures 4](#pbio-0020199-g004){ref-type="fig"} and [S2](#sg002){ref-type="supplementary-material"}), and this is particularly pronounced for motif AT. As with the mononucleotide patterns, there is often marked 5′ to 3′ asymmetry (for example, cassette T/T, dinucleotide AT; [Figure 4](#pbio-0020199-g004){ref-type="fig"}B). Frequency plots for the 16 cassette types reveal patterns that fall into classes similar to those observed for the mononucleotides, summarised in [Table 2](#pbio-0020199-t002){ref-type="table"}. Both the presence of patterning and the degree of 5′ to 3′ asymmetry show a strong dependence on cassette type.
::: {#pbio-0020199-g004 .fig}
Figure 4
::: {.caption}
###### Flanking Sequence Frequency Distributions for Three Representative Dinucleotide Motif--Cassette Combinations for (AC)~5~ Microsatellites
(See Figure S4 for the four other patterns). In each panel, the microsatellite is centrally placed, represented as a gap at position zero, and the cassette type, dinucleotide motif, and number of sequences considered *(n)* are given. Frequency distributions are plotted with separate 95% confidence intervals for odd- and even-numbered positions (shading). Horizontal lines indicate mean frequencies for the 3′ and 5′ flanking regions, calculated separately. A summary of how all seven patterns are distributed among all dinucleotide motif--cassette combinations is given in [Table 2](#pbio-0020199-t002){ref-type="table"}.
:::
:::
::: {#pbio-0020199-t002 .table-wrap}
Table 2
::: {.caption}
###### Summary of Patterns in Flanking Sequence Dinucleotide Frequencies by Cassette
:::
:::
From [Table 2](#pbio-0020199-t002){ref-type="table"} it seems that patterning occurs most commonly where the 5′ cassette base is T or the 3′ cassette base is A and least commonly where the 5′ cassette base is either G or A. In almost all cases where patterning is recorded, the dinucleotide involves one or both bases present in the cassette. However, it is unclear whether the flanking pattern is simply an extension of the cassette bases. For example, although 5′ AT or TA periodicity only occurs where the 5′ cassette base is T, CC deviations occur where the 5′ cassette base is A (cassette A/C).
As with the mononucleotide patterning, the periodicity in dinucleotide frequencies changes in amplitude and width (5′ to 3′) as repeat number increases. For most cassettes, the paucity of long microsatellites precludes study of how the patterning changes with repeat number. However, cassette T/A is sufficiently abundant for the progression to be described, and cassettes A/A and C/A, although less common, nonetheless yield meaningful results. [Figure 5](#pbio-0020199-g005){ref-type="fig"}A--[5](#pbio-0020199-g005){ref-type="fig"}F shows, for cassette T/A, how the patterning of motif AT first becomes detectable by eye around (AC)~3~, then increases towards a peak in strength at (AC)~9~ before diminishing as AC repeat number increases further. Cassettes A/A and C/A show similar trends and together suggest that, where patterning occurs, it is apparent by (AC)~6~. When data were plotted without first categorizing them by cassette type, although a number of the dinucleotide patterns in [Table 1](#pbio-0020199-t001){ref-type="table"} were seen, deviations from expectation were weaker (for example, about half as strong for dinucleotide AT).
::: {#pbio-0020199-g005 .fig}
Figure 5
::: {.caption}
###### Dependence of Dinucleotide Flanking Sequence Patterning on AC Repeat Number
Plots are as described in [Figure 4](#pbio-0020199-g004){ref-type="fig"}. The progression for dinucleotide AT is illustrated for the commonest cassette type, (T/A). (A--F) depict AT dinucleotide frequencies, where patterning is most extreme, and show how periodicity and amplitude increase towards a maximum at around (AC)~10~ and decline thereafter.
:::
:::
Other Repetitive Elements in the Genome {#s2d}
---------------------------------------
An artefactual appearance of convergent evolution could arise if microsatellites within major classes of interspersed repetitive elements such as LINE and Alu repeats are treated as independent observations. Such loci will often share a common origin, and hence appear more similar to each other than expected. To address this problem, we used the program RepeatMasker ([@pbio-0020199-Smit1]) to divide Chromosome 1 into sequences related or not related to known interspersed repeats. Just under half (45%) of all isolated microsatellites were found within interspersed repeats, but only a minority contained microsatellites as long or longer than (AC)~5~. Classifying loci by cassette type, length, and whether they occurred in LINE/L1, SINE/Alu, or unique sequence DNA yielded in most classes sample sizes too low to be of use. However, where sample sizes were adequate, that is, cassette T/A, dinucleotide AT, patterning in unique sequence loci was indistinguishable from that in LINE and SINE microsatellites ([Figure S3](#sg003){ref-type="supplementary-material"}). If the among-locus similarities were due to shared evolutionary history, we would expect flanking sequences in the three classes to differ. That they do not, suggests an evolutionary process that depends little if at all on original context.
Microsatellites in the Flanking Sequence {#s2e}
----------------------------------------
The strongest two-base periodicities we find tend to involve motif AT. Such periodicity might arise in two main ways: either because AT motifs in phase with the microsatellite tend to expand through slippage to form (AT)*~n~* microsatellites, or because mutation biases favour the formation of AT motifs in phase with the AC tract. Consequently, we examined the extent to which patterning could be reduced by filtering the flanking sequences for the presence of AT motifs, focusing on cassette T/A and (AC)~5~ to provide strong patterning and large sample sizes. The results of deleting all flanking sequences containing AT microsatellites with *n* or more repeats, where *n* = 2, 3, 4, and 5, are given in [Figure 6](#pbio-0020199-g006){ref-type="fig"}. This filtering effectively abolishes patterning in all but the region immediately adjacent to the AC repeat tract. Here, patterning extends as far as the maximum value of *n* allowed, suggesting that the dominant AT patterning results from (AT)*~n~* microsatellites developing immediately adjacent to the AC tract.
::: {#pbio-0020199-g006 .fig}
Figure 6
::: {.caption}
###### Dependence of Dinucleotide Pattern Strength on the Presence of Repeat Clusters
Beginning with the dataset from the scenario showing strong patterning and large sample size (cassette T/A, dinucleotide AT, (AC)~5~; see [Figure 5](#pbio-0020199-g005){ref-type="fig"}C), flanking sequences containing (AT)*~x~* were excluded, where *x* equalled 2 or more (A), 3 or more (B), 4 or more (C), and 5 or more (D). Plotting conventions are the same as for [Figure 4](#pbio-0020199-g004){ref-type="fig"}.
:::
:::
We next examined whether the phase of AT dinucleotides was determined solely by their tendency to form clusters next to (AC)*~n~* repeats. This is an important test given that our strict definition of a microsatellite restricts our analysis to pure AC repeats and allows the possibility that compound repeats, for example, (AT)*~n~*(AC)*~n~*(AT)*~n~* are included. To do this, we plotted the distribution of single AT motifs around (AC)~3+~ microsatellites in the subset of flanking sequences with no (AT)*~n~* microsatellites, where *n* \> 1. Summed over all AC microsatellite lengths, single AT dinucleotides are overrepresented in 5′ sequences at odd numbered positions and at even numbered positions in 3′ sequences ([Figure 7](#pbio-0020199-g007){ref-type="fig"}). Excesses (or deficits) in AT occur up to six bases away from the microsatellites, suggesting that the periodic patterns we see do indeed occur in the flanking sequence over and above the generation of AT microsatellites adjacent to the microsatellite itself. In other words, the patterning we see is generated beyond any tendency for our strict definition of a pure AC repeat to include compound repeats.
::: {#pbio-0020199-g007 .fig}
Figure 7
::: {.caption}
###### Location of Single AT Dinucleotide Motifs Relative to the Central AC Microsatellite in Flanking Sequences Lacking (AT)~2+~ Microsatellites
Figure shows frequency of AT dinucleotides around all length classes of AC repeat microsatellites longer than (AC)~2~ (5′ number of sequences, *n* = 2,924; 3′ number of sequences, *n* = 3,309), with significantly greater numbers at odd positions 5′ and even positions 3′. Data are for cassette T/A only. Error bars show upper 95% confidence limit.
:::
:::
Convergence of Flanking Sequence Pattern {#s2f}
----------------------------------------
To assess the level of any convergent evolution, we used a simple assignment test to determine how often individual sequences resemble others flanking unrelated microsatellites of similar length (see [Materials and Methods](#s4){ref-type="sec"}). [Figure 8](#pbio-0020199-g008){ref-type="fig"} summarises these results. If all sequences were evolving divergently, any given sequence would be assigned to each of the microsatellite length classes with equal probability of around 5%. Sequences not associated with (AC)~2~ or longer were assigned back to their own class 57% of the time, showing that these sequences consistently differ from those near to AC repeat tracts. Remarkably, this figure falls to almost half (32%) for sequences flanking (AC)~2~, indicating that, even with just two repeats, similarities to other microsatellite flanking sequences already exist. The same pattern extends to other length microsatellites, with flanking sequences tending to be preferentially assigned back to their own or to an adjacent length class. When a flanking sequence is not assigned back to its own class, it is usually assigned to one of three other classes: the '1' class of random sequences, the '9' class where patterning is strongest, or to the longest class, class 21 (unpublished data).
::: {#pbio-0020199-g008 .fig}
Figure 8
::: {.caption}
###### Cross-Locus Similarity among Sequences Flanking Microsatellites of Similar Length
Length classes are as follows: class 1, randomly selected sequences not containing (AC)~2+~; classes 2--20, (AC)~2~--(AC)~20~; and class 21, (AC)~21--25~. Figure shows proportion of flanking sequences assigned on the basis of sequence similarity to their own AC repeat number class (dark grey), to the class above (grey), or to the class below (white). Expectation for assignment to self is shown by the horizontal line. Data are for cassette T/A only. Asterisks denote significant overassignment back to the same class or to an adjacent class, tested using *χ^2^* tests (*p* \< 0.05 using sequential Bonferroni corrections).
:::
:::
These analyses reveal a tendency for microsatellite flanking sequences to be similar to each other, but fail to quantify the level of sequence change involved. To do this, we sought to estimate similarity among three classes of sequence: (1) blocks of 50 bp lying immediately adjacent to a microsatellite; (2) blocks of 50 bp chosen randomly to lie between 500 and 600 bases downstream from a microsatellite (the random selection aims to remove possible complications of exact position and phase with the microsatellite); and (3) blocks of 50 bp randomly selected from around the genome. Comparisons within class 3 define the average level chance similarity in the genome, here estimated at 12.77 ± 3.28 (sd) bases out of 50. Comparisons within class 1 estimate how much convergent evolution is apparent at any given repeat number, and reveal a profile that rises to a maximum of 14.31 at a length of seven repeats, followed by a gentle decline with increasing length thereafter ([Figure 9](#pbio-0020199-g009){ref-type="fig"}A). Similarity is significantly above random for all but the very shortest microsatellites. As controls, we also made comparisons between class 1 and class 2 within a locus ([Figure 9](#pbio-0020199-g009){ref-type="fig"}B), between class 1 and class 2 among loci ([Figure 9](#pbio-0020199-g009){ref-type="fig"}C), and between class 1 and class 3 ([Figure 9](#pbio-0020199-g009){ref-type="fig"}D). Each of these comparisons reveals above random similarity in a profile that approximates that of the class 1--class 1 comparisons but peaking at lower levels.
::: {#pbio-0020199-g009 .fig}
Figure 9
::: {.caption}
###### Dependence of Sequence Similarity among Flanking Sequences on AC Repeat Number
The average number of matches shown (± standard error) quantifies similarity among three classes of sequence: (1) blocks of 50 bp lying immediately adjacent to a microsatellite; (2) blocks of 50 bp chosen randomly to lie between 500 and 600 bases downstream from a microsatellite; and (3) randomly selected blocks of 50 bp from around the genome. Average level of chance similarity in the genome is shown by a black line in each plot (comparison among class 3). 5′ and 3′ sequences are shown separately. Comparisons among sequence classes are shown for class 1 to class 1 (A), class 1 to class 2 for sequences at the same locus (B), class 1 to class 2 for sequences at different loci (C), and class 1 to class 3 (D).
:::
:::
Thus, in all cases, sequences immediately flanking a microsatellite show greater similarity to each other, to sequences nearby, and to sequences elsewhere in the genome than randomly selected sequences do to each other, a trend that is maximal for microsatellites around 7--10 repeats in length. We believe these similarities are generated primarily by the enhanced simplicity of microsatellite flanking regions and their tendency to gain AT motifs. Such characteristics allow unusually high matches when compared with random blocks of 50 bases that have high simplicity or contain polyA tracts. Over and above this background level of elevated similarity, proximity to any microsatellite appears to increase similarity, implying that microsatellites tend to lie more generally in regions of similar base composition. This might reflect either a tendency for microsatellites to arise preferentially in certain broad sequence contexts, or modification of the local base composition by the microsatellite itself. Moreover, similarity is further enhanced when a flanking sequence is compared with a neighbouring block. This suggests a local context effect such as might arise through the isochore structure of the genome, with neighbouring blocks being located in the same isochore and hence 'coloured' by the same nucleotide biases.
How Big Is the Sphere of Influence of a Microsatellite? {#s2g}
-------------------------------------------------------
To define more precisely the regions where convergent evolution is occurring, we repeated the assignment test but instead of using the full 50 bases either side we now used a symmetric pair of moving 25-base windows placed either side of the AC microsatellite ([Figure 10](#pbio-0020199-g010){ref-type="fig"}). Close to the microsatellite, the assignment probability is similar to but a little greater than that observed for the full 50-base analysis. As expected, this value declines as the window is moved away from the microsatellite. However, overassignment of (AC)~2~ flanking sequences to their own class is significant up to ten bases away from the microsatellite (*χ^2^* = 9.7, d.f. = 1, *p* \< 0.05 with sequential Bonferroni correction), and only when the window reaches 24 bases from the microsatellite does the assignment level fall to the value of 4.8% expected of random sequences.
::: {#pbio-0020199-g010 .fig}
Figure 10
::: {.caption}
###### Relationship between the Probability of Assigning (AC)~2~ Microsatellite Flanking Sequences to Self and Proximity to the AC Microsatellite
Solid line shows the probability of assignment back to self. Analysis is restricted to (AC)~2~ flanking sequences and is based on an assignment window 25 nucleotides wide on each side of the microsatellite. Dotted line indicates assignment probability expected of random DNA sequences.
:::
:::
Do Microsatellites Increase the Local Rate of Evolution? {#s2h}
--------------------------------------------------------
So far we have considered only the nature of the mutations that affect flanking sequences, and not their rate. To examine whether mutation rates are affected by the presence of a microsatellite, we used Megablast (National Center for Biotechnology Information (NCBI); <ftp://ftp.ncbi.nih.gov/blast/executables>) to compare microsatellites identified in completed sections of the chimpanzee genome against homologous loci identified in humans. If homologous loci are identified through comparisons among their immediate flanking sequences, an element of circularity will be introduced, since loci with lower rates of evolution will be identified preferentially. To circumvent this problem, we used a region 300 bases in length and 220 bases downstream of the microsatellite to conduct each Megablast search.
A total of 8218 chimpanzee loci were identified, and they yielded 5537 unique human homologues. Since microsatellite flanking sequences may contain insertions or deletions, we adopted the following approach so as to minimise problems of alignment. The chimpanzee flanking sequence was divided into 20 contiguous blocks of 20 bases, ten on each side of the microsatellite. Each block was then compared against 1,000 bases of human sequence, downstream from the region identified by Megablast, to find the best possible match. To filter sequences with major rearrangement we required both that all 20 blocks match with at least 15/20 bases, and that matched blocks all lie in the same order as their homologues. Differences between pairs of homologous flanking sequences were then quantified, at each of the 20-block positions, as the proportion of perfectly matching blocks and the average number of matches within each block ([Figure S4](#sg004){ref-type="supplementary-material"}).
After the above filtering, our data base contained 5017 sequences (91% of the original number), and among these, approximately 77% of the blocks were identical. Around (AC)~2--3~ there is little apparent variation in either measure of similarity with block position apart from a small tendency for 5′ blocks to show an increasing average percentage match closer to the microsatellite. However, although the trends are by no means strong, around longer microsatellites similarity of blocks near to the microsatellite is reduced, particularly when measured using average percentage matches ([Figure S4C and S4D](#sg004){ref-type="supplementary-material"}).
Our analysis of dinucleotide frequencies around microsatellites shows patterns of similarity on a smaller scale than might be revealed with 20-base blocks of sequence. To investigate the number of changes in the immediate flanking bases, we also calculated the proportion of mismatches occurring at a given base for each 5′ and 3′ sequence in the immediate flanking blocks (blocks −1 and +1; [Figure S5](#sg005){ref-type="supplementary-material"}). The average proportion of mismatches is relatively constant along the flanking sequence around short microsatellites, with a possible rise immediately 3′ of the microsatellites. However, the clear overabundance of mutations in the immediate flanking region of long microsatellites, with a higher than average proportion of mismatches −9 bases 5′ and +4 bases 3′, indicates that the regions closest to the microsatellites are indeed experiencing elevated mutation rates. The dearth of mismatches further away from the microsatellites, and a similar overall mean proportion of mismatches to that around short microsatellites (0.012 versus 0.011, respectively), suggests that a higher mutation rate close to microsatellites is occurring at the expense of the number of mutations further from microsatellites.
Discussion {#s3}
==========
We have studied very large numbers of (AC)*~n~* microsatellite flanking regions culled from the human genome and asked questions about the extent that these evolve in any consistent and unusual manner. Patterning is present in the form of over- and underrepresentation of bases and dinucleotide motifs at odd and even positions either side of the microsatellite. Pattern strength is maximal around (AC)~9~, but appears present even around sequences as short as (AC)~2~ and may extend as many as 50 bases either side. Some patterning is more or less symmetrical, but we also found several examples showing strong 5′ to 3′ asymmetry, implying that the two ends of a microsatellite are by no means equivalent. The net result is that sequences flanking microsatellites of a given length tend to be more similar to each other than to random sequences or to sequences flanking microsatellites of different lengths. Thus, there appears to be convergent evolution. Finally, we compared large numbers of homologous flanking sequences between humans and chimpanzees, and found evidence that mutation rates near microsatellites tend to be somewhat elevated.
Sequences surrounding (AC)*~n~* tracts exhibit remarkable levels of patterning, with any given dinucleotide motif tending to be much more likely to occur at even numbered positions rather than odd, or vice versa. For several reasons, we believe that the patterning arises due to the structural properties of the microsatellite (see below), becoming more pronounced as repeat number increases. These reasons include the following: the consistently central placement of microsatellites within the patterning, the dependence of the strength of patterning on AC repeat number, the similarity between microsatellites in LINE and SINE elements and those elsewhere, the weakness of the patterning around (AC)~2~, and the strong influence of cassette type on the form of patterning. Unfortunately, it is surprisingly difficult to eliminate the alternative hypothesis, namely that the patterning arises due to some other force and that AC repeats then either form or expand more rapidly when placed centrally within the pattern. This ambiguity is particularly relevant to the question of cassette distribution, where it seems reasonable both that (AC)*~n~* tracts might cause biased interconversion between cassettes and that certain cassettes may allow slippage more than others. For example, while the structural properties of AC repeats are known to generate mutational biases in adjacent bases ([@pbio-0020199-Timsit1]) capable of changing cassette type, minisatellite mutation rate can depend critically on the presence of a particular base in the flanking sequence ([@pbio-0020199-Monckton1]).
The relationship between an AC microsatellite and its flanking sequences begins surprisingly early, with (AC)~2~ already showing a small but significant bias in the distribution of cassette types and greater similarity to other sequences flanking AC microsatellites than to random sequences. In addition, the moving window assignment test indicates that significant convergence exists even when the ten bases closest to the microsatellite are excluded. Such a wide influence around such a common, short motif is remarkable and suggests that a high proportion of the genome may be affected by these and similar forces. To illustrate, (AC)~2~ is expected to occur every 250 bases, as is (GT)~2~. Taking the sphere of influence on each side as ten bases plus half the 25-bp window yields a value of 45. This predicts that over 30% (approximately 45 bases of every 125) of the genome will be affected by (AC)~2~ on one strand or the other, a figure that will only increase with inclusion of longer arrays and other microsatellite motifs.
As AC repeat number increases, so does the strength of patterning, becoming pronounced by (AC)~5~ and peaking in strength at (AC)~9~. Although patterning is seen in several different dinucleotide motifs, even in the human genome there are insufficient data to study any but the commonest cassette--motif combinations over a wide range of microsatellite lengths. Focusing on the motif AT, we found evidence that the strongest patterning was due to the development of AT microsatellites abutting AC tracts. However, this is not the only effect. After removal of all (AT)~2~ or longer microsatellites, there remains a significant tendency for single AT motifs to appear in phase with AC tracts, suggesting that mutation bias as well as slippage is involved.
Given the increase in strength of patterning between (AC)~2~ and (AC)~9~, it might seem logical that the pattern would become stronger and stronger as repeat number increases further. Instead, (AC)~9~ appears to be the peak strength, with longer microsatellites showing lower amplitude but a broader spread of patterning. It is interesting that this peak coincides with the length at which microsatellites begin to become polymorphic: a common rule of thumb for marker development in mammals is that primers are designed for loci carrying ten or more repeats ([@pbio-0020199-Weber1]). This may be mere coincidence or may reflect, for example, a change in mutation process associated with individuals who are heterozygous for alleles carrying different repeat numbers ([@pbio-0020199-Rubinsztein1]; [@pbio-0020199-Amos2]; [@pbio-0020199-Amos1]). Again, there are parallels with minisatellites, where many mutations occur by the transfer of material from one homologous chromosome to the other ([@pbio-0020199-Jeffreys2]).
The rich patterning we find presumably arises through local mutation biases. Previous work on mutation biases has tended to reveal either generic effects such as isochors ([@pbio-0020199-Bernardi1]), where some bases are favoured over others in large regions of the genome, or specific but highly localised biases where one or two bases may influence what happens to their immediate neighbours ([@pbio-0020199-Blake1]; [@pbio-0020199-Morton1]; [@pbio-0020199-Goodman1]; [@pbio-0020199-Zavolan1]). The patterns we find suggest a somewhat intermediate process in which mutational dependency appears to extend over distances of 30 bases or more. At the same time, the patterning is position dependent, in that it involves not just, for example, a favouring of A over other bases, but, instead, a favouring of A over other bases at even numbered sites.
The actual mechanism that causes patterning remains unclear, but our data suggest a model based on the structural properties of AC repeat tracts. Local variation in DNA structure is known to be associated with mutational biases ([@pbio-0020199-Morton1]) and variation in mutation rate ([@pbio-0020199-Petruska1]; [@pbio-0020199-Goodman1]), as well as possibly influencing the mismatch repair process ([@pbio-0020199-Werntges1]; [@pbio-0020199-Marra1]). Tracts of repeating AC motifs tend to exhibit unusual structural properties with high propeller twist and shifted base pairing ([@pbio-0020199-Timsit1]), and hence may be considered prime candidates for sequences capable of influencing the evolution of their immediate surroundings. Indeed, crystallographic studies indicate that sequences like (AC)*~n~* and (A)*~n~* induce local mutation biases ([@pbio-0020199-Timsit1]).
The unusual structure of microsatellite DNA may generate mutational biases in at least two ways. First, in AC repeat tracts, each base interacts unusually strongly with the neighbour of its complement base in a way that may lead to misincorporation of incoming nucleotides toward the ends of the microsatellite or in the immediate flanking region. Second, AC tract structure may influence the efficiency of the mismatch repair machinery in correcting either noncomplementary bases or loops resulting from slipped strand misalignment of repetitive DNA. Given that the mismatch repair system is strongly implicated in moderating the otherwise high rates of slippage mutation at microsatellite loci ([@pbio-0020199-Levinson1]; [@pbio-0020199-Schlotterer1]), it seems possible that even a small bias in the repair of loop structures might be responsible for the patterning we observe. However, although variation in mismatch repair efficiency may depend to some extent on DNA structure, the effect of sequence context on repair is not well understood ([@pbio-0020199-Marra1]). Unfortunately, with current understanding, none of these mechanisms would generate mutation biases that extend tens of bases away from the microsatellite, and hence this aspect must await further research.
An alternative explanation for some of the patterning, for example, the tendency for single AT motifs to lie in phase with the microsatellite, could be that these elements represent the remnants of a longer and now eroded (AC)*~n~* repeat tract. Under this scenario, point mutations at specific positions along the microsatellite would presumably interrupt the repeats. Given a strong bias toward transition mutations, we can explain both the existence of strong AT pattern, with C to T transition mutations dominating over C to R (purine) or A to Y (pyrimidine) transversion mutations, and also the increase in pattern strength around longer microsatellites, with interruptions in longer arrays more likely to be internal to the repeat tract and hence be excluded from the analysis. However, we suggest that this model is unlikely for two reasons. First, such a model fails to accommodate the strong asymmetry in patterning that is observed for some dinucleotides and specific cassette bases around the (AC)*~n~* repeat tract. Polarity has been noted for minisatellite mutations, with mutational processes differing between the two ends of the repeat tract ([@pbio-0020199-Armour1]; [@pbio-0020199-Jeffreys1]), but a microsatellite is much simpler in structure than a minisatellite and any polarity would have to affect some dinucleotides but not others. Second, the commonest and strongest patterning is observed for dinucleotide AT, and this would require high rates of C to T transitions but effectively no A to G transitions. More generally, the microsatellite erosion model predicts that flanking sequence patterning should be dominated by purine/pyrimidine, and this is not the case (see [Table 2](#pbio-0020199-t002){ref-type="table"}).
The patterning we describe appears to represent an important component of the forces that shape genome evolution, both in terms of its ubiquity and the absolute strength of its effect. It follows that there are many possible practical and theoretical implications. For example, even very short microsatellites appear able to cause some level of convergent sequence evolution, and hence to confound phylogenetic analyses. Similarly, microsatellites near genes may increase local mutation rates and influence the spectrum of new mutations that arise. To explore the size of these effects we designed experiments both to measure absolute convergence and to ask about evidence for changes in mutation rate.
To measure convergence, we made various comparisons between blocks of 50 bases chosen randomly, lying next to a microsatellite and lying near a microsatellite. We found an ordered progression of similarity from 12.77/50 bases for random--random through to a maximum of 14.31/50 bases between blocks adjacent to microsatellites 7--10 repeats long, an increase of 12% similarity. Although modest, trends are highly significant, with all comparisons showing a dependency on microsatellite length that peaks at around 7--10 repeats. The most parsimonious explanation for these similarities is that sequences flanking AC microsatellites tend to be AT-rich and to exhibit increased simplicity. Both these characteristics would increase the chance of flanking sequences being unusually similar both to each other and to random sequences that may contain polyA tails or other sources of simplicity. At the same time, the high scores gained by (AC)~7--10~ both for assignment to their own class and for similarity to each other relative to random blocks provide a clear indication that convergent sequence evolution is occurring. Interestingly, any given flanking sequence tends to be more similar to a block 500 bases away than to a similarly placed block near a different microsatellite, suggesting longer range patterning such as might arise through placement within the same isochore ([@pbio-0020199-Bernardi1]). Furthermore, our attempts to measure variation in mutation rate indicate reduced similarity between homologous human and chimpanzee sequences, implying a higher rate of evolution, at least for a region in the order of ten bases around the microsatellite. On a scale of blocks of 20 bases the trends are less convincing. Having said this, it seems likely that any genuine variation in mutation rate would be to some extent masked by the convergent evolution, and hence that this aspect would benefit from further investigation.
In conclusion, previous studies of microsatellite flanking sequences have identified several features, including a tendency to harbour other microsatellites, a locally increased mutation rate, and, conversely, conservation over unexpectedly large tracts of evolutionary time. Our analyses support all these trends and provide a possible resolution for the apparent contradiction between faster evolution but at the same time greater sequence conservation. Although there is evidence that mutation rates near microsatellites are elevated, we also find evidence of convergent evolution. Consequently, the increased rate of change may be to some extent neutralised and perhaps even reversed by the tendency for similar changes to occur in related lineages. Furthermore, the greatest changes appear to occur in flanking sequences around microsatellites that are below the length used as markers, at least in humans. Overall, therefore, we have been able to formalise previous anecdotal evidence and hence to document a remarkably widespread source of directional change and nonrandom evolution that undoubtedly plays an important role in shaping the make-up of our genomes.
Materials and Methods {#s4}
=====================
{#s4a}
### Dataset. {#s4a1}
Our dataset of (AC)*~n~* dinucleotide repeats was extracted from the human genome (build 33, NCBI Reference Sequences; NCBI, Bethesda, Maryland, United States) using a custom macro written in Visual Basic. Only microsatellites separated by at least 100 bp from the nearest (AC)~2~ or longer were included in the dataset. Thus (AC)~2~AT(AC)~10~ would not be included in the dataset, whereas ACAT(AC)~10~ would be included as (AC)~10~. Flanking sequences are here defined as the 50 bases lying either side of a microsatellite. No attempt was made to translate TG repeats with complementary AC repeats on the opposite strand. Consequently, all our microsatellites are 5′-(AC)*~n~*-3′.
### Flanking sequence base composition. {#s4a2}
For each frequency estimate, 95% confidence intervals were derived based on the binomial distribution (*n* \< 200 observations) or a normal approximation (*n* ≥ 200 observations). Bases used to define cassette type were excluded from all calculations, and expected frequencies were taken as the average frequency across all positions.
### Convergence of flanking sequence pattern: assignment test. {#s4a3}
Microsatellites were divided into 21 classes according to repeat number. Class 1 was the control set, comprising 5,000 randomly selected, non-microsatellite-associated sequences from Chromosome 1. All other classes contained flanking sequences from single-length microsatellites, except class 21, which contained combined data from microsatellites 21--25 repeats long. Analysis was restricted to the most abundant cassette class, T/A, yielding sample sizes that peaked at 1,087 for (AC)~5~ and declined to 175 for (AC)~20~ (see [Figure 2](#pbio-0020199-g002){ref-type="fig"}).
As an index of similarity, we calculated the log likelihood of observing a given sequence based on its position-specific dinucleotide motif composition:
where *f~ijk~* is the frequency of dinucleotide *i* at position *j* (*j* ≠ −1 or 0, with position 0 including the microsatellite and its cassette bases) in flanking sequences of class *k*. To avoid bias, when a sequence was compared with its own class, its contribution to the dinucleotide frequencies was first removed. For each sequence in turn, *A* was calculated for every class and the sequence was then assigned to the class that yielded the highest index value. Under convergent evolution, we expect sequences to tend to be assigned to their own or similar length classes.
### Convergence of flanking sequence pattern: quantifying sequence change. {#s4a4}
Sequences were again divided into length classes 2 to 21, and each sequence contributed four 50-bp blocks of sequence, one from each side immediately adjacent to the microsatellite but excluding the cassette bases (class 1), and one from each side displaced by a randomly selected number 500--600 bases distal (class 2). In addition, we also generated a database of 5,000 non-microsatellite-associated sequences. When making comparisons within a class, nonindependence was avoided by randomising the sequence order and then comparing sequence 1 with sequence 2, 2 with 3, ..., (n − 1) with n. Our index of similarity was simply a count of the number of matching bases. A few pairs of sequences (less than 0.1%) gave high similarity scores of over 30/50 matching bases, presumably because these loci have been duplicated or lie in repetitive elements. Such sequences were discarded. As with all other analyses, sequences containing base ambiguities (marked base N) were also discarded.
### Rate of evolution around microsatellites. {#s4a5}
(AC)*~n~* repeat microsatellites were extracted from the available chimpanzee finished-quality high-throughput genomic sequence (NCBI) as outlined above for humans. A 300-base region 220 bases upstream from each chimpanzee microsatellite was used by Megablast (Win32 version 2.2.6, NCBI) to identify homologous human loci in the finished genome sequence. Sequences with multiple high-scoring hits were discarded, as they presumably occur because a locus is found in repetitive elements or has been duplicated. Those nonoverlapping hits with at least 280/300 matching bases and an expectation (e-value) greater than five times that of any other hit to the same sequence were thus retained, giving a dataset of 5,537 sequences.
Supporting Information {#s5}
======================
Figure S1
::: {.caption}
###### Flanking Sequence Nucleotide Frequency Distributions Illustrating Three Classes of Patterning with Neither Strong Periodicity Nor Asymmetry
From little structure of any kind (A) to complicated aperiodic clustering (C). Plots are as described in [Figure 3](#pbio-0020199-g003){ref-type="fig"}.
(1.7 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S2
::: {.caption}
###### Flanking Sequence Frequency Distributions for Four Dinucleotide Motif--Cassette Combinations Further to Those Shown in [Figure 4](#pbio-0020199-g004){ref-type="fig"}
Plots are as described in [Figure 4](#pbio-0020199-g004){ref-type="fig"}.
(3.0 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S3
::: {.caption}
###### Dinucleotide Flanking Sequence Patterning in Interspersed Repeats and Unique Sequence DNA
Figure depicts equivalent patterns of asymmetry in AT dinucleotide frequencies for the commonest cassette type, (T/A), around microsatellites in unique sequence DNA (A), LINE/L1 elements (B), and SINE/Alu elements (C). Plotting conventions are the same as for [Figure 4](#pbio-0020199-g004){ref-type="fig"}.
(1.4 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S4
::: {.caption}
###### Dependence of Differences among Homologous Loci on Location of Microsatellite
Block position is relative to the central microsatellite (not shown).
(A and B) Proportion of exact matches (with 95% binomial confidence intervals) and average number of matches, excluding exact matches (± standard error), with block position around (AC)~2--3~ microsatellites (*n* = 4,593).
(C and D) As (A and B) but for (AC)~4+~ microsatellites (*n* = 356). Average proportion of exact matches and number of matches, calculated separately for 5′ and 3′ blocks around (AC)~2--3~ microsatellites, are shown by a black line in (A) and (C), and (B) and (D), respectively. Average percentage match rather than average match is plotted in (B) and (D) because overlapping blocks were truncated to exclude overlapping regions from the analysis, with the result that not all blocks contained 20 bases.
(2.9 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S5
::: {.caption}
###### Mean Proportion of Mismatches along Homologous Flanking Sequences
The proportion of mismatches occurring at a given base in a flanking sequence are averaged over (AC)~2--3~ microsatellite loci (A) and over (AC)~4+~ microsatellite loci (B). Shown ± standard error. The microsatellite at base position 0 is not shown. Expectation, calculated separately for 5′ and 3′ sequences around (AC)~2--3~ microsatellites, is shown by a black line in both plots.
(3.4 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Work was funded under a Natural Environment Research Council studentship. We are grateful for help with computing facilities from David Judge (Department of Genetics, Cambridge University).
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** EJV and WA conceived and designed the experiments. EJV performed the experiments and analyzed the data. EJV and WA wrote the paper.
Academic Editor: David Penny, Massey University
Citation: Vowles EJ, Amos W (2004) Evidence for widespread convergent evolution around human microsatellites. PLoS Biol 2(8): e199.
NCBI
: National Center for Biotechnology Information
| PubMed Central | 2024-06-05T03:55:46.730646 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509290/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e199",
"authors": [
{
"first": "Edward J",
"last": "Vowles"
},
{
"first": "William",
"last": "Amos"
}
]
} |
PMC509291 | Imagine a fledgling biology instructor at a university in the developing world. Heading into her first semester of teaching, she is armed with nothing but her college degree, some old notebooks, and---if she is lucky---a late-edition textbook. Forging a curriculum that is both current and engaging for her students could be a daunting challenge.
But what if that same young instructor was given free and open access to a syllabus, complete lecture notes, and problem sets and solutions from two members of the faculty of the Massachusetts Institute of Technology (MIT)? And not just any faculty members, but David Page---the recipient of a MacArthur Foundation Prize Fellowship in 1986, a Searle Scholar\'s Award in 1989, and the Amory Prize for advances in reproductive biology from the American Academy of Arts and Sciences in 1997---and Chris Kaiser, who won a 1999 fellowship from MIT that recognizes his teaching excellence?
This is the premise of MIT\'s OpenCourseWare project.
Utilizing the Internet, MIT OpenCourseWare (MIT OCW) has opened MIT\'s curriculum and educational materials to a global audience of teachers and learners---an instructor at a new engineering university in Ghana, a precocious highschool biology student in suburban Chicago, a political scientist in Poland, a literature professor in upstate New York---who are all now able to use the same materials that MIT\'s professors rely on to teach their full-time students.
The Makings of a Movement {#s2}
=========================
Ten years from now, we expect that MIT OCW will have become firmly planted in MIT\'s educational landscape. But MIT OCW was just a leap of faith when the concept was originally proposed by a group of faculty four years ago. In the fall of 1999, Provost Robert A. Brown asked the faculty committee to provide strategic guidance on how the institute should position itself in the e-learning environment. At first, many members of the group assumed that their work would lead to an "MIT.com" venture. But after a year of analysis, market research, and development of business scenarios, the committee concluded that a revenue-generating distance-education model was not desirable for MIT.
The committee went back to the drawing board and, convinced that open software and open systems were the wave of the future, came to a very simple conclusion: that MIT should use the Internet to give its teaching materials away.
Brown and MIT President Charles Vest instantly recognized the simplicity and brilliance of the idea. "It seemed to me that it would be a way to advance education, by constantly widening access to our information and inspiring other institutions to do the same with theirs," Vest said.
While the 701 courses currently available represent just a third of the ultimate goal of 2,000 courses by the year 2008, MIT OCW has already had an impact on MIT\'s campus. We have published teaching materials from almost half of MIT\'s 950 faculty members, and a significant portion of the faculty have told us that they are already using materials available on MIT OCW---the lecture notes, syllabi, problem sets, and exams of their colleagues---to prepare for their classes, do research, and help their students.
But the real payoff of what we hope will become the "opencourseware movement" will be its effect on educators and learners around the world. Our goal is to create a model that other universities can follow and improve upon. Ultimately, the trend toward open knowledge will help bring people of all backgrounds together and promote improved educational systems across the globe.
Measuring Success {#s3}
=================
Since April 2001, we have received more than 20,000 e-mail messages from around the world endorsing the vision and potential benefits of sharing knowledge freely. A typical message came from Andrew Wilson in the United Kingdom in October 2003: There can be no greater hope for humankind than the belief that wisdom generated through increased learning will ultimately lead to a better world. With OCW, MIT has taken an ethical stand against the belief that knowledge should only be accessible to those who can pay for it or are in proximity to it."
Just after its "official launch" in fall 2003, MIT began a rigorous data collection process to find out who is accessing MIT OCW, why and how they use it, and what difference the initiative makes. The results of this first baseline evaluation confirm what we have heard anecdotally through those e-mails: that educators, students, and self-learners around the world are using our course materials, and that, overwhelmingly, they find the materials useful in meeting their own learning and teaching goals.
Who Is Accessing MIT OCW? {#s3a}
-------------------------
On average, MIT OCW clocks over 11,000 visits per day, with nearly a quarter-million unique visitors per month. About 45% of these visitors are from the United States and Canada. Outside North America, the top countries of origin are China, the United Kingdom, Germany, India, and Brazil. About 52% of visitors identify themselves as "self-learners," 31% as "students" enrolled in a formal course of study, and 13% as "educators." We view educators as a particularly important target audience because it is through them that MIT course materials can touch the greatest number of people and have the most profound impact on education around the world.
Why and how are they using it? {#s3b}
------------------------------
MIT OCW asked visitors their primary purpose in using MIT course materials. Of educators who responded, about 57% answered that they use it for course or curriculum development, 33% to enhance their subject matter understanding or support research, and 7% for student advising. Elements of MIT materials have been adapted for classroom use by 47% of educators who answered our survey, while 41% report they are considering it.
Critical Mass {#s4}
=============
Among the 33 academic disciplines available are 15 courses from the MIT Department of Biology (see [Figure 1](#pbio-0020200-g001){ref-type="fig"}), 63 from the Department of Brain and Cognitive Sciences, and 13 from the Harvard--MIT Division of Health Sciences and Technology.[](#pbio-0020200-g001){ref-type="fig"}
::: {#pbio-0020200-g001 .fig}
Figure 1
::: {.caption}
###### Sample MIT OCW Course Homepage for Graduate Biochemistry Course
(Image of DNA courtesy of Lawrence Livermore National Laboratory.)
:::
:::
Educators, students, and self-learners from a wide variety of fields will find materials they can use in their teaching and learning activities. And as the opencourseware concept spreads to other colleges and universities, we expect that access to the work of faculty from diverse disciplines and institutions will increase, by an order of magnitude, the benefits to educators and learners who (whether for reasons of geography, cost, or culture) would not otherwise have access to such materials.
History has proved that education and discovery are best advanced when knowledge is shared openly. Our agenda must evolve to shape the future, and to respond to new challenges and opportunities.
At MIT, we believe the idea of opencourseware is one such opportunity, which we must seize during the next decade.
*For more information about MIT OCW, please contact Jon Paul Potts, MIT OCW Communications Manager, at E-mail:* <jpotts@mit.edu> or 617-452-3621.
Anne H. Margulies is Executive Director of the Massachusetts Institute of Technology\'s OpenCourseWare initiative. E-mail: <amarguli@mit.edu>
MIT
: Massachusetts Institute of Technology
MIT OCW
: MIT OpenCourseWare
| PubMed Central | 2024-06-05T03:55:46.734758 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509291/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e200",
"authors": [
{
"first": "Anne H",
"last": "Margulies"
}
]
} |
PMC509292 | Introduction {#s1}
============
Plants produce many secondary metabolites, of which some are thought to function as direct defenses against pathogens and herbivores by reducing their performance, survival, and reproduction. Numerous plant allelochemicals with antiherbivore properties are classified according to their mode of action (e.g., toxins, antifeedants, antidigestive proteins, etc.) ([@pbio-0020217-Bennett1]) and have been used in agriculture to control insect pests ([@pbio-0020217-Hedin1]). The fact that a secondary metabolite reduces herbivore performance does not by itself demonstrate that the endogenously expressed metabolite functions defensively in the plant\'s natural environment ([@pbio-0020217-Bell1]), because the evolutionary interaction between herbivores and their host plants may have reduced the defensive efficacy of the metabolite. Phytophagous insects have evolved various strategies to cope with allelochemicals ([@pbio-0020217-Karban1]) and tend to tolerate, or even co-opt, plant defenses for their own defenses ([@pbio-0020217-Wink1]).
Pharmacological studies demonstrating a resistance effect of metabolites applied to plants or artificial diets ([@pbio-0020217-Yamamoto1]; [@pbio-0020217-Bowers1]), and studies using heterologously expressed genes in agricultural systems ([@pbio-0020217-Carozzi1]; [@pbio-0020217-Hilder1]), represent a first step in evaluating the defensive function of a secondary metabolite. The interpretation of these studies is confounded by both the altered ecological context in which the resistance is measured and the altered chemical milieu, which is also known to influence the defensive function of a metabolite. Stronger evidence for resistance effects of allelochemicals arises from studies establishing correlations between plant resistance against herbivores and the genetically variable accumulation of secondary metabolites ([@pbio-0020217-Berenbaum1]; [@pbio-0020217-Shonle1]) or from studies demonstrating the defensive role played by a suite of elicited metabolites ([@pbio-0020217-Orozco-Cardenas1]; [@pbio-0020217-Baldwin2]; [@pbio-0020217-Halitschke1]). Ideally, the benefits of a putative defense trait should be determined in plants differing only in a single gene that controls the expression of a resistance trait and are otherwise identical ([@pbio-0020217-Bergelson1]). To date, studies measuring resistance of "near isogenic" lines with altered metabolite accumulations ([@pbio-0020217-Jackson1]) provide the strongest evidence for their resistance, but these lines, which are created by repetitive backcrossing, are likely to differ in many loci linked to the target locus, which may also affect resistance. Such problems of genetic linkage have been overcome through the use of genetic transformation to explore the fitness effects of herbicide resistance ([@pbio-0020217-Bergelson2]; [@pbio-0020217-Purrington1]) and pathogen resistance ([@pbio-0020217-Tian1]) in field populations of *Arabidopsis*. In this study, we use transgenic silencing to alter a single putative resistance trait---the production of nicotine---and thereby establish its contribution to plant resistance in the field.
The pyridine alkaloid nicotine is one of the best-studied putative plant resistance traits. Because it can interact with the acetylcholine receptors in the nervous systems of animals, nicotine is extremely toxic to most herbivores and, consequently, was one of the first insecticides used to control pests in agriculture ([@pbio-0020217-Schmeltz1]). Evidence for the resistance value of nicotine arises from the agricultural practice of using nicotine sprays and genotypes of cultivated tobacco differing in nicotine levels ([@pbio-0020217-Jackson1]). Although nicotine is widely toxic, insects adapted to nicotine-producing plants have evolved resistance to this alkaloid ([@pbio-0020217-Glendinning1]). The tobacco specialist Manduca sexta (tobacco hornworm) tolerates doses of nicotine that are fatal to unadapted herbivores but grows more slowly on high-nicotine diets ([@pbio-0020217-Appel1]; [@pbio-0020217-Wink1]). Other studies suggest that M. sexta might even be better defended by dietary nicotine against its parasitoid, *Cotesia congregata,* which suffers higher mortality when parasitizing larvae fed on high- rather than low-nicotine diets ([@pbio-0020217-Barbosa1]; [@pbio-0020217-Thorpe1]). Thus, the coevolutionary arms race between nicotine-producing plants and their adapted herbivores may have reduced the defensive value of nicotine.
In the native tobacco species Nicotiana attenuata and *N. sylvestris,* nicotine is the most abundant alkaloid. Elicitation of N. attenuata with jasmonic acid methyl ester (MeJA) in its native habitat increases nicotine content, which is correlated with enhanced plant fitness when plants are attacked ([@pbio-0020217-Baldwin2]). However, herbivore attack and MeJA elicitation (as well as the plant\'s endogenous jasmonic acid cascade \[[@pbio-0020217-Halitschke1]\]) regulate many resistance traits, including trypsin protease inhibitors (TPIs), diterpene glycosides, and volatile emissions involved in indirect defense. Hence, nicotine is only one of a suite of putative defense traits elicited by herbivore attack, and its specific role remains to be determined.
In laboratory trials, resistance benefits of nicotine production against M. sexta larvae were established using transgenic N. sylvestris plants silenced in their nicotine biosynthesis by antisense expression of putrescine N-methyl transferase (PMT). Plant consumption and the performance of M. sexta larvae were negatively correlated with constitutive nicotine levels in laboratory feeding trials ([@pbio-0020217-Voelckel1]); whether this result applies to plants in their natural habitat is unclear. To examine the resistance effect of nicotine, we transformed N. attenuata with inverted-repeat *pmt* (IR*pmt*) and antisense *pmt* constructs and found that only IR*pmt* plants had strongly reduced nicotine content. We characterized the defense and growth phenotypes of two independently transformed homozygous IR*pmt* lines and found that measured direct and indirect defenses did not differ from those of the wild-type (WT) plants, except for a dramatic reduction (greater than 95%) of MeJA-elicited and constitutive nicotine production and an increase in anatabine content. In pulse-chase experiments with D~4~-nicotinic acid (NA) ethyl ester, we demonstrated that the increased anatabine likely results from a dimerization of the NA that would normally have been used in nicotine biosynthesis. In feeding trials, M. sexta larvae preferred and grew faster on IR*pmt* than WT leaves. We transplanted WT and IR*pmt* plants into N. attenuata\'s native habitat in southwestern Utah and elicited a subset with MeJA. Several naturally occurring herbivore species attacked and damaged unelicited IR*pmt* plants more than unelicited or elicited WT and elicited IR*pmt* plants. These results demonstrate that nicotine functions as an effective resistance trait under natural conditions.
Results/Discussion {#s2}
==================
IR*pmt* Constructs Silence Nicotine Production {#s2a}
----------------------------------------------
Nicotine accumulation was not reduced in most of the independent lines transformed with antisense *pmt* constructs (25 lines of pNATPMT1 and six lines of pCAMPMT1) compared to WT ([Figure 1](#pbio-0020217-g001){ref-type="fig"}A). None of the five lines with lower nicotine accumulation in the T~1~ screen had nicotine levels lower than those of WT in the homozygous T~2~ generation. In contrast, 29 of 34 independently transformed lines with the IR*pmt* construct pRESC5PMT had dramatically reduced constitutive and MeJA-induced nicotine accumulations ([Figure 1](#pbio-0020217-g001){ref-type="fig"}B). The suppression of nicotine accumulation was stable during plant development and when plants were grown in the glasshouse or in the field in Utah. Clearly, inverted-repeat constructs are more efficient at silencing the expression of endogenous genes, as has been previously described ([@pbio-0020217-Wesley1]).
::: {#pbio-0020217-g001 .fig}
Figure 1
::: {.caption}
###### Comparison of Antisense and Inverted-Repeat Silencing of *pmt*
Nicotine content (mean of 5--6 plants/line) normalized to mean of WT of unelicited (control) N. attenuata plants and plants 5 d after elicitation with 150 μg of MeJA per plant from independent lines transformed with (A) antisense *pmt* constructs and (B) an IR*pmt* construct. In contrast to the 31 lines transformed with the antisense *pmt* construct, 29 of the 34 IR*pmt* lines had dramatically reduced constitutive and MeJA-induced nicotine levels. T, terminator; P, promoter; I, spliceable intron; arrow, 950-bp consensus fragment of *pmt1* and *pmt2*. For details of transformation constructs see Protocol S1.
:::
:::
Genomic and Transcriptional Characterization {#s2b}
--------------------------------------------
Two homozygous T~2~ IR*pmt* lines (108 and 145) with reduced nicotine levels were further characterized. Southern blot analysis using a probe hybridizing to the selective marker in the IR*pmt* construct demonstrated that both lines contained a single insertion ([Figure S1](#sg001){ref-type="supplementary-material"}). Transformation with a pRESC transformation vector allowed the transferred DNA (T-DNA) and flanking DNA at the insertion site to be recovered from the plant genomic DNA. These experiments demonstrated that the T-DNA integrated into the N. attenuata genome at a single site in each line, since all sequenced clones from a line (108, *n* = 4; 145, *n* = 5) contained the same flanking sequence (see [Figure S1](#sg001){ref-type="supplementary-material"} and [Protocol S1](#sd001){ref-type="supplementary-material"}).
Transcripts of the *pmt* genes in the two lines were significantly reduced to approximately 10% of the constitutive and MeJA-induced WT mRNA levels ([Figure 2](#pbio-0020217-g002){ref-type="fig"}A), demonstrating that the targeted genes were successfully silenced.
::: {#pbio-0020217-g002 .fig}
Figure 2
::: {.caption}
###### PMT Transcript and Alkaloid Levels of IR*pmt* Lines
Mean (± SE) relative PMT mRNA transcript levels in the roots (A), and leaf levels of (B) nicotine and (C) anatabine, in two independent lines of IR*pmt*-transformed (108 and 145) and WT N. attenuata plants. Elicited (150 μg of MeJA) and unelicited (control) plants were harvested at 10 h for transcript (A) and at 4 d for alkaloid (B and C) quantification. Both IR*pmt* lines had significantly reduced PMT transcript and nicotine but featured anatabine not present in WT plants. Lowercase letters signify differences at *p* ≤ 0.01, Bonferroni corrected (\[A\] *n* = 3, ANOVA: F~2,12~ = 12.55; \[B\] *n* = 8--10, ANOVA: F~2,50~ = 135.4; \[C\] *n* = 8--10, ANOVA: F~2,50~ = 39.611\]. n.d., not detected.
:::
:::
Metabolic Consequences of *pmt* Silencing in N. attenuata {#s2c}
---------------------------------------------------------
Consistent with the observed silencing of *pmt* transcripts, the constitutive and induced nicotine levels in transformed plants of both lines were dramatically reduced to 3%--4% of the levels found in WT plants ([Figure 2](#pbio-0020217-g002){ref-type="fig"}B). All 29 IR*pmt* lines with reduced nicotine levels accumulated the alkaloid anatabine, which was not detected in WT plants. Constitutive and MeJA-induced total (nicotine, anabasine, and anatabine) alkaloid contents of the two IR*pmt* lines were about one-half and one-third of the WT levels, respectively, of which anatabine comprised 30% and 23% ([Figure 2](#pbio-0020217-g002){ref-type="fig"}C). Levels of anabasine representing 20% of the constitutive and 8% of the MeJA-elicited total alkaloid contents in WT plants were unchanged in IR*pmt* plants ([Figure S2](#sg002){ref-type="supplementary-material"}). Elevated anatabine levels were also found in recently published studies with antisense *pmt* transformation of *N. tabacum;* elevated anatabine levels did not affect transcript levels of other genes encoding enzymes involved in alkaloid metabolism ([@pbio-0020217-Chintapakorn1]).
Anatabine consists of a pyridine and a piperideine ring. Both are likely derived from NA, which is also the precursor of the pyridine ring of nicotine ([@pbio-0020217-Leete1]). Disrupting nicotine biosynthesis at the formation of the pyrrolidine ring by silencing PMT activity might cause an oversupply of the NA used in the biosynthesis of anatabine. Feeding the roots of hydroponically grown MeJA-elicited WT plants with NA ethyl ester resulted in formation of anatabine at levels of about a third of the total alkaloids (nicotine and anatabine) ([Figure 3](#pbio-0020217-g003){ref-type="fig"}); in the IR*pmt* lines, anatabine constitutes 98% of the total alkaloids. Feeding plants with D~4~-NA ethyl ester results in the formation not only of D~4~-nicotine and D~4~-anatabine but also of D~8~-anatabine, demonstrating that the last integrates two D~4~-NA units. When these experiments are conducted with WT plants, about half of the anatabine is labeled, suggesting that the unlabeled half was formed from endogenous unlabeled NA. In addition, about one-fourth of the WT nicotine was D~4~-nicotine. In IR*pmt* plants, in contrast, only traces of D~4~-nicotine were found, but one-third of the anatabine was either D~4~- or D~8~-labeled. In summary, exogenously supplied NA is taken up by the roots of N. attenuata plants and used in alkaloid biosynthesis, and an oversupply of NA results in the formation of anatabine. These results support the hypothesis that the silencing of *pmt* disrupts nicotine biosynthesis, causing an oversupply of NA and the subsequent formation of anatabine.
::: {#pbio-0020217-g003 .fig}
Figure 3
::: {.caption}
###### Alkaloid Biosynthesis and the Consequences of a NA Oversupply
Biosynthesis scheme and proportion of unlabeled (M^+^) and labeled (M^+^+4, M^+^+8) nicotine and anatabine in the leaves of two independently transformed N. attenuata IR*pmt* lines (108 and 145) and WT plants 5 d after elicitation with 150 μg of MeJA per plant. Plants were grown in hydroponic solutions and supplied with either unlabeled or D~4~-ring-labeled NA ethyl ester (1 mM) 24 h after elicitation (*n* = 3 or 4). The oversupply of NA resulted in the formation of anatabine even in WT plants from both labeled exogenous and unlabeled endogenous NA pools.
:::
:::
IR*pmt* plants did not differ from WT plants in any other measured secondary metabolite or growth parameter. Constitutive or MeJA-induced levels of caffeoylputrescine, chlorogenic aid, rutin ([Figure S2](#sg002){ref-type="supplementary-material"}), TPI activity, or the release of *cis*-α-bergamotene ([Figure S3](#sg003){ref-type="supplementary-material"}) in IR*pmt*-transformed plants did not differ from those of WT plants. Rosette-stage and elongation-stage growth in individual pots in both the glasshouse and the field ([Figure S4](#sg004){ref-type="supplementary-material"}) did not differ between WT and IR*pmt* lines, and transformed lines were not visually or morphologically distinguishable from WT plants. Hence, the IR*pmt* plants represent an ideal construct with which to examine the ecological consequences of nicotine production.
Effects of Nicotine Silencing on N. attenuata Herbivores {#s2d}
--------------------------------------------------------
M. sexta larvae reared on IR*pmt* plants in the glasshouse gained significantly more mass and changed instars faster than larvae reared on WT plants (*n* = 17--20; ANOVA: *p* \< 0.01, *p* ~WT-PMT108~ \< 0.02, *p* ~WT-PMT145~ \< 0.01). The differences were comparable to those observed for M. sexta larvae reared on nicotine-enriched artificial diets ([@pbio-0020217-Parr1]; [@pbio-0020217-Appel1]) or on nicotine-enhanced WT ([@pbio-0020217-Baldwin1]) or antisense-*pmt*--transformed N. sylvestris plants ([@pbio-0020217-Voelckel1]). Two-thirds of freshly eclosed M. sexta larvae, given the choice between leaf material from WT or IR*pmt* (108) plants, preferred to initiate feeding on the latter (*n* = 43; Chi^2^ = 6.7, *p* \< 0.01). Such behavior suggests that nicotine plays an important role in determining feeding sites of M. sexta larvae*,* as has been suggested in a study with cultivated tobacco ([@pbio-0020217-Kester1]). While the relative toxic effects of anatabine and nicotine remain unstudied, these results are likely to underestimate the influence of nicotine on M. sexta choice and performance, because IR*pmt* plants had enhanced levels of anatabine.
Since secondary metabolism is known to be sensitive to environmental parameters that differ between glasshouse and field conditions (e.g., UV-B influence; [@pbio-0020217-Caldwell1]), nicotine, anatabine, and TPI levels of WT and IR*pmt* plants grown in the field plantation were analyzed: they were found not to differ from plants grown under laboratory conditions ([Figure 4](#pbio-0020217-g004){ref-type="fig"}A). A M. sexta feeding choice test evaluating the larvae\'s choice between field-grown WT and IR*pmt* plants (*n* = 57; Chi^2^ = 7.74, *p* \< 0.01) verified the results described above for the same experiment conducted with glasshouse-grown plants. Thus, the phenotype of glasshouse-grown IR*pmt* plants was not altered by growth under field conditions. In addition, choice tests with field-collected *D. undecimpunctata,* which was observed colonizing only IR*pmt* plants in the field plantation, revealed that 77% of these beetles preferred the nicotine-deficient IR*pmt* leaf material over WT (*n* = 35; Chi^2^ = 10.31, *p* \< 0.001). Another beetle species observed occasionally on WT plants, *Trichobarus mucorea,* does not distinguish between WT and IR*pmt* leaf material in choice tests (*n* = 19; Chi^2^ = 0.05, *p* = 0.8).
::: {#pbio-0020217-g004 .fig}
Figure 4
::: {.caption}
###### Herbivore Damage to IR*pmt* and WT N. attenuata Plants in Nature
\(A) Leaf alkaloids (nicotine and anatabine) and TPIs 7 wk after transplantation (*n* = 6). Mean (± SE) percentage total leaf area damaged by (B) all herbivores and (C) only by Spodoptera exigua on WT N. attenuata plants and plants transformed with an IR*pmt* construct (108) that were either untreated (solid lines) or elicited (dotted lines; asterisk) with MeJA 7 d after plants were transplanted into a field plot in a native habitat. Differences between 108 and WT, 108\*, and WT\* are significant at *p* ≤ 0.05 (*n* ~PMT~ = 36, *n* ~WT~ = 50, *n* ~PMT\*~ = 28, *n* ~WT\*~ = 27; \[B\] ANOVA: F~3,822~ = 5.73, *p* = 0.001; \[C\] ANOVA: F~3,822~ = 4.6, *p* = 0.004). Plants of the nicotine-deficient transformed line 108 suffered significantly higher leaf area damage than did WT plants, but when line 108 was elicited, leaf damage by all herbivores was reduced to WT levels.
:::
:::
In the field plantation, IR*pmt* plants lost significantly more leaf area to herbivores than did WT plants ([Figure 4](#pbio-0020217-g004){ref-type="fig"}B), demonstrating that nicotine indeed functions as a direct resistance trait of N. attenuata in its native habitat. Over a period of 16 d, IR*pmt* plants exposed to naturally occurring herbivores lost 16% of their total leaf area to herbivores, an amount that is more than double the amount of damage incurred by WT plants. In order to meet compliance requirements described in the Code of Federal Regulations (7CFR340.3c) for the introduction of organisms altered through genetic engineering, flowers were removed as they matured, and therefore we could not directly measure the fitness consequences of this greater herbivore load. However, in other experiments with N. attenuata plants grown in natural populations, leaf area damage is negatively correlated with capsule number ([@pbio-0020217-Baldwin2]; [@pbio-0020217-Kessler1]), suggesting that the strongly enhanced herbivore damage of the nicotine-deficient IR*pmt* plants translates into a fitness loss.
IR*pmt* plants were attacked by a variety of insect herbivores. About half of the total herbivore damage resulted from S. exigua feeding ([Figure 4](#pbio-0020217-g004){ref-type="fig"}C). One-third of the total herbivore damage was damage from grasshoppers of the genus *Trimerotropis,* which followed the same general pattern of distribution as S. exigua damage, but the differences between unelicited IR*pmt* and WT plants were not significant. The damage caused by Epetrix hirtipennis was variable but significantly higher for unelicited IR*pmt* compared to WT plants (ANOVA: F = 2.81, df = 3, *p* = 0.04, *p* ~PMT-WT~ \< 0.05).
MeJA elicitation significantly reduced the damage of IR*pmt* plants to levels found on WT plants, suggesting that MeJA treatment elicits defense traits that are as efficient as the constitutive levels of nicotine in protecting plants. MeJA elicitation of N. attenuata plants is known to induce a diverse suite of transcriptional responses and secondary metabolites including TPIs, phenolics, flavonoids, phenolic putrescine conjugates, diterpene sugar esters, and volatile organic compounds ([@pbio-0020217-Halitschke1]; [@pbio-0020217-Roda1]), some of which apparently function as resistance traits. Which component of this complex suite of elicited metabolites is as effective as nicotine remains to be determined. It should be noted that the overall amounts of leaf area lost to herbivores was relatively low during the field experiments. Only 5% of the canopy area was lost from control and MeJA-elicited WT plants. In previous experiments ([@pbio-0020217-Baldwin2]), fitness differences were observed between control and MeJA-elicited WT plants in populations that had lost approximately 40% of their canopy area to herbivores.
Altogether, these results provide direct evidence for the defensive value of nicotine. In a field trial, we established that a native tobacco, which produces large amounts of nicotine, is better defended against its natural herbivores than are nicotine-deficient transformants of the same genetic background. This is likely mediated by the reduction of herbivore performance and by the fact that these phytophagous insects prefer low-nicotine diets. In contrast to studies demonstrating genetic correlations between the production of secondary metabolites and herbivore resistance ([@pbio-0020217-Berenbaum1]; [@pbio-0020217-Shonle1]), the resistance effects established in this study can be directly attributed to the altered traits. The fact that the silencing of one enzyme in the nicotine biosynthetic pathway redirects metabolite flux, resulting in the accumulation of an apparently less toxic alkaloid, anatabine, underscores the importance of characterizing single-gene transformants for secondary effects.
Conclusion {#s2e}
----------
Plant secondary metabolites are widely accepted as essential components of a plant\'s direct defenses against its natural enemies, but unambiguous proof has been lacking, mainly because of the difficulty of altering the expression of single traits in plants and testing the consequences of these manipulations under natural conditions. Transformation technology has provided biologists with the ability to manipulate and study the ecological consequences of single-gene manipulations. To date, the technology has largely been used for the heterologous expression of resistance genes (e.g., *Bacillus thuringiensis d-endotoxin*) in agricultural systems (see [@pbio-0020217-Tian1] for an elegant exception), and therefore has provided little evidence for the defensive value of endogenously expressed traits against a plant\'s native herbivore community. The scientific value of transgenically silencing endogenous genes in native plants to understand the ecological function of particular genes has been undermined by the polarized attitudes towards the use of genetically modified organisms in agriculture. Transgenic down-regulation of nicotine demonstrates that N. attenuata is under relentless herbivore pressure. Disabling this resistance trait, even in a year of low herbivore abundance, results in a large increase in opportunistic herbivory and supports the conclusion that secondary metabolites play an important role in explaining why the earth is largely green ([@pbio-0020217-Hairston1]).
Materials and Methods {#s3}
=====================
{#s3a}
### Plant material and transformation {#s3a1}
N. attenuata Torr. ex Watson (synonymous with N. torreyana Nelson and Macbr.; Solanaceae) grown from field-collected seeds ([@pbio-0020217-Baldwin2]) and inbred 11 or 14 generations were used for transformation and all experiments. Seed germination and the Agrobacterium tumifaciens (strain LBA 4404)--mediated transformation procedure are described in [@pbio-0020217-Krugel1]. In order to silence the expression of the two *N. attenuata pmt* genes, plants were transformed with pCAMPMT1 and pNATPMT1 vectors, which contain a gene fragment of *pmt1* (which has 95% identity to *pmt2*) in an antisense orientation, and pRESC5PMT, which contains the *pmt* gene fragment twice in an inverted orientation separated by intron 3 of the Flaveria trinervia gene *pyruvate orthophosphate dikinase (pdk)* (for vector construction and plasmids see [Figure S5](#sg005){ref-type="supplementary-material"} and [Protocol S1](#sd001){ref-type="supplementary-material"}). T~1~ plants were screened for hygromycin resistance (*hygromycin phosphotransferase II* gene of the vector pCAMBIA-1301) and constitutive and induced nicotine accumulation; homozygosity was determined by resistance screening of the T~2~ plants. Two independently transformed homozygous IR*pmt* lines (108 and 145) were further characterized by Southern blot analysis and by the rescuing of the transformation vector from genomic DNA into Escherichia coli to identify copy number and insertion site of the T-DNA (see [Figure S1](#sg001){ref-type="supplementary-material"} and [Protocol S1](#sd001){ref-type="supplementary-material"}).
### PMT mRNA accumulation and secondary metabolites. {#s3a2}
Transformed lines (108 and 145) and WT plants were grown in 1-l hydroponic vessels in a climate chamber as described in [@pbio-0020217-Hermsmeier1], and 4-wk-old rosette-stage plants were treated (elicited) on the first two fully expanded (source) leaves with 150 μg of MeJA per plant applied in 20 μl of lanolin paste, or left untreated. Approximately 200 mg of young roots was harvested and frozen in liquid nitrogen 10 h after elicitation, and RNA was extracted with Tri Reagent (Sigma, Taufkirchen, Germany) according to the manufacturer\'s instructions (*n* = 3/line/treatment). PMT transcript accumulation was analyzed by real-time PCR (ABI PRISM 7000; Applied Biosystems, Darmstadt, Germany). cDNA was generated from 20 ng of RNA with MultiScribe reverse transcriptase (Applied Biosystems), and amplified using the qPCR core reagent kit (Eurogentec, Searing, Belgium) and a probe and primers that were gene-specific (for sequences see [Figure S6](#sg006){ref-type="supplementary-material"}). For analysis of secondary metabolites, leaves growing one node above the sink-source transition leaf and young root tissue were harvested 4 d after elicitation (*n* = 8--10/line/treatment). Samples were analyzed by HPLC for alkaloids, caffeoylputrescine, chlorogenic acid, and rutin ([@pbio-0020217-Keinanen1]; [@pbio-0020217-Halitschke1]). A peak occurring in IR*pmt* alkaloid extracts but not in extracts of WT N. attenuata was collected and identified by nuclear magnetic resonance imaging as anatabine (for spectra and method, see [Protocol S1](#sd001){ref-type="supplementary-material"}).
To determine whether a NA oversupply was responsible for the formation of anatabine in the transformed lines, we supplied 4-wk-old plants with either unlabeled or D~4~-NA ethyl ester (1 mM) in their hydroponic solution 24 h after MeJA elicitation (*n* = 4/line/treatment). After 4 d, the treated leaf was harvested and extracted as above, but analyzed by LC/MS/MS to detect incorporation of the deuterium into nicotine and anatabine (for instrument conditions, see [Protocol S1](#sd001){ref-type="supplementary-material"}).
To examine the release of *cis*-α-bergamotene in the transformed lines compared to WT, volatiles from hydroponically grown plants (*n* = 3--5/line/treatment) enclosed in open-top volatile collection chambers were collected for an 8 h period starting 24 h after MeJA elicitation of the first two source leaves, and analyzed by GC/MS ([@pbio-0020217-Halitschke2]). TPI activity in the MeJA-treated leaf 3 d after elicitation was analyzed in plants (*n* = 5/line/treatment) by radial diffusion activity assay ([@pbio-0020217-van1]).
### *M. sexta* performance and feeding choice {#s3a3}
In the glasshouse, 2-wk-old seedlings were planted individually into 2-l pots with potting soil (C 410; Stender, Schermbeck, Germany) at 26--28 °C under 16-h supplemental light from Philips Sun-T Agro 400- or 600-W Na lights. For analysis of performance, newly eclosed M. sexta larvae (North Carolina State University, Raleigh, North Carolina, United States) were placed on the first-stem leaf of 8-wk-old WT and IR*pmt* (108 and 145) plants and allowed to feed for 14 d. Larval mass was recorded at 8, 10, 12, and 14 d.
The first feeding choice of M. sexta was determined by placing newly eclosed larvae in the center of a 3-cm--diameter cup containing, on opposite sides, 1.5-cm^2^ WT and IR*pmt* (108) leaf pieces and recording the leaf on which larvae started feeding (*n* = 44).
### Resistance of WT and IR*pmt* plants to herbivores in the natural habitat. {#s3a4}
In a field plantation (15 m × 18 m; GPS: lat 37°08′45′′N, long 114°01′12′′) in N. attenuata\'s natural habitat in southwest Utah, transformed IR*pmt* (108) and WT plants were exposed to naturally occurring herbivores dispersing from adjacent populations. To allow for spatial heterogeneity, plants were transplanted in a paired design (with 0.3 m and 1.5 m between plants of a pair and between pairs, respectively) in which plants were matched for equal rosette diameters. Plants were grown in soil (Potting Mix; Miracle-Gro, Marysville, Ohio, United States) for 5 wk after germination ([@pbio-0020217-Krugel1]), and were transplanted into the field plot (10 columns by 15 lines) in their 3.8-l pots. Seven days after transplantation, 30 WT and IR*pmt* plants were elicited with 150 μg of MeJA per plant applied in 20 μl of lanolin paste to the two youngest rosette leaves. Starting 4 d after transplantation, each plant was examined for damage and insects (including predators and eggs) every other day for 14 d. Damage amount was estimated as a percentage of the total leaf area, and the characteristic damage caused by caterpillars, beetles, grasshoppers, myrids, and leafhoppers was noted separately. The most abundant herbivores observed in the field plantation during the release were S. exigua, *Trimerotropis* spp., *E. hirtipennis,* and *D. undecimpunctata. M. sexta* and M. quinquemaculata occurred in the season only rarely, and no eggs were laid in the plantation during the 14 d. As plants began to elongate and produce flowers, they were examined daily, and all flowers were removed before opening and anthesis to meet the performance standards in the Code of Federal Regulations (7CFR340.3c). Consequently, direct fitness measures were unobtainable in this experiment.
For analysis of alkaloids and TPIs under field conditions, leaf samples of WT and IR*pmt* plants in the plot (*n* = 6) were taken 7 wk after transplantation and frozen (dry ice). To determine if the herbivore phenotype of IR*pmt* plants observed in glasshouse-grown plants was retained in plants grown under natural light conditions, the M. sexta choice experiment was repeated. The first feeding choices of freshly eclosed M. sexta larvae (North Carolina State University) and of adults of field-collected D. undecimpunctata and Trichobarus mucorea (Chrysomelidae and Curculionidae) found on N. attenuata were determined as described above.
Supporting Information {#s4}
======================
Figure S1
::: {.caption}
###### Copy Number of T-DNA in the Two Studied IR*pmt* Lines
\(A) Southern blot analysis of two independently transformed N. attenuata IR*pmt* lines (108 and 145) and WT plants. Genomic DNA (15 μg) from individual plants of the three genotypes and the plasmid used for transformation pRESC5PMT (4 ng) were digested with EcoRV and blotted onto nylon membranes ([@pbio-0020217-Winz1]). The blot was hybridized with a PCR fragment of the *hygromycin phosphotransferase II* gene from pCAMBIA-1301, which is specific for the selective marker on the T-DNA and signifies one insertion in each of the two lines.
\(B) Ethidium bromide staining of the DNA revealed an overload of the DNA of the IR*pmt* lines and therefore loading of the WT was controlled by stripping and rehybridization with a PMT probe, which clearly revealed the endogenous *pmt1* and *pmt 2* genes described ([@pbio-0020217-Winz1]) (unpublished data).
(6.3 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S2
::: {.caption}
###### Secondary Metabolite Levels in the Studied IR*pmt* Lines
Inverted-repeat silencing of *pmt* did not change the levels of (A) anabasine, (B) caffeoylputrescine, (C) chlorogenic acid, and (D) rutin (mean ± standard error \[SE\]) in two independently transformed N. attenuata lines (108 and 145) compared to WT plants. Plants were harvested 4 d after receiving one of four treatments: untreated control (Con), wounding (W), wounding and regurgitate application (W+R), and application of 150 μg of MeJA per plant applied in a lanolin paste. Plants were treated at the first two fully expanded (source) leaves and wounding was performed by generating three rows of puncture wounds on each leaf side using a pattern wheel. Subsequently, 10 μl per leaf of either water or M. sexta regurgitate diluted 1:1 (v:v) was dispersed over the puncture wounds (*n* = 8--10).
(179 KB PPT).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S3
::: {.caption}
###### Proteinase Inhibitor and Volatile Emission of the Studied IR*pmt* Lines
Levels of (A) TPI and (B) *cis-*α-bergamotene emission (mean ± SE) in two independently transformed N. attenuata IR*pmt* lines (108 and 145) did not differ from WT plants 4 d (for TPI) and 10 h (for *cis-*α-bergamotene) after receiving one of four treatments (as described for S2): untreated control (Con), wounding (W), wounding with additional regurgitate application (W+R), and MeJA elicitation. IS, internal standard.
(73 KB PPT).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S4
::: {.caption}
###### Growth Parameters Under Glasshouse and Field Conditions of the Studied IR*pmt* Lines
N. attenuata plants transformed with an IR*pmt* construct (108 or 145) did not differ in (A) stalk length \[*n* ~PMT~ = 43, *n* ~WT~ = 57, *n* ~PMT\*~ and *n* ~WT\*~ = 28\] and (B) rosette diameter \[*n* = 8\] from WT grown under either field (A) or glasshouse (B) conditions. Plants in (A) were untreated or elicited (\*) with MeJA 7 d after plants were transplanted into a field plot in a native habitat.
(98 KB PPT).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S5
::: {.caption}
###### Transformation Vectors
This figure shows plasmids used for the generation of N. attenuata lines with reduced levels of two PMTs due to posttranscriptional gene silencing. Both (A) pCAMPMT1 (10.7 kb) and (B) pNATPMT1 (9.7 kb) allow the synthesis of *pmt* antisense RNA. (C) pRESC5PMT (12.4 kb) was used for the synthesis of *pmt* RNA capable of forming an inverted repeat. Functional elements: *bla*, beta-lactamase gene from plasmid pUC19; *hptII*, gene for hygromycin resistance from pCAMBIA-1301; LB and RB, left and right border of T-DNA; *nptIII*, aminoglycoside phosphotransferase of type III from Streptococcus faecalis; ori ColE1, origin of replication from pUC19; ori pVS1, origin of replication from plasmid pVS1; P~CaMV~ and T~CaMV~, 35S promoter and terminator of cauliflower mosaic virus; *pdk i3*, intron 3 of *pdk; pmt1*, gene fragment of *pmt1* (95% identical with *N. attenuata pmt2*); P~NOS~ and T~NOS~, promoter and terminator of the *nopaline synthase* gene; *repA* pVS1, replication protein gene from pVS1; *sat-1*, nourseothricin resistance gene; *staA* pVS1, partitioning protein gene from pVS1. Displayed restriction sites mark the borders of functional elements, which are displayed in gray if on the T-DNA and in black if outside the T-DNA.
(56 KB PPT).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S6
::: {.caption}
###### PMT Sequences and TaqMan Probe
Nucleotide sequences of *N. attenuata pmt1* and *pmt2* mRNA ([@pbio-0020217-Winz1]) aligned with ClustalW. Primers and probe (underlined) used for real-time PCR of *pmt* mRNA are highlighted and bold.
(396 KB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Protocol S1
::: {.caption}
###### Molecular and Analytical Methods
(58 KB DOC).
:::
::: {.caption}
######
Click here for additional data file.
:::
Accession Numbers {#s4a8}
-----------------
GenBank accession numbers for the genes discussed in this paper are *bla* from puc19 (L09137), *hygromycin phosphotransferase II* from pCAMBIA-1301 (AF234297), *pdk* (X79095), *pmt1* (AF280402), and *pmt2* (AF280403).
We thank M. Lim and A. Wissgott for outstanding plant transformation services; B. Schneider for NMR analysis; A. Kessler and D. Kessler for species determination; E. Wheeler for editing; the Brigham Young University for use of their awesome field station, the Lytle Preserve; L. Rausing for helping us promote the discussion of the scientific value of transformed plants and J. White and the Animal Plant Health Inspection Service personnel for facilitating their safe use in nature; and the Max-Planck-Gesellschaft for financial support.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** AS and ITB conceived and designed the experiments. AS, RH, and ITB performed the experiments. AS, BK, RH, and ITB analyzed the data. KG contributed reagents/materials/analysis tools. AS and ITB wrote the paper.
Academic Editor: Michael Levine, University of California at Berkeley
Citation: Steppuhn A, Gase K, Krock B, Halitschke R, Baldwin IT (2004) Nicotine\'s defensive function in nature. PLoS Biol 2(8): e217
IR*pmt*
: inverted-repeat *putrescine N-methyl transferase;* MeJA
NA
: nicotinic acid
*pdk*
: *pyruvate orthophosphate dikinase;* PMT
SE
: standard error
T-DNA
: transferred DNA
TPI
: trypsin protease inhibitor
WT
: wild-type
| PubMed Central | 2024-06-05T03:55:46.735632 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509292/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e217",
"authors": [
{
"first": "Anke",
"last": "Steppuhn"
},
{
"first": "Klaus",
"last": "Gase"
},
{
"first": "Bernd",
"last": "Krock"
},
{
"first": "Rayko",
"last": "Halitschke"
},
{
"first": "Ian T",
"last": "Baldwin"
}
]
} |
PMC509293 | Introduction {#s1}
============
There is currently a significant focus on strategies to promote regeneration in adult mammals and therefore a renewed interest in the mechanisms that underlie regeneration in urodele amphibians. An adult salamander such as the newt or axolotl can regenerate its limbs and tail, jaws, ocular tissues such as the lens, and small sections of the heart ([@pbio-0020218-Goss1]; [@pbio-0020218-Eguchi1]; [@pbio-0020218-Oberpriller1]; [@pbio-0020218-Okada1]; [@pbio-0020218-Ghosh1]; [@pbio-0020218-Brockes1]; [@pbio-0020218-Nye1]). A key feature of urodele regeneration is the local plasticity of differentiated cells at the site of tissue injury or removal ([@pbio-0020218-Brockes2]; [@pbio-0020218-Odelberg1]; [@pbio-0020218-Del1]; [@pbio-0020218-Tanaka1]). This has been investigated for pigment epithelial cells of the iris ([@pbio-0020218-Eguchi1]; [@pbio-0020218-Simon1]; [@pbio-0020218-Imokawa1]; [@pbio-0020218-Imokawa2]), cardiomyocytes ([@pbio-0020218-Oberpriller2]; [@pbio-0020218-Bettencourt-Dias1]), and skeletal myofibers and myotubes ([@pbio-0020218-Hay1]; [@pbio-0020218-Lo1]; [@pbio-0020218-Tanaka2], [@pbio-0020218-Tanaka3]; [@pbio-0020218-Kumar1]; [@pbio-0020218-Echeverri1]), all of which reenter the cell cycle during regeneration, in contrast to their mammalian counterparts. A second aspect of plasticity is the ability of multinucleate skeletal muscle cells to fragment into viable mononucleate cells that then contribute to the regenerate. This process, sometimes referred to as cellularisation, was described in classical studies of limb regeneration ([@pbio-0020218-Thornton1]; [@pbio-0020218-Hay1]), but was first analysed with marked cells by implantation of cultured newt myotubes labelled by microinjection with a lineage tracer ([@pbio-0020218-Lo1]) or by an integrated retrovirus ([@pbio-0020218-Kumar1]). The myotubes were effectively converted to mononucleate cells that proliferated in the blastema, and this process occurred in cells that were blocked from cell cycle reentry ([@pbio-0020218-Velloso1]), thus showing that the two aspects of plasticity are not linked mechanistically. In an important recent contribution, myofibers were labelled in situ by microinjection in the tail of the larval axolotl ([@pbio-0020218-Echeverri1]). After amputation of the tail, the myofibers fragmented into viable mononucleate cells, thus establishing that cellularisation occurs during regeneration and contributes to the proliferative zone or blastema.
Our understanding of this intriguing process has received considerable impetus from the recognition of two manipulations that induce mammalian myotubes to undergo fragmentation. The first is exposure to myoseverin, a trisubstituted purine derivative isolated from a combinatorial library ([@pbio-0020218-Rosania1]). It evokes depolymerisation of microtubules, apparently by interacting directly with tubulin, as well as inducing changes in the expression of genes that are implicated in tissue remodelling and wound healing. The second is the conditional expression of the homeobox gene *Msx1* in mouse myotubes ([@pbio-0020218-Odelberg2]). *Msx1* has been studied as an antagonist of myogenic and osteogenic differentiation (reviewed in [@pbio-0020218-Bendall1]) and is expressed in the migrating precursor cells of limb muscle during chick development, apparently to prevent precocious differentiation ([@pbio-0020218-Bendall2]). The expression in mouse C2C12 myotubes evokes two aspects of plasticity that occur in 5%--10% of the cells; the first is cleavage of the cells into smaller multinucleated myotubes, which remain viable, and the other is the formation of mononucleate cells capable of division ([@pbio-0020218-Odelberg2]). In the latter case, the clonal progeny of a single myotube were shown to be capable of several pathways of mesenchymal differentiation.
The studies on cellularisation by myoseverin and *Msx1* have underlined that mammalian as well as urodele cells are capable of this response ([@pbio-0020218-Rosania1]; [@pbio-0020218-Odelberg1]). *Msx1* is expressed during urodele limb regeneration ([@pbio-0020218-Carlson1]; [@pbio-0020218-Koshiba1]), as well as during fin ([@pbio-0020218-Akimenko1]; [@pbio-0020218-Nechiporuk1]) and heart regeneration in the zebrafish ([@pbio-0020218-Raya1]), along with other *Msx* family genes. Therefore, it becomes important to investigate whether it controls cellularisation during regeneration. Although prior studies of this process have used the multinucleate myotube as the target cell in culture, the critical target during epimorphic regeneration is the more differentiated striated myofiber. The regeneration of muscle fibers in vertebrates proceeds by the mobilisation of reserve satellite cells ([@pbio-0020218-Charge1]), and these have been described in myofibers of larval salamander limbs ([@pbio-0020218-Popiela1]). Their participation in cellularisation was excluded in the earlier experiments on urodele cells by selective injection of a lineage tracer into myotubes in culture ([@pbio-0020218-Lo1]) or myofibers in the salamander tail ([@pbio-0020218-Echeverri1]). In order to address these various questions, we have established a culture system in which striated myofibers are dissociated from the limb of larval salamanders and attach to a culture substrate where they can be observed by time-lapse microscopy. The fibers are found to be activated by dissociation to undergo cellularisation and viable fragmentation, and this depends on expression of *Msx1*.
Results {#s2}
=======
Dissociated Myofibers in Culture {#s2a}
--------------------------------
In order to obtain striated myofibers, tissue was isolated from the limbs of two species of larval salamander (Ambystoma maculatum or Ambystoma mexicanum), which have been used interchangeably with comparable results. After removing the epidermis, the tissue was dissociated by proteolytic digestion, filtered through a sieve to remove most of the mononucleate cells, and plated in serum-free medium. The striated myofibers readily attached to the culture dish ([Figure 1](#pbio-0020218-g001){ref-type="fig"}; [Figure S1](#sg001){ref-type="supplementary-material"}) and were found to express myosin heavy chain (MHC) and titin after antibody staining. In view of the potential contribution of satellite type cells to the issues under investigation, cultures were treated with a viable nuclear stain and the myofibers were examined carefully at 2 d after plating. Of 1,290 fibers examined in seven independent cultures, there were only 46 examples of mononucleate cells adherent to myofibers, and such cells were not observed in the cases of plasticity that are discussed here.
::: {#pbio-0020218-g001 .fig}
Figure 1
::: {.caption}
###### A Live Striated Myofiber from the Larval Salamander
Photomicrograph of a live striated myofiber dissociated from the larval limb musculature and adhering to the culture dish in serum-free medium. This cell has the appearance of a normal quiescent fiber and was photographed with VAREL optics at 48 h after plating. Scale bar, 50 μm.
:::
:::
When cultures were labelled with tritiated thymidine, no labelled nuclei were observed in multinucleate cells after labelling for 24 h (540 myofibers, five cultures) or 48 h (263 myofibers, three cultures), while 16% of the mononucleate cells were labelled in the latter case. It is noteworthy that the absence of S-phase entry in nuclei within multinucleate cells includes the population of myofibers that undergoes the events of cellularisation or fragmentation described below.
Cellularisation of Myofibers after Implantation {#s2b}
-----------------------------------------------
The dissociation of viable myofibers has allowed us to evaluate their plasticity after implantation into a limb blastema, a procedure that has previously been performed only on myotubes formed in cell culture. Fibers were labelled with a cell tracker dye in suspension after dissociation, and single fibers were examined to verify the absence of any adherent mononucleate cells and were drawn into a glass micropipette ([Figure 2](#pbio-0020218-g002){ref-type="fig"}A). A few fibers (see [Materials and Methods](#s4){ref-type="sec"}) were injected from the pipette into the early forelimb regenerate of a larval axolotl. The regenerating limbs were sectioned 2--4 d later, and many examples were observed of mononucleate cells labelled with the tracker dye ([Figure 2](#pbio-0020218-g002){ref-type="fig"}B). Such cells were clearly mononucleates, as determined by analysis of serial sections, and were observed in 17 out of 23 animals implanted with labelled myofibers. We conclude that these cells readily undergo cellularisation in the environment of the limb blastema.
::: {#pbio-0020218-g002 .fig}
Figure 2
::: {.caption}
###### Cellularisation of Striated Myofibers after Implantation into a Larval Limb Blastema
\(A) Schematic diagram of procedure. After dissociation of larval limb musculature, the cells were loaded with a cell tracker dye and single myofibers taken up into a suction micropipette, prior to injection into a larval limb blastema as detailed in the [Materials and Methods](#s4){ref-type="sec"}.
\(B) Section of a limb at 48 h after implantation of CellTracker Orange-labelled myofibers. The section has been counterstained with the nuclear stain Sytox green. Note the dye-labelled mononucleate cells (arrowed). Scale bar, 20 μm.
:::
:::
Plasticity in Culture {#s2c}
---------------------
After 48 h in culture, some of the striated fibers remained viable, but showed no significant change in morphology and retained the appearance of the cell shown in [Figure 1](#pbio-0020218-g001){ref-type="fig"}. The remainder of the fibers showed various changes in morphology, and these were investigated either by microinjection of single fibers with the lineage tracer Texas red (TR)--dextran and subsequent analysis of the progeny cells or by sequential digital time-lapse observation.
### Cellularisation. {#s2c1}
Approximately 10% of the total population of myofibers underwent changes in nuclear localisation within the cells such that a lobulated or 'cauliflower' structure formed in the middle or end of the cell ([Figure 3](#pbio-0020218-g003){ref-type="fig"}A and [3](#pbio-0020218-g003){ref-type="fig"}B). This occurred without labelling by tritiated thymidine or any participation by adherent mononucleate cells, which were rarely present on such fibers. The lobules, each of which contained a nucleus, were rapidly resolved into adherent mononucleate cells. In order to analyse these events, single myofibers were microinjected with TR--dextran so as to fill the cells with tracer ([Figure 3](#pbio-0020218-g003){ref-type="fig"}C). We employed the 70 kDa dextran, which is not transferred across gap junctions ([@pbio-0020218-Coelho1]; [@pbio-0020218-Landesman1]). In cases in which fibers formed the cauliflower structure and underwent cellularisation, the mononucleate cells in the initial colony were labelled with the tracer in a rim of cytoplasm around the nucleus ([Figure 3](#pbio-0020218-g003){ref-type="fig"}D). In some cases, adjacent fibers were injected and the progeny of the myofibers gave rise after 5--7 d to overlapping dense colonies with many labelled cells ([Figure 3](#pbio-0020218-g003){ref-type="fig"}E). These cells did not express detectable levels of MHC after staining by indirect immunofluorescence.
::: {#pbio-0020218-g003 .fig}
Figure 3
::: {.caption}
###### Plasticity of Isolated Myofibers
\(A) Phase-contrast micrograph of a live cell at 3 d after plating, showing a lobulated structure in the middle of the fiber.
\(B) Micrograph of a live fiber at 2 d after plating, showing budding of nuclei at one end. The cell has been counterstained with Syto 13.
\(C) Fluorescence micrograph of a myofiber at 24 h after microinjection with TR--dextran. The cell has been counterstained with Syto 13 dye to show the nuclei.
\(D) Fluorescence micrograph of a colony formed from a single myofiber injected 24 h earlier with TR--dextran. The cell has flattened on the substrate and the nuclei are stained with Syto 13 dye.
\(E) Fluorescence micrograph of a colony formed from the progeny of several myofibers in proximity that were injected 5 d earlier with TR--dextran.
\(F) Analysis of the DNA content of cells derived from myofibers injected 5 d earlier with TR--dextran. The DNA content was determined by image analysis of the nuclei of mononucleate TR-positive cells that had been stained with Hoechst (see [Materials and Methods](#s4){ref-type="sec"}). The green arrow is the value for G~0~ nuclei in quiescent myofibers, while the blue arrow is the G~2~/M value determined for mononucleate cells with anti-phosphohistone H3. The red arrow is the G~1~ value determined for mononucleate cells.
\(G) Photomicrograph of a live myofiber, 48 h after plating, showing a binucleate bud formed at the end. The cell was stained as for (B).
\(H) Fluorescence micrograph of a bud containing three nuclei stained with Syto13 (yellow) derived from a myofiber that contained at least five nuclei and that was injected with TR--dextran (red).
Scale bars: (B), (C), and (G), 100 μm; (A), (E), and (H), 50 μm; and (D), 10 μm.
:::
:::
We have analysed the DNA content of single Hoechst-stained nuclei by normalised measurements of fluorescence intensity in TR-labelled cells within such colonies, and an example of a representative distribution for a single colony is shown in [Figure 3](#pbio-0020218-g003){ref-type="fig"}F. This also shows the corresponding values (shown by arrows in [Figure 3](#pbio-0020218-g003){ref-type="fig"}F) for G~0~ nuclei in myofibers, G~1~ nuclei in mononucleate cells, and G~2~/M nuclei in mononucleate cells labelled with antibody to phosphohistone H3. The histogram of DNA content for cells in the colony is comparable to that previously observed for cycling newt mononucleate cells ([@pbio-0020218-Tanaka2]). The relatively long S-phase in urodele cells leads to a prominent contribution of cells with DNA content between 2N and 4N. In addition, there were examples of TR-labelled mononucleate cells in M-phase, as determined with anti-phosphohistone H3. We conclude that the progeny mononucleate cells are able to traverse S-phase and enter mitosis after cellularisation.
### Fragmentation. {#s2c2}
In a second aspect of plasticity, which was shown by 40%--70% of the total population of myofibers, the initial stages also involved the migration of nuclei to form local aggregates, often of two or three nuclei, within the fiber. The migration of nuclei into a terminal aggregate is illustrated by selected images from a time-lapse video analysis ([Figure 4](#pbio-0020218-g004){ref-type="fig"}A; [Video S1](#sv001){ref-type="supplementary-material"}). The series of [Figure 4](#pbio-0020218-g004){ref-type="fig"}B illustrates a trinucleate terminal aggregate that fragments from the body of the myofiber (yellow arrows). This fragment remained adherent and extended cytoplasmic processes. In some cases, the nuclear aggregate formed a bud that was discharged into the medium. An example of a multinucleate bud formed at the end of a fiber is shown in [Figure 3](#pbio-0020218-g003){ref-type="fig"}G. In cases in which fibers containing at least five nuclei had been injected with TR--dextran, such buds were often observed to adhere as viable bi- or trinucleate-labelled cells (see [Figure 3](#pbio-0020218-g003){ref-type="fig"}H). The multinucleate progeny resulting from these processes did not label with tritiated thymidine or undergo division.
::: {#pbio-0020218-g004 .fig}
Figure 4
::: {.caption}
###### Analysis of Nuclear Migration and Fragmentation by Time-Lapse Microscopy
\(A) Single frames illustrating the migration of three nuclei (yellow arrows) along a myofiber, of which two are incorporated into a terminal aggregate by 11.4 h. One nucleus (green arrow) remained stationary during this period.
\(B) Single frames illustrating the production of viable multinucleate fragments from a myofiber. Note the presence of a trinucleate aggregate (arrowed green) that separates after lateral breakage of the fiber (0 min, arrowed yellow). This fragment subsequently extends cytoplasmic processes (14.3 and 15.4 h) and migrates over the culture substratum.
Series (A) and (B) begin at 6 h after plating. Scale bars: (A) 50 μm; (B) 200 μm.
:::
:::
### Inhibition by taxol. {#s2c3}
In view of the evidence that implicates microtubules as a target for myoseverin, we stained the cultures with antibody to β-tubulin. Although tubulin was polymerised in microtubules parallel to the axis of the fibers, the regions of nuclear aggregation were associated with depolymerised tubulin ([Figure 5](#pbio-0020218-g005){ref-type="fig"}A). In order to assess the functional relevance of depolymerisation, we exposed the cultures to 2 μM taxol, an agent that stabilises microtubules and inhibits division of mononucleate urodele cells without effects on cell viability or adhesion of myofibers to the culture substrate. Whereas 80% of the control fibers showed the morphologies associated with plasticity, as described above (see [Materials and Methods](#s4){ref-type="sec"} for the criteria), only 16% were observed in the case treated with taxol ([Figure 5](#pbio-0020218-g005){ref-type="fig"}B), although the total number of adherent cells was unaffected. This suggests that localised depolymerisation of microtubules may be a significant target for the regulation of these responses.
::: {#pbio-0020218-g005 .fig}
Figure 5
::: {.caption}
###### Plasticity and Microtubule Depolymerization
\(A) The distribution of microtubules surrounding a multinucleate aggregate on a myofiber, as analysed by staining with anti-β-tubulin. Note the relatively disordered state of the tubulin (arrowed) in the vicinity of the nuclei. The fiber was stained at 48 h after plating. Scale bar, 50 μm.
\(B) Taxol inhibits the activation of myofibers after dissociation. Myofibers were dissociated and cultured in the presence of taxol as described in the [Materials and Methods](#s4){ref-type="sec"}. The number of active fibers was determined as described.
:::
:::
Expression of *Msx1* {#s2d}
--------------------
The cultures were reacted with a digoxygenin-substituted antisense riboprobe to axolotl *Msx1*. The mononucleate cells and inactive myofibers showed little or no reactivity, but active fibers showed strong reactivity with the probe in the vicinity of nuclear aggregations ([Figure 6](#pbio-0020218-g006){ref-type="fig"}A and [6](#pbio-0020218-g006){ref-type="fig"}B). Several control probes were negative on both classes of fibers, while the quiescent fibers as well as the active ones were reactive to antisense probes to urodele *EF1a* and *Nrad* ([Figure 6](#pbio-0020218-g006){ref-type="fig"}C). In view of the relationship between *Msx1* expression and plasticity in mouse myotubes ([@pbio-0020218-Odelberg2]), the expression in the active fibers is suggestive of a role in the endogenous urodele programme.
::: {#pbio-0020218-g006 .fig}
Figure 6
::: {.caption}
###### Analysis of mRNA Expression in Myofibers at 48 h by In Situ Hybridisation
\(A) Expression of *Msx1* mRNA in active myofiber. Note the accumulation of reaction product around nuclear aggregates (arrowed).
\(B) Absence of significant *Msx1* mRNA expression in a quiescent fiber. This image is taken from the same culture as (A).
\(C) Expression of *NRad* mRNA in nuclei of quiescent fibers (arrowed). Comparable intensity was observed for *NRad* expression by active fibers.
\(D) Expression of *Msx1* mRNA in nuclei of fibers (arrowed) made quiescent by culture in taxol. Note the difference in *Msx1* expression levels between the taxol-induced inactive fibers and normal quiescent myofibers in (B). Scale bar, 50 μm.
:::
:::
In an initial investigation of this possibility, cultures were arrested as before by treatment with taxol, followed by reaction with the *Msx1* antisense probe. In parallel control cultures, only 4.2% of the inactive fibers (*n* = 404) showed any reaction with the probe, whereas 51% of all myofibers (*n* = 820) showed reactivity. After treatment with taxol, 56.3% of inhibited fibers (*n* = 765) were positive, whereas 63% of all myofibers (*n* = 901) showed expression of *Msx1*. It is clear, therefore, that the arrest of nuclear mobilisation does not prevent the early expression of *Msx1* in activated myofibers. These data are consistent with an upstream role for *Msx1* in the activation of plasticity, but direct evidence for functional activity has come from antisense perturbation.
Activity of Morpholino-Substituted Oligonucleotides {#s2e}
---------------------------------------------------
In order to evaluate the uptake of morpholino-substituted oligonucleotides, larval myofibers were dissociated as usual in the presence of 10 μM biotinylated morpholinos or underivatised morpholinos. The cells were cultured for 48 h and then analysed by a detection procedure involving tyramide signal amplification (see [Materials and Methods](#s4){ref-type="sec"}). Approximately 70%--90% of the fibers showed uptake of the biotinylated oligonucleotides in different experiments ([Figure 7](#pbio-0020218-g007){ref-type="fig"}A), and no signal was detectable in the absence of oligonucleotide or with underivatised morpholinos ([Figure 7](#pbio-0020218-g007){ref-type="fig"}B). The cells are thus effectively loaded by dissociation in the presence of morpholinos.
::: {#pbio-0020218-g007 .fig}
Figure 7
::: {.caption}
###### Analysis of the Functional Role of Msx1 Expression by Exposure to Morpholino Antisense Oligonucleotides
(A and B) Uptake of morpholino by myofibers. Myofibers were dissociated in the presence of biotinylated (A) or control (B) morpholinos and analysed by tyramide signal amplification at 24 h after plating. Note the positive signal in (A), dependent on the presence of biotin moiety. In three different experiments 70%--90% of the fibers were loaded as determined with this assay. Scale bar, 50 μm.
\(C) Functional effect of loading various morpholinos. Note that loading *Msx1* antisense leads to a specific decrease in the proportion of active fibers relative to controls.
(D--G) Staining of myofibers with antibody to Msx1 protein. (D and E) Fluorescence micrograph of a nucleus in a quiescent myofiber stained with Hoechst for DNA (D) and Msx1 protein (E). (F and G) Fluorescence micrograph of a nucleus in an active myofiber stained for DNA (F) and Msx1 protein (G). These images (D--G) were taken from the same culture. Scale bar, 20 μm.
\(H) Distribution of fluorescence intensity of nuclei in myofibers after staining with antibody to Msx1. The distributions for control active fibers and control quiescent fibers were determined for cells in the same culture and are significantly different (ANOVA, *p* \< 0.001 at 95% confidence level). The distribution for antisense-treated quiescent fibers is not significantly different from that for control quiescent fibers.
:::
:::
Limb tissue was dissociated in the presence of control morpholinos or a morpholino antisense reagent directed at the translation initiation sequence of axolotl *Msx1*. The resulting cultures were analysed in parallel for the proportion of active fibers and the antisense reagent reproducibly and specifically decreased this by 60%--70% ([Figure 7](#pbio-0020218-g007){ref-type="fig"}C). This led, as expected from the mechanism of such reagents, to the presence of inhibited fibers that expressed *Msx1* mRNA after in situ hybridisation, and the proportion of such *Msx1* positive and inhibited cells was increased by 5-fold relative to parallel cultures incubated with control oligonucleotides.
The myofiber cultures were stained by indirect immunofluorescence with a rabbit antibody to Msx1 in order to evaluate the level of expression of the homeoprotein in the nuclei. There was a significant difference is staining of nuclei between active and quiescent fibers in the same culture ([Figure 7](#pbio-0020218-g007){ref-type="fig"}D--[7](#pbio-0020218-g007){ref-type="fig"}G). The level of expression in nuclei of different fibers was estimated by quantitative image analysis, and the distribution of intensities is shown in [Figure 7](#pbio-0020218-g007){ref-type="fig"}H. There was a significant difference in the fluorescence intensity of nuclei in active and quiescent fibers in control cultures ([Figure 7](#pbio-0020218-g007){ref-type="fig"}H), consistent with the difference in mRNA levels observed by in situ hybridisation (see [Figure 6](#pbio-0020218-g006){ref-type="fig"}A and [6](#pbio-0020218-g006){ref-type="fig"}B). The distribution of intensities for nuclei in quiescent fibers in parallel cultures treated with antisense oligonucleotides to Msx1 was not significantly different from the control quiescent distribution ([Figure 7](#pbio-0020218-g007){ref-type="fig"}H). It should be noted that more than half of the quiescent fibers were inhibited as a result of the antisense treatment, thus indicating that the antisense distribution reflects a significant decrease in protein expression in the nucleus relative to active fibers. We conclude that expression of a critical level of Msx1 protein is necessary for the fibers to exhibit plasticity in this culture system.
Discussion {#s3}
==========
The plasticity of isolated urodele myofibers as described here has not been observed in previous work on dissociated mouse myofibers ([@pbio-0020218-Rosenblatt1]; [@pbio-0020218-Blaveri1]). These apparently retain their morphological identity in culture without undergoing viable fragmentation or cellularisation. In preliminary work on myofibers dissociated from the forelimb of *Xenopus* tadpoles (stages 56--63), we have observed fragmentation comparable to that described here for fibers of the larval salamander, but no cellularisation. It is possible, therefore, that there is a gradation in the degree of plasticity after dissociation, and this may be related to the ability to undergo reversal during regeneration, although more work is required to investigate these comparative issues. It is interesting that the mononucleate progeny of cellularisation were observed to reenter the cell cycle, while multinucleate fragments retained the postmitotic arrest of the parental fibers. At least half of the salamander fibers were activated after dissociation and could be scored by morphological criteria as an index of plasticity, as well as by analysis of gene expression in situ. The occurrence of cellularisation did not reflect the activation of adherent mononucleate cells since the injection of a nontransferable tracer into the fibers resulted in labelling of the mononucleate progeny, and furthermore the mobilisation of nuclear aggregates occurred without any detectable S-phase reentry. It is probable that the process of enzymatic and mechanical dissociation mimics the activation events after amputation, either in terms of mechanical factors sensed by the fibers or the release of signals from the tissue or matrix. Earlier experiments on microinjected fibers in the larval tail have explored the stimuli required to trigger cellularisation and concluded that activation apparently required both 'clipping' at the end of the fiber as well as tissue injury in the vicinity ([@pbio-0020218-Echeverri1]). It has also been reported that crude extracts from early regenerates of the adult newt limb are able to induce cellularisation of newt and mouse myotubes in culture ([@pbio-0020218-McGann1]). The precise nature of the signal(s) that couples tissue injury to activation of this response remains an important subject for future investigation, particularly as it may be a key difference between urodeles and mammals.
One striking consequence of fiber activation is the appearance of the *Msx1* transcript, and our work strongly supports the hypothesis that *Msx1* is a pivotal regulator of plasticity in differentiated cells. Although taxol treatment is able to block the internal reorganisation in activated fibers, it does not inhibit the induction of *Msx1*, suggesting that microtubule depolymerisation, while being a direct target of myoseverin ([@pbio-0020218-Rosania1]), may also be a downstream target for regulation by *Msx1*. The striated myofibers are more highly differentiated than the newt A1 myotubes employed for implantation or the C2C12 mouse myotubes used to assay myoseverin and *Msx1*. The events of cellularisation, cleavage, or budding off from myofibers are preceded by migration of nuclei to generate local concentrations, reminiscent of the events leading to formation of the neuromuscular junction ([@pbio-0020218-Merlie1]; [@pbio-0020218-Englander1]), although mouse myotubes seem to undergo lateral breakage without such reorganisation ([@pbio-0020218-Rosania1]). This migration is inhibited by taxol, and nuclear migration in other contexts is dependent on microtubule function ([@pbio-0020218-Morris1]). All of the events described for the myofibers occur without entry into S-phase, as determined previously for cellularisation of myotubes after implantation ([@pbio-0020218-Velloso2]). The formation of mononucleate cells is followed by rapid division and loss of myosin expression, and these cells are presumably the culture equivalent of muscle-derived blastemal cells.
The activity of the *Msx1* gene has recently been implicated in digit tip regeneration in fetal and neonatal mice by comparing regeneration in normal and *Msx1* mutant animals ([@pbio-0020218-Reginelli1]; [@pbio-0020218-Han1]). It has also been shown that transgenic expression of an activated Msx1 protein can induce tail regeneration in larval *Xenopus* during the refractory period between stages 45 and 47 ([@pbio-0020218-Beck1]). This evidence, taken in conjunction with the present study and that of [@pbio-0020218-Odelberg2], indicates that this gene is an important regulator of regeneration. Various activities have been associated with the protein, including a role as a repressor of transcription (reviewed in [@pbio-0020218-Bendall1]), for example, of various myogenic differentiation genes in C2C12 myotubes ([@pbio-0020218-Odelberg2]) and also as a positive regulator of genes that promote cell cycling such as cyclin D ([@pbio-0020218-Hu1]). Our analysis of the myofiber cultures provides evidence for its ability to mobilise a postmitotic cell, for example, by nuclear migration and cellularisation, without S-phase reentry in the syncytium, and this suggests a different aspect of its activity as a regulator. Studies on mammalian myotubes should continue to be informative, while the present system, with its ready incorporation of antisense oligonucleotides, should be helpful for relating such studies to the endogenous programme of urodele regeneration. This in turn should assist the long-term goal of promoting the reversal of cellular differentiation as a strategy for mammalian regeneration ([@pbio-0020218-Charge1]).
Materials and Methods {#s4}
=====================
{#s4a}
### Tissue dissociation and culture of myofibers {#s4a1}
The forelimbs and hind limbs of the larval spotted salamander (A. maculatum) or axolotl (A. mexicanum) (3--5 cm size) were removed, the epidermis was peeled off, and the tissue was rinsed in serum-free amphibian MEM (AMEM) ([@pbio-0020218-Ferretti1]) prior to dissociation for 3 h at 26 °C in PBS containing 0.15% collagenase (Type 1A, Sigma, St. Louis, Missouri, United States), 0.8% Dispase II (Roche, Basel, Switzerland), 0.15% crystalline bovine serum albumin, 0.3% D-glucose, and 0.15 mg/ml DNase I (Roche). After 30 min of incubation, the tissues were gently triturated through a fire-polished glass pasteur pipette to aid the detachment of myofibers from the bone. After incubation, the suspension was triturated several times, centrifuged at 400 rpm for 10 min, resuspended in AMEM, and filtered through a 35 μm sieve (VWR International, Poole, United Kingdom) to remove most of the mononucleate cells. The retentate was rinsed with AMEM and plated on 35 mm Falcon Primaria (Becton-Dickinson, Palo Alto, California, United States) tissue culture dishes. Cultures were maintained at 25 °C with 2.5% CO~2~ in a humidified incubator as described elsewhere ([@pbio-0020218-Ferretti1]). After attachment of the myofibers to the culture dish by overnight incubation, the culture media was replaced with serum-free AMEM or AMEM supplemented with 10% foetal bovine serum.
### Labelling and implantation of myofibers {#s4a2}
Myofibers were dissociated as above, retained in suspension in a sterile bacteriological dish (Bibby Sterilin, Stone, United Kingdom), and incubated with 10 μM CellTracker Orange CMTMR (Molecular Probes, Eugene, Oregon, United States) for 30 min at 25 °C. The labelling was terminated by addition of 10% AMEM, and the cells were incubated for 45 min at 25 °C to permit enzymatic activation of the dye. The cell suspension was diluted several fold to allow observation of myofibers at low density. The forelimbs of axolotl larvae (7--10 cm size) were amputated at mid humerus level under tricaine (0.1%) anaesthesia 48 h before injection of labelled myofibers (see [Figure 2](#pbio-0020218-g002){ref-type="fig"}A). The animals were anaesthetized, and the forelimbs were positioned under a stereo zoom microscope. The myofiber suspension was placed under inverted microscope, and the myofibers were drawn into a glass micropipette (30 μm tip diameter) using an oil-driven manual microinjector (Sutter Instruments, Novato, California, United States) mounted on a Narishige (Tokyo, Japan) MMO-1 micromanipulator. The skin was punctured with a tungsten needle in order to introduce the blunt end of the micropipette. Three to eight myofibers were picked, examined carefully to verify the absence of any adherent mononucleate cells, and injected into each limb regenerate. Contralateral limbs were mock injected with medium from the suspension. The regenerates were removed at 48 h and 96 h after injection, fixed in 4% paraformaldehyde (PFA), and processed ([@pbio-0020218-Kumar1]). Serial longitudinal sections of 60 μm were cut on a cryostat (Leica, Solms, Germany), air dried, dehydrated in PBS, and counterstained with 2.5 μM Sytox Green (Molecular Probes). The sections were observed under epifluorescence on an Axiophot microscope (Zeiss, Jena, Germany).
### Microinjection of cultured myofibers with conjugated dextran {#s4a3}
Myofibers were incubated in AMEM containing 2,3-butanedionne monoxime (BDM) (4 mM) for 30 min to prevent contraction of the myofibers ([@pbio-0020218-Bettencourt-Dias1]) and maintained in the same medium during microinjection. The culture dishes were placed under a Zeiss Axiovert microscope and microinjected with TR-conjugated dextran (TR--dextran, 70 kDa; Molecular Probes). The medium was changed immediately after injection and the cultures were returned to the incubator. To identify the labelled myofibers and their mononucleate progeny, cultures were counterstained in Syto13 (200 nM; Molecular Probes) live nucleic acid stain for 30 min and observed under fluorescence microscope with a dual band pass (FITC/TRITC) filter.
### Live imaging of myofiber plasticity {#s4a4}
To record the coordinates of the myofibers in culture, the dish was scored underneath with a scalpel, and cells in each grid square were observed daily and images were acquired with a CCD camera (Sony, Tokyo, Japan). For time-lapse microscopy, myofiber cultures were placed under an Axiovert microscope fitted with an incubation chamber maintained at 26 °C and 3% CO~2~, and phase contrast or variable relief contrast (VAREL) (Zeiss) images were acquired using a digital camera controlled through Image-Pro Plus software (Media Cybernetics, Silver Spring, Maryland, United States). A sequence gallery was created using Image Pro-Plus and images of interest were selected, digitally enhanced, and processed in Adobe Photoshop 6.0 (Adobe, San Jose, California, United States).
### \[^3^H\]thymidine labelling. {#s4a5}
Myofibers were incubated in 1 μCi/ml \[^3^H\] thymidine (Amersham Biosciences, Little Chalfont, United Kingdom) for 24 h, fixed in 1% glutaraldehyde, and processed for autoradiography ([@pbio-0020218-Velloso1]).
### DNA cytometry {#s4a6}
DNA content in myofiber nuclei and TR--dextran-labelled mononucleate progeny was measured quantitatively after fixation and staining of the nuclei with Hoechst 33258 (2 μg/ml; Sigma). Baseline values for nuclear DNA content in cycling axolotl mononucleate cells were measured in parallel after incorporation of 5-deoxy-2′-bromouridine (BrdU) (1 μM; see [@pbio-0020218-Tanaka2]). The BrdU-labelled cells were processed for double immunofluorescence with monoclonal antibody against BrdU and rabbit antibody to anti-phosphohistone H3 ([@pbio-0020218-Velloso1]; [@pbio-0020218-Bettencourt-Dias1]). The nuclei were counterstained with Hoechst dye as above. All images were acquired using 12-bit cooled CCD camera (Photonic Sciences, Robertsbridge, United Kingdom), maintaining camera and microscope settings identical between various samples, corrected for uneven illumination and background using software functions, and processed using classification and measuring routines in Image-Pro Plus software.
### Scoring of plasticity in myofibers {#s4a7}
A viable nucleic acid stain such as Syto 13 or Hoechst 33342 (Molecular Probes) was routinely used in cultures to visualize and score the myofibers. The quiescent or inactive myofiber nuclei were aligned along the fiber (see [Figures 1](#pbio-0020218-g001){ref-type="fig"} and [7](#pbio-0020218-g001){ref-type="fig"}D), and the cell did not show any cytoplasmic extensions from the axis. The nuclei in active fibers moved along the axis of the fiber to form aggregations that were localized either in the middle or towards the end of the cell (see [Figure 3](#pbio-0020218-g003){ref-type="fig"}A, [3](#pbio-0020218-g003){ref-type="fig"}B, and [3](#pbio-0020218-g003){ref-type="fig"}G). In most cases, this resulted in formation of localised cytoplasmic protrusions in the vicinity of the nuclei. Myofibers were classified and counted based on the above criteria.
### Taxol inhibition assay. {#s4a8}
Dissociated myofibers were plated in medium containing taxol (2 μM; Sigma). Parallel control cultures were incubated in vehicle (DMSO) in a similar way. The cultures were fixed at 48 h after treatment and processed for tubulin immunofluorescence or in situ hybridisation.
### Functional assay for Msx-1 using morpholino antisense oligonucleotides {#s4a9}
Morpholino-based antisense oligonucleotides of 25 oligomere were prepared to target the translation initiation site of axolotl *Msx1* gene (5′-CGGTCTGCATCCTCTGCTTGCTTAG-3′) by Gene Tools Inc. (Corvallis, Oregon, United States). Invert control oligos of *Msx1* (5′-GATTGCTTCGTCTCCTAGCTCTGGC-3′) and standard control oligos (5′-CCTCTTACCTCAGTTACAATTTATA-3′) from the supplier were used as controls. 3′-Biotin-end-labelled standard control oligos were used for evaluating the uptake of morpholino oligonucleotides by myofibers. Oligo stock solutions were prepared according to guidelines from the manufacturer and stored at 4 °C. The morpholino oligos were added at a concentration of 10 μM to the dissociation cocktail and the myofibers were dissociated as described. After purification of myofibers, fresh morpholino oligos were added to the culture medium. After sequential washes to remove any adherent morpholino ([@pbio-0020218-McKeon1]), cultures were fixed at 48 h after treatment, prior to analysis.
### Detection of morpholino uptake by immunofluorescence. {#s4a10}
Tyramide signal amplification (PerkinElmer Life Sciences Inc., Wellesley, Massachusetts, United States) coupled with enzyme-linked immunofluorescence (ELF97, Molecular Probes) was employed to localize the uptake of morpholino oligos in cultured myofibers. Myofiber cultures were fixed at 48 h in 0.5% PFA containing 0.05% glutaraldehyde for 15 min on ice. The fixative was replaced with freshly made 0.1% NaBH~4~ solution and incubated for 5 min. The manufacturer\'s protocol was employed for TSA amplification with the ELF97 modification. The samples were developed in ELF reaction buffer under fluorescence microscope for 10--20 s and images were acquired using a cooled digital camera.
### In situ hybridisation {#s4a11}
The axolotl *Msx1* cDNA (a kind gift from H. Ide, Tohoku University, Sendai, Japan) was cloned into Bluescribe vector (Stratagene, La Jolla, California, United States), and probes were generated as described elsewhere ([@pbio-0020218-Koshiba1]). A 0.7 kb axolotl EF-1α fragment (kindly provided by D. Gardiner and S. Bryant, University of California, Irvine, United States) was cloned into PCR II vector (Invitrogen, Carlsbad, California, United States) and linearised with XhoI (antisense), and a riboprobe was generated with SP6 RNA polymerase. Newt *Rad* (*NRad*, a gift from K. Yoshizato, Hiroshima University, Hiroshima, Japan) probe was generated from a fragment of approximately 400 bp from Bluescribe vector after linearising with either HindIII (antisense) or EcoRI (sense), and riboprobes were synthesized using T3 and T7 RNA polymerase respectively ([@pbio-0020218-Shimizu-Nishikawa1]). Axolotl *EF1α* and *NRad* probes were used as positive controls, while neomycin ([@pbio-0020218-Cash1]), *NRad* sense, and *Msx1* sense probes served as negative controls. For in situ hybridisation, the myofiber cultures were incubated in BDM (4 mM), fixed in chilled 1% glutaraldehyde for 15 min, postfixed in 4% PFA, and washed in 0.3% PBT. In situ hybridisation was essentially as described elsewhere ([@pbio-0020218-Kumar1]), with minor modifications in the hybridisation temperature.
### Antibodies and immunofluorescence {#s4a12}
Myofiber cultures were routinely fixed in ice-cold 0.5% PFA containing 0.05% glutaraldehyde for 10 min on ice. For β-tubulin staining, 5 μM Taxol (Sigma) was incorporated into the fixative. After fixation, the culture was treated with freshly prepared 0.1% NaBH~4~ for 5 min to reduce nonspecific fluorescence. The samples were post-fixed in ice-cold methanol at −20 °C for 10 min, washed three to four times in 0.3% PBT, and blocked in PBT containing 10% goat serum. The primary antibodies were to MHC and titin, and BrdU monoclonal antibody and rabbit polyclonal antibodies to phosphohistone H3, were all as described elsewhere ([@pbio-0020218-Tanaka2]; [@pbio-0020218-Kumar1]; [@pbio-0020218-Velloso1]; [@pbio-0020218-Bettencourt-Dias1]). For localisation of β-tubulin, the culture was fixed and washed overnight in 0.3% PBT and incubated with mouse monoclonal β-tubulin antibody (1:100; clone TUB 2.1; Sigma) overnight at 4 °C. The samples were washed extensively in GS/PBT and incubated in TR-conjugated goat anti-mouse antibody (1 μg/ml; Molecular Probes). The nuclei were counterstained with Hoechst 33258 (2 μg/ml). A rabbit polyclonal antibody raised against the full-length mouse Msx1 homeoprotein was used to detect expression of Msx1 protein (BAbCO, Richmond, California, United States). When a full-length expression construct of axolotl *Msx1* was expressed in mouse cells by transient transfection, the antibody gave strong and specific staining of nuclei in transfected cells ([Figure S2](#sg002){ref-type="supplementary-material"}). The samples were fixed and processed as before and incubated with Msx1 antibody (1:1000) overnight at 4 °C. After several washes, the cultures were incubated with FITC-conjugated goat anti-rabbit antibodies (1:100; DakoCytomation, Cambridgeshire, United Kingdom), and the nuclei were counterstained with Hoechst. A control rabbit polyclonal antibody was processed in parallel to obtain a baseline value for quantitative fluorescence measurements on immunostained nuclei.
The myofiber cultures stained for β-tubulin, MHC, or titin, or cultures injected with TR--dextran were observed under confocal laser scanning microscope (Leica). The images were acquired as *z*-stacks, and composite maximum projection images were generated through Leica software. Samples stained for Msx1 protein were observed under a Zeiss Axioplan microscope and images were acquired with an Axiocam digital camera. The fluorescence intensity in myofiber nuclei was measured in Axiovision software (Zeiss), and the data were analysed by one-way analysis of variance (ANOVA) followed by multiple range test using Instat (Graphpad Software Inc., San Diego, California, United States).
Supporting Information {#s5}
======================
Figure S1
::: {.caption}
###### Live Striated Myofiber Dissociated from the Limb of a Larval Salamander
The myonuclei incorporate Syto13 live nuclear stain. The myofiber was observed with VAREL optics at 24 h after plating. Scale bar, 100 μm.
(4.1 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S2
::: {.caption}
###### Expression of Newt *Msx1* in Mouse PS Cells by Transient Transfection
Nuclear localisation of Msx1 protein (green) was detected with a rabbit polyclonal antibody generated against the full-length mouse Msx1 homeoprotein.
(5.6 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Video S1
::: {.caption}
###### Time-Lapse Video Analysis of Nuclear Migration in a Myofiber
Time-lapse sequence was begun 6 h after plating of the myofiber on to a culture dish. The images were taken at 6 min intervals under 32× VAREL objective magnification.
(110 KB AVI).
:::
::: {.caption}
######
Click here for additional data file.
:::
We thank Phillip Gates for plasmid construction, Tim Landy and Karel Liem for comments and suggestions on the manuscript, and Daniel Ciantar (Confocal Unit, Department of Anatomy, University College London) for his help. This work was funded by a Programme Grant from the Medical Research Council to JPB.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** AK and JPB conceived and designed the experiments. AK, CV, and YI performed the experiments. AK, CV, YI, and JPB analyzed the data. CV, YI, and JPB contributed reagents/materials/analysis tools. AK and JPB wrote the paper.
Academic Editor: Brigid Hogan, Duke University Medical Center
Citation: Kumar A, Velloso CP, Imokawa Y, Brockes JP (2004) The regenerative plasticity of isolated urodele myofibers and its dependence on Msx1. PLoS Biol 2(8): e218
AMEM
: amphibian minimum essential medium
ANOVA
: analysis of variance
BDM
: 2,3-butanedionne monoxime
BrdU
: 5-deoxy-2′-bromouridine
MHC
: myosin heavy chain
PFA
: paraformaldehyde
TR
: Texas red-conjugated dextran
VAREL
: variable relief contrast
| PubMed Central | 2024-06-05T03:55:46.739330 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509293/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e218",
"authors": [
{
"first": "Anoop",
"last": "Kumar"
},
{
"first": "Cristiana P",
"last": "Velloso"
},
{
"first": "Yutaka",
"last": "Imokawa"
},
{
"first": "Jeremy P",
"last": "Brockes"
}
]
} |
PMC509294 | Introduction {#s1}
============
The cerebral cortex is the seat of consciousness and the means by which we carry out abstract reasoning. Understanding how the cortex is assembled during embryonic development will give deeper insights into how this marvelous machine functions and provide the basis for therapy and repair. Although a diversity of approaches will be needed to answer all of our questions, an important starting point in studying events in development is often the careful analysis of mutant phenotypes. Much of what we know about cortical development has emerged through the study of mutations in mice and humans. For example, spontaneous mutations in mice such as *reeler* and *scrambler* have helped to tease apart the regulation of the radial migrations that create the cortical layers. Other important insights have come from the study of spontaneous mutations that cause radial migration defects and lead to lissencephaly and similar cortical defects in humans. Our understanding of radial migration and many other aspects of cortical development have also benefited enormously from the application of gene knockout approaches in mice. Despite this progress, many aspects of cortical development remain to be explored and would benefit enormously from additional mutant resources. The tangential migrations of cortical interneurons, for example, are regulated differently from the radial migrations of projection neurons, and only a few mutations have been described that disrupt interneuron migration.
Forward genetic approaches in the mouse, although technically feasible for many years, have become increasingly attractive with the availability of a dense genetic map and a nearly complete genomic sequence. These tools allow the process of gene identification, which was once very cumbersome, to be relatively straightforward. With the initial resurgence of interest in genetic screens, large-scale screens aimed at identifying mutations in broadly defined phenotypic categories were established at mutagenesis centers in Germany, the United Kingdom, the United States, and other countries ([@pbio-0020219-Hrabe1]; [@pbio-0020219-Nolan1]). More recently, smaller, more focused screening efforts have had notable success ([@pbio-0020219-Vitaterna1]; [@pbio-0020219-Eggenschwiler1]; [@pbio-0020219-Kapfhamer1]; [@pbio-0020219-Garcia-Garcia1]; [@pbio-0020219-Hoebe1]), generally in situations where the effects of mutation on a specific cellular or biochemical process can be readily ascertained. Further development of forward genetics in the mouse as an approach with general utility will require the validation of focused screening strategies that allow for the identification of mutations disrupting specific processes in diverse situations, as has been done in other model organisms. In *Drosophila,* deletion strains and chromosomal inversions have been used to identify mutations within a specific region of the genome, and this approach has been elegantly adapted for use in the mouse ([@pbio-0020219-Juriloff1]; [@pbio-0020219-Kile1]). The development of screening strategies for the mouse that focus on the identification of mutations affecting a biological process rather than mapping to a certain genomic region will further expand the questions that can be addressed.
Here we describe a focused genetic screen in the mouse that takes advantage of a transgenic reporter line that labels the ganglionic eminences of the ventral forebrain and migrating cortical interneurons with β-galactosidase. The use of this reporter gene allows the isolation of mutations that alter growth and morphogenesis of the cortex very efficiently and, more specifically, allows the identification of mutations disrupting interneuron migration from the ganglionic eminences into the cortex. In this screen, we isolated four mutations that affect tangential migration. We also isolated nine novel mutations affecting other aspects of cortical development; these mutations include three that represent novel alleles of genes that have been shown to have a role in cortical development. These results illustrate the power of a forward genetic approach, borrowed from other model organisms, that can be applied to various questions of mammalian biology.
Results {#s2}
=======
The *Dlx-LacZ* Transgene Labels the Ganglionic Eminences and Migrating Interneurons {#s2a}
-----------------------------------------------------------------------------------
The expression of Dlx family homeodomain transcription factors is necessary for differentiation of neurons in the striatum and for migration of many, if not all, of the interneuron precursors that arise in the ganglionic eminences of the embryonic telencephalon ([@pbio-0020219-Marin1], [@pbio-0020219-Marin2]). A transgenic line in which the expression of β-galactosidase is driven by transcriptional control sequences from the *Dlx5/6* intergenic region faithfully recapitulates most aspects of Dlx5 expression in the central nervous system, including strong expression in the subventricular zone (SVZ; a secondary progenitor zone) of the ganglionic eminences, in immature interneurons as they migrate tangentially through the intermediate zone (IZ) or marginal zone (MZ) of the cortex ([Figure 1](#pbio-0020219-g001){ref-type="fig"}A--[1](#pbio-0020219-g001){ref-type="fig"}C), and in mature cortical GABAergic interneurons ([@pbio-0020219-Stuhmer1]). The dispersed interneuron precursors label the cerebral cortex, and the underlying high-level expression in the ganglionic eminences provides a useful adjunct to the morphological landmarks, allowing cortical defects to be identified in whole-mount stained embryos. We took advantage of the Dlx-LacZ line as a background upon which to screen for mutations. Mutations were induced using ethyl-nitroso-urea (ENU), a mutagen that induces primarily single-base substitutions with very little bias ([@pbio-0020219-Russell1]; [@pbio-0020219-Vrieling1]; [@pbio-0020219-Nivard1]), and animals were bred in order to detect recessive mutations that disrupted the distribution of Dlx-LacZ--labeled cells in the developing cortex at embryonic day 14.5 (E14.5) ([Figure 1](#pbio-0020219-g001){ref-type="fig"}D). In total, 705 litters with an average of seven embryos each, and representing 305 lines of mice, were stained using β-galactosidase histochemistry, and then examined in whole mount; higher-resolution analysis on 100-μm coronal vibratome sections of brains was also performed on a third (225) of the litters.
::: {#pbio-0020219-g001 .fig}
Figure 1
::: {.caption}
###### The Strategy for Isolation of Recessive Mutations Disrupting Cortical Development
\(A) Migrating interneuron precursors appear to migrate tangentially (yellow arrows) through both the MZ and the IZ/SVZ. Precursors also migrate radially (pink arrows) to reach the developing cortical plate (CP).
\(B) Migrating interneuron precursors expressing β-galactosidase can be seen in a whole-mount preparation of an E14.5 Dlx-LacZ mouse as diffuse cortical staining.
\(C) In coronal section, the densely labeled SVZ of the LGE can be seen, as can the streams of migrating interneuron precursors in the cortex.
\(D) To identify recessive mutations, male C57BL6/J mice were treated with ENU and then crossed to FVBN/J females that were homozygous for the *Dlx-LacZ* transgene. Male offspring of this cross were backcrossed to produce female offspring that were then backcrossed to their fathers. Embryonic litters from these backcrosses were stained and examined for defects at E13.5 or E14.5.
:::
:::
Dlx-LacZ Allows Efficient Identification of Cortical Mutants {#s2b}
------------------------------------------------------------
During the course of screening we identified eight mutants with defective growth or patterning of the cerebral cortex, four mutants with defects in the migration of interneuron precursors into the cortex, and one mutant in which thalamocortical axons fail to invade the cortex ([Table 1](#pbio-0020219-t001){ref-type="table"}). Lines of mice carrying each mutation were established, in which the phenotype was propagated as a recessive trait with Mendelian inheritance. Preliminary mapping, together with the overt phenotype, pointed us toward a likely locus for three of the mutants ([Figure 2](#pbio-0020219-g002){ref-type="fig"}). The other ten mutations appear to identify loci whose role in forebrain development has not previously been described.
::: {#pbio-0020219-g002 .fig}
Figure 2
::: {.caption}
###### Novel Alleles of *scribble, megalin,* and *Rfx4*
(A and B) Comparison of E12.5 WT (A) and mutant (B) embryos showing the severe craniorachischisis of line 90 embryos.
\(C) Sequencing chromatograms show the thymine in the WT sequence (top) that is an adenine in line 90 (bottom). This thymine-to-adenine substitution changes an isoleucine codon to a lysine codon within a conserved LRR domain of SCRIBBLE.
(D and E) Ribbon diagrams of an LRR fold, illustrating the predicted position (red arrowheads) of the line 90 amino acid substitution. The substitution is in a linker region between two stretches of β-sheet. An LRR fold has a β-sheet on the inside, and linker regions on the outside, of a broad half-circular curve.
\(F) The predicted domain structure of SCRIBBLE indicating where the line 90 and the *Circletail* alleles alter the protein relative to the three LRR and four PDZ domains.
(G and H) Dorsal views of the cortex of E17.5 WT (G) and line 267 mutant (H) embryos stained to reveal the expression of the *Dlx-LacZ* transgene. The red arrowheads point to the choroid plexus of the third ventricle, which is stained because of its endogenous β-galactosidase expression, and which is greatly enlarged in the mutant. The cortex of the mutant is longer along the rostrocaudal axis and is altered in shape in the caudal portion (yellow arrows). The olfactory bulbs (blue arrowheads) are also altered in shape.
\(I) The choroid plexus persists in its hypertrophic state after birth and can be seen as a pinkish lump on the head of this postnatal day 14 (P14) pup.
\(J) A coronal section through the dorsal portion of the diencephalon of a P0 pup.
\(K) Sequencing chromatograms show the thymine in the WT sequence (top) that is an adenine in line 267 (bottom).
\(L) The predicted domain structure of MEGALIN indicating where the new stop codon is introduced by the line 267 mutation.
(M and N) Dorsal views of the cerebral cortex of E14.5 WT (M) and line 269 mutant (N) embryos stained to reveal the expression of the *Dlx-LacZ* transgene.
(O and P) Coronal sections of E14.5 WT (O) and line 269 mutant (P) forebrains. Red arrowheads indicate the apparent span over which dorsal midline structures are lost in the mutant.
\(Q) Sequence chromatograms showing the thymine in the WT sequence (top) that is a cytosine in line 269 (bottom).
\(R) The predicted domain structure of RFX4 indicating where the line 269 substitution alters the protein relative to the DNA binding and the extended (B, C, and Dim) dimerization domains.
:::
:::
::: {#pbio-0020219-t001 .table-wrap}
Table 1
::: {.caption}
###### Summary of Cortical Mutants
:::
^a^ See supporting information
^b^ Scored on the basis of polydactyly
^c^ These two mutants fail to complement and are likely to be allelic
^d^ Scored on the basis of migration defects
^e^ These two mutants have very different phenotypes. Since the interval is large the two mutants seem likely to represent two different genes
:::
A Novel *scribble* Allele {#s2c}
-------------------------
The three mutations in previously characterized loci produce alleles that differ from the existing ones in informative ways ([Figure 2](#pbio-0020219-g002){ref-type="fig"}). Mice homozygous for the line 90 mutation have an open neural tube in the spinal cord and hindbrain region, or craniorachischisis, and a disorganized and hyperplastic neuroepithelium in the cortex and other parts of the central nervous system ([Figure 2](#pbio-0020219-g002){ref-type="fig"}A and [2](#pbio-0020219-g002){ref-type="fig"}B). An essentially identical phenotype is seen in homozygous *Loop-tail and Circletail* mutants ([@pbio-0020219-Kibar1]; [@pbio-0020219-Murdoch1]a, [@pbio-0020219-Murdoch2]b, [@pbio-0020219-Murdoch3]), both of which also have dominant tail defects, a phenotype that is not seen in line 90. Both *Loop-tail* and *Circletail* mice have mutations in genes that regulate planar cell polarity. *Loop-tail* mice carry a mutation in the *strabismus-1* gene *(Str-1,* also known as *Ltap/Lpp1),* and *Circletail* mice have a mutation in the *scribble* gene *(Scrb1).* Mapping of the line 90 mutation places it on Chromosome 15 in the region of *Scrb1. Scrb1* encodes a protein of 1,665 amino acids with three leucine-rich repeat (LRR) domains near the amino terminus and four centrally located PDZ domains ([Figure 2](#pbio-0020219-g002){ref-type="fig"}F). Sequencing of RT-PCR products from line 90 mice identified a missense mutation in *Scrb1* that causes an isoleucine-to-lysine substitution in the third LRR domain ([Figure 2](#pbio-0020219-g002){ref-type="fig"}C--[2](#pbio-0020219-g002){ref-type="fig"}F).
In *Drosophila,* a critical role for a *Scrb1* homolog, *scribbled,* in establishment of apical basal polarity in epithelia has been described ([@pbio-0020219-Bilder1]; [@pbio-0020219-Bilder2], [@pbio-0020219-Bilder3]). Analysis of mice carrying the *Circletail* allele of *Scrb1* show a loss of planar cell polarity in the hair cells of the inner ear, indicating that *Scrb1* is required for the establishment of planar cell polarity rather than apical-basal polarity in the mouse ([@pbio-0020219-Montcouquiol1]). Interestingly, a strong genetic interaction has been observed between the *Circletail* allele of *Scrb1* and *Loop-tail* mutants ([@pbio-0020219-Murdoch2]). The interaction is strong enough that compound heterozygotes for the two mutations show a severe phenotype that is indistinguishable from the individual homozygous phenotypes. The molecular nature of the *Circletail* allele of *Scrb1,* with a frameshift mutation that causes a premature stop codon after the first two PDZ domains ([@pbio-0020219-Murdoch3]), is substantially different from that of line 90. To see whether interaction between the two loci was a unique attribute of the *Circletail* allele, we crossed line 90 with *Loop-tail* mice. As with the *Circletail* allele, a strong genetic interaction was seen between the line 90 allele of *Scrb1* and the *Ltap/Lpp1* mutation, such that embryos indistinguishable from homozygotes of the individual mutations were recovered (unpublished data). This indicates quite clearly that the integrity of the LRR domains is critical for the tight coordination between *Scrb1* and *Ltap/Lpp1* in establishment of planar cell polarity.
A Novel *megalin* Allele {#s2d}
------------------------
We also identified a mutation that caused an enlarged cortex, hypertrophy of the choroid plexus of the third ventricle, and abnormalities in the dorsal diencephalon ([Figure 2](#pbio-0020219-g002){ref-type="fig"}G--[2](#pbio-0020219-g002){ref-type="fig"}J). Mapping of this mutation places it on Chromosome 2 in a region containing the *megalin* gene (also known as *Lrp2*). Prolapse of the third ventricle choroid plexus was described in a knockout allele of *megalin* ([@pbio-0020219-Willnow1]), suggesting that this might be the responsible locus in this case. On this basis we sequenced RT-PCR products from the *megalin* gene and identified a base substitution producing a premature stop codon rather than the tyrosine codon at residue 2721 ([Figure 2](#pbio-0020219-g002){ref-type="fig"}K and [2](#pbio-0020219-g002){ref-type="fig"}L). The ENU-induced allele is predicted to express a truncated MEGALIN protein consisting of the amino-terminal portion of the extracellular domain, whereas the knockout is a null. Unlike the case of *Scrb1,* where the phenotypes produced by the two alleles are essentially identical, the two *megalin* alleles show phenotypic differences. Although both alleles produce a hypertrophic choroid plexus, this defect is more pronounced with the ENU-induced allele. We have found that the choroid plexus defects are also associated with an expansion and inhibition of differentiation in the dorsal neuroepithelium of the diencephalon that would ordinarily form the subcommissural organ and of the pineal gland just caudal to the defective choroid plexus (see [Figure 2](#pbio-0020219-g002){ref-type="fig"}I--[2](#pbio-0020219-g002){ref-type="fig"}J; A. Ashique, unpublished data). It is not possible to say from the published characterization of the knockout allele whether a similar condition occurs there, but it is clear that the knockout allele causes holoprosencephaly ([@pbio-0020219-Willnow1]), a phenotype that we have not seen in line 267. Indeed, the ENU-induced allele causes an enlarged cortex ([Figure 2](#pbio-0020219-g002){ref-type="fig"}G and [2](#pbio-0020219-g002){ref-type="fig"}H) without any obvious deficiency of midline structures such as would be expected in even mild holoprosencephaly.
A Novel *Rfx4* Allele {#s2e}
---------------------
A morphologically identifiable dorsal midline is absent from the cerebral cortex of line 269 homozygotes ([Figure 2](#pbio-0020219-g002){ref-type="fig"}M--[2](#pbio-0020219-g002){ref-type="fig"}P). This is an unusual defect that is similar to that seen in a transgene insertion mutation that disrupts the *Rfx4* gene ([@pbio-0020219-Blackshear1]). The line 269 mutation was mapped to Chromosome 10 in the region containing *Rfx4*. Sequencing of *Rfx4* revealed a base substitution that changes an evolutionarily conserved leucine residue to proline ([Figure 2](#pbio-0020219-g002){ref-type="fig"}Q and [2](#pbio-0020219-g002){ref-type="fig"}R). RFX4 is a member of the Rfx subfamily of winged-helix transcription factors ([@pbio-0020219-Emery1]) and can form dimers with two of the other family members, RFX2 and RFX3 ([@pbio-0020219-Morotomi-Yano1]). The amino acid substitution affects the conserved C domain of RFX4\'s large dimerization domain ([@pbio-0020219-Katan-Khaykovich2]). The transgene insertion mutation, on the other hand, selectively eliminates the expression of a neural-specific transcript of the gene, and so behaves as a tissue-specific null ([@pbio-0020219-Blackshear1]). Dimerization is not necessary for DNA binding activity of Rfx-class transcription factors ([@pbio-0020219-Katan1]; [@pbio-0020219-Katan-Khaykovich1]). Instead, dimerization appears to determine whether the bound transcription factor mediates transcriptional activation or repression. The fact that the line 269 allele produces a phenotype that is apparently identical to that of a null allele provides clear evidence that RFX4-containing dimers regulate important transcriptional regulatory events during formation of the cortical midline.
Dlx-LacZ Allows Efficient Identification of Tangential Migration Mutants {#s2f}
------------------------------------------------------------------------
Four mutants were identified in which the morphology of the cortex was normal at E14.5, but in which the distribution of LacZ-expressing cells in the cortex was altered. Three mutants, 154, 239, and 275, were identified on the basis of defects that are apparent in whole-mount preparations. Defects in the fourth mutant, 251, were only apparent upon examining sections. All four mutations are, for unknown reasons, perinatal lethal when homozygous, and this has prevented us from analyzing adult phenotypes. In addition, the line 154, 239, and 275 mutations appear to cause an increase in spontaneous seizures and mortality in young adult heterozygotes. Because the seizures are sporadic, their basis has not yet been studied.
Line 154 has the most severe defects. Affected embryos often have fewer labeled cells in the cortex except in the most rostral regions, where they form abnormal aggregates ([Figure 3](#pbio-0020219-g003){ref-type="fig"}A and [3](#pbio-0020219-g003){ref-type="fig"}B). Abnormalities in the LacZ expression pattern that are associated with the failure of cells to invade the developing cortex can also be found in the subcortical telencephalon of line 154 embryos ([Figure 3](#pbio-0020219-g003){ref-type="fig"}C--[3](#pbio-0020219-g003){ref-type="fig"}H). Here, prominent, radially oriented columns of cells form near or at boundaries between subcortical subdivisions: the dorsal LGE, the ventral LGE, and the medial ganglionic eminence. To confirm that the abnormal pattern of LacZ expression was due to a perturbation in the distribution of interneurons in the cortex and not the result of ectopic expression of the transgene, we examined the expression of an interneuron marker, *glutamic acid decarboxylase 1 (Gad67).* Interneurons are GABAergic and so express GAD67, an enzyme involved in GABA synthesis. Glutamatergic projection neurons do not express GAD67. Whole-mount in situ analysis of GAD67 at E15.5 showed that the distribution of interneurons was abnormal, as predicted by the aberrant expression of the transgene ([Figure 3](#pbio-0020219-g003){ref-type="fig"}I and [3](#pbio-0020219-g003){ref-type="fig"}J).
::: {#pbio-0020219-g003 .fig}
Figure 3
::: {.caption}
###### Severe Disruption of Interneuron Migration in Line 154 Mutants
\(A) Disseminated immature interneurons are seen as diffuse cortical (Cx) staining in WT embryos. Subcortical expression in the SVZ of the LGE can be seen as a darkly stained, inverted crescent.
\(B) Embryos homozygous for the line 154 mutation have little or no cortical staining, and the subcortical staining has aberrant streaks (pink arrowheads) and spots (white arrowhead), particularly in the frontal cortex.
(C, E, and G) In this rostral-to-caudal series of coronal sections from WT embryos, the normal MZ and IZ/SVZ migratory streams are diffusely labeled (red arrowheads), and a sharp cortical-subcortical boundary (black arrowheads) is marked by the abrupt transition between the densely stained SVZ of the LGE and the diffuse staining of the migrating interneuron precursors.
(D, F, and H) A rostral-to-caudal series of coronal sections from a line 154 mutant embryo shows the rostral spots (white arrowheads) visible in whole mount to be aggregates of cells in the cortex, and the streaky subcortical staining to be radially directed linear aggregates (pink arrowheads). The SVZ of the LGE is also noticeably thinner, and there is not a well-defined cortical-subcortical transition in the staining pattern.
(I and J) Rostral views of *Gad67* whole-mount in situ hybridization show the pattern of migration abnormalities in the cortex and significant defects in population of the olfactory bulb by GABAergic neurons.
:::
:::
Three Migration Mutants Have Defects in a Common Process {#s2g}
--------------------------------------------------------
Lines 239, 251, and 275 share features that suggest that the loci involved may have roles in a common regulatory process. All three mutations produce defects that are most apparent in the rostral cortex at E14.5, and all three cause anterior polydactyly ([Figure 4](#pbio-0020219-g004){ref-type="fig"}A--[4](#pbio-0020219-g004){ref-type="fig"}C; unpublished data). The limb defects might be only superficially similar or they could have a common developmental basis. If the latter explanation were true, it would strongly suggest that the neuronal migration phenotypes of the three mutants are similar because the same regulatory mechanism is defective in all of them. Initial mapping uncovered linkage to Chromosome 7 for the line 251 mutation and to Chromosome 10 for the other two, indicating that at least two distinct loci are involved ([Table 1](#pbio-0020219-t001){ref-type="table"}). To determine the number of loci involved, complementation tests were carried out using crosses between all three lines. The line 251 mutation complemented the other two as expected, but crosses between line 239 and line 275 produced mutant embryos with both limb and forebrain defects that were indistinguishable from either parental line. Further study will be required to determine whether the two lines carry the same or two independent mutations.
::: {#pbio-0020219-g004 .fig}
Figure 4
::: {.caption}
###### Limb Patterning Defects in Tangential Migration Mutants
Anterior is to the right for all limbs.
(A--C) Left hindlimbs are shown from E14.5 WT (A), line 251 (B), and line 239 (C). Yellow arrowheads point to extra digits on the anterior (thumb) side of the limb in mutants. Line 275 mutants also have anterior polydactyly. The mutants have a slight developmental delay that causes some differences in appearance of the limb buds at E14.5, when the limbs are growing rapidly.
\(D) This diagram illustrates components of the *Shh/Fgf* feedback loop that maintains *Fgf4* expression in the AER.
(E--J) In situ hybridization on E11.5 limb buds. Unlike in WT (E), expression of the posterior patterning gene *Hoxd13* in left forelimb buds extends ectopically into an anterior domain (yellow arrowheads) in 251 (F) and 239 (G) mutants. *Fgf4* expression in left hindlimb buds, restricted to the posterior AER in WT (H), has an ectopic expression domain at the far anterior edge of the AER (yellow arrowheads) in 251 (I) and 239 (J) mutants.
:::
:::
To identify the developmental basis of the limb defects, we examined the expression of genes involved in limb patterning. A regulatory network involving *Shh* and *Fgf4* regulates the expression of *Hoxd13* and controls patterning of the limb ([Figure 4](#pbio-0020219-g004){ref-type="fig"}D). We first investigated the expression of *Hoxd13* as a molecular marker of anterior-posterior (A-P) pattern in the limb at early stages. *Hoxd13* is ectopically expressed in the anterior portion of line 239 and line 251 mutant limbs ([Figure 4](#pbio-0020219-g004){ref-type="fig"}E--[4](#pbio-0020219-g004){ref-type="fig"}G), indicating that the anterior polydactyly is caused by a defect in A-P patterning. We next examined the expression of the elements of the network that regulate *Hoxd13* expression ([Figure 4](#pbio-0020219-g004){ref-type="fig"}D) to see whether their expression was perturbed. The expression of *Shh*, *Ptc1, gremlin* (also known as *Cktsf1b1*), and *Bmp4* are not distinguishable from wild-type (WT) (unpublished data). In contrast, an ectopic domain of FGF4 persists in the anterior apical ectodermal ridge (AER) ([Figure 4](#pbio-0020219-g004){ref-type="fig"}H--[4](#pbio-0020219-g004){ref-type="fig"}J). Thus the defects in limb patterning result from disruption of a step downstream of *Shh, Ptc1, gremlin,* and *Bmp4,* and upstream of *Fgf4*. This does not allow a specific molecular mechanism to be invoked, yet the combination of similar defects in both the limb and in the pattern of interneuron migration is strong evidence that the same molecular mechanism is disrupted in all three mutants.
Migrating Cells Persist Abnormally in the IZ of 239, 251, and 275 {#s2h}
-----------------------------------------------------------------
The 239, 251, and 275 mutants have clusters of LacZ-expressing cells in the IZ and/or SVZ of the rostral cortex, whereas more caudally, defects appear milder ([Figure 5](#pbio-0020219-g005){ref-type="fig"}). In WT animals there is a clear demarcation, at the corticostriatal junction, between the SVZ of the dorsal LGE and the stream of cells migrating into the cortex (black arrowheads in [Figure 5](#pbio-0020219-g005){ref-type="fig"}C--[5](#pbio-0020219-g005){ref-type="fig"}E). The crispness of this boundary is lost in the 239 and 275 lines (yellow ovals in [Figure 5](#pbio-0020219-g005){ref-type="fig"}F--[5](#pbio-0020219-g005){ref-type="fig"}H and [5](#pbio-0020219-g005){ref-type="fig"}L--[5](#pbio-0020219-g005){ref-type="fig"}N), although not in 251. In all three mutants, occasional linear aggregates of LacZ-expressing cells extend from the IZ/SVZ to the MZ (red arrowheads in [Figure 5](#pbio-0020219-g005){ref-type="fig"}I and [5](#pbio-0020219-g005){ref-type="fig"}N).
::: {#pbio-0020219-g005 .fig}
Figure 5
::: {.caption}
###### Line 239, 251, and 275 Mutants Have Similar Migration Defects
\(A) Frontal view of an E14.5 WT embryo.
\(B) Frontal view of an E14.5 embryo homozygous for the line 239 mutation. The stream of cells leaving the SVZ of the LGE is streaky and aggregated in the rostral cortex of the mutant.
(C--E) A rostral-to-caudal series of coronal sections from an E14.5 WT embryo. WT embryos have diffuse cortical staining and a sharp boundary (black arrowheads) between the subcortical and the cortical telencephalon.
(F--H) Coronal sections from E14.5 line 239 mutant forebrains. Yellow circles indicate the area of the cortical-subcortical boundary where a large excess of migrating cells can be seen in the IZ/SVZ area. Orange arrowhead indicates aggregated cells in the IZ/SVZ of the lateral wall of the cortex. White arrowheads indicate aggregates in the medial wall. The staining in the MZ appears normal. Defects become less apparent in the more caudal sections.
(I--K) Sections from an E14.5 line 251 mutant embryo. Orange arrowheads indicate aggregates in the lateral wall of the cortex. In (I), a linear aggregate of stained cells can be seen extending from the IZ/SVZ to the MZ (red arrowhead). The cortical-subcortical boundary is well defined in line 251 mutants.
(L--N) Sections from an E14.5 line 275 mutant embryo. Yellow circles indicate aberrant staining in the cortical-subcortical boundary region. The white arrowhead indicates aggregates in the medial wall of the cortex, and the red arrowhead points to a radially directed aggregate of cells extending from the IZ/SVZ to the MZ.
:::
:::
To study the effects of the mutations at later stages of cortical interneuron development, we examined near-term (E18.5) animals ([Figure 6](#pbio-0020219-g006){ref-type="fig"}). We focused our analysis on line 251 and evaluated the expression of the *Dlx-LacZ* transgene and *Gad67*. As at E14.5, aggregation of *Gad67^+^; LacZ^+^* cells in the IZ/SVZ is a prominent feature of the phenotype (black arrowheads, [Figure 6](#pbio-0020219-g006){ref-type="fig"}B, [6](#pbio-0020219-g006){ref-type="fig"}F, and [6](#pbio-0020219-g006){ref-type="fig"}J). Despite the severity of the defects observed in the IZ/SVZ, Dlx-LacZ--expressing interneuron precursors in the MZ and in the cortical plate do not form aggregates (yellow and blue arrowheads in [Figure 6](#pbio-0020219-g006){ref-type="fig"}I and [6](#pbio-0020219-g006){ref-type="fig"}J), suggesting that the mutation inhibits the ability of migrating interneurons to leave the IZ/SVZ, but does not significantly impact other aspects of their migration.
::: {#pbio-0020219-g006 .fig}
Figure 6
::: {.caption}
###### Dlx-LacZ and GAD67 Expression Show that Interneuron Precursors Persist in the IZ/SVZ of 251 Mutant Cortices
\(A) Coronal section through the forebrain of an E18.5 WT embryo stained for β-galactosidase and counterstained with nuclear fast red.
\(B) A similar section from a line 251 mutant embryo. Unusual accumulations of stained cells can be seen in the cortex just dorsal to the striatum (white arrowheads). Aggregates of cells in the IZ/SVZ can also be seen (black arrowhead).
(C and D) Sections adjacent to those in (A) and (B) hybridized with a probe for *Gad67* mRNA. Arrowheads in (D) point to the same features that are seen in (B).
(E--H) Higher-magnification views of the dorsal portions of the sections in (A--D).
(I and J) Higher-magnification view of cortex. In the WT cortex (I), cells can be seen dispersed through the cortical plate (yellow arrowheads) and scattered through the MZ (blue arrowheads). Similar distributions of labeled cells can be seen in the mutant cortex (J). In contrast to the WT, however, aggregates of cells are found in IZ/SVZ (black arrowheads).
:::
:::
A Mutant with Defects in Invasion of the Cortex by Thalamocortical Axons {#s2i}
------------------------------------------------------------------------
In addition to the seven mutants described in the previous sections, six other mutants were isolated. Most of these have not been characterized in detail, but one, line 412, illustrates the range of phenotypes that the *Dlx-LacZ* transgene allowed us to identify. In line 412, the mutant phenotype was detectable in whole mount as a subtle but consistent defect near the cortical-subcortical boundary. Upon sectioning, this defect was revealed to be a delamination in the region of the external capsule ([Figure 7](#pbio-0020219-g007){ref-type="fig"}A--[7](#pbio-0020219-g007){ref-type="fig"}D). Thalamocortical fibers ordinarily enter the cortex at this point, and this defect can occur when fewer axon tracts cross this zone ([@pbio-0020219-Hevner1]). Indeed, the 412 mutant has fewer thalamocortical fibers traversing the corticostriatal boundary at E14.5, as evidenced by the reduced density of L1^+^ axons in this location ([Figure 7](#pbio-0020219-g007){ref-type="fig"}E and [7](#pbio-0020219-g007){ref-type="fig"}F). Examination of late-stage embryos shows that the lack of cortical invasion by thalamocortical fibers at E14.5 is due to a persistent inhibition and not merely a delay in innervation (unpublished data). The IZ of the cortex often appears thinner in mutants, consistent with the absence of thalamocortical axons. Interestingly, TAG1^+^ corticofugal axons are still present in the cortex ([Figure 7](#pbio-0020219-g007){ref-type="fig"}G and [7](#pbio-0020219-g007){ref-type="fig"}H). Analysis of markers for the dorsal thalamus, where the thalamocortical axons originate, and for the striatum, which they must traverse, does not reveal any obvious defects (A. Ashique, personal communication). This, together with the observation that corticofugal fibers appear to be intact, suggests that the mutation may disrupt a molecule that is directly involved in pathfinding by thalamocortical axons.
::: {#pbio-0020219-g007 .fig}
Figure 7
::: {.caption}
###### Corticostriatal Delamination and Lack of the Thalamocortical Projection in Line 412 Mutants
(A--D) Coronal hemisections of E15.5 WT (A and C) and line 412 (B and D) mutant embryos stained for the *Dlx-LacZ* transgene. The cortex is thinner in 412 mutants, as can be seen in both the rostral (B) and the caudal (D) sections. Delamination of the corticostriatal boundary can be seen in the region between the red arrowheads in (D).
(E and F) Immunostaining for L1 antigen labels the thalamocortical fibers in coronal hemisections from E14.5 WT (E) and mutant (F) embryos. The striatum and corticostriatal areas are shown. In the WT section, the thalamocortical fibers can be seen traversing the striatum through the internal capsule and coursing into the cortex. In the mutant section, a few fibers enter the striatum but do not traverse it to reach the cortex. Corticostriatal delamination can be seen as a hole in the right side of the section.
(G and H) Immunostaining for TAG1 antigen (blue arrowheads) reveals corticofugal fibers.
:::
:::
Discussion {#s3}
==========
Novel ENU-Induced Alleles {#s3a}
-------------------------
ENU overwhelmingly induces single-basepair substitution mutations. The mutant alleles that are produced often have relatively selective effects on protein function, and so provide valuable probes of the function of different protein domains. This is illustrated by two of the alleles described here, *Rfx4* and *Scrb1*. Each of these alleles is a missense mutation that appears to disrupt specific domains in a selective fashion.
The SCRB1 protein has two sets of recognizable domains: three amino-terminal LRRs and four, more carboxy-terminal, PDZ domains. The previously described allele of *Scrb1,* the *Circletail* allele, has a stop codon that terminates translation after the first two PDZ domains. In contrast, the line 90 allele of *Scrb1* is predicted to encode a charged lysine in place of a hydrophobic isoleucine on the outside of the third LRR domain (see [Figure 2](#pbio-0020219-g002){ref-type="fig"}D and [2](#pbio-0020219-g002){ref-type="fig"}E). The homozygous phenotypes produced by the line 90 and the *Circletail* alleles are apparently identical, and both show strong genetic interactions with a mutation in *Str-1,* a cell-surface protein that regulates planar cell polarity in both flies and mammals. From this we can conclude that both the LRR and the PDZ domains are required for SCRB1\'s role in the establishment of epithelial polarity.
The missense allele of *Rfx4* is predicted to have very subtle effects on the structure of the protein. The mutation causes a proline to be substituted for a leucine residue in a dimerization domain of the protein. Proline residues are not compatible with α-helices, but in this case the substitution appears to be at the beginning of a β-turn that links two helices. Presumably, the substitution causes the linkage between the α-helices to be stiffened or abnormally constrained in some way. Whatever the exact molecular consequences are, the nature of the allele and the genetics suggest that the mutation prevents dimerization. Ordinarily RFX4 can dimerize with itself and with the related transcription factors RFX2 and RFX3 ([@pbio-0020219-Morotomi-Yano1]). If inactive dimers were produced by the missense allele, it would seem likely to cause a dominant phenotype, which it does not. Studies are underway that will test these ideas as well as determine whether the failure of the dorsal midline to form is the result of a loss of a dorsal signaling center or an inability of the cortical neuroepithelium to respond to those signals.
The *megalin* allele provides a contrast to these first two cases. Despite its molecular severity as a premature stop codon, its phenotypic consequences are less pronounced than the knockout allele. The knockout mutation produces early-head-fold--stage embryos with reduced neuroepithelium in the anterior midline ([@pbio-0020219-Willnow1]). These defects originate during gastrulation, and by mid-gestation result in holoprosencephaly. The absence of these early defects in the ENU-induced allele could be interpreted to mean that the defective protein retains some function and supplies the MEGALIN activity that is required during gastrulation. Alternatively, it is possible that the deleted region in the knockout is required for the proper expression of a neighboring gene and that the gastrulation phenotype does not reflect an early role for *megalin*.
In each of these three cases, the ENU-induced allele provides novel information about the role of the gene in cortical development and suggests avenues for further exploration. As we determine the molecular nature of the other mutations, it is likely that the selective nature of the ENU-induced alleles will provide important insights into the function of other proteins that regulate cortical development.
Nature of the Tangential Migration Defects {#s3b}
------------------------------------------
The migration of immature interneurons has been followed using lipophilic dyes, tissue chimeras, transfection with a green fluorescent protein (GFP) expression vector, and, recently, using a *Gad67-GFP* knockin mouse ([@pbio-0020219-de1]; [@pbio-0020219-Anderson1], [@pbio-0020219-Anderson2]; [@pbio-0020219-Tamamaki1]; [@pbio-0020219-Denaxa1]; [@pbio-0020219-Polleux1]; [@pbio-0020219-Ang1]). These studies have identified tangentially migrating cells in both the IZ/SVZ and the MZ. Some studies have concluded that the cells in the IZ/SVZ and the MZ are two independent migratory streams ([@pbio-0020219-Lavdas1]). In contrast, the use of a *Gad67-GFP* knockin allele to follow interneuron precursors ([@pbio-0020219-Tanaka1]) led to the conclusion that the general pattern of cell migration is tangentially through the IZ and thence radially outward from the IZ to the MZ. Cells in the MZ also migrate tangentially but at a slower rate than those in the IZ ([@pbio-0020219-Polleux1]; [@pbio-0020219-Tanaka1]), in a process that may be a search for the proper location at which to invade the cortical plate ([@pbio-0020219-Ang1]). These two models for tangential migration produce different interpretations of the pattern of defects that we see in our novel mutants. In the first model, obvious defects in the IZ but not the MZ would imply that the mutations are disrupting a process used to regulate migration through the IZ but not through the independently regulated MZ. In the second model, the predominance of defects in the IZ/SVZ would indicate an inhibition at an early step of the migratory process. The presence of cell aggregates extending from the IZ to the MZ is consistent with this being the step that is defective, whereas the presence of properly dispersed cells in the MZ and cortical plate would indicate that the mutations inhibit, but do not completely block, this early step.
Concordance of Limb Patterning and Migration Defects {#s3c}
----------------------------------------------------
Three out of the four tangential migration defects also result in anterior polydactyly. The association of limb patterning and neuronal migration defects has not been previously reported and, given that a great deal more is known about limb patterning than about tangential migration, it is tempting to speculate on what this may imply about the migration mutants. One possibility is that shared mechanisms are used to pattern the telencephalon and the limb. It is equally possible that genes that regulate patterning in the limb regulate cell migration decisions more directly. Mutations in an *arista-less* homolog, *Arx,* cause profound defects in interneuron migrations, and mutations in another *arista-less* homolog, *Alx4,* cause anterior polydactyly. However, as the limbs of *Alx4* mutants, unlike the 239, 251, and 275 mutants, have ectopic *Shh* expression in the anterior limb bud, it is unlikely that the mutations described here disrupt an *arista-less* pathway. *Shh* maintains the expression of *Fgf4* in the posterior AER of WT limb buds in a process that requires *gremlin*. Genes that act in this process downstream of *Bmp4* are plausible candidates for the line 239, 251, and 275 mutations.
General Implications for Genetic Screens in the Mouse {#s3d}
-----------------------------------------------------
The work described here demonstrates clearly that genetic screening strategies in the mouse need not be limited to general or broad-based phenotyping approaches; a focused genetic screening strategy can provide a powerful means of dissecting a specific aspect of mammalian biology. The strategy of broad-based approaches has been an effective one for the past several years as the collection of mutants using chemically induced mutations resurged in popularity. In other genetic systems, strategies have evolved from general toward focused screens that allow mutations affecting a specific process to be identified, and it is likely that mouse genetics will progress in a similar fashion. An additional concern with mice, however, is the cost of breeding and housing, which is higher than that for other model genetic organisms. We have shown, nonetheless, that a laboratory-based, focused screening strategy is a productive pursuit. The costs of such a screen could be shared by the careful combination of reporters so that screening for several distinct processes could be carried out at once. This general idea is highlighted by our isolation of mutations in which the growth and patterning of the cortex is defective. With the exception of line 90, the identification of these mutants benefited from the easy visualization of cortical size and structure by expression of the transgene. In several cases, the interneuron migration mutants being a good example, the identification of the mutant phenotype without the transgene would have been very unlikely. Independent reporters could be used to pursue the simultaneous identification of several different classes of mutants more directly. For example, reporters that label migrating neurons with GFP, and thalamocortical axons with β-galactosidase or alkaline phosphatase, would allow both migration and axonal pathfinding mutations to be identified in the same screen. Given the large number of reporter and indicator strains that have been made over the last few years and the powerful tools that exist for gene identification, it is clear that focused screens in the mouse will provide the resources to address many questions in the coming years.
Materials and Methods {#s4}
=====================
{#s4a}
### Animals and breeding. {#s4a1}
Male C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, Maine, United States) and treated with three intraperitoneal injections of 100 mg/kg ENU (Sigma, St. Louis, Missouri, United States) spaced at 7-d intervals. Eight weeks after the last injection, the ENU-treated males were set up in breeding pairs with FVB/NJ females homozygous for the *Dlx-LacZ* transgene. Male offspring of this cross (G1 males) were backcrossed to the transgenic line, and female offspring (G2 females) were saved. For each line, one to six of the G2 females were backcrossed to their fathers to generate timed pregnancies. Embryos were harvested either 13 d (the first 100 lines) or 14 d (all subsequent lines) after the vaginal plug was identified.
### Screening. {#s4a2}
Embryos were dissected in phosphate-buffered saline (PBS) and fixed for 45 min at room temperature in 4% paraformaldehyde (PFA) in PBS. Subsequently, embryos were washed in detergent rinse (0.1 M phosphate buffer \[pH 7.3\], 2 mM MgCl~2~, 0.01% sodium deoxycholate and 0.02% Nonidet P-40). Embryos were then stained for 48--72 h at room temperature on a rocking platform using X-gal as a substrate for the detection of β-galactosidase activity. Staining was terminated after visual inspection by repeated washing in PBS. Embryos were then fixed again and stored in 4% PFA in PBS until further examined. All litters were examined in whole mount and approximately one-half were selected for sectioning on a vibratome (VT1000S; Leica, Wetzlar, Germany). For sectioning, the heads of all pups from a litter were separated from the torsos and mounted aligned in the same orientation in an agarose block. Sections 100 μm thick were collected in PBS and mounted in Kaiser\'s glycerol gelatin (Merck, Darmstadt, Germany) on slides. All phenotypes reported here were seen in more than ten litters resulting from both G1 male × G2 female and G2 male × G2 female crosses.
### Histology. {#s4a3}
Whole-mount in situ hybridization was carried out according to [@pbio-0020219-Henrique1]. *Gad67* in situ hybridization on sections was carried out following standard protocols ([@pbio-0020219-Dagerlind1]) with a digoxigenin-labeled antisense RNA probe generated by in vitro transcription using a plasmid obtained from Brian G. Condie ([@pbio-0020219-Maddox1]). In brief, embryos were dissected in diethylpyrocarbonate-treated PBS, and the heads were removed and fresh-frozen on dry ice. Tissue was sectioned at 20 μm on a cryostat (Leica CM3050). Slide-mounted cryosections were warmed to room temperature and fixed in 4% PFA in PBS. Deacetylation was performed for 10 min by immersion in 0.1 M triethanolamine containing 25 mM acetic anhydride followed by rinsing in 2× saline sodium citrate and dehydration through an increasing alcohol series (60%, 75%, 95%, and 100%). Sections were hybridized with the riboprobe under stringent conditions (50% formamide, 10% dextran sulfate, 20 mM Tris-HCl, 0.3 M NaCl, 5 mM EDTA, 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, 0.02% BSA, 0.5 mg/ml tRNA, 0.2 mg/ml carrier DNA, and 200 mM DTT) for 16--20 h at 63 °C. After hybridization, sections were washed four times in 4× saline sodium citrate solution and incubated for 30 min in RNase buffer (10 mM Tris-HCl \[pH 7.5\], 0.5 M NaCl, and 5 mM EDTA \[pH 8.0\]) containing 20 μg/ml RNase A at 37 °C. High-stringency washes were performed twice for 30 min at 63 °C. Incubation with the anti-digoxigenin antibody (Roche, Basel, Switzerland) was carried out in maleic acid buffer (100 mM maleic acid, 150 mM NaCl, and 1% blocking reagent \[Roche\]). Finally, staining was performed with BM purple substrate (Roche) terminated by repeated washes in PBS, and sections were coverslipped using Kaiser\'s glycerol gelatin. Nuclear fast red counterstaining for X-gal--stained sections was performed with pre-made staining solution (Vector Laboratories, Burlingame, California, United States). Staining was differentiated in 70% ethanol.
Immunohistochemistry using anti-TAG1 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, United States) and anti-L1 (Chemikon, Mönchengladback, Germany) antibodies was performed on 100-μm free-floating sections following the instructions and using the reagents of the Vectastain ABC Kit (Vector). Primary antibodies were used at a dilution of 1:500. Horseradish peroxidase activity from secondary antibodies was revealed by diaminobenzidine as a substrate in staining buffer (0.5 mg/ml diaminobenzidine, 20 mM sodium cacodylate, and 30 mN acetic acid).
### Mapping. {#s4a4}
Initial linkage was established using 12 DNA samples from both carriers and mutant embryos. We scored a set of 82 simple sequence repeat markers. The panel of markers was selected from the set at the Center for Inherited Disease Research Website (<http://www.cidr.jhmi.edu/mouse/mouse.html>). Markers were chosen that could be scored easily on agarose gels. All chromosomal assignments reported are the result of LOD scores significantly greater than three. The intervals that are listed in [Table 1](#pbio-0020219-t001){ref-type="table"} were derived following the initial establishment of linkage by haplotype analysis that included additional recombinant chromosomes from mutant animals or obligate carriers. The phenotypes of 239 and 275 are very similar, and the fact that they map to the same interval likely reflects allelism. The localization of both 351 and 357 to the same interval seems more likely to be coincidental, because the phenotypes are quite different.
### Sequencing. {#s4a5}
For sequencing of *Rfx4* and *Scrb1* genes, RT-PCR samples were prepared from cDNA prepared using E10.5 to E16.5 embryos. For *megalin,* exons were amplified using genomic DNA samples. Primer sequences are available upon request. In both cases sequencing was done by the University of California, Berkeley, DNA Sequencing Facility.
Supporting Information {#s5}
======================
Figure S1
::: {.caption}
###### Cortical Size Is Altered in Lines 152 and 351
\(A) Dorsal views of the cortex of WT (left) and line 152 mutant (right) embryos stained for expression of the Dlx-LacZ transgene. The line 152 mutation reduces the size of the cortex.
(B and C) Coronal sections through the cortex of E14.5 WT (B) and line 351 mutant (C) embryos. The cortex has a characteristic high-domed shape and is thinner in the mutant.
(D and E) Dorsal (D) and ventral (E) views of adult brains of WT (left) and line 351 mutant (right) brains. The cortex is overall larger in the mutant than in the WT. The olfactory bulbs are present in the mutant but are tucked under the cortex, making them less visible.
(9.1 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S2
::: {.caption}
###### Central Nervous System Defects and Cleft Upper Jaw in Lines 366 and 357
\(A) Lateral view of an E14.5 WT embryo.
\(B) The cleft upper jaw of a line 366 mutant is visible at the left. The telencephalon, including the cortex, is significantly shortened along the rostrocaudal axis.
(C and D) Lateral and front views of the cleft and reduced upper jaw of an E18.5 embryo homozygous for the line 366 mutation.
(E and F) Lateral views of WT (E) and line 357 homozygote (F) embryos at E13.5 showing the reduced telencephalon and relatively expanded midbrain.
\(G) A front view of the embryo in (F) shows the cleft upper jaw.
(H and I) Sagittal sections through E13.5 WT (H) and line 357 (I) embryos. The overgrown midbrain in the mutant embryo has forced the neuroepithelium into folds.
(9.9 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Figure S3
::: {.caption}
###### Line 407 Mutants Have Dorsoventral Defects in the Cortical Primordia and Facial Midline Defects
\(A) Lateral view of an E14.5 mutant embryo showing edema and hemorrhage suggestive of vascular defects. Frontal views of WT (B) and mutant (C) embryos illustrate the narrowed frontonasal process, maxilla, and mandible of the mutant. (D) shows coronal hemisections in a WT embryo. (E) shows the accumulation of Dlx-LacZ--positive cells in a SVZ-like area dorsal to the LGE.
(4.3 MB TIF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Accession Numbers {#s5a4}
-----------------
Accession numbers of the genes discussed in this paper are available at LocusLink (<http://www.ncbi.nih.gov/LocusLink>, and are as follows: *Alx4* (11695), *Arx* (11878), *Bmp4* (12159), *Dlx5/6* (13395/13396), *Fgf4* (14175), *Gad67* (14415), *gremlin* (23892), *Hoxd13* (15433), *L1* (16728), *Lrp2* (14725), *Ltap/Lpp1* (93840), *Ptc1* (19206), *Rfx4* (71137), *Scrb1* (105782), *scribbled* (44448), *Shh* (20423), and *Tag1* (21367).
We thank Dr. Amir Ashique for permission to cite unpublished work, Dr. Viktor Kharazia for help and advice with histological analysis, and Marlene Lane for help with many aspects of animal breeding and husbandry and for help with the early stages of the screen. We also thank Deanna Louie, Kristina Bray, Tyler Wai Chan, and William Tseng for their assistance.
This research was supported in part by funds provided by the National Institutes of Health/National Institute of Mental Health, award number R21 MH068525 (ASP); a Human Frontiers Science Program Grant, number RG0160/2000-B (ASP); a Wills Foundation Fellowship (KZ); and a Broad Foundation Fellowship (SM). It was also supported in part by funds provided by the State of California for medical research on alcohol and substance abuse through the University of California, San Francisco.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** ASP conceived the screen. ASP, KZ, SRM, and YS designed and performed the experiments. KZ, SRM, YS, JLRR, and ASP analyzed the data. ME contributed reagents/materials/analysis tools. ASP, KZ, and SRM wrote the paper.
Academic Editor: Joshua R. Sanes, Harvard University
Citation: Zarbalis K, May SR, Shen Y, Ekker M, Rubenstein JLR, Peterson AS (2004) A focused and efficient genetic screening strategy in the mouse: Identification of mutations that disrupt cortical development. PLoS Biol 2(8): e219.
AER
: apical ectodermal ridge
A-P
: anterior-posterior
E\[number\]
: embryonic day \[number\]
ENU
: ethyl-nitroso-urea
GFP
: green fluorescent protein
IZ
: intermediate zone
LGE
: lateral ganglionic eminence
LRR
: leucine-rich repeat
MZ
: marginal zone
P\[number\]
: postnatal day \[number\]
PBS
: phosphate-buffered saline
PFA
: paraformaldehyde
*Shh*
: *sonic hedgehog;* SVZ
WT
: wild-type
| PubMed Central | 2024-06-05T03:55:46.743006 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509294/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e219",
"authors": [
{
"first": "Konstantinos",
"last": "Zarbalis"
},
{
"first": "Scott R",
"last": "May"
},
{
"first": "Yiguo",
"last": "Shen"
},
{
"first": "Marc",
"last": "Ekker"
},
{
"first": "John L. R",
"last": "Rubenstein"
},
{
"first": "Andrew S",
"last": "Peterson"
}
]
} |
PMC509295 | Given the overwhelming complexity of the immune system, it\'s no wonder that unraveling the mechanisms responsible for immunological disease has proved so difficult. The factors that trigger autoimmunity---which involves a breakdown in the body\'s ability to tolerate its own molecules---are not well understood, though animal studies show that genetic predisposition greatly increases risk. And that\'s where the real challenge begins. For certain diseases, individuals with defects in both copies of a specific gene invariably develop the disease. But more often, diseases with an inherited component result from the complex interplay of a variety of genes, each contributing a small effect that is typically dependent both on the expression of other genes and on both random and environmental factors.
Researchers have increasingly turned to mice to model autoimmunity in humans and have found the same genetic complexity at work, with different strains of lupus-prone mice having different genes predisposing them to autoimmune disease. One model involves targeted disruption of candidate immune system genes to study their role in disease. Gene targeting experiments modify or remove a gene of interest and then watch for corresponding effects on the organism\'s physiology. Interpretations of results from these experiments have traditionally been predicated on the assumption that the "background," or nontargeted, genes do not contribute to observed physiological changes. Yet in some studies, mice without targeted mutations develop an unexpected susceptibility to the autoimmune disease under study.[](#pbio-0020220-g001){ref-type="fig"}
::: {#pbio-0020220-g001 .fig}
::: {.caption}
###### Genetic background can induce autoimmunity in knock-out mice
:::
:::
To investigate what effects background genes might be having in these mouse models, Marina Botto and colleagues compared the genomes of three hybrid strains of the most commonly used genetic background---the 129 and C57BL/6 hybrid mice. One of the hybrids, which carries a mutation in both copies of the *Apcs* gene, was chosen as an example of a gene-targeted model that develops a lupus-like disease, offering an opportunity to examine the relative contributions of the targeted versus background genes. *Apcs* is a candidate gene for human systemic lupus erythematosus (SLE), a form of autoimmunity marked by chronic inflammation resulting from a sustained attack on antibodies throughout the body.
Botto and colleagues found that several genomic regions from both the 129 and C57BL/6 mice contributed to autoimmunity, even in the absence of gene-targeted mutations. All of the hybrid strains developed autoimmunity, though disease was more severe in mice with *Apcs* mutations. Disease outcome in gene-targeted mice, it turns out, can be influenced not just by disease-susceptible (or disease-resistant) gene variants near the targeted gene (in this case, *Apcs*) but by the random heterogeneity of the hybrid genetic background: multiple combinations of genes in the hybrids can produce the same result.
These results fall in line with mounting evidence that background genes are not silent partners in gene-targeted disease models, but can themselves facilitate expression of the disease. This finding underscores the notion that genes are not solitary, static entities; their expression often depends on context. With genetically complex diseases, having the requisite combination of susceptibility genes does not always lead to disease. Much work remains to be done to identify the triggers that cause the immune system to turn on itself. For more on mouse models and autoimmunity, see the primer by Morel in this issue.
| PubMed Central | 2024-06-05T03:55:46.747922 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509295/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e220",
"authors": []
} |
PMC509296 | Grafting is an ancient technique used by farmers and gardeners to combine desired attributes of the rootstock with those of the donor plant shoot, or scion. Grafting essentially saved European wine making: when the insect Dactylosphera vitifoliae devastated European grapewine varieties over the course of the late 1800s and early 1900s, the varieties were saved by grafting them onto resistant rootstocks from the New World. Since then, these rootstocks have been used to maintain the susceptible Old World cultivars. But grafting is also an excellent tool for scientists studying systemic signals traveling between the rootstock and distal parts of the plants, and vice versa. For example, two important studies ([@pbio-0020224-Palauqui1]; [@pbio-0020224-Voinnet1]) used grafting to demonstrate the spreading of RNA silencing in plants. However, it was a subsequent paper ([@pbio-0020224-Crete1]) that followed up on certain inconsistencies in the grafting results that pointed to subtleties important for both experimental design and understanding systemic signaling in plants.
RNA silencing (termed posttranscriptional gene silencing in plants, quelling in fungi, and RNA interference in animals) refers to the phenomenon whereby specific gene transcript levels are reduced in the presence of a related RNA. From studies of RNA silencing in several systems, much is now known about the mechanisms involved ([@pbio-0020224-Matzke1]; [@pbio-0020224-Mlotshwa1]), but the systemic spreading in plants is still a bit of a mystery. Posttranscriptional gene silencing spreads systemically throughout the individual plants in a very characteristic manner reminiscent of viral spread. This has led to the hypothesis of a systemic silencing signal that is produced in the tissues where silencing is initiated and is then transmitted to the distant parts of the plant where it can initiate silencing in a sequence-specific manner. The sequence specificity of the silencing strongly implies that the signal is a nucleic acid, most likely an RNA, but the identity of the signal remains unknown. Silencing spreads mainly in the direction from carbon source to carbon sink, that is, from tissues such as leaves that export the sugar products of photosynthesis, to tissues such as roots that import these products, and it can take up to several weeks until it is established in the whole plant ([@pbio-0020224-Palauqui1]; [@pbio-0020224-Palauqui2]; [@pbio-0020224-Voinnet1]; [@pbio-0020224-Sonoda1]).
As expected, the discovery of this process triggered a quest for the "systemic inducer" of the process: a signal that travels through the plant and is able to initiate silencing in a remote location within the plant. Grafting was an obvious tool to use in the quest for this signal, as it allowed silencing source and sink tissues to be of different origin.
[@pbio-0020224-Palauqui1] were the first to unambiguously demonstrate that silencing spreads from a silenced rootstock to a nonsilenced scion. They used as a stock a transgenic tobacco carrying an additional copy of the endogenous nitrate reductase gene, *Nia*. Some of the transgenic lines generated always showed higher levels of *Nia* transcripts than the wild type---as expected from the presence of an additional gene---and were termed class I lines. However, other transgenic lines had undergone silencing for both the endogenous and exogenous *Nia* genes and those were termed class II lines. [@pbio-0020224-Palauqui1] found that silenced class II rootstocks were able to silence class I scions. This was true even in a "sandwich graft," where a wild-type (nontransgenic) segment was grafted between the silenced stock and the nonsilenced scion. Spreading of silencing was unidirectional from stock to scion. Though not explicitly stated, it was implied that it took more than 3 wk after grafting for systemic silencing to occur in the scion. The reported rate of transmission was 100%.
Related experiments by [@pbio-0020224-Voinnet1] used scions that transgenically expressed green fluorescent protein grafted onto plants with established silencing of the same transgene. There, too, silencing spread through the graft to the nonsilenced scion, even when a wild-type section was grafted between transgenic rootstock and scion. Unlike the *Nia* transgene, which has an endogenous counterpart in the wildtype plant, green fluorescent protein has no homolog in nontransgenic lines. Therefore, silencing spreading in the wild-type "spacer" in the sandwich grafts could not be assisted by an endogenous sequence. Rather, the systemic signal must have traveled all the way to the scion and induced gene silencing there. The establishment of systemic silencing took 4 wk in the "direct" grafts and 6 wk in the sandwich grafts. However, silencing spread to the scion only in some of the grafts: in ten out of 16 direct grafts and five out of 11 sandwich grafts.
I found these papers were very important not only for what they proved---the existence of a systemic signal of silencing---but also because they gave an unequivocal answer to the scientific questions they posed, using relatively simple methodology. Although excited by these successful examples of the transmission of silencing, I kept coming back to two questions: (1) what prevents transmission in some of grafts, and (2) why does it take longer to transmit silencing to the scion than it takes systemic silencing to reach the most remote parts of an intact plant?
When we started working on the silencing signal ourselves, we repeated some of the above experiments but found somehow lower efficiencies in the initiation of silencing in the scions. We soon realized that our results were influenced by the developmental stage of our scions. A paper from Jeff Meins\'s laboratory in Switzerland shed light on some aspects of the grafting puzzle. Researchers there introduced additional chitinase genes using bolistics, in sense or antisense orientation under the control of a strong promoter (35S) into chitinase transformant lines of tobacco that never exhibited spontaneous gene silencing ([@pbio-0020224-Crete1]). In lines bombarded late in plant development, triggering of silencing was rarely observed. However, when the transgene was introduced earlier in development, a large portion of the lines showed a substantial decrease and eventually full suppression of the chitinase mRNA levels. Lines that showed silencing were used as rootstocks and nonsilencing lines were used as scions in three types of grafting experiments. In the first type of grafting experiment, called top grafting, a 5-cm scion cut into a wedge at the bottom was inserted into the vascular ring at the cut surface of a 50-cm-high rootstock ([Figure 1A](#pbio-0020224-g001){ref-type="fig"}). In the second type of experiment, reciprocal transverse grafts of 50-cm-tall plants were exchanged between class I and class II plants ([Figure 1B](#pbio-0020224-g001){ref-type="fig"}). Finally, the third type of experiment involved plug grafts, which were made by exchanging transverse plugs of stems cut with a 5-mm-diameter cork borer from an internode approximately in the middle of a 50-cm plant ([Figure 1C](#pbio-0020224-g001){ref-type="fig"}).[](#pbio-0020224-g001){ref-type="fig"}
::: {#pbio-0020224-g001 .fig}
Figure 1
::: {.caption}
###### Grafting Method May Influence the Spreading Efficiency of the Silencing Signal (According to [@pbio-0020224-Crete1])
\(A) Top grafting, the most effective in transmitting the silencing signal. (B) Reciprocal transverse graft. (C) Plug graft.
:::
:::
Surprisingly, only top grafting resulted in scions that were systemically silenced by a rootstock signal. Furthermore, even transmission after top grafting was less effective than expected; in one stock/scion combination only 27 out of 71 grafts exhibited transmission of the silencing signal. The authors also found that antisense-induced silencing was never transmitted to the scion. These findings do not answer the many questions about the mechanisms underlying systemic silencing, but they point us in certain directions.
The individual parts of a whole plant are, in terms of import and export, in an equilibrium that changes with development. When grafting takes place, how this equilibrium is altered depends on the individual "parts" that contribute to the "new" whole plant. In addition, there is now indirect evidence ([@pbio-0020224-Fagard1]) that what is source tissue and what is sink tissue in terms of sugar transport affects what is source and what is sink in terms of the systemic silencing signal. Taking into account the above findings and choosing the right combination of stock/scion, we have managed to significantly increase the efficiency of graft-transmitted silencing, a prerequisite for continuing the search for the systemic signal.
From the grafting experiments to date, it is now evident that the transporting capacity of the vascular tissue bypass that is formed at the graft junction does not fully reach the level of the original vascular tissue. The basis for these restrictions is not known. In a way, the graft interface functions as an unintentional filter. If the specificities of this "filter" were known, it would help us comprehend some transmission inconsistencies. Keeping in mind these limitations, grafting remains an invaluable tool in the search for the systemic silencing signal.
Kriton Kalantidis is at the Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology--Hellas, Crete, Greece. E-mail: <kriton@imbb.forth.gr>
*Nia*
: nitrate reductase gene
| PubMed Central | 2024-06-05T03:55:46.748480 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509296/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e224",
"authors": [
{
"first": "Kriton",
"last": "Kalantidis"
}
]
} |
PMC509297 | Introduction {#s1}
============
For more than a century, the phenomenon of retrograde amnesia (RA)---the loss of memory for events that occur prior to a variety of precipitating brain insults---has provided the foundation for theories of memory consolidation and the locus of trace storage ([@pbio-0020225-McGaugh1]; [@pbio-0020225-Davis1]; [@pbio-0020225-Dudai1]). However, RA may also reflect the inability of a memory system to access a trace---a failure of memory retrieval ([@pbio-0020225-Warrington1]). This very dilemma was noted by [@pbio-0020225-Ribot1], p. 475) in his seminal discussion of RA: "*"Two suppositions are equally warranted, viz., that either the registration of the prior states has been effaced; or that the retention of the anterior states persisting, their aptitude for being revived by associations with the present is destroyed. We are not in a position to decide between these two hypotheses."*"
Studies of RA have favoured a memory-consolidation interpretation in instances in which systematic variation of the time interval between experience or training and the subsequent brain insult has revealed a temporal gradation of RA ([@pbio-0020225-Squire1]). Computational models also point to the need for a rapid encoding and storage system, together with a slower interleaving mechanism that is thought to underlie systems-level consolidation and long-term storage in the cortex (e.g., [@pbio-0020225-McClelland1]). However, the existence of some amnesic patients with long, flat gradients of RA extending for years or decades into periods of their life when memory function was normal provided some of the first evidence that RA might be due to retrieval failure ([@pbio-0020225-Sanders1]). This perspective on RA was initially supported by studies indicating that, in the anterograde domain, impaired memory could be alleviated by partial cues ([@pbio-0020225-Warrington1]). However, these observations were later construed as reflecting the operation of a separate memory phenomenon called priming ([@pbio-0020225-Graf1]). Several animal studies have also indicated that a variety of 'reminder\' treatments delivered prior to retention testing can realize the expression of lost memories ([@pbio-0020225-Gold2]; [@pbio-0020225-Miller2]; [@pbio-0020225-Spear1]; [@pbio-0020225-Gold1]; [@pbio-0020225-Riccio1]; [@pbio-0020225-Sara1]), but it is not easy to distinguish priming-induced memory from explicit recall and recognition in animal studies. Experimental resolution of the consolidation-versus-retrieval controversy has been notoriously difficult, and no consensus has been achieved. A key methodological issue, and the focus of the new technique described here, concerns the need to demonstrate that the memory observed after a reminder treatment results from the reactivation of an existing memory ([@pbio-0020225-Miller1]), rather than a facilitation of new learning ([@pbio-0020225-Gold2]).
In studies of spatial memory using the watermaze, amnesia for the location of the escape platform in posttraining probe trials (PTs) has generally been interpreted as a failure of learning, consolidation, or storage ([@pbio-0020225-DaHooge1]). To investigate the alternative possibility of retrieval failure, we deliberately created conditions that should maximize the possibility of seeing such an effect. This involved training rats to find an escape platform in a specific location followed by partial lesioning of the hippocampus. We reasoned that this would weaken but not completely disrupt the memory of the correct location by damaging a subset of the ensemble of stored traces. The animals\' memory was tested and observed to be undetectable. This same memory test provided, however, the opportunity to remind animals that escape from the water was possible via an escape platform in the correct or incorrect location. One hour later, the animals\' memory was tested again. We observed that memory was now detectably above chance and was equally strong when the animals had previously been given correct or potentially misleading information about the current location of the platform. Additional control procedures, and the performance of other groups with sham or complete hippocampal lesions, established that the earlier failure of memory must have been due, at least in part, to retrieval failure.
Results {#s2}
=======
A summary of the experimental design is provided in [Figure 1](#pbio-0020225-g001){ref-type="fig"} (see [Materials and Methods](#s4){ref-type="sec"}).
::: {#pbio-0020225-g001 .fig}
Figure 1
::: {.caption}
###### Experimental Design
Outline of the different phases of testing. The platform position used during training is indicated by a red circle in the NE quadrant of the pool (large blue circle), although in practice platform locations were counterbalanced between NE and SW locations. The novel location, to which a subset of rats was exposed during reminding, is indicated by a black circle in the SW quadrant. This position was always opposite to that used during training. PT1 and PT2: probe test 1 and 2. The hatched areas represent the original training quadrant irrespective of the position of the platform (i.e., original or novel) during retention testing. PT~n~1 and PT~n~2: PTs during new context learning in the second pool.
:::
:::
Training Prior to the Lesions {#s2a}
-----------------------------
During cued pretraining, the rats quickly learned to search for, and climb onto, the visually cued escape platform. In the main spatial training phase, the animals rapidly learned to locate and raise the platform in order to escape from the pool ([Figure 2](#pbio-0020225-g002){ref-type="fig"}), as indicated by the highly significant reduction in latencies over trials (*F*\[7.78, 412\] = 30.4, *p* \< 0.001). Only animals that reached the acquisition criterion received lesions (69 out of 73 rats trained). The prospective lesion groups, trained as normal animals, did not differ (*F* \< 1, *n* = 59; see Surgery below).
::: {#pbio-0020225-g002 .fig}
Figure 2
::: {.caption}
###### Training
Mean latencies to escape from the water and climb onto the hidden platform during task acquisition. Data are averaged in blocks of five trials and grouped according to the lesion made at the end of training; note that all animals were unoperated during acquisition. Only rats that reached criterion (mean escape latency less than 15 s over the last ten trials) and whose lesions were considered acceptable (see Results: Surgery) are presented. Animals rapidly learned to locate the escape platform, and prospective lesion groups did not differ.
:::
:::
Surgery {#s2b}
-------
Of the 69 animals that received lesions, one died after surgery and nine were excluded based on strict histological criteria, leaving a total of 59 animals (22 sham lesions, 19 complete hippocampal lesions, and 18 partial hippocampal lesions; see [Figure 3](#pbio-0020225-g003){ref-type="fig"}).
::: {#pbio-0020225-g003 .fig}
Figure 3
::: {.caption}
###### Lesion Analysis
Representative photomicrographs of cresyl-violet-stained coronal brain sections taken from subjects belonging to each of the three lesion groups---partial hippocampal lesion (A), sham lesion (B), and complete hippocampal lesion (C). In each case, sections corresponding to anterior, middle, and posterior levels of the hippocampus are displayed. The mean area of spared hippocampal tissue in each group (see [Materials and Methods](#s4){ref-type="sec"} for calculation) is plotted below in (D). Note that the volumes of spared tissue in the septal and temporal halves of the hippocampus are plotted separately, but these values are still expressed as percentages of the entire hippocampal volume---hence the value of 50% per half in shams. The cartoon hippocampi accompanying the graph indicate lesioned tissue in dark grey, and spared tissue in light cream. As intended, partially lesioned rats exhibited substantial sparing only in the septal (dorsal) half of the hippocampus, and rats with complete hippocampal lesions exhibited minimal sparing (less than 5% at either pole).
:::
:::
Retention Testing {#s2c}
-----------------
The key new findings are shown in [Figures 4](#pbio-0020225-g004){ref-type="fig"} and [5](#pbio-0020225-g005){ref-type="fig"} using two separate but related measures of memory retrieval: percentage time in quadrant ([Figure 4](#pbio-0020225-g004){ref-type="fig"}) and a more sensitive measure, percentage time in a zone centred on the platform location ([Figure 5](#pbio-0020225-g005){ref-type="fig"}; see [Materials and Methods](#s4){ref-type="sec"}). An overall analysis of variance (ANOVA) of percentage time in the training (where the platform was located during training) and the opposite quadrants of the pool revealed a significant quadruple interaction (*F*\[2, 53\] = 7.66, *p* \< 0.01) involving two between-subject factors: lesion group and platform location during the reminder treatment (original versus novel), and two within-subject factors: PT (PT1 and PT2) and quadrant (training versus opposite). In both figures, the initial memory expressed during PT1 is shown in the left lane. This reveals that the partially lesioned rats were at chance, whereas the sham-lesioned rats could remember the location of the platform (*t* = 6.15, *df* = 21, *p* \< 0.005, paired-sample t-test, training versus opposite quadrant). The complete-lesioned animals were at chance. Analysis of percentage time in zone ([Figure 5](#pbio-0020225-g005){ref-type="fig"}) likewise confirmed that memory was detectable in the sham lesion group (*t* = 4.18, *df* = 21, *p* \< 0.005, one-sample t-test, comparison with chance = 50%), but not in the two lesion groups.
::: {#pbio-0020225-g004 .fig}
Figure 4
::: {.caption}
###### Retention Testing: Quadrant Analysis
Percentage time during PT1 and PT2 spent in the training and opposite quadrants of the pool (left and right lanes) and the reminder treatment (grey central lane). The training location is represented as a red circle in the NE quadrant, and the novel location (novel subgroups only) as a black circle in the SW quadrant. In practice, NE and SW quadrants were counterbalanced. Rats with partial hippocampal lesions were unable to remember the platform location on PT1 but could be reminded of the training location by exposure, at the end of PT1, to a platform in the original or a novel location. (Note that the 'reminder\' lane simply refers to this exposure to a platform---PT1 is itself the 'reminder trial.\') The key finding is that the improvement in PT2 occurred irrespective of the platform location during reminding. In contrast, sham-lesioned animals exhibited some reversal learning upon exposure to the platform in a novel location. Complete-lesioned rats did not remember the platform location during either PT1 or PT2. \**p* \< 0.05; \*\**p* \< 0.01; n.s. = nonsignificant; comparisons with chance = 50%; one-sample t-tests. Representative swim paths are included.
:::
:::
::: {#pbio-0020225-g005 .fig}
Figure 5
::: {.caption}
###### Retention Testing: Zone Analysis
Percentage time in PT1 (left) and PT2 (right) spent within a zone, 20 cm in radius, centred on either the original training location (broken circle; grey) or an equivalent location in the opposite quadrant (broken circle; yellow), expressed as a percentage of the total time spent in both of the zones. The reminder treatment is again shown as the grey central lane and as the location where the hidden platform became available at the end of PT1 within these zones (original = red; novel = black). Consistent with [Figure 4](#pbio-0020225-g004){ref-type="fig"}, rats with partial hippocampal lesions were amnesic in PT1 but could be reminded of the correct location, even by exposure to the platform in a novel location. \**p* \< 0.05; \*\**p* \< 0.01; n.s. = nonsignificant; comparisons with chance = 50%; one-sample t-tests.
:::
:::
PT1 ended with the animals finding the platform in the original training location, or in a novel location in the 'opposite\' quadrant of the pool (middle lane in [Figures 4](#pbio-0020225-g004){ref-type="fig"} and [5](#pbio-0020225-g005){ref-type="fig"}; see [Materials and Methods](#s4){ref-type="sec"} for explanation of terminology). These different events at the end of the swim trial potentially served both as a reminder of what happens in a watermaze, namely, escape from the water at a particular location, and/or as an opportunity for new learning. We reasoned that if the reward of escaping from the water served only to support new learning, animals capable of learning would show an enhanced bias towards the training location after finding the platform in the original location, but a reduced bias after finding it in the opposite novel location. Conversely, if these events served only as reminder cues, they might be equally effective in reminding the rats of the original training location.
The key new finding is that the partial lesion group displayed a bias for the training quadrant that was equivalent whether the animals had found the platform in the original training location or in the novel opposite location, at the end of PT1. The overall ANOVA of the PT2 quadrant data revealed a triple interaction of lesion group × quadrant (training versus opposite) × platform location during reminding (novel versus original) (*F*\[2, 53\] = 19.28, *p* \< 0.001). With respect to the performance of the partial lesion group alone on this quadrant measure (see [Figure 4](#pbio-0020225-g004){ref-type="fig"}, right lane), there was a significant improvement between PT1 and PT2 (*F*\[1, 16\] = 7.98, *p* \< 0.02) and no difference between novel and original reminding locations (*F* \< 1). The partial lesion group also showed a highly significant preference for the training quadrant versus the opposite quadrant on PT2 (*F*\[1, 16\] = 16.83, *p* \< 0.001). The same pattern of results is apparent in the zone data (see [Figure 5](#pbio-0020225-g005){ref-type="fig"}) where, overall, the partial lesion group displayed a significant improvement between PT1 and PT2 (*F*\[1, 16\] = 7.64, *p* \< 0.02) that also did not differ between 'novel\' and 'original\' groups (*F* \< 1). Because a bias for the training location appeared even in the animals that were exposed to a novel platform position, memory on PT2 cannot be attributed to relearning of the platform location.
In contrast, sham-lesioned animals behaved quite differently in PT2 as a function of whether the platform was presented in the original or the novel location during the reminder treatment. Performance showed a further bias towards the training location between PT1 and PT2 following the event of climbing onto the escape platform in its original location, but exposure to the novel location resulted in a reduction in time spent in the training zone---a partial reversal. Supported by significant interactions in the overall ANOVA, analysis of time spent in the training quadrant revealed that, as expected, sham-lesioned animals reexposed to the original location increased their time there between PT1 and PT2 (*F*\[1, 11\] = 12.41, *p* \< 0.005). Conversely, sham-lesioned animals exposed to the novel location exhibited modest reversal learning, increasing their time in the opposite quadrant (*F*\[1, 9\] = 9.35, *p* \< 0.02). The same pattern of results was obtained from the analysis of time in the training zone ([Figure 5](#pbio-0020225-g005){ref-type="fig"}), for which a significant interaction between PT (PT1 or PT2) and platform location during reminding (original versus novel) was observed (*F*\[1, 20\] = 5.46, *p* \< 0.05).
Complete-lesioned rats performed at chance during all PTs (see [Figures 4](#pbio-0020225-g004){ref-type="fig"} and [5](#pbio-0020225-g005){ref-type="fig"}, left and right lanes). That is, their behaviour during the retention tests before and after the reminder treatment showed no impact of that treatment.
Novel Context Learning {#s2d}
----------------------
As an independent test of whether the reminder treatment of escape onto a platform could support new learning, all animals were taken to a second ('downstairs\') watermaze and given two PTs ([Figure 6](#pbio-0020225-g006){ref-type="fig"}). This was a novel environment, and, therefore, there was no reason to expect the animals to perform at better than chance levels in the first of these PTs in a novel environment (PT~n~1). However, escape from the water at the end of this PT might be sufficient to support new one-trial learning. Such learning was absent in the partial hippocampal lesion group (*F* \< 1). The sham lesion group, in contrast, did learn (*F*\[1, 21\] = 4.51, *p* \< 0.05), performing significantly better than the lesioned groups on PT~n~2 (post hoc Ryan--Einot--Gabriel--Welsch range test, *p* \< 0.005). The complete lesion group again showed no evidence of learning in a new environment (*F* \< 1).
::: {#pbio-0020225-g006 .fig}
Figure 6
::: {.caption}
###### Novel Context Learning
Percentage time spent in the target quadrant containing the escape platform during one-trial new learning in a different pool. \**p* \< 0.05; n.s. = nonsignificant; comparison of percentage time spent in training zone during PT~n~1 and PT~n~2; paired-sample t-tests. New learning was observed only in sham-lesioned rats.
:::
:::
Discussion {#s3}
==========
The key finding of this study is that rats with partial lesions of the hippocampus can be reminded of a preoperatively learned escape location in a watermaze by both correct and potentially misleading information. Whereas sham-lesioned rats showed new one-trial learning towards or away from the originally trained quadrant as a function of the type of reminder treatment to which they were exposed, partially lesioned animals were unable to learn. Instead, the first PT served only as a reminder of the original platform location irrespective of where in the pool the platform was raised at the end of this trial. Rats with complete hippocampal lesions showed neither new learning nor reminding.
There is an extensive classic literature on the nature and effectiveness of reminder treatments ([@pbio-0020225-Riccio1]). Exposure to the training context, noncontingent stimuli, or additional training trials are just some examples of methods successfully used to remind animals of a prior training experience ([@pbio-0020225-Zinkin1]; [@pbio-0020225-Miller2]; [@pbio-0020225-Mactutus1]; [@pbio-0020225-Gisquet1]; [@pbio-0020225-Przybyslawski1]). Controversy did, however, surround studies that interpreted memory following a reminder treatment as evidence that the original amnesia was the result of a retrieval deficit ([@pbio-0020225-Zinkin1]; [@pbio-0020225-Miller2]). It was argued that a reminding trial simply strengthens a weak memory that is behaviourally unobservable, similar to what happens during initial learning ([@pbio-0020225-Cherkin1]; [@pbio-0020225-Gold2]; [@pbio-0020225-Haycock1]; [@pbio-0020225-Gold1]), or that, when amnesia is complete, it results in one-trial learning or response generalization. However, manipulations that are unlikely to produce new learning can also serve as effective reminders. Examples include pharmacological manipulations of the internal state ([@pbio-0020225-Mactutus2]; [@pbio-0020225-Concannon1]) and reexposure to the amnestic agent prior to retention testing ([@pbio-0020225-Thompson1]; [@pbio-0020225-Hinderliter1]). In many such studies, however, the use of inhibitory avoidance as a memory test makes it difficult to determine the cognitive 'content\' (cf. [@pbio-0020225-Riccio1]) of the behaviour expressed during retention testing. Although memory reactivation may have occurred when a rat inhibits movement that previously led to electric shock, an alternative interpretation is that a generalized fear state has been induced. The issue of whether and when amnesia reflects a storage or retrieval deficit was, thus, left unresolved.
Two features are distinctive about our study. First, unlike in many previous studies, the reactivated memory involves the recall and expression of highly specific information---a discriminable position in space, and not just a faster escape latency, or greater freezing. Second, despite exposure to a novel platform location leading to reversal learning in the sham lesion group, the partial lesion group displayed only reminding of the original platform location. This distinction is important because, with the current revival of interest in memory retrieval, our protocol circumvents the ambiguities involved in the use of relearning as an index of retention. One example of a study that used a reacquisition rather than a true reminding protocol ([@pbio-0020225-Land1]) revealed that a reminder prior to retention testing could alleviate amnesia in animals with hippocampal lesions. However, it is difficult to distinguish between 'pure\' reminding and the facilitation of new learning using reacquisition alone.
Nonetheless, the watermaze task is deceptively complex, and successful performance depends on the operation of several distinct memory systems ([@pbio-0020225-Bannerman1]; [@pbio-0020225-Whishaw1]; [@pbio-0020225-Warburton1]; [@pbio-0020225-Eichenbaum1]; [@pbio-0020225-White2]). Accordingly, while no new learning of the platform location occurs in the partial and complete lesion groups, some 'procedural\' learning may take place during PT1; this may enhance a weak, subthreshold spatial memory to a point at which it can be expressed in PT2. However, for this argument to be plausible, one would expect there to be minimal retention of the procedural components in PT1. This was clearly not the case, as rats with both partial and complete hippocampal lesions did not behave like naïve animals during PT1. They searched at an appropriate distance from the pool walls and readily climbed onto the escape platform when it was eventually made available. Procedural learning is also generally well retained over time and, being slow, unlikely to change much in one trial. We also doubt that the recovery of memory on PT2 reflects the emergence of latent memory mediated solely by an extrahippocampal structure, but not expressed during PT1. For example, rats with complete hippocampal lesions have been shown to learn a spatial conditioned-cued preference mediated by the amygdala ([@pbio-0020225-White1]), a form of memory that is partially masked by hippocampus-dependent learning in normal rats ([@pbio-0020225-McDonald1]). However, seeing reminding in partial but not complete hippocampus-lesioned animals argues against this possibility in this case. Finally, the recovery of a simple stimulus--response strategy based on approaching single cues is unlikely, as novel start locations were always used during retention testing (cf. [@pbio-0020225-Eichenbaum2]; see [Materials and Methods](#s4){ref-type="sec"}). Under these circumstances, it is reasonable to interpret the apparently complete amnesia observed in PT1 as, at least in part, a failure of spatial memory retrieval.
Our use of partial hippocampal lesioning introduces several other issues. First, it is a technique that is arguably more relevant to human amnesia, in which damage to a structure is typically incomplete. Second, it is also relevant to the many studies in which a pharmacological intervention is applied at a single site within a brain region---microinfusion into the dorsal hippocampus, for instance, is likely to have minimal effects on ventral hippocampal tissue (see [@pbio-0020225-Steele1]). Third, and perhaps most interesting, is the question of where memory traces are located. Given that reminding only occurs in partially lesioned rats, it is reasonable to suppose that spatial memory traces are either located (and reactivated) within the hippocampus, or that the hippocampus is required for the process of reactivation or expression of a reactivated memory stored elsewhere. According to the latter hypothesis, spatial memory traces might be stored in cortex but require fast synaptic transmission in the hippocampus to be retrieved (cf. [@pbio-0020225-Teyler1])---at least during the period after training and before the completion of systems-level consolidation. Alternatively, some hippocampal tissue might be required for cortically expressed memory to gain access to striatal motor planning and executive systems. Findings reported by [@pbio-0020225-Virley1] suggest that this retrieval hypothesis might not be implausible. In this study, monkeys with CA1 pyramidal cell lesions were amnesic for a preoperatively acquired visuospatial discrimination. Subsequent grafting of CA1 pyramidal cells resulted in the recovery of memory for a second preoperatively acquired discrimination. As the grafted tissue cannot contain specific memory traces, the implication is that the recovery of some aspects of CA1 cellular function is sufficient for the information processing mediating the retrieval of memories stored elsewhere.
In raising many more questions than they answer, the present findings open a potential avenue of research into the neural dynamics of memory reactivation and retrieval. Specific interventions such as local AMPA receptor blockade (cf. [@pbio-0020225-Riedel1]) might be directed at the hippocampus or cortex during PT1 or PT2. Such a study could provide information about the role of these structures---and their network interactions---in the reactivation of apparently lost memories, and in their subsequent retrieval. For example, hippocampal neural activity may be necessary for effective retrieval, but perhaps not for the reminding-induced reactivation of memory, even for an ostensibly hippocampus-dependent task (cf. [@pbio-0020225-Land1]). Similarly, the necessity for hippocampal neural activity during retrieval might vary as a function of time after memory consolidation. In addition, the determinants of the reminder phenomenon itself remain unclear. It would be useful to establish whether reinforcement in the form of an escape platform is, in fact, necessary during PT1, or indeed whether a reminder trial in a separate pool would have been effective. Experiments involving partial versus complete sets of cues might also provide valuable insights into the reminding process (cf. [@pbio-0020225-OaKeefe1]). These and related analyses will be the subject of future studies.
Dissociating the storage and retrieval functions of the hippocampus in memory is central to our understanding of the role of hippocampo--cortical connections. Many theories of hippocampal function are based on the idea that the hippocampus acts as a mediating link between different cortical regions during the interval before systems consolidation is complete ([@pbio-0020225-Teyler1]; [@pbio-0020225-Squire2]). Paradoxically, the same features that point to the alternative possibility---that the hippocampal formation is a site of encoding and long-term storage of complex multimodal memories within its distributed intrinsic circuitry ([@pbio-0020225-Moscovitch1])---also place this group of structures in an ideal position to help reactivate memories from traces distributed over several cortical structures, perhaps via a mechanism such as pattern completion (see [@pbio-0020225-Marr1]; [@pbio-0020225-Nakazawa1]). It is possible that, when the hippocampus is partially damaged and the cortico--hippocampal network is therefore degraded, retrieval is only possible once a more complete recreation of the training situation, possibly including reexposure to a platform, is provided. Although comparisons across different species and forms of memory should be viewed with caution, this scenario is reminiscent of Tulving\'s encoding specificity principle ([@pbio-0020225-Tulving1]; [@pbio-0020225-Thomson1]) in that exposure to similar cues during encoding and retrieval phases permits the recovery of the original memory, despite the provision of incorrect information about the target location itself. Paradoxically, the poor learning abilities of partially lesioned rats might explain why a trial ending with exposure to a novel spatial location can serve as a reminder for the original location---by limiting new learning of the new location, a reactivated memory for the old location is unmasked.
Materials and Methods {#s4}
=====================
{#s4a}
### Subjects {#s4a1}
We used a total of 73 male Lister Hooded rats obtained from a commercial supplier (Charles River Laboratories, United Kingdom). They were pair-housed in plastic cages with sawdust bedding and ad libitum access to food and water. Their care and maintenance and all experimental procedures were carried out in accordance with United Kingdom Home Office Regulations.
Behavioural testing was conducted using two separate circular pools, 2.0 m in diameter and 60 cm high, each located in well-lit rooms with numerous distal visual cues. One pool was used for training and retention ('upstairs\') and the other for new context learning ('downstairs\'). The pools were filled with water at 25 °C ± 1 °C made opaque by the addition of 200 ml of latex liquid (Cementone-Beaver, Buckingham, United Kingdom). We used the 'Atlantis platform\' ([@pbio-0020225-Spooner1]), a polystyrene platform that becomes available by rising from the bottom of the pool only if the animals swim to and stay within a specified 'dwell radius\' centred on the correct location for a predetermined 'dwell time.\' When risen, the top of the platform remained 1.5 cm below the water surface. The animals\' swimming was monitored by an overhead video camera connected to a video recorder and an online data acquisition system (Watermaze, Watermaze Software, Edinburgh, United Kingdom; [@pbio-0020225-Spooner1]) located in an adjacent room. This system digitizes the path taken by an animal and computes various parameters such as escape latency, time spent in a zone overlying the platform, and other conventional measures of watermaze performance.
### Training protocol {#s4a2}
Testing was carried out according to the schedule illustrated in [Figure 1](#pbio-0020225-g001){ref-type="fig"}.
### Cued pretraining {#s4a3}
This phase consisted of a single day of nonspatial cued training in the 'upstairs\' watermaze (curtains drawn around the pool to occlude extramaze cues, with ten trials in two sessions of five trials each (intertrial interval ≈ 20 min; intersession interval ≈ 3 h). The visible cue was suspended approximately 25 cm above the platform, which was moved every two trials to one of four possible locations, according to a pseudorandom schedule; the dwell radius was set at 20 cm, and the dwell time was 1 s.
### Training {#s4a4}
Training on a spatial reference memory task began 3 d later in the same watermaze. Rats received ten trials/day, in two sessions of five consecutive trials each (intersession interval ≈ 2 h), for 4 d. The dwell time was set to 0.5 s throughout training, but the dwell radius was gradually reduced over days (day 1: 20 cm; day 2: 15 cm; days 3 and 4: 13 cm). This schedule was intended to promote accurate and focused searching, but without generating the highly perseverative strategy that typically results from the use of long dwell times ([@pbio-0020225-Riedel1]). Rats were given a maximum of 120 s to find an escape platform located at the centre of either the NE or SW quadrant, after which they remained on the platform for 30 s On the rare trials in which a rat failed to escape within 2 min, the experimenter placed a hand above the correct location in order to guide the animal to the platform. For each animal, the platform position remained constant throughout training, but start locations (N, S, E, or W) were varied pseudorandomly across trials. Only those animals achieving the acquisition criterion of mean escape latencies of 15 s or less on day 4 of training proceeded to the next phase of testing.
### Surgery {#s4a5}
Surgery took place 1--2 d after the end of training. Rats were given either partial or complete bilateral neurotoxic lesions of the hippocampal formation (DG and CA fields), or sham surgery. Complete lesions were intended to remove 85% or more of the total hippocampal volume. Partial lesions targeted the temporal two-thirds of the hippocampus, sparing the septal (dorsal) third of the structure. The rats were assigned to groups of equivalent mean performance on the basis of their escape latencies during the final day of training. Lesions were made with ibotenic acid (Biosearch Technologies, Novato, California, United States; dissolved in 0.1 M phosphate-buffered saline \[pH 7.4\] at 10 mg/ml) following the protocol of [@pbio-0020225-Jarrard1]. The animals were anaesthetized with an intraperitoneal injection of tribromoethanol (avertin) and placed in a Kopf Instruments (Tujunga, California, United States) stereotaxic frame such that Bregma and Lambda lay on the same horizontal plane. Rats received nine or 13 injections of ibotenic acid (partial and complete lesion groups, respectively; 0.05 μ1, 0.08 μ1, or 0.1 μ1 per injection) at different rostrocaudal and dorsoventral levels via an SGE syringe secured to the stereotaxic frame (see [@pbio-0020225-de1]). The injection rate was 0.1 μ1/min, and the needle was removed very slowly 90 s after the injection. A total of 0.65 μ1 or 0.91μ1 per hemisphere was necessary for the partial and complete lesions, respectively. The coordinates were modified from [@pbio-0020225-Jarrard1] to suit the slightly different brain size of Lister Hooded rats and to achieve the desired amount of partial hippocampal damage (see [@pbio-0020225-de1]). Sham lesions were made in the same way, with the injections replaced by a piercing of the dura (intended to cause comparable neocortical damage).
### Retention testing {#s4a6}
This phase began 14 d after the end of training. It consisted of two PTs (PT1and PT2) spaced 1 h apart, with a reminder treatment occurring at the end of PT1.
Each PT (PT1 and PT2) began with a standard 60-s swim with the platform unavailable. In each PT, the rats were placed into the pool in either the adjacent right or the adjacent left quadrants with respect to the training quadrant. Start positions were counterbalanced across PTs and across rats. At the end of the 60 s the platform was raised and the animals were allowed to find and climb onto it. The rats were allowed a further 60 s to locate the platform once risen (but still hidden just below the water surface); if unsuccessful within this period, they were guided to the platform. They then remained on the platform for 30 s.
The raising of the platform at the end of PT1 constituted the reminder treatment; thus PT1 is sometimes referred to as the 'reminder trial.\' A key variable was that the platform was raised in either the original training location (half the animals) or in a novel location in the centre of the opposite quadrant of the pool (the other half). Note that reminding using the original location always occurred in the training quadrant, and reminding using the novel location always occurred in the opposite quadrant. However, whereas the terms 'training\' and 'opposite\' are used to refer to physical areas of the pool, 'novel\' and 'original\' refer also to separate groups that received each type of reminder.
For analysis of the different behavioural phases, several measures of performance were assessed, including escape latency, swim speed, and time spent within defined regions of the pool. Memory retention during PTs is inferred from the time spent in each quadrant of the pool as a percentage of the 60-s duration of the PT. A more sensitive measure can be obtained by analysing percentage time spent within a specified radius (zone) centred on the platform location ([@pbio-0020225-Moser1]). When time in zone is presented, it is expressed as a percentage of the total time spent in both the original training zone and the novel opposite zone. Statistical analysis (SPSS, Chicago, Illinois, United States) began with an ANOVA followed by appropriate post hoc comparisons. Numerical data are reported as mean ± standard error (s.e.m.) throughout.
### Novel context learning {#s4a7}
New learning was assessed the next day in a separate 'downstairs\' watermaze that constituted a novel context. The protocol was identical to that used during 'upstairs\' retention testing, i.e., two rewarded PTs (PT~n~1 and PT~n~2) spaced 1 h apart.
### Lesion analysis {#s4a8}
At the end of behavioural testing, rats were perfused intracardially with saline followed by 10% formalin under terminal pentobarbitone anaesthesia (Euthatal, 1 ml). Their brains were removed and stored in 10% formalin for 24 h before being blocked and embedded in egg yolk. The embedding procedure is described in [@pbio-0020225-de1]. Coronal, 30-μm sections through the hippocampus and other structures were cut using a cryostat: every fifth section was recovered, mounted on a slide, and stained with cresyl violet (see [Figure 3](#pbio-0020225-g003){ref-type="fig"}A--[3](#pbio-0020225-g003){ref-type="fig"}C).
The relative volume of spared tissue was calculated by measuring the area of hippocampus spared in each section of a particular brain according to the following protocol: Each coronal section containing hippocampus was placed under a photomacroscope (Wild, Heerbrugg, Switzerland), and the image taken by a mounted video camera was imported into NIH Image 1.63 (National Institutes of Health, Bethesda, Maryland, United States). The area of spared hippocampal tissue in each section was then outlined and automatically calculated. Surrounding fibres such as the fimbria were excluded on the grounds that they would not be considered in a section were all the hippocampal cells dead. The sections were spaced 150 μm apart, yielding up to 32 sections in a sham lesion animal, and fewer in animals with acceptable partial lesions. For each rat, the total hippocampal 'volume\' was calculated by adding the area of hippocampal tissue spared in each successive section. The proportion of hippocampus spared for each lesioned animal was expressed as a percentage of the mean hippocampal 'volume\' for sham-lesioned animals. Values for the left and right hippocampi were initially calculated separately and then averaged (see [Figure 3](#pbio-0020225-g003){ref-type="fig"}D).
Strict criteria for acceptance of a lesion were used. The lesion had to be confined to the hippocampus in all cases, and leave intact tissue volumes of 25%--50% in the septal hippocampus with minimal sparing (less than 10%) elsewhere in the structure in the case of partial lesions, or less than 15% total hippocampal sparing in the case of complete lesions. Animals with minimal subicular damage, typically located at medial levels of the structure, were accepted.
We would like to thank Jane Knox for histology, Andrew Bernard for animal care, and David Foster for helpful discussion. This research was supported by a Medical Research Council (MRC) Programme Grant held by RGMM and an MRC Research Fellowship held by LdH.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** LdH, SJM, and RGMM conceived and designed the experiments. LdH and SJM performed the experiments. LdH and SJM analysed the data. LdH, SJM, and RGMM wrote the paper.
Academic Editor: Howard B. Eichenbaum, Boston University
Citation: de Hoz L, Martin SJ, Morris RGM (2004) Forgetting, reminding, and remembering: The retrieval of lost spatial memory. PLoS Biol 2(8): e225.
ANOVA
: analysis of variance
PT
: probe trial
PT~n~
: probe trial in a novel environment
RA
: retrograde amnesia
| PubMed Central | 2024-06-05T03:55:46.749924 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509297/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e225",
"authors": [
{
"first": "Livia",
"last": "de Hoz"
},
{
"first": "Stephen J",
"last": "Martin"
},
{
"first": "Richard G. M",
"last": "Morris"
}
]
} |
PMC509298 | The regeneration of lost body parts and injured organs has captured the human imagination since the time of the ancient Greeks. The deep-seated roots of this early fascination can be seen in Greek mythology. The many-headed Hydra nearly defeated the hero Heracles by growing two new heads for every one that Heracles cut off, and the liver of Prometheus, devoured by a ravenous eagle each night, regenerated every morning. Aristotle, who lived from 384--322 BC, noted that the tails of lizards and snakes, as well as the eyes of swallow-chicks, could regenerate ([@pbio-0020232-Aristotle1]). This fascination became a legitimate area of scientific inquiry in 1712, when the French scientist René-Antoine Ferchault de Réaumur published his seminal work on crayfish limb and claw regeneration ([@pbio-0020232-Reaumur1]). Soon thereafter, several other prominent scientists of the eighteenth century, including Abraham Trembley, Charles Bonnet, Peter Simon Pallas, and Lazzaro Spallanzani, discovered remarkable regenerative abilities in a variety of organisms. Hydra, earthworms, and planarians could regenerate their heads and tails ([@pbio-0020232-Pallas1]; [@pbio-0020232-Lenhoff1]); salamanders could regenerate their limbs, tails, and jaws; premetamorphic frogs and toads could regenerate their tails and legs; slugs could regenerate their horns; and snails could regenerate their heads ([@pbio-0020232-Spallanzani1]). This last discovery caused quite a stir in eighteenth-century France, leading to an "unprecedented assault" on snails as both naturalists and the general public participated in the quest for scientific knowledge by reproducing Spallanzani\'s intriguing results ([@pbio-0020232-Newth1]).
Stem Cells Versus Dedifferentiation {#s2}
===================================
During the nineteenth century and for most of the twentieth century, regeneration research primarily focused on the phenomenology of regeneration and its cellular basis. Many important discoveries were made during this period, which led in part to the general conclusion that progenitor cells are required for most regenerative processes. However, the origin of these progenitor cells varies between regenerating systems. In some cases, such as the regeneration of skin, blood, muscle, and bone in mammals and the replacement of lost tissues in the flatworm planarian, the progenitor cells pre-exist as reserve cells or stem cells that only need to be activated in response to injury or tissue depletion. In other cases, the progenitor cells can be created de novo through a process in which fully differentiated cells reverse their normal developmental processes and revert to proliferating progenitor cells. This latter process, known as cellular dedifferentiation, is especially prominent in vertebrates with exceptional regenerative abilities, such as salamanders. For example, during salamander limb regeneration, cells from muscle, bone, cartilage, nerve sheath, and connective tissues participate in the dedifferentiation process to form a pool of proliferating progenitor cells known as the regeneration blastema ([Figure 1](#pbio-0020232-g001){ref-type="fig"}) ([@pbio-0020232-Chalkley1]; [@pbio-0020232-Bodemer1]; [@pbio-0020232-Hay1]; [@pbio-0020232-Wallace1]; [@pbio-0020232-Lo1]; [@pbio-0020232-Kumar2]). It has not yet been determined whether pre-existing stem cells or reserve cells also contribute to the pool of progenitor cells---nor whether the blastemal cells are multipotent (capable of differentiating into multiple cell types), are committed to a particular cell lineage, or are a mix of multipotent and committed progenitor cells. Regardless, these blastemal cells will later redifferentiate to form all the internal tissues of the regenerated limb other than the peripheral nerve axons. This extraordinary degree of cellular plasticity distinguishes those vertebrates that can replace entire anatomical structures, such as limbs, from vertebrates with more limited regenerative abilities.[](#pbio-0020232-g001){ref-type="fig"}
::: {#pbio-0020232-g001 .fig}
Figure 1
::: {.caption}
###### Dedifferentiation of Limb Cells During Salamander Limb Regeneration
Brown nuclei are a result of BrdU incorporation during DNA synthesis, and therefore mark cells that are progressing through the cell cycle. Abbreviations: e, epidermis; d, dermis; m, muscle; b, bone; bl, blastema; aec, apical epithelial cap.
\(A) Unamputated right forelimb of a newt and coronal section of the stylopodium. The only cells actively synthesizing DNA are those in the basal layer of the epidermis (bone marrow cells also actively synthesize DNA in the unamputated limbs but are not shown here). Note the long myofibers in the nonregenerating newt limb and the distant spacing between the muscle nuclei.
\(B) Seven-day limb regenerate and coronal section of the distal regenerating tip. Note that the muscle cells have lost their normal architecture and that the nuclei are more closely spaced and have begun to synthesize DNA.
\(C) Twenty-one-day limb regenerate and coronal section of the distal regenerating tip. The nuclei of the blastema are spaced closely together, and many nuclei are actively synthesizing DNA. The bone is also being broken down in the vicinity of the blastema.
:::
:::
The public has recently exhibited a renewed interest in regeneration research, due in large part to stem cell research, which has provided promising avenues for the field of regenerative medicine. In addition, celebrities such as Christopher Reeve and Michael J. Fox have given a human face to the many people who could benefit from effective regenerative therapies. The political, ethical, and religious controversies surrounding the use of human embryonic stem cells for therapeutic purposes have only served to increase the public\'s awareness of the promising potential of regenerative medicine. But this interest in using scientific knowledge to enhance the regenerative capacity in humans is not new. Spallanzani closed his 1768 monograph on regeneration, *An Essay on Animal Reproductions*, with a series of questions--- which, except for the antiquated language, could be asked by citizens of the twenty-first century: But if the abovementioned animals, either aquatic or amphibious, recover their legs, even when kept on dry ground, how comes it to pass, that other land animals, at least such as are commonly accounted perfect, and are better known to us, are not endued with the same power? Is it to be hoped they may acquire them by some useful dispositions? \[A\]nd should the flattering expectation of obtaining this advantage for ourselves be considered entirely as chimerical?
Although most of the current interest in regenerative medicine focuses on the potential benefits of either embryonic or adult stem cells, there are several investigators who are now taking an entirely different approach to the problem. These researchers think that although stem cells may offer some benefits in the relatively near future, a more comprehensive approach will be required to meet all of our regenerative needs. To achieve this goal, they must first learn how nature has already solved the problem of regeneration and then use this information to enhance the regenerative capacity in mammals. These studies seek to understand the biology of regeneration, especially the cellular and molecular mechanisms that govern regenerative processes. The experimental systems range from the unicellular protozoa to complex vertebrates, such as salamanders and mice.
The Molecular Biology of Regeneration {#s3}
=====================================
With the technological advances that followed the advent of molecular biology, researchers acquired the basic tools to begin to unravel the molecular basis for cellular plasticity and regeneration. However, progress in this arena has been slow, given that most organisms with marked regenerative abilities are not yet amenable to routine genetic manipulation. Recent advances, such as the application of mutagenic screens to study fin regeneration in zebrafish ([@pbio-0020232-Johnson1]; [@pbio-0020232-Poss2]) and the application of RNAi knockdown technology to study regeneration in planarians ([@pbio-0020232-Sanchez1]; [@pbio-0020232-Newmark1]), are quite promising and could largely ameliorate this deficiency. Nevertheless, results from several recent studies have converged on a set of genes that appear to play an important role in regeneration, and evidence is accumulating that suggests some of these genes may function to control regenerative cellular plasticity. Three such genes are *Msx1, BMP4*, and *Notch1*. These genes encode, respectively, a transcriptional repressor, a signaling ligand, and a signaling cell surface receptor.
Numerous studies over the past three decades have shown that mammals, including humans, can regenerate their digit tips provided the amputation plane is distal to the terminal phalangeal joint ([@pbio-0020232-Douglas1]; [@pbio-0020232-Illingworth1]; [@pbio-0020232-Borgens1]; [@pbio-0020232-Singer1]). However, *Msx1*-deficient mice exhibit impaired fetal digit-tip regeneration, a phenotype that can be rescued in ex vivo cultures in a dose-dependent manner by application of exogenous BMP4 ([@pbio-0020232-Han1]). Recently, it has been demonstrated that Xenopus tadpoles are unable to regenerate their tails during a refractory period of development between stages 45 and 47 ([@pbio-0020232-Beck1]). If tails are amputated during this refractory period, genes that are normally expressed during the early stages of tadpole tail regeneration, such as *BMP4, Msx1*, and *Notch-1*, are not expressed. However, transgenic frogs carrying a hyperactive form of *Msx1* or constitutively active *ALK3* (a receptor for BMP4) are able to regenerate their tails during the refractory period. Transgenic frogs carrying a constitutively active *Notch-1* receptor will regenerate their notochords and spinal cords but exhibit little or no muscle regeneration, suggesting that Notch-1 signaling alone cannot rescue complete regenerative capacity in frog tadpoles ([@pbio-0020232-Beck1]). Results from expression studies in a variety of organisms are consistent with these in vivo gene function studies. *Msx* genes are upregulated in regenerating salamander limbs and regenerating zebrafish fins and hearts ([@pbio-0020232-Simon1]; [@pbio-0020232-Poss1]; [@pbio-0020232-Raya1]), while *notch-1b* and its ligand, *deltaC*, are upregulated during zebrafish heart and fin regeneration ([@pbio-0020232-Raya1]).
*Msx1* and Cellular Plasticity {#s4}
==============================
Although these functional and expression studies indicate that *Msx1, Bmp4*, and *Notch-1* are important for a variety of regenerative processes, they do not address the mechanism by which these genes exert their effects. However, several in vitro studies suggest that *Msx1* may be involved in regulating cellular plasticity. Ectopic expression of *Msx1* can inhibit the differentiation of a variety of mesenchymal and epithelial progenitor cell types ([@pbio-0020232-Song1]; [@pbio-0020232-Hu1]), suggesting that this gene may play a role in maintaining cells in an undifferentiated state. Furthermore, *Msx1* may be functioning not only to prevent differentiation of progenitor cells but also to induce dedifferentiation of cells that have already differentiated. Ectopic expression of *Msx1* in mouse myotubes (differentiated muscle cells that are multinucleated and are able to contract), coupled with serum stimulation, can induce these multinucleated cells to reduce their levels of myogenic proteins and undergo a cell cleavage process that produces proliferating mononucleated cells (a process known as cellularization) ([@pbio-0020232-Odelberg1]). Clonal populations of these dedifferentiated cells can redifferentiate into cells expressing markers for cartilage, fat, and bone cells, as well as myotubes. These results suggest that the combination of ectopic *Msx1* expression and serum stimulation can induce differentiated muscle cells to dedifferentiate into proliferating multipotent progenitor cells. Given this degree of cellular plasticity, it is not surprising that *Msx1* can also induce muscle progenitor cells, known as myoblasts, to dedifferentiate to multipotent progenitor cells ([@pbio-0020232-Odelberg1]).
Cellularization of myotubes and myoblast dedifferentiation can also be induced by at least two synthetic trisubstituted purines. Myoseverin is a trisubstituted purine that binds to and disassembles microtubules, leading to the cellularization of multinucleated myotubes ([@pbio-0020232-Rosania1]). The resulting mononucleated cells proliferate when stimulated with serum and redifferentiate into myotubes following serum starvation. A second trisubstituted purine, reversine, induces myoblasts to dedifferentiate into progenitor cells with adipogenic and osteogenic potential ([@pbio-0020232-Chen1]). Therefore, reversine and *Msx1* appear to have a similar effect on mouse myoblasts, although no reports have yet addressed whether reversine might induce dedifferentiation of multinucleated myotubes.
In this issue of *PLoS Biology*, [@pbio-0020232-Kumar1] present data linking *Msx1* function to microtubule disassembly during the process of salamander myofiber cellularization and fragmentation (myofibers are formed from myotubes and represent the completely mature form of the differentiated skeletal muscle cell). Their results suggest that *Msx1* expression induces microtubule disassembly, which then leads to myofiber cellularization or fragmentation. If *Msx1* function is markedly reduced in salamander myofibers by preventing the efficient synthesis of the Msx1 protein, cellularization or fragmentation of the myofiber is inhibited, suggesting that Msx1 is required for this process. Thus, this study complements previous work ([@pbio-0020232-Odelberg1]) showing that ectopic *Msx1* expression, coupled with serum stimulation, is sufficient to induce cleavage, cellularization, and dedifferentiation of mouse myotubes. The two studies point to an essential role for *Msx1* in regenerative cellular plasticity and when combined with previous in vivo studies, raise the possibility that BMP or Notch signaling might also play a role in this process.
Results from these and other similar studies are beginning to give researchers a glimpse into the molecular mechanisms that control regeneration and cellular plasticity. With the new tools available to identify candidate genes and assess their function, the next few decades appear promising for scientists engaged in regeneration research. Elucidating the molecular basis for regeneration may prove to be an essential step in devising effective methods for enhancing regeneration in mammals and may well usher in a golden era for regenerative medicine.
Accession Numbers {#s5}
=================
The Mouse Genome Informatics (<http://www.informatics.jax.org/>) accession numbers of the genes discussed in this paper are *ALK3* (MGI: 1338938), *BMP4* (MGI: 88180), *Msx1* (MGI: 97168), and *Notch1* (MGI: 97363). The GenBank (<http://www.ncbi.nih.gov/GenBank/>) accession numbers of the genes discussed in this paper are *deltaC* (NM 130944), *notch1b* (Y10352), Ambystoma mexicanum *Msx1* (AY525844), Danio rerio msxb (U16311; partial sequence), D. rerio msxc (NM 131272), Homo sapiens *ALK3* (Z22535), and Mus musculus *ALK3* (Z23154).
Work in our laboratory on identifying newt genes that regulate cellular plasticity is supported by grants from the National Institutes of Health (R01NS43878 and R01NS43878S1).
Shannon J. Odelberg is with the Cardiology Division of the Department of Internal Medicine at the University of Utah, Salt Lake City, Utah, United States of America. E-mail: <odelberg@howard.genetics.utah.edu>
| PubMed Central | 2024-06-05T03:55:46.753449 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509298/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e232",
"authors": [
{
"first": "Shannon J",
"last": "Odelberg"
}
]
} |
PMC509299 | Introduction {#s1}
============
Retroviral replication requires reverse transcription of the viral RNA genome and integration of the resulting DNA copy into a chromosome of the host cell. A topic of long standing interest has been the chromosomal and nuclear features dictating the location of integration target sites (reviewed in [@pbio-0020234-Coffin1]; [@pbio-0020234-Bushman2]). Integration site selection has also gained increased interest because of its importance for human gene therapy. Retroviral vectors have been used extensively to deliver therapeutic genes carried in retroviral backbones. However, retroviral integration can take place at many locations in the genome, on occasion resulting in insertional activation of oncogenes (reviewed in [@pbio-0020234-Coffin1]; [@pbio-0020234-Bushman2]). Recently, two patients undergoing gene therapy for X-linked severe combined immunodeficiency developed leukemia-like illnesses associated with integration of a therapeutic retroviral vector in or near the LMO2 proto-oncogene ([@pbio-0020234-Check1]; [@pbio-0020234-Hacein-Bey-Abina1]). Insertional activation of oncogenes by retroviral vectors has also been detected in animal models ([@pbio-0020234-Li1]).
With the availability of the complete human genome sequence, large-scale sequence-based surveys of integration sites have become possible ([@pbio-0020234-Schroder1]; [@pbio-0020234-Laufs1]; [@pbio-0020234-Wu1]). [@pbio-0020234-Schroder1] investigated targeting of human immunodeficiency virus (HIV) and HIV-based vectors in a human lymphoid cell line (SupT1) and found that genes were favored integration targets. Global analysis of transcription in SupT1 cells showed that active genes were favored for integration, particularly those that were active after infection with the HIV-based vector. [@pbio-0020234-Wu1] examined targeting of murine leukemia virus (MLV) in human HeLa cells and found that MLV does not strongly favor integration in transcription units, but rather favors integration near sequences encoding mRNA 5′ ends. Here we contrast integration targeting by three retroviruses, avian sarcoma-leukosis virus (ASLV), HIV, and MLV, taking advantage of 1,462 new integration site sequences and matched transcriptional profiling data. We find that ASLV does not favor integration near transcription start sites, nor does it strongly favor active genes. For HIV, we find that active genes are favored for integration in two primary cell types, extending findings from previous studies of transformed cell lines. Cell-type-specific transcription was found to result in cell-type-specific biases in integration site placement. We also reanalyzed the MLV data from Burgess and coworkers ([@pbio-0020234-Wu1]) in parallel, confirming that MLV integration is favored near transcription start sites. Thus it appears that each retrovirus studied to date has a unique pattern of integration site selection within the human genome, suggesting that there may be local recognition of chromosomal features unique to each virus.
Results {#s2}
=======
Integration Site Datasets Used in this Study {#s2a}
--------------------------------------------
The origins of the 3,127 integration sites studied are summarized in [Table 1](#pbio-0020234-t001){ref-type="table"}. All were generated by acute infection of cells with retroviruses or with viruses generated from retroviral vectors. To isolate integration sites, DNA from infected cells was isolated, cleaved with restriction enzymes, then ligated to DNA linkers. Integration sites were amplified using one primer that bound to the viral DNA end and another that bound the linker, then amplification products were cloned and sequenced ([@pbio-0020234-Schroder1]; [@pbio-0020234-Wu1]). Integration sites were mapped on the draft human genome sequence ([Figure 1](#pbio-0020234-g001){ref-type="fig"}) and local features at integration sites quantified.
::: {#pbio-0020234-g001 .fig}
Figure 1
::: {.caption}
###### Relationship between Integration Sites and Transcriptional Intensity in the Human Genome
The human chromosomes are shown numbered. HIV integration sites from all datasets in Table 1 are shown as blue "lollipops"; MLV integration sites are shown in lavender; and ASLV integration sites are shown in green. Transcriptional activity is shown by the red shading on each of the chromosomes (derived from quantification of nonnormalized EST libraries, see text). Centromeres, which are mostly unsequenced, are shown as grey rectangles.
:::
:::
::: {#pbio-0020234-t001 .table-wrap}
Table 1
::: {.caption}
###### Integration Site Datasets Used in This Study
:::
DOI: [10.1371/journal.pbio.0020234.t001](10.1371/journal.pbio.0020234.t001)
:::
Three integration site datasets were newly determined in this study. Integration by an ASLV vector was analyzed in 293T-TVA cells, which are human 293T cells engineered to express the subgroup A avian retrovirus receptor. Integration by an HIV-based vector was characterized in two types of primary human cells, peripheral blood mononuclear cells (PBMCs) and IMR90 lung fibroblasts. Several previously described datasets were also subjected to further analysis in parallel---HIV integration sites in three transformed cell lines (SupT1 \[[@pbio-0020234-Schroder1]\], H9, and HeLa \[[@pbio-0020234-Wu1]\]) and MLV integration sites in HeLa cells ([@pbio-0020234-Wu1]).
The use of restriction enzymes to cleave cellular DNA during the cloning of integration sites could potentially introduce a bias in favor of isolating integration events closer to restriction sites. Previous work suggested that integration site surveys were not strongly biased. In one study, an experimental control based on integration in vitro indicated that the cloning and analytical methods used did not detectably bias the conclusions ([@pbio-0020234-Schroder1]). In addition, HIV integration sites cloned by different methods showed a similar preference for active genes ([@pbio-0020234-Carteau1]; [@pbio-0020234-Schroder1]; [@pbio-0020234-Wu1]), including sites isolated using several different restriction enzymes to cleave cellular DNA prior to linker ligation, and isolation using inverse PCR instead of ligation-mediated PCR.
In this study we have added a computational method to address possible biased isolation. Each integration site was paired with ten sites in the human genome randomly selected in silico that were constrained to be the same distance from a restriction site of the type used for cloning as the experimentally determined integration site. Statistical tests were then carried out by comparing each experimentally determined integration site to the ten matched random control sites. In this way any bias due to the placement of restriction sites in the human genome was accounted for in the statistical analysis. All the collections of integration sites were analyzed in this manner, including data previously published in ([@pbio-0020234-Schroder1]; [@pbio-0020234-Wu1]). However, direct analysis of the distribution of integration sites without this correction yielded generally similar conclusions, suggesting that restriction site placement did not introduce a strong bias. A detailed description of the statistical analysis is presented in [Protocol S1](#sd001){ref-type="supplementary-material"} (p. 2).
Integration in Transcription Units {#s2b}
----------------------------------
For HIV the frequency of integration in transcription units ranged from 75% to 80%, while the frequency for MLV was 61% and for ASLV was 57%. For comparison, about 45% of the human genome is composed of transcription units (using the Acembly gene definition). Analysis using the different catalogs of human genes suggests that somewhat different fractions of the human genome are transcribed, and new information indicates that an unexpectedly large fraction of the human genome may be transcribed into noncoding RNAs ([@pbio-0020234-Cawley1]). However, for comparisons using any catalog of the human genes, the rate of integration in human transcription units determined experimentally was substantially higher than in the matched random control sites ([Protocol S1](#sd001){ref-type="supplementary-material"}, p. 3--11).
We next assessed the placement of integration sites within genes and intergenic regions. A previous study revealed that integration by MLV is favored near transcription start sites, but no such bias was seen for HIV ([@pbio-0020234-Wu1]). We investigated this issue for ASLV and reanalyzed all of the available data by comparison to the matched random control dataset ([Figure 2](#pbio-0020234-g002){ref-type="fig"}). For sites within genes, MLV showed a highly significant bias in favor of integration near transcription start sites (*p* = 1.4 × 10^−14^). No such bias was seen for ASLV (*p \>* 0.05). For the HIV datasets, two of the four sets showed modest bias (HIV/PBMC, *p =* 0.0007; HIV/H9 and HeLa combined, *p =* 0.027). For HIV, the observed statistical significance was sensitive to the details of analytical approach used and is of questionable biological importance. Thus, ASLV and HIV do not strongly favor integration at transcription start sites as was seen with MLV.
::: {#pbio-0020234-g002 .fig}
Figure 2
::: {.caption}
###### Integration Intensity in Genes and Intergenic Regions
Genes or intergenic regions were normalized to a common length and then divided into ten intervals to allow comparison. The number of integration sites in each interval was divided by the number of matched random control sites and the value plotted. A value of one indicates no difference between the experimental sites and the random controls. Viruses and cell types studied are as marked above each graph. The direction of transcription within each gene is from left to right. Note that our normalization method de-emphasizes favored MLV integration events just upstream of gene 5′ ends (outside transcription units), as reported by [@pbio-0020234-Wu1]. We carried out an analysis specially designed to identify this effect and confirmed that the regions just upstream of gene 5′ ends are favored for MLV integration when reanalyzed with the matched random control data (unpublished data).
:::
:::
Effects of Transcriptional Activity on Integration {#s2c}
--------------------------------------------------
Transcriptional profiling analysis was carried out in some of the cell types studied, allowing the influence of transcriptional activity on integration site selection to be assessed. Transcriptional profiling was carried out on infected cells so that the data reflected the known influence of infection on cellular gene activity ([@pbio-0020234-Corbeil1]; [@pbio-0020234-Schroder1]; [@pbio-0020234-Mitchell1]; [@pbio-0020234-van1]). Prior to isolating RNA for microarray analysis, the SupT1, PBMC, and IMR90 cells were each infected with the HIV-based vector used for HIV integration site isolation. The 293T-TVA cells were infected with the RCAS ASLV vector prior to RNA isolation. RNA samples were harvested 24--48 h after infection. For analysis of MLV and HIV integration sites in HeLa cells, published transcriptional profiling data from uninfected cells were used ([@pbio-0020234-Tian1]).
The median expression level (average difference value) of genes hosting integration events was consistently higher than the median of all genes assayed on the microarrays. The ratios (targeted genes/all genes assayed) for HIV ranged from 1.6 to 3.0, indicating that integration targeting in human primary cells (PBMC and IMR90) favored active genes, as shown previously for transformed cell lines ([@pbio-0020234-Schroder1]; [@pbio-0020234-Wu1]). For MLV and ASLV the ratios were each 1.3, lower than for HIV but still greater than the chip average. The analysis shown in [Figure 3](#pbio-0020234-g003){ref-type="fig"} reveals that the association of integration sites with gene activity was statistically significant for all the HIV datasets. For MLV and ASLV, there was a weak tendency for integration to favor active genes, but the trend did not achieve statistical significance.
::: {#pbio-0020234-g003 .fig}
Figure 3
::: {.caption}
###### Influence of Gene Activity on Integration Frequency
Expression levels were assayed using Affymetrix HU-95Av2 or HU-133A microarrays and scored by the average difference value as defined in the Affymetrix Microarray Suite 4.1 software package. All the genes assayed by the chip were divided into eight "bins" according to their relative level of expression (the leftmost bin in each panel is lowest expression levels and the rightmost the highest). Genes that hosted integration events were then distributed into the same bins, summed, and expressed as a percent of the total. The y-axis indicates the percent of all genes in the indicated bin. *P* values were determined using the Chi-square test for trends by comparison to a null hypothesis of no bias due to expression level. All average difference values were ranked prior to analysis, and the analysis was carried out on the ranked data. This was done to avoid possible complications due to differential normalization or other data processing differences arising during work up of the microarrays.
:::
:::
All three HIV datasets showed reduced integration in the most highly expressed category of genes analyzed, suggesting that although transcription favors integration, very high level transcription may actually be less favorable ([Figure 3](#pbio-0020234-g003){ref-type="fig"}). A fourth dataset monitoring infection by an HIV-based vector in a T-cell line also showed this trend (M. Lewinski, P. S., J. R. E., and F. D. B., unpublished data). Possibly this finding is related to that of a previous study of integration by ALV in a model gene that also suggested that high level transcription may disfavor integration ([@pbio-0020234-Weidhaas1]).
Tissue-Specific Transcription Results in Tissue-Specific Biases in HIV Integration Site Selection {#s2d}
-------------------------------------------------------------------------------------------------
We next investigated the effects of cell-type-specific transcription on integration site selection. For this analysis we used only the three HIV integration site datasets for which we had transcriptional profiling data from infected cells (i. e., SupT1, PBMC, and IMR90), to allow us to control for the effects of infection on transcription. Pairwise comparisons of the microarray datasets for the three cell types showed that the correlation coefficients ranged only from 0.64 to 0.79, indicating that transcriptional activity indeed differed among cell types. We reasoned that since active transcription favors integration, then the genes targeted by integration should on average be more highly expressed in the cell type that hosted the integration event than in either of the other two. Statistical analysis ([Figure 4](#pbio-0020234-g004){ref-type="fig"}) showed that transcription of targeted genes was higher on average in the cell type hosting the integration event than in either of the other two tested (all comparisons attain *p \<* 0.05 using the Chi-square test for linear trend in proportions). We note, however, that the differences were quantitatively modest, perhaps because much of the cellular program of gene activity is shared among many cell types ([@pbio-0020234-Caron1]; [@pbio-0020234-Mungall1]; [@pbio-0020234-Versteeg1]).
::: {#pbio-0020234-g004 .fig}
Figure 4
::: {.caption}
###### Effects of Tissue-Specific Transcription on Integration Site Selection in Different Cell Types
Genes hosting integration events by the HIV vector were analyzed for their expression levels in transcriptional profiling data from IMR90, PBMC, and SupT1 cells. For each gene hosting an integration event, the expression values from the three cell types were then ranked lowest (red), medium (orange), and highest (yellow). The values were summed and displayed separately for each set of integration sites: (A) IMR90 sites, (B) PBMC sites, and (C) SupT1 sites. In each case there was a significant trend for the cell type hosting the integration events to show the highest expression values relative to the other two (*p \<* 0.05 for all comparisons).
:::
:::
Integration Site Selection and Transcriptional Domains {#s2e}
------------------------------------------------------
We next analyzed factors influencing the placement of integration sites at the chromosomal level, taking into account both gene density and expression (see [Figure 1](#pbio-0020234-g001){ref-type="fig"}). Transcriptional activity was quantified by counting the EST sequence copies for each gene present in a collection of nonnormalized EST libraries ([@pbio-0020234-Mungall1]). EST sequences from many tissues were used to build up the map of transcriptional activity, thus focusing the analysis on transcriptional patterns common to many cell types ([@pbio-0020234-Caron1]; [@pbio-0020234-Mungall1]; [@pbio-0020234-Versteeg1]). A detailed comparison of the relationship between EST frequency and integration frequency of HIV, MLV, and ASLV is shown for Chromosome 11 in [Figure 5](#pbio-0020234-g005){ref-type="fig"}. Integration frequency for HIV closely parallels the transcriptional intensity deduced from EST counts. Fewer sites are available for analyzing MLV and ASLV, but MLV may show a related trend, while it is unclear whether ASLV does so or not. Similar analysis of the other human chromosomes yields similar conclusions (unpublished data).
::: {#pbio-0020234-g005 .fig}
Figure 5
::: {.caption}
###### Comparison of Transcriptional Intensity to Integration Intensity on Human Chromosome 11
All data were quantified in 2-Mb intervals. The top line shows summed EST data documenting the "transcriptional intensity" for each chromosomal interval (data from [@pbio-0020234-Mungall1]). The bottom three lines show the summed frequency of integration site sequences in each interval. The numbers of ESTs (top) or integration sites (bottom three) are shown on the y-axis.
:::
:::
Statistical analysis was carried out comparing integration frequencies to (1) gene density or (2) transcriptional intensity, as measured by the EST counts. All analyses incorporated a comparison to the matched random control set of integration sites. Each type of vector showed a significant positive correlation with gene density (HIV, *p =* 1.8 × 10^−12^ to 3.2 × 10^−38^, depending on the dataset; MLV, *p =* 2.4 × 10^−22^; ASLV, *p =* 3.2 × 10^−9^) and a stronger association with transcriptional intensity (HIV, *p =* 3.8 × 10^−19^ to 9.7 × 10^−46^; MLV, *p =* 5.1 × 10^−35^; ASLV, *p =* 1.7 × 10^−11^). Overall, ASLV showed the weakest association with gene density and transcriptional intensity.
Thus, the analysis of transcriptional activity in the context of chromosomal location revealed significant effects of transcription on MLV and ASLV integration. This is in contrast to the study based on transcriptional profiling alone, in which the effect was not statistically significant---however, a similar trend was evident and the general conclusions similar (see [Figure 3](#pbio-0020234-g003){ref-type="fig"}). It appears that adding information on chromosomal position to the gene expression data allowed quantitatively modest effects to reach statistical significance.
Substructures within Chromosomal Regions Favorable for Integration {#s2f}
------------------------------------------------------------------
Two lines of evidence indicated that the chromosomal regions favorable for integration can be subdivided into favorable and unfavorable segments. In the first study, a computational analysis was carried out to determine the length of the chromosomal segments yielding the best fit between transcriptional intensity and integration intensity. The sizes of the chromosomal regions analyzed were varied systematically from 25 kb to 32,000 kb, and the statistical significance determined for the correlations. This analysis revealed that the segment length yielding the best correlation was comparatively short, around 100--250 kb, the length of one or a few human genes. These conclusions held for HIV, ASLV, and MLV ([Protocol S1](#sd001){ref-type="supplementary-material"}, p. 16--65).
An analysis of integration frequency near CpG islands also indicated substructure within regions favorable for integration. CpG islands are chromosomal regions enriched in the rare CpG dinucleotide. These regions commonly correspond to gene regulatory regions containing clustered transcription factor binding sites---consequently, CpG islands are more frequent in gene-rich regions. Previously [@pbio-0020234-Wu1] reported that CpG islands were positively associated with MLV integration sites but that for HIV integration sites there was no influence. An analysis of the effects of proximity to CpG islands on integration frequency incorporating the matched random control dataset is shown in [Figure 6](#pbio-0020234-g006){ref-type="fig"}. The relative integration frequency near CpG islands was found to be much higher than expected by chance for MLV, as reported previously, and slightly higher than expected by chance for ASLV. For HIV, the region surrounding CpG islands was actually disfavored, and this was statistically significant in three out of four datasets. Thus for HIV, broadly favorable gene-dense chromosomal regions actually contain a mixture of favorable clusters of active genes and unfavorable CpG islands. For MLV, in contrast, CpG islands are quite favorable.
::: {#pbio-0020234-g006 .fig}
Figure 6
::: {.caption}
###### The Effects of Proximity to CpG Islands Differs for HIV, MLV, and ASLV Integration
The viral vectors and cell types studied are indicated by color. A value of one indicates no bias, less than one indicates disfavored integration, and more than one indicates favored integration. The x-axis (from plus or minus 1 kb to 50 kb) indicates distance from the edge of a CpG island in either direction along the genome. The statistical analysis specifically removed the favorable effects of being in a gene and being in a region containing expressed genes to highlight the effects of CpG islands alone. When effects of gene density and activity are left in, HIV integration goes from disfavored at short distances (less than 1 kb) to favored at longer distances (more than 10 kb). This is because at longer distances the association with genes is significant---many CpG islands are within 10 kb of a gene, and genes are favored targets for HIV integration. To carry out this analysis, the numbers of experimentally determined and matched control sites were fitted according to whether they were near a CpG island, whether they were in genes, and the level of the expression density variable. Each variable contributes a "multiplier" for the ratio of the number of experimental to control sites. The multiplier for "near CpG island" is shown ([see Protocol S2](#sd002){ref-type="supplementary-material"}, p. 9--12).
:::
:::
High gene density in the human chromosomes is known to correlate with several other features, including high levels of gene expression, high densities of CpG islands, the occurrence of Giemsa-light chromosomal bands, and high G/C content ([@pbio-0020234-Caron1]; [@pbio-0020234-Lander1]; [@pbio-0020234-Venter1]; [@pbio-0020234-Mungall1]; [@pbio-0020234-Versteeg1]). The effects of chromosomal banding pattern and G/C content were analyzed statistically and found to favor integration, as expected from the correlation with other favorable features ([Protocol S2](#sd002){ref-type="supplementary-material"}).
A Quantitative Model for Integration Intensity {#s2g}
----------------------------------------------
A statistical model was constructed to examine the relative contributions to integration intensity of (1) gene density, (2) gene activity, (3) proximity to CpG islands, (4) G/C content, and (5) location within genes ([Protocol S2](#sd002){ref-type="supplementary-material"}, p. 13--15). Inclusion of each of these parameters improved the fit of the model to the observed experimental datasets, but the quantitative contribution of each parameter varied among the different retrovirus types. The effects of being in a gene or a region with many expressed genes were most important for HIV and ASLV. For MLV, the distance from the transcription start site was the most important parameter. ASLV differed significantly from each of the other datasets (*p \<* 0.0001), and the model based on the above parameters predicted the placement of ASLV integration sites least well. Thus ASLV is least responsive to the effects of the parameters so far known to affect integration site selection in human cells.
Integration Frequency in the Individual Human Chromosomes {#s2h}
---------------------------------------------------------
[Figure 7](#pbio-0020234-g007){ref-type="fig"} presents the frequency of integration in each of the different human chromosomes for HIV, MLV, and ASLV. For HIV, each of the datasets is shown separately. A statistical analysis was carried out comparing the observed frequency of integration in each chromosome to that expected from the matched random control ([Protocol S1](#sd001){ref-type="supplementary-material"}, p. 3). As can be seen from the figure, the frequencies were quite different among the different chromosomes. For example, the gene-rich Chromosome 19 showed more integration than expected by chance, while the gene-poor Chromosome 18 showed less integration. The differences in integration frequencies among chromosomes are in part a function of gene density. However, for unknown reasons, the datasets also differed significantly from each other (*p* \< 2.22 × 10^−16^). Evidently there are factors---so far unknown---affecting integration targeting that operate at the level of whole chromosomes(a conclusion also reached by [@pbio-0020234-Laufs1]).
::: {#pbio-0020234-g007 .fig}
Figure 7
::: {.caption}
###### Frequency of Integration in Human Chromosomes
Human chromosome numbers are indicated at the bottom of the figure. The number of integration events detected in each chromosome was divided by the number expected from the matched random control. The line at one indicates the bar height expected if the observed number of integration events matched the expected number. Higher bars indicate favored integration, lower bars, disfavored integration. Most of the cell types studied were from human females; too little data were available for the Y chromosome for meaningful analysis.
:::
:::
Discussion {#s3}
==========
We report that ASLV, MLV, and HIV have quite different preferences for integration sites in the human chromosomes. HIV strongly favors active genes in primary cells as well as in transformed cell lines. MLV favors integration near transcription start regions and favors active genes only weakly. ASLV shows the weakest bias toward integration in active genes and no favoring of integration near transcription start sites. We expect that these same patterns will be seen for MLV and ASLV integration in different human cell types, because all four HIV datasets yielded similar results, though more data on additional cell and tissue types will be helpful to further evaluate the generality.
One of the earliest models for chromosomal influences on integration targeting proposed that condensed chromatin in inactive regions disfavored integration, thereby concentrating integration in more open active chromatin ([@pbio-0020234-Panet1]; [@pbio-0020234-Vijaya1]; [@pbio-0020234-Rohdewohld1]). Integration by HIV, ASLV, and MLV all showed at least a weak bias in favor of integration in active genes, consistent with the idea that open chromatin at active genes favors integration. Also consistent with this idea, heterochromatic regions at human centromeres and telomeres were found to disfavor integration.
However, it seems unlikely that relative accessibility is the only feature directing integration site selection, because HIV, ASLV, and MLV each show such distinctive target sequence preferences. Studies of the Ty retrotransposons of yeast, close relatives of retroviruses, have revealed that interactions with bound chromosomal proteins can tether the Ty integration machinery to chromosomes and thereby direct integration to nearby sites ([@pbio-0020234-Boeke1]; [@pbio-0020234-Bushman3]; [@pbio-0020234-Sandmeyer1]; [@pbio-0020234-Zhu1]). Such a model may explain integration targeting by retroviruses as well ([@pbio-0020234-Bushman3]). HIV integration complexes might bind to factors enriched at active genes, while MLV complexes could bind to factors bound near transcription start sites. In support of this idea, in vitro studies have established that retroviral integrase enzymes fused to sequence-specific DNA-binding domains can direct integration preferentially to local regions when tethered at specific DNA sites ([@pbio-0020234-Bushman1]; [@pbio-0020234-Goulaouic1]; [@pbio-0020234-Katz1]).
The analysis of chromosomal regions favored for integration also suggested a role for locally bound proteins. Chromosomal regions enriched in active genes were generally favorable, but further analysis revealed interleaved favorable and unfavorable regions. Statistical tests indicated that favorable regions were typically short (100--250 kb), and for HIV these were interspersed with unfavorable regions near CpG islands. CpG islands are thought to be regulatory regions that bind distinctive sets of transcription factors. Thus, a simple model to explain targeting is that a distinctive set of sequence-specific DNA-binding proteins bound at or near CpG islands disfavor HIV integration, while proteins bound in active transcription units are favorable. For MLV, the proteins bound at CpG islands instead favor integration.
For ASLV, it is possible that the viral integration machinery does not interact with factors bound in or near genes, explaining the more random distribution of integration sites in the genome. Such a pattern might have evolved to minimize disruption to the host cell chromosomes due to integration. Another possibility, however, is that ASLV does have stricter target site preferences during normal integration in chicken cells, but the targeting system does not function properly in the human cells studied here. According to this idea, putative chicken chromosomal proteins normally bind ASLV integration complexes and direct integration, but the homologous human proteins may be too different to interact properly. It should be possible to investigate this possibility by characterizing ASLV integration in chicken cells, now that the draft chicken genome sequence is completed ([@pbio-0020234-Ren1]).
One consequence of the above findings is that integration will differ from tissue to tissue as a consequence of cell-type-specific transcription. To assess effects of tissue-specific transcription, we analyzed HIV integration in three different cell types (SupT1, PBMC, and IMR90). Transcriptional profiling data showed that transcription was significantly different among the three. This allowed an analysis of integration targeting, which showed that highly expressed genes particular to each tissue were favored for integration in that tissue. However, the magnitude of the tissue-specific biases on integration were modest, probably because most of the cellular transcriptional program appears to be common among cell types ([@pbio-0020234-Caron1]; [@pbio-0020234-Mungall1]; [@pbio-0020234-Versteeg1]).
Additional mechanisms could also contribute to targeting. For example, we and others have detected statistically significant biases in integration frequency in whole chromosomes that do not appear to be fully explained by gene density or gene activity ([@pbio-0020234-Schroder1]; [@pbio-0020234-Laufs1]; data reported here). Perhaps the intranuclear position of chromosomes may have an influence, since this has been proposed to be relatively fixed for cells of specific types but may differ among cell types ([@pbio-0020234-Boyle1]; [@pbio-0020234-Chubb1]).
Our data indicate that ASLV has integration site preferences that may make it attractive as a vector for human gene therapy. MLV-based vectors have the unfavorable preference for integration near transcription start sites ([@pbio-0020234-Wu1]). The adverse events arising during X-linked severe combined immunodeficiency gene therapy involved integration of an MLV vector near the transcription start region of the LMO2 proto-oncogene. HIV-based vectors strongly favor integration in active genes, which is also likely to be disruptive to the host cell genome ([@pbio-0020234-Schroder1]; [@pbio-0020234-Wu1]). ASLV, in contrast, shows only weak favoring of integration in active genes, and no favoring of integration near transcription start sites. A quantitative model based on gene density, expression, and proximity to transcription start regions fit the ASLV data least well, indicating that ASLV has the weakest bias toward integration in these unfavorable locations. ASLV vectors are known to infect a variety of human cell types (e. g., [@pbio-0020234-Valsesia-Wittmann1]; [@pbio-0020234-Federspiel1]; [@pbio-0020234-Hatziioannou1]; [@pbio-0020234-Katz2]) and can transduce nondividing cells ([@pbio-0020234-Hatziioannou1]; [@pbio-0020234-Katz2]), adding to their possible utility. More generally, this study, together with previous work ([@pbio-0020234-Schroder1]; [@pbio-0020234-Wu1]), indicates that the selection of different retroviral integration systems can modulate the selection of integration target sites, and this may potentially be exploited for safer gene therapy.
Materials and Methods {#s4}
=====================
{#s4a}
### Oligonucleotides used in this study {#s4a1}
Each oligonucleotide is described by its name, sequence (written 5′ to 3′), and use, in that order. *Hinc*II adaptor, GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGC, adapter for use with DNA cleaved by 6-cutter restriction enzymes, top strand; mNheIAvrIISpeII adaptor, P-CTAGGCAGCCCG-NH~2~, adapter for 6-cutter restriction enzymes, bottom strand; ASB-9, GACTCACTATAGGGCACGCGT, adapter primer for PCR for I/SupT1, PBMC, and IMR-90; SB-76, GAGGGATCTCTAGTTACCAGAGTCACA, HIV primer for PCR for I/SupT1; ASB-19, GAGATTTTCCACACTGACTAAAAGGGTC, HIV primer for PCR for I/PBMC and IMR-90; ASB-16, GTCGACGGCCCGGGCTGCCTA, adapter primer for nested PCR for I/SupT1, PBMC, and IMR-90; ASB-1, AGCCAGAGAGCTCCCAGGCTCAGATC, HIV primer for nested PCR for I/SupT1; ASB-20, CTGAGGGATCTCTAGTTACCAGAGTCA, HIV primer for nested PCR forPBMC and IMR-90; MseI linker +, GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC, MseI linker, top strand; MseI linker −, P-TAGTCCCTTAAGCGGAG-NH~2,~ MseI linker, bottom strand; MseI linker primer, GTAATACGACTCACTATAGGGC, MseI linker primer for first round of PCR; MseI linker nested primer, AGGGCTCCGCTTAAGGGAC, MseI linker nested primer for second round of PCR; BB389, GATGGCCGGACCGTTGATTC, inner ASLV LTR primer for second round of "nested" PCR; BB390, CGATACAATAAACGCCATTTGACCATTC, outer ASLV LTR primer for first round of PCR.
### Preparation of the ASLV- and HIV-based vectors {#s4a2}
To produce HIV vector particles, 293T cells were cotransfected with three plasmids: one encoding the HIV vector segment (p156RRLsinPPTCMVGFPWPRE) ([@pbio-0020234-Follenzi1]), the second, the packaging construct (pCMVdeltaR9) ([@pbio-0020234-Naldini1]), and the third, the gene for *VSV-G* (pMD.G) ([@pbio-0020234-Naldini1]). Forty-eight hours after transfection, supernatants were collected, centrifuged to pellet cellular debris, then filtered through 0.45-μm filters. Viral particles were further purified by centrifugation at 23,000g and resuspended in 1/17 volume of fresh medium. ASLV particles were generated by transfecting the DF-1 chicken embryonic fibroblast cell line (ATCC CRL-12203) with the plasmid RCASBP(A)GFP (from Steve Hughes, National Cancer Institute, Frederick, Maryland). Supernatant was removed from the cells 4 d post transfection (when cells were nearly 100% GFP positive) and filtered though a 0.45-μm syringe filter.
### Infections. {#s4a3}
PBMCs were separated from human blood using a ficoll gradient (Amersham Biosciences, Little Chalfont, United Kingdom). 1 × 10^7^ PHA, IL-2 prestimulated PBMCs, or IMR-90 cells (passage \#36) at 30%--50% confluency (1--2 × 10^6^ cells) were infected with the HIV-based vector at an moi of 10 (60 ng p24 per 5 × 10 ^5^ cells). The vector was added to the cells with DEAE-dextran at a final concentration of 5 μg/ml. Forty-eight hours after infection, the cells were pelleted. For RNA isolation, cells were resuspended in 250 μl of PBS and 750 μl of TRIzol and frozen in liquid nitrogen. To determine the extent of infection, cells were analyzed by flow cytometry. For ASLV, supernatant containing RCASBP(A)GFP particles was added to 293T-TVA cells (293T 0.8 cells; a gift from John Young, Salk Institute) at 30%--50% confluency. Forty-eight hours post infection, green fluorescence was seen in approximately 30% of the cells, as determined by examination of the cultures with a fluorescence microscope. DNA was harvested at this point (DNeasy, Qiagen, Valencia, California, United States). RNA from infected cells was also isolated at 48 h post infection (TRIzol) and stored at −80 °C until used for transcriptional profiling analysis. RNA was isolated from infected cell cultures, and samples from each were used for hybridization on one Affymetrix (Santa Clara, California, United States) microarray.
### Integration site determination. {#s4a4}
HIV integration sites were cloned by ligation-mediated PCR essentially as described in [@pbio-0020234-Schroder1]. ASLV integration site determination using ligation-mediated PCR was carried out essentially as described in [@pbio-0020234-Wu1]. Oligonucleotides used are summarized above. All novel integration site sequences are deposited at the National Center for Biotechnology Information (NCBI) (accession numbers CL528318--CL529767). Integration sites from earlier studies were reanalyzed on the November 2002 freeze of the human genome sequence (using the University of California at Santa Cruz browser), and a few were excluded because they did not find matches of sufficiently high quality on the new draft sequence, accounting for slightly different numbers than in previous reports. This study used primarily the Acembly and Ensemble human gene catalogs; similar results were generally obtained when the Unigene, RefGene, or GeneScan catalogs of the human genes were used ([Protocol S1](#sd001){ref-type="supplementary-material"}, p. 3--10).
### Transcriptional profiling analysis {#s4a5}
Transcriptional profiling was carried out using Affymetrix microarrays as described in [@pbio-0020234-Schroder1]. Gene expression levels (average difference values) were analyzed using Affymetrix Microarray Suite 4.1 software. All novel transcriptional profiling datasets reported here are deposited at NCBI (GEO dataset numbers GSE1407, GSE1408, GSE1409, and GSE1410). For the analysis in [Figure 3](#pbio-0020234-g003){ref-type="fig"}, a complication was introduced by the fact that the HU95A chips used have multiple probe sets for some genes but not others. In our analysis all probe sets were accepted and analyzed in these cases.
### Statistical analysis {#s4a6}
A detailed description of the statistical methods used is presented in Protocols [S1](#sd001){ref-type="supplementary-material"} and [S2](#sd002){ref-type="supplementary-material"}.
Supporting Information {#s5}
======================
Protocol S1
::: {.caption}
###### Association of Genomic Features with Integration---Part 1
(322 KB PDF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Protocol S2
::: {.caption}
###### Association of Genomic Features with Integration---Part 2
(128 KB PDF).
:::
::: {.caption}
######
Click here for additional data file.
:::
Accession Numbers {#s5a3}
-----------------
The GenBank (<http://www.ncbi.nlm.nih.gov/>) accession numbers for the novel integration site sequences discussed in this paper are CL528318--CL529767. The GenBank GEO dataset numbers for the novel transcriptional profiling datasets reported here are GSE1407, GSE1408, GSE1409, and GSE1410.
We thank members of the Bushman laboratory for helpful discussions, and Dr. John Young for the 293T-TVA cells. This work was supported by National Institutes of Health grants AI52845 and AI34786, the James B. Pendleton Charitable Trust, the Berger Foundation, Robin and Frederic Withington (grant to FDB), and the Fritz B. Burns Foundation (grant to JRE). ARWS was supported by a grant from the Deutsche Forschungsgemeinschaft.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** RSM, BFB, ARWS, CCB, JRE, and FDB conceived and designed the experiments. RSM, BFB, ARWS, PS, HC, CCB, JRE, and FDB performed the experiments. RSM, BFB, ARWS, PS, HC, CCB, HCB, JRE, and FDB analyzed the data. RSM, BFB, CCB, JRE, and FDB wrote the paper.
Academic Editor: Michael Emerman, Fred Hutchinson Cancer Research Center
Citation: Mitchell RS, Beitzel BF, Schroder ARW, Shinn P, Chen H, et al. (2004) Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol 2(8): e234.
ASLV
: avian sarcoma-leukosis virus
HIV
: human immunodeficiency virus
MLV
: murine leukemia virus
PBMC
: peripheral blood mononuclear cell
| PubMed Central | 2024-06-05T03:55:46.755343 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509299/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e234",
"authors": [
{
"first": "Rick S",
"last": "Mitchell"
},
{
"first": "Brett F",
"last": "Beitzel"
},
{
"first": "Astrid R. W",
"last": "Schroder"
},
{
"first": "Paul",
"last": "Shinn"
},
{
"first": "Huaming",
"last": "Chen"
},
{
"first": "Charles C",
"last": "Berry"
},
{
"first": "Joseph R",
"last": "Ecker"
},
{
"first": "Frederic D",
"last": "Bushman"
}
]
} |
PMC509300 | Introduction {#s1}
============
In eukaryotes, the endoplasmic reticulum (ER) serves as the first station of the secretory pathway, through which all secreted and membrane proteins must pass. Within the ER, proteins are folded into their native structure and multisubunit protein complexes are assembled. The ER is a dynamic organelle, capable of sensing and adjusting its folding capacity in response to increased demand: when misfolded proteins accumulate in the ER, a signaling pathway, termed the unfolded protein response (UPR), is activated (reviewed in [@pbio-0020235-Ma1]; [@pbio-0020235-Patil1]; [@pbio-0020235-Kaufman1]; [@pbio-0020235-Ron1]). The UPR activates the expression of genes that enable the cell to adapt to and survive the stress, including those encoding ER-resident chaperones ([@pbio-0020235-Lee1]; [@pbio-0020235-Kozutsumi1]), key enzymes in lipid biosynthesis ([@pbio-0020235-Cox3]), members of the ER-associated degradation (ERAD) machinery, and other components of the secretory system ([@pbio-0020235-Ng1]; [@pbio-0020235-Travers1]; [@pbio-0020235-Urano1]).
In yeast, the UPR is controlled by a binary switch imposed by a nonconventional splicing reaction that governs the production of the Hac1p transcription factor responsible for the activation of UPR target genes ([@pbio-0020235-Cox2]; [@pbio-0020235-Kohno1]; [@pbio-0020235-Cox1]; [@pbio-0020235-Mori1], [@pbio-0020235-Mori3]). In uninduced cells, direct base pairing between the 5′ untranslated region (UTR) and an intron at the 3′ end of the mRNA prevents *HAC1* mRNA translation ([@pbio-0020235-Chapman1]; [@pbio-0020235-Ruegsegger1]). Accumulation of unfolded proteins activates the ER-resident transmembrane kinase/endoribonuclease Ire1p, which then cleaves the *HAC1* mRNA at two precise splice junctions, excising the intron ([@pbio-0020235-Cox2]; [@pbio-0020235-Mori2]; [@pbio-0020235-Sidrauski1]). The two *HAC1* exons are then joined by tRNA ligase, allowing translation of Hac1p ([@pbio-0020235-Sidrauski2]).
To date, Ire1-dependent *HAC1* mRNA splicing is the only identified way by which signals from the ER lumen affect transcription in yeast. By contrast, in metazoan cells three mechanistically distinct pathways are known that operate in parallel, although their relative importance in different tissues remains to be determined (reviewed in [@pbio-0020235-Ma1]). Hints that further complexity also exists in yeast comes from data presented in the accompanying paper ([@pbio-0020235-Patil2]): these data demonstrate that Hac1p activity is modulated by interaction with Gcn4p, a transcription factor central to regulation of amino acid biosynthesis. The UPR, therefore, may integrate signals from more than one source to compute a transcriptional output appropriate for the physiological conditions of the cell.
In this paper, we show that *HAC1* mRNA transcription is regulated, resulting in control of Hac1p abundance. Thus the on/off switch provided by *IRE1*-dependent splicing is not the only regulatory step of the UPR. This regulation responds to a bipartite signal that emanates from the ER and is communicated by an Ire1p-independent pathway. As a consequence, an alternate transcriptional program is triggered, with specific alterations to the normal UPR allowing the cell to survive. Thus, quantitative modulation of Hac1p imposes gain control on a binary switch in the UPR circuitry and, in collaboration with an additional signaling input, transforms a discrete transcriptional response into a more complex signaling function.
Results {#s2}
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Secretory Stress Boosts *HAC1* mRNA Abundance {#s2a}
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To define the basic circuitry of signal transduction in the UPR, we evaluated the *HAC1* mRNA processing step in a quantitative manner. To this end, we induced the UPR with either dithiothreitol (DTT) or tunicamycin (both agents that cause protein misfolding selectively in the ER) and monitored *HAC1* mRNA by Northern blot analysis ([Figure 1](#pbio-0020235-g001){ref-type="fig"}A). In agreement with previous results, we observed rapid and efficient splicing of *HAC1* mRNA, as apparent from the conversion of unspliced *HAC1^u^* mRNA (*u* for UPR-*u*ninduced) to spliced *HAC1^i^* mRNA (*i* for UPR-*i*nduced). Quantitation of the results shows that the relative abundance of *HAC1* mRNA (the sum of *HAC1^u^* and *HAC1^i^* mRNAs) remained unchanged over at least 12 h ([Figure 1](#pbio-0020235-g001){ref-type="fig"}A; unpublished data). These data demonstrate that acute induction of unfolded proteins triggers a simple on/off switch that controls *HAC1* mRNA splicing.
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Figure 1
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###### ER-Distal Secretory Stress Boosts *HAC1* mRNA Abundance
\(A) Determination of *HAC1* mRNA abundance during the UPR. The UPR was induced in WT cells by addition of either 6 mM DTT (lanes 1--4) or 1 μg/ml tunicamycin (lanes 5--8) for the times indicated. Total RNA was harvested at the indicated intervals, and the relative abundance of *HAC1* and *ACT1* mRNAs was analyzed by Northern blot analysis (see [Materials and Methods](#s4){ref-type="sec"}). Splicing was calculated at the ratio of spliced *(HAC1^i^)* to total *(HAC1^i^ + HAC1^u^)* mRNA.
\(B) Determination of *HAC1* mRNA abundance during ER-distal secretory stress. WT, *sec12--1, sec14--3,* and *sec1--1* strains were grown at 23 °C and shifted to 37 °C.
\(C) Determination of *HAC1* mRNA abundance during ER-proximal secretory stress. WT, *sec14--3, sec61--101, sec62--101,* and *sec63--201* strains were grown at 23 °C and shifted to 37 °C.
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In light of these observations, we were surprised to find that blocking the secretory pathway distal to the ER resulted in a pronounced increase in *HAC1* mRNA abundance. As shown in [Figure 1](#pbio-0020235-g001){ref-type="fig"}B, *HAC1* mRNA levels increased 3- to 4-fold in mutant strains compromised at various steps in the secretory pathway when shifted to the nonpermissive temperature (*sec12--1:* ER → Golgi, lanes 5--8; *sec14--1:* intra-Golgi, lanes 9--12; and *sec1--1:* Golgi → plasma membrane, lanes 13--16) ([@pbio-0020235-Novick1]). Splicing was also induced, albeit to a lesser degree than was observed with DTT or tunicamycin treatment. The observed splicing suggests that blockages in ER-distal compartments of the secretory pathway lead to activation of Ire1p in the ER. Temperature shift alone only transiently induced *HAC1* mRNA splicing and had no effect on *HAC1* mRNA abundance ([Figure 1](#pbio-0020235-g001){ref-type="fig"}B, lanes 1--4). To determine if any disruption of the secretory pathway had similar consequences, we blocked earlier stages of protein traffic. Mutations that blocked protein entry into the ER had no effect ([Figure 1](#pbio-0020235-g001){ref-type="fig"}C: *sec62--101,* lanes 13--16; *sec63--201,* lanes 17--20) or only a mild effect (*sec61--101,* lanes 9--12) on *HAC1* mRNA abundance.
Thus, a surveillance pathway operates to adjust *HAC1* mRNA levels in response to altered conditions in the secretory pathway. In the experiments described above, we observed *HAC1* mRNA induction only in *sec* mutants that block transport distal to the ER, not in those that block protein entry into the ER. One common consequence of blocking the secretory pathway at later stages is that proteins in transit will eventually back up into the ER ([@pbio-0020235-Rose1]; [@pbio-0020235-Chang1]). This condition results in protein folding defects, thereby activating Ire1p, as indicated by the observed *HAC1* mRNA splicing. From the data discussed above ([Figure 1](#pbio-0020235-g001){ref-type="fig"}A), however, we know that an accumulation of unfolded proteins alone is insufficient to trigger an upregulation of *HAC1* mRNA, suggesting that an additional inducing signal is required.
*HAC1* mRNA Induction Requires a Bipartite Signal {#s2b}
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To determine the nature of this second signal, we sought conditions that induce *HAC1* mRNA when combined with ER protein misfolding drugs. Canvassing different conditions, we found two scenarios under which wild-type (WT) cells can be induced to upregulate *HAC1* mRNA: (1) ER protein misfolding combined with a temperature shift from 23 °C to 37 °C ([Figure 2](#pbio-0020235-g002){ref-type="fig"}A) and (2) ER protein misfolding combined with inositol starvation ([Figure 2](#pbio-0020235-g002){ref-type="fig"}B). Intriguingly, while ER protein misfolding and inositol starvation each activated the UPR individually (as shown by the activation of *HAC1* mRNA splicing; [Figure 2](#pbio-0020235-g002){ref-type="fig"}A, lanes 5--8; [Figure 2](#pbio-0020235-g002){ref-type="fig"}B, lanes 1--4 and 5--8), neither stress alone was sufficient to cause *HAC1* mRNA upregulation. Similarly, the temperature shift reproducibly caused a transient UPR induction (see [Figure 1](#pbio-0020235-g001){ref-type="fig"}B, lanes 1--4; [Figure 2](#pbio-0020235-g002){ref-type="fig"}A, lanes 1--4) but by itself did not affect *HAC1* mRNA levels. Only the combination of ER stress with either temperature shift ([Figure 2](#pbio-0020235-g002){ref-type="fig"}A, lanes 9--12) or inositol starvation ([Figure 2](#pbio-0020235-g002){ref-type="fig"}B, lanes 9--12) led to an increase in *HAC1* mRNA abundance. Subjecting cells to both temperature shift and inositol deprivation had no additive effect, nor did treating cells with both DTT and tunicamycin (unpublished data). Thus, *HAC1* mRNA induction requires a bipartite signal, consisting of one input provided by unfolded proteins in the ER (UP signal), and the other input provided by inositol starvation or temperature shift (I/T signal).
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Figure 2
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###### *HAC1* mRNA Induction Requires a Bipartite Signal and Is *IRE1*-Independent
\(A) Determination of *HAC1* mRNA abundance during ER stress and temperature shift. WT cells were grown at 23 °C and shifted to 37 °C (lanes 1--4 and 9--12) or kept constant at 30 °C (lanes 5--8). DTT was added as indicated (lanes 5--8 and 9--12).
\(B) Determination of *HAC1* mRNA abundance during ER stress and inositol deprivation. WT cells were grown at 30 °C in synthetic medium supplemented with inositol and shifted to synthetic medium lacking inositol (lanes 1--4 and 9--12), or continuously grown in medium supplemented with inositol (lanes 5--8). Tunicamycin was added to a final concentration of 1 μg/ml as indicated (lanes 5--8 and 9--12).
\(C) Distinction between heat shock response and *HAC1-*mRNA-inducing conditions. WT (lanes 1--4 and 9--12) and *HSF1^c^* (lanes 5--8) strains were grown at 23 °C and shifted to 37 °C (lanes 1--4 and 5--8) or continuously grown at 37 °C (lanes 9--12), and DTT added as indicated.
\(D) Analysis of *IRE1* pathway for a role in *HAC1* mRNA induction. Δ*ire1* cells were grown at 23 °C and shifted to 37 °C (lanes 1--4 and 9--12) or continuously grown at 30 °C (lanes 5--8), and DTT was added as indicated (lanes 5--8 and 9--12). Note that in Δ*ire1* cells, *HAC1* mRNA is modestly induced in response to DTT alone (lanes 5--8). This observation is indicative of feedback regulation, whereby a block in the UPR induces the I/T signal.
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The heat shock response is transiently induced by shifting cells from 23 °C to 37 °C. To determine whether the heat shock response is an important component of the I/T signal, we tested whether continued growth at 37 °C or expression of a constitutively active allele of the heat shock factor Hsf1p ([@pbio-0020235-Sorger1]; [@pbio-0020235-Bulman1]) would substitute for the temperature shift described above. Constitutive expression of active Hsf1p ([Figure 2](#pbio-0020235-g002){ref-type="fig"}C, lanes 5--8) led to upregulation of *SSA1,* a known target of the heat shock response ([@pbio-0020235-Slater1]), but did not substitute for the I/T signal for *HAC1* upregulation. In contrast, continued growth at 37 °C ([Figure 2](#pbio-0020235-g002){ref-type="fig"}C, lanes 9--12) allowed for modest induction of *HAC1* mRNA. Thus, elevated temperature elicits effects other than heat shock, which are important for *HAC1* mRNA upregulation.
*HAC1* Induction Is *IRE1*-Independent {#s2c}
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The UP signal was experimentally induced by DTT or tunicamycin treatment of the cells. As Ire1p is a sensor of folding conditions within the ER lumen, we tested next whether Ire1p was required to transmit this signal. Surprisingly, it was not. *HAC1* mRNA abundance was induced 2.6-fold in Δ*ire1* cells ([Figure 2](#pbio-0020235-g002){ref-type="fig"}D, lanes 9--12), similar to the 3-fold induction observed in WT cells ([Figure 2](#pbio-0020235-g002){ref-type="fig"}A, lanes 9--12). These results show that a previously unrecognized Ire1p-independent surveillance mechanism must exist that monitors protein folding in the ER.
*HAC1* mRNA Abundance Is Regulated Transcriptionally {#s2d}
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Increase of *HAC1* mRNA abundance could result from increased transcription, reduced degradation, or both. To distinguish between these possibilities, we constructed a reporter gene consisting of the *HAC1* promoter driving transcription of the open reading frame encoding the green fluorescent protein (GFP) flanked by *ACT1* untranslated regions *(HAC1pro-GFP).* The resulting heterologous *GFP* mRNA therefore contained no *HAC1* mRNA sequences. Under conditions providing both the UP and I/T signals, the change in abundance of the *GFP* mRNA ([Figure 3](#pbio-0020235-g003){ref-type="fig"}A, lanes 5--8) mirrored that of the endogenous *HAC1* mRNA ([Figure 3](#pbio-0020235-g003){ref-type="fig"}A, lanes 1--4), both in the kinetics and magnitude of the response. These data demonstrate that the observed increase in *HAC1* mRNA abundance was caused by increased transcriptional activity of the *HAC1* promoter.
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Figure 3
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###### Activation of the *HAC1* Promoter Controls Increase in *HAC1* mRNA Abundance
\(A) Analysis of *HAC1* promoter activity during bipartite stress conditions. Δ*hac1* cells containing either a construct restoring *HAC1* expression (lanes 1--4) or a construct expressing *GFP* driven by the *HAC1* promoter (lanes 5--8) were grown at 23 °C and shifted to 37 °C concurrent with addition of DTT as indicated.
\(B) Determination of mRNA half-life during *HAC1*-mRNA-inducing conditions. *polII^ts^* cells were grown at 23 °C and were shifted to 37 °C either in the absence (open symbols) or presence (filled symbols) of DTT. *HAC1* mRNA abundance (squares) and *ACT1* mRNA abundance (circles) are normalized to the abundance of the PolIII transcript *SCR1*.
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To further test this notion, we compared the rate of decay of *HAC1* mRNA under both *HAC1*mRNA-inducing and noninducing conditions. To this end, we employed a strain bearing a temperature-sensitive allele of RNA polymerase II, which was subjected to either elevated temperature alone, or to both elevated temperature and DTT treatment. In both cases, polymerase II transcription ceased upon temperature shift, and mRNA decay was measured. As shown in [Figure 3](#pbio-0020235-g003){ref-type="fig"}B, the rate of decay of *HAC1* mRNA was indistinguishable under the two conditions. Therefore, the increase in *HAC1* mRNA abundance in response to the combination of UP and I/T signals is due solely to activation of the *HAC1* promoter.
*HAC1* Promoter Regulation Is Required to Survive Certain Stress Conditions {#s2e}
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The results presented so far define a novel regulatory mechanism whereby cells adjust the amount of *HAC1* mRNA. This mRNA is the substrate for the Ire1p-mediated splicing reaction, which in turn produces *HAC1^i^* mRNA that is translated to produce Hac1p transcription factor. We therefore asked whether elevated levels of *HAC1* mRNA led to a proportional increase in the level of Hac1p. Quantitative Western blot analysis showed that this is indeed the case: when cells were treated with DTT and concomitantly shifted to 37 °C, the levels of Hac1p increased 3-fold ([Figure 4](#pbio-0020235-g004){ref-type="fig"}A, lanes 5--8), relative to the Hac1p levels observed in cells subjected to DTT treatment alone ([Figure 4](#pbio-0020235-g004){ref-type="fig"}A, lanes 1--4). Therefore, the transcriptional induction of *HAC1* mRNA combined with Ire1p-mediated splicing results in elevated Hac1p levels, characterizing a new physiological state. Henceforth, we refer to this state as the "Super-UPR" (S-UPR).
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Figure 4
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###### *HAC1* Promoter Regulation Is Required to Survive Stress
\(A) Determination of Hac1p levels during either ER stress alone or during both ER stress and temperature shift. WT cells were either grown at 30 °C and treated with DTT (lanes 1--4) or grown at 23 °C and simultaneously shifted to 37 °C and treated with DTT (lanes 5--8). Protein lysates were prepared, and protein levels were analyzed by Western blot analysis. The relative Hac1p/Pgk1p ratio is normalized to the DTT-treated sample (lane 4).
\(B) Characterization of *HAC1* expression in strain used to approximate basal *HAC1* expression. Cells expressing *HAC1* from the endogenous promoter (lanes 1--4) or the *ADH1* promoter (lanes 5--8) were grown at 30 °C in synthetic medium supplemented with inositol and shifted to synthetic medium lacking inositol simultaneous with the addition of tunicamycin.
\(C) Reduced viability of strains unable to express *HAC1* at elevated levels. The strains described in (B) were plated in serial dilutions (left to right) on synthetic medium lacking inositol ("−ino") and synthetic medium lacking inositol and containing tunicamycin ("−ino +TM").
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To assess the physiological role of the S-UPR, we sought conditions that would allow us to directly monitor the consequences of changes in *HAC1* mRNA levels under otherwise identical growth conditions. To this end, we engineered a yeast strain unable to transcriptionally upregulate *HAC1*. In these cells, *HAC1* mRNA expression was removed from the control of the *HAC1* promoter and was instead driven by the heterologous *ADH1* promoter (*ADH1pro*-*HAC1*), at levels closely approximating the uninduced *HAC1* state ([Figure 4](#pbio-0020235-g004){ref-type="fig"}B, compare *ADH1pro*-*HAC1*, lanes 5--8, to *HAC1pro*-*HAC1*, lanes 1--4). Expression from the *ADH1* promoter was constitutive, and the levels of *HAC1* mRNA did not change significantly under the various inducing conditions described above. As expected, induction of the UPR in these strains led to efficient *HAC1* mRNA splicing and Hac1p production. This strain therefore allowed us to fix the cellular Hac1p concentration to a level closely approximating the basal *HAC1* expression state observed during the UPR.
We next assessed whether we could identify physiological conditions under which elevated *HAC1* mRNA levels were required for cell growth. Therefore, we subjected WT cells and the engineered strain described above to the combinations of stresses described in [Figure 2](#pbio-0020235-g002){ref-type="fig"}. Cells expressing *HAC1* from the endogenous or from the *ADH1* promoter grew equally well on plates lacking inositol ([Figure 4](#pbio-0020235-g004){ref-type="fig"}C, left, first and third rows). This condition induces the UPR and requires the expression of at least a minimal amount of *HAC1* mRNA, as Δ*hac1* cells fail to grow ([Figure 4](#pbio-0020235-g004){ref-type="fig"}C, left, second row). In contrast, only WT cells, which are able to upregulate *HAC1* mRNA production, grew on plates lacking inositol and also containing tunicamycin. Cells expressing *HAC1* mRNA only at the basal levels from the *ADH1* promoter were nonviable on these plates ([Figure 4](#pbio-0020235-g004){ref-type="fig"}C, right, third row). As shown previously in [Figure 2](#pbio-0020235-g002){ref-type="fig"}B, this combination of stresses induces the S-UPR. The data therefore reveal that regulation provided by the *HAC1* promoter is necessary for cells to survive certain stress conditions that otherwise are lethal.
Differential UPR Target Gene Induction by Elevated Hac1p Levels {#s2f}
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To begin to characterize the cause for increased viability, we next determined differences in UPR target gene expression resulting from either UPR or S-UPR induction.
To this end, we used DNA microarray chip analysis to determine the complete mRNA profile of cells grown under UPR and S-UPR conditions. The results of this analysis are shown in [Figure 5](#pbio-0020235-g005){ref-type="fig"}A. Each spot represents the fold induction of a UPR target under UPR conditions (x-axis) or S-UPR conditions (y-axis) (see [Materials and Methods](#s4){ref-type="sec"} for definition of the UPR target set used in this analysis). UPR target genes for which the S-UPR has no additional effect should undergo equal induction under both conditions, and are expected to scatter around the diagonal, indicated by the dashed line. This was the case for many UPR targets. However, induction of a substantial number of genes was skewed to the top of the graph, indicating stronger induction under S-UPR conditions than under UPR conditions. These same data are displayed in [Figure 5](#pbio-0020235-g005){ref-type="fig"}B to highlight and categorize these differences. In the histogram, the x-axis represents the ratio of the induction of a target gene during S-UPR and UPR conditions, and the y-axis shows the number of genes with a given ratio. We have operationally divided UPR target genes into three classes, based on their fold induction during the S-UPR compared to their fold induction during the UPR. (1) Class 1 targets ([Figure 5](#pbio-0020235-g005){ref-type="fig"}, red bars) exhibit little if any difference in induction during the UPR and S-UPR (S-UPR induction / UPR induction \< 2). Thus, the increased Hac1p during the S-UPR does not lead to enhanced transcription, indicating that for these genes the response is already saturated at UPR Hac1p levels. Class 1 targets include many of the known genes encoding ER lumenal chaperones (including *KAR2, SCJ1, LHS1,* and *JEM1*) and redox proteins (including *PDI1, EUG1,* and *ERO1*). (2) Class 2 targets ([Figure 5](#pbio-0020235-g005){ref-type="fig"}, blue bars) are induced to a 2- to 4-fold greater extent during S-UPR than during the UPR. Transcription of these genes is therefore roughly proportional to the Hac1p levels in the cell. Class 2 targets include *YIP3,* involved in ER-to-Golgi transport, *OPI3,* encoding a phospholipid methyltransferase, and the hexose transporters *HXT12, HXT15, HXT16,* and *HXT17*. (3) Class 3 targets ([Figure 5](#pbio-0020235-g005){ref-type="fig"}, green bars) are induced by the S-UPR greater than 4-fold more than by the UPR. Class 3 contains the UPR targets *DER1,* involved in ER-associated degradation ([@pbio-0020235-Knop1]; [@pbio-0020235-Ng1]; [@pbio-0020235-Travers1]), and *INO1,* critical for membrane biogenesis ([@pbio-0020235-Hirsch1]).
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Figure 5
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###### Differential UPR Target Gene Induction by Elevated Hac1p Levels
\(A) Comparison of UPR target gene induction under either UPR or S-UPR conditions. Whole-genome mRNA expression analysis was carried out on WT cells harvested after 60 min of treatment, either grown at 30 °C and treated with 6 mM DTT (x-axis), or grown at 23 °C and simultaneously shifted to 37 °C and treated with 6 mM DTT (y-axis). Fold changes in gene expression are in reference to the untreated (*t* = 0) samples. Shown are only those genes designated as targets of the UPR (see [Materials and Methods](#s4){ref-type="sec"}). The dashed diagonal line represents equal induction under both conditions.
\(B) Comparison of UPR target gene induction under either UPR or S-UPR conditions (alternate display). The data from (A) were analyzed to generate a ratio (x-axis) for each gene, dividing the induction during S-UPR-inducing conditions by the induction during UPR-inducing conditions, with target genes of similar ratio grouped together (y-axis).
\(C) Characterization of *HAC1* expression in a strain constitutively expressing *HAC1* at high levels. Cells expressing *HAC1* from the endogenous promoter (WT; lanes 1 and 2), or a modified promoter constitutively expressing *HAC1* at high levels (*HAC1pro^HI^*; lanes 3 and 4) were treated with 6 mM DTT for 60 min. Although the basal transcription of *HAC1pro^HI^* is elevated, the promoter is still capable of further induction during the S-UPR (unpublished data).
\(D) Determination of Hac1p level in a strain constitutively expressing *HAC1* at high levels. Protein lysates were prepared from the strains described in (C), and protein levels were analyzed by Western blot analysis. The relative Hac1p/Pgk1p ratio is normalized to the WT DTT-treated (*t* = 60) sample from [Figure 4](#pbio-0020235-g004){ref-type="fig"}A.
\(E) Transcriptional response of different classes of UPR targets to high levels of Hac1p. Whole-genome mRNA expression analysis was carried on *HAC1pro^HI^* and WT cells treated with 6 mM DTT and harvested after 60 min. For the genes in each of the three classes of UPR targets defined in (B), a ratio (x-axis) is calculated by dividing the fold induction in DTT-treated *HAC1pro^HI^* cells by the fold induction in DTT-treated WT cells. This ratio is plotted against the number of genes with a similar ratio (y-axis). The Class 2 target *YFR026C* (asterisk), which is DTT-induced approximately 10-fold more in *HAC1pro^HI^* than in WT cells, is of unknown function. *a, DER1; b, INO1; c, YOR289W; d, YHR087W*.
\(F) Transcriptional response of different classes of UPR targets to UMF. Whole-genome mRNA expression analysis was carried on *ADH1pro-HAC1* cells grown at 23 °C and simultaneously shifted to 37 °C and treated with 6 mM DTT, and WT cells treated with 6 mM DTT, both harvested after 60 min. For the genes in each of the three classes of UPR targets defined in (B), a ratio (x-axis) is calculated by dividing the fold induction in *ADH1pro-HAC1* cells under S-UPR-inducing conditions by the fold induction in WT cells under UPR-inducing conditions. This ratio is plotted against the number of genes with a similar ratio (y-axis). *a, DER1; b, INO1; c, YOR289W; d, YHR087W*.
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Role for a Putative UPR Modulatory Factor {#s2g}
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The increased transcriptional output under S-UPR conditions could occur for two reasons. It could be due to increased Hac1p concentrations in the cell, or it could result because an additional S-UPR-specific transcription factor is produced or activated (perhaps the same that regulates *HAC1* transcription). It could also be due to a combination of these two scenarios. To distinguish among these possibilities, we determined the target gene induction profile in cells in which the *HAC1* mRNA concentration was artificially elevated to a similar level as that found after S-UPR induction. We took advantage of a specific 15-bp deletion in the *HAC1* promoter *(HAC1pro^HI^),* which increases basal expression by about 3-fold, as compared to the endogenous promoter ([Figure 5](#pbio-0020235-g005){ref-type="fig"}C). In cells bearing a *HAC1pro^HI^*-*HAC1* gene ("*HAC1pro^HI^* cells"), splicing of *HAC1* mRNA was somewhat reduced upon UPR induction (47%, compared to 67% for WT); however, even with this reduction, *HAC1pro^HI^* cells produced approximately 2.5-fold more spliced *HAC1^i^* mRNA than WT cells ([Figure 5](#pbio-0020235-g005){ref-type="fig"}C, compare lanes 3 and 4 to lanes 1 and 2). The increased levels of *HAC1^i^* mRNA led to a corresponding increase in Hac1p ([Figure 5](#pbio-0020235-g005){ref-type="fig"}D, compare lanes 3 and 4 to lanes 1 and 2). The amount of Hac1p produced by DTT induction of *HAC1pro^HI^* cells is approximately the same as the amount of Hac1p produced during the S-UPR (compare [Figure 5](#pbio-0020235-g005){ref-type="fig"}D, lanes 2 and 4 with [Figure 4](#pbio-0020235-g004){ref-type="fig"}A, lanes 4 and 8).
The ability to set *HAC1* mRNA levels to S-UPR levels allowed us to compare directly UPR target gene induction with the cellular Hac1p concentration being the only variable. We induced the UPR in both WT and *HAC1pro^HI^* cells with DTT and determined the mRNA expression profiles. For each class of UPR target defined above, the expression analysis of UPR-induced WT and *HAC1pro^HI^* cells is shown in [Figure 5](#pbio-0020235-g005){ref-type="fig"}E. In the histograms, the x-axis shows the ratio of target gene induction during the UPR driven by a high level of Hac1p from *HAC1pro^HI^* cells compared to induction during the UPR in WT cells. The y-axis shows the number of genes at any given ratio. As expected, Class 1 targets ([Figure 5](#pbio-0020235-g005){ref-type="fig"}E, top panel) did not further respond to the higher levels of Hac1p produced in *HAC1pro^HI^* cells. The majority of Class 2 and Class 3 targets ([Figure 5](#pbio-0020235-g005){ref-type="fig"}E, middle and bottom panels) also did not respond to higher levels of Hac1p (ratio less than 2), indicating that only raising the Hac1p concentration in cells is not sufficient to account for their full increased induction during the S-UPR. By contrast, ten of the 32 Class 2 and Class 3 targets were significantly induced (ratio greater than 2) in cells expressing high levels of Hac1p. For the Class 3 target *DER1,* high levels of Hac1p were sufficient to elevate expression to S-UPR levels (compare 8-fold induction in DTT-treated *HAC1pro^HI^* cells to 9-fold induction in WT cells during the S-UPR). Otherwise, however, high levels of Hac1p did not fully reconstitute the induction seen during the S-UPR. For example, while the Class 3 gene *INO1* was induced 7.5-fold more in the S-UPR than in the UPR, it was induced only 3-fold more by high levels of Hac1p, compared to normal levels. We conclude that elevated Hac1p levels are sufficient to selectively increase the induction of a few UPR targets, but that the full transcriptional program of the S-UPR predicts the production or activation of an additional transcriptional activator, which we term UPR modulatory factor (UMF).
To dissect further the UMF contribution during the S-UPR, we sought conditions under which UMF activity was the only variable. To this end, we induced the S-UPR in *ADH1pro*-*HAC1* cells, which are prevented from achieving high level Hac1p expression, and compared the mRNA expression profile against the UPR in WT cells. In this analysis, Hac1p levels were approximately equivalent in the two conditions, so variations from the normal UPR transcriptional program reflect the activity of UMF. The results are shown in [Figure 5](#pbio-0020235-g005){ref-type="fig"}F, with the data displayed similarly to [Figure 5](#pbio-0020235-g005){ref-type="fig"}E: the x-axis shows the ratio of target gene induction during the S-UPR in *ADH1pro*-*HAC1* cells, compared to induction during the UPR in WT cells, and the y-axis shows the number of genes at any given ratio. Not surprisingly, the induction of Class 1 targets ([Figure 5](#pbio-0020235-g005){ref-type="fig"}F, top panel) was unaffected: these are targets that are fully induced by even low levels of Hac1p and are not more induced during the S-UPR. Two Class 3 targets, *YOR289W* and *YHR087W* (both of unknown function) reach near WT S-UPR induction levels, without elevated levels of Hac1p; for these targets, UMF likely plays a leading role in their induction, with Hac1p having less influence. Most Class 2 and Class 3 targets ([Figure 5](#pbio-0020235-g005){ref-type="fig"}F, middle and bottom panels), however, do not reach full S-UPR induction levels in the absence of elevated Hac1p levels. For example, the Class 3 target *INO1* is induced roughly 25-fold in *ADH1pro*-*HAC1* cells during S-UPR conditions; while this is roughly twice the induction observed during the UPR, it falls far short of the 75-fold S-UPR induction in WT cells.
These results reinforce the in vivo requirement for high levels of Hac1p to survive S-UPR stress, demonstrated in [Figure 4](#pbio-0020235-g004){ref-type="fig"}C. Taken together with the data shown in [Figure 5](#pbio-0020235-g005){ref-type="fig"}E, we conclude that the full S-UPR transcriptional program results from a collaboration between elevated Hac1p levels and UMF, with the relative contribution from each varying among different target genes.
Discussion {#s3}
==========
The Circuitry of the UPR {#s3a}
------------------------
In this paper, we describe a novel ER surveillance pathway in yeast that modulates the UPR, resulting in a new physiological state that we term the S-UPR. In response to a bipartite signal transmitted from the ER by an *IRE1*-independent pathway, the *HAC1* promoter is activated, resulting in increased *HAC1* mRNA levels that, upon splicing, yield more Hac1p. The increased Hac1p concentration, in conjunction with an additional postulated factor(s) produced or activated by the S-UPR (UMF), allows the cell to mount a modified transcriptional response to cope with the inducing stress conditions.
[Figure 6](#pbio-0020235-g006){ref-type="fig"} shows the UPR as a circuit diagram utilizing multiple logical operations to integrate various signals. In the "classical UPR" (in red), basal transcription of *HAC1* produces *HAC1^u^* mRNA, which is translationally inactive due to the presence of the inhibitory intron. In response to unfolded proteins, Ire1p performs an on/off operation, excising the intron from *HAC1^u^* mRNA to generate spliced *HAC1^i^* mRNA, which is translated to produce the Hac1p transcription activator. The S-UPR provides another layer of regulation superimposed on the UPR (in blue). If ER folding stress is combined with either a shift to elevated temperature or inositol starvation, an AND gate integrates this bipartite signal and boosts *HAC1* mRNA levels. In turn, this regulation causes increased Hac1p production. Together with UMF, Hac1p induces UPR target genes, with particular genes responding differentially to differences in Hac1p and UMF concentration. Thus the S-UPR can be seen as an adaptation of the classical (or basal) UPR, fine-tuning the activation of select targets to produce a response suited to the challenge faced by the cell.
::: {#pbio-0020235-g006 .fig}
Figure 6
::: {.caption}
###### A Schematic of the Circuitry of the UPR
The model depicts the circuitry of the UPR (red) and the S-UPR (blue). Transcriptional control of *HAC1* is indicated by an icon representing a rheostat affording gain control of the UPR; Ire1p-dependent *HAC1^u^* mRNA splicing is indicated by an icon representing an on/off switch. The I/T and UP signals in the S-UPR are integrated by an AND gate (semicircle, top right), i.e., both conditions must be met to propagate the S-UPR signal. The putative UMF may collaborate with Hac1p to control transcription of UPR target genes (shown) and also be involved in regulating *HAC1* transcription (not shown); alternatively, different factors may be involved. The collaboration of Hac1p and UMF is indicated by the diamond-shaped icon, which integrates the information coming from both Hac1p and UMF concentration and activity.
:::
:::
In the accompanying paper, [@pbio-0020235-Patil2] describe a third signaling element, which additionally modifies the transcriptional program of the yeast UPR. The authors show that the transcriptional activator Gcn4p collaborates with Hac1p at the promoters of UPR targets, providing an additional opportunity for integration of information about the physiological state of the cell. Gcn4p is a highly regulated transcription regulator that responds to metabolic conditions, such as amino acid availability. Gcn4p is not UMF, as S-UPR induction of *HAC1* proceeds normally in Δ*gcn4* cells (unpublished data). A recent report from Ogawa and coworkers ([@pbio-0020235-Ogawa1]) demonstrates autoregulation of *HAC1* expression under conditions of extreme and prolonged ER stress, mediated by Hac1p binding to its own promoter. Because the S-UPR can be triggered in Δ*ire1* cells that do not produce Hac1p, autoregulation and the S-UPR are distinct pathways. The existence of multiple mechanisms of *HAC1* regulation reinforces the notion that multiple cellular stimuli become integrated to fine-tune an appropriate response.
Bipartite Signal Requirement for S-UPR Activation {#s3b}
-------------------------------------------------
Presently, the molecular details of the pathway by which the S-UPR signal exerts transcriptional control are not known. In particular, it will be of interest to determine where in the cell the two branches of the S-UPR signal are integrated, i.e., how the AND gate is constructed and where it resides. One possibility is that this signal integration event occurs close to the source at the ER membrane. Both temperature shift and inositol starvation can equally induce the I/T signal pathway, and it is conceivable that both conditions affect ER membrane properties similarly. Inositol is an essential precursor for phosphatidylinositol, a major structural phospholipid in yeast that is required for proper functioning of the secretory system ([@pbio-0020235-White1]; [@pbio-0020235-Zinser1]; [@pbio-0020235-Greenberg1]). Previous work has demonstrated an intimate link between inositol regulation and the UPR, presumably to coordinate the concentration of ER lumenal and membrane components ([@pbio-0020235-Cox3]). A similar sensing mechanism operates in cholesterol homeostasis, with sterol composition in ER membranes affecting the activity of SCAP, a membrane-bound regulator of SREBP intramembrane proteolysis ([@pbio-0020235-Espenshade1]). It is likely that elevated temperatures also affect ER membrane properties, such as fluidity ([@pbio-0020235-Laroche1]). If such a property were sensed, it would explain how the temperature effect contributing to the I/T signal is separate from the heat shock response. ER membranes distressed by either inositol deprivation or elevated temperature (the I/T signal) might then control the activity of a membrane-bound component of a signal transduction machine that also senses protein folding conditions (the UP signal) in the ER lumen.
Alternatively, the AND gate might be well removed from the ER membrane, with I/T and UP signals traveling separately through the cell and meeting possibly as late as at the promoters of the affected target genes. Components that map onto either signaling pathway need to be identified and placed into the circuit to distinguish between these possibilities.
The Transcriptional Output of the S-UPR {#s3c}
---------------------------------------
The transcriptional response elicited by the S-UPR reveals different classes of UPR targets. During the S-UPR, the further activation of UPR targets is not simply proportional to the increase in Hac1p concentration; rather, we observe a multitude of complex responses. Some targets are already maximally transcribed during UPR conditions and are not induced further during the S-UPR, while other targets become significantly more induced. For some targets (a minority), elevated Hac1p concentrations are sufficient to increase transcriptional induction, while for others, S-UPR-derived UMF is also required. We find evidence for both kinds of regulation. The promoters of target genes, therefore, display differential responsiveness to Hac1p concentration and UMF activity.
The production of different levels of Hac1p allowed us to isolate and directly assess the responsiveness of target genes to Hac1p concentration under otherwise identical conditions. Those target genes that undergo equivalent activation under both conditions likely have promoters that are saturated by the lower amount of Hac1p, and thus reach full activation more readily. For UPR targets at the other end of the spectrum, induction continues to increase as Hac1p levels increase; lower concentrations of Hac1p are inadequate for full stimulation of these genes, which may have lower affinity for Hac1p. Because genes respond differentially to Hac1p levels, regulation of *HAC1* mRNA abundance can be used as a gene-specific gain control for target activation. This control is similar to that observed in regulation of phosphate metabolism, where the differential affinity of certain Pho4p phosphoforms for target promoters allows for the selective activation of a subset of phosphate-responsive genes ([@pbio-0020235-Springer1]).
For most target genes, however, the S-UPR further enhances the transcriptional activity even in the presence of high concentrations of Hac1p. For example, *INO1* is induced over 75-fold by the S-UPR in WT cells, compared to 33-fold during the UPR in *HAC1pro^HI^* cells, while the amount of Hac1p produced in both cases is approximately the same. This added induction during the S-UPR is dependent on Hac1p, as *ADH1pro*-*HAC1* cells treated with DTT and shifted to elevated temperature show significantly reduced induction of *INO1*. The simplest interpretation of these findings is that S-UPR-induced UMF, which may or may not be identical to the transcription factor regulating *HAC1* mRNA, collaborates with Hac1p to further boost transcription of these genes.
The *cis* determinants that instruct genes to behave as Class 1, 2, or 3 targets are unknown. One attractive possibility is that target gene promoters have differential affinity for Hac1p and/or UMF. Promoters with stronger affinity for Hac1p would be maximally occupied and fully activated during a normal UPR and would not further respond to increased Hac1p levels (i.e., Class 1 targets). Promoters with lesser affinity for Hac1p would increase in occupancy, and hence transcriptional activation, as Hac1p levels rose during the S-UPR, and would possibly achieve full transcriptional activity only with the additional binding of UMF (i.e., Class 2 and 3 targets). Such a mechanism of promoter-encoded differential responsiveness to transcription factor concentration would explain the selective regulation of subsets of UPR target genes.
Links with the Metazoan UPR {#s3d}
---------------------------
Higher eukaryotes possess three separate pathways to sense ER stress and direct overlapping but distinct transcriptional outputs (reviewed in [@pbio-0020235-Ma1]). In the first branch, Ire1p senses unfolded proteins in the ER lumen and directs the cleavage of an intron from the mRNA encoding the XBP-1 transcription factor, analogous to the splicing of *HAC1* in yeast ([@pbio-0020235-Yoshida1]; [@pbio-0020235-Calfon1]). In a second branch, the transmembrane kinase PERK phosphorylates and inactivates the eIF2-α translation initiation factor ([@pbio-0020235-Harding1]). This attenuates global protein synthesis, but selectively increases the translation of a small number of proteins including the ATF-4 transcriptional activator. Interestingly, ATF-4 is the metazoan ortholog of Gcn4p, the yeast transcription factor demonstrated by [@pbio-0020235-Patil2] to collaborate with Hac1p. Finally, in a third branch, activation of the UPR in metazoans allows for the ER-to-Golgi transit of the membrane-tethered ATF-6 protein. In the Golgi apparatus, ATF-6 undergoes proteolytic cleavage within its membrane-spanning domain, and the soluble fragment subsequently travels to the nucleus as an active transcription factor ([@pbio-0020235-Haze1]; [@pbio-0020235-Ye1]). XBP-1, ATF-4, and ATF-6 all activate separate but overlapping transcriptional programs that enable the cell to respond to changing conditions in the ER. Notably, the XBP-1 promoter is a target of ATF-6 activation ([@pbio-0020235-Yoshida1]), reminiscent of the circuitry described here for yeast. Conceptually, therefore, *HAC1* mRNA upregulation by the S-UPR pathway in yeast takes the place of XBP-1 upregulation by the ATF-6 fragment in metazoans. Moreover, ATF-6 and XBP-1 can heterodimerize ([@pbio-0020235-Lee2]), reminiscent of the proposed collaboration of UMF and Hac1p. Thus, intriguing parallels between yeast and metazoans in the wiring that connects the elements of the UPR signaling circuit are beginning to come to light.
These findings suggest a common strategy among all eukaryotic cells for responding to challenges to the secretory system. Maintaining separate ER surveillance pathways creates the potential for cells to integrate multiple signals that, in principle, could convey precise information regarding the nature of the imbalance to afford finely tailored corrective measures. In this view, the UPR operates as a homeostatic control circuit, in which such regulation ensures that components of the secretory apparatus are produced according to need. The challenge now at hand is to decipher the logic between the UPR inducing conditions and the transcriptional output to add physiological explanations to the complex regulation of the response that we observe experimentally.
Materials and Methods {#s4}
=====================
{#s4a}
### Yeast strains. {#s4a1}
The WT strain W303--1A, the Δ*ire1* strain CS165, and the Δ*hac1* strain JC408 are as described previously ([@pbio-0020235-Cox2]; [@pbio-0020235-Cox1]). All *sec* strains used in this study were provided by Robert Fuller (University of Michigan, Ann Arbor, Michigan, United States) and are otherwise genotypically identical to W303. The *HSF1^c^* strain was a kind gift of Hillary Nelson (University of Pennsylvania, Philadelphia, Pennsylvania, United States) and contains the R222A allele of *HSF1* ([@pbio-0020235-Bulman1]) replacing the chromosomal locus in a W303 background. Strains used in the experiments described in [Figure 3](#pbio-0020235-g003){ref-type="fig"}A were Δ*hac1* transformed with pPW598 (*HAC1pro-HAC*1*, HA*-tagged *HAC1* \[[@pbio-0020235-Cox1]\] under its own promoter and with native *HAC1* flanking sequences, in a pRS304 background) or with pPW599 *(HAC1pro-GFP,* the *GFP* ORF, driven by the *HAC1* promoter \[defined as the region starting at the mapped start site of *HAC1* transcription ([@pbio-0020235-Ruegsegger1]) and extending 500 bp upstream\] and flanked by 5′ UTR and 3′ UTR sequences from *ACT1).* Strains used in experiments described in [Figure 4](#pbio-0020235-g004){ref-type="fig"} were *HAC1pro-HAC1* and Δ*hac1* (described above) and Δ*hac1* transformed with pPW600 (*ADH1pro-HAC1, HA*-tagged *HAC1* with 5′ and 3′ UTR *HAC1* sequence subcloned into the p414 ADH expression vector \[[@pbio-0020235-Mumberg1]\] and transferred to a pRS304 backbone). In [Figure 5](#pbio-0020235-g005){ref-type="fig"}, *HAC1pro^HI^* (pPW601) was made by subjecting *HAC1pro-HAC1* to QuikChange mutagenesis (Stratagene, La Jolla, California, United States) following the manufacturer\'s protocol, using oligonucleotides to remove the 15 bp at coordinates −338 to −323 (+1 representing the start site of transcription).
### Cell culture and plates {#s4a2}
Yeast cultures were grown in YPD medium (unless otherwise specified) at the indicated temperatures to midlog phase (OD~600~ **≈** 0.5). For temperature shift experiments, cultures were transferred to a preheated 37 °C water bath shaking incubator. DTT (Roche, Basel, Switzerland) was added to a final concentration of 6 mM, and tunicamycin (Boehringer Mannheim, Indianapolis, Indiana, United States) was added to a final concentration of 1 μg/ml. For experiments involving inositol deprivation in liquid medium, yeast cells were grown in liquid complete synthetic medium described by [@pbio-0020235-Sherman1], supplemented with myo-inositol (Sigma, St. Louis, Missouri, United States) to a final concentration of 100 μg/ml. Cells were then harvested by filtration, washed three times in prewarmed complete synthetic medium lacking inositol, and then filter-transferred to a flask containing prewarmed complete synthetic medium lacking inositol.
For the experiment described in [Figure 4](#pbio-0020235-g004){ref-type="fig"}C, yeast strains were grown in YPD to midlog phase (OD~600~ ≈ 0.5), transferred to a 96-well microtiter plate, and serially 5-fold diluted in fresh YPD. Using a liquid transfer prong ("frogging") tool (Aladin Enterprises, San Francisco, California, United States), approximately 3 μl of all serial dilutions of all strains was simultaneously transferred to complete synthetic plates lacking inositol (described above), either in the absence or presence of 0.2 μg/ml tunicamycin. After approximately 2 d of incubation at 30 °C, plates were photographed using the Epi Chemi II Darkroom GelDoc system (UVP, Upland, California, United States).
### RNA analysis {#s4a3}
Isolation of total RNA from yeast cells was carried out with the modified hot-phenol extraction method described in [@pbio-0020235-Ruegsegger1]. For Northern blot analysis, 10 μg of total RNA was separated on a 1.5% w/v agarose gel and transferred to a Duralon-UV nylon membrane (Stratagene), which was incubated with a probe directed against the 5′ exon of *HAC1*. The mRNA abundance was quantitated using a PhosporImager (Molecular Dynamics, Sunnyvale, California, United States). The membranes were then stripped with two serial washes using 0.1% SDS at 65 °C for 60 min each and incubated with a probe directed against the 3′ exon of *ACT1,* and mRNA abundance was again quantitated. To control for the variable strength of Northern blot probes across multiple experiments, the relative *HAC1*/*ACT1* mRNA abundance ratio is always normalized to the untreated (*t* = 0) sample. For the detection of other mRNAs, membranes were incubated with the additional relevant probes *(GFP, SSA1)* concurrent with the *HAC1* probe. All data shown are an average of at least two independent experiments.
PolyA+ mRNA was isolated from total RNA using the PolyATract system (Promega, Madison, Wisconsin, United States) according to the manufacturer\'s instructions. Microarray analysis, using yeast ORF arrays printed at the University of California, San Francisco, Core Center for Genomics and Proteomics (<http://derisilab.ucsf.edu/core/>, was performed as in [@pbio-0020235-Carroll1] using protocols and reagents described at <http://microarrays.org/>. All array data are the average of two independent experiments. For this study, we were obliged to evaluate UPR targets differently than in [@pbio-0020235-Travers1], as we considered *HAC1*-independent responses, whereas the former study specifically isolated genes induced by unfolded proteins via Hac1p (*z-*score ≥ 3.6 σ). Here, UPR targets were defined as those genes that met the following three criteria in a parallel set of microarray experiments using WT (W303) and Δ*hac1* (JC408) strains. First, induction (log~2~ of the fold change in gene expression) in WT cells treated with DTT must be at least one standard deviation greater than the mean (\[*induction* ^WT,DTT^ − *μ* ^WT,DTT^\]/*σ* ^WT,DTT^ ≥ 1). Second, induction in WT cells treated with tunicamycin must be at least one standard deviation greater than the mean (\[*induction* ^WT,tunicamycin^ − *μ* ^WT,tunicamycin^\]/*σ* ^WT,tunicamycin^ ≥ 1). Third, induction in Δ*hac1* cells treated with DTT must be at least one standard deviation less than the induction in WT cells treated with DTT (or, more awkwardly, \[(\[(*induction* ^WT,DTT^ − *μ* ^WT,DTT^)/*σ* ^WT,DTT^\] − \[(*induction* ^Δ*hac1*^ ^,DTT^ − *μ* ^Δ*hac1*^ ^,DTT^)/*σ* ^Δ*hac1*,DTT^\]) − *μ* ^WT,DTT\ −\ Δ*hac1*,DTT^\]/*σ* ^WT,DTT\ −\ Δ*hac1*,DTT^ ≥ 1).
### Isolation and detection of protein from yeast cells {#s4a4}
Cells were collected by filtration, frozen in liquid nitrogen, and disrupted in 150 μl of 8 M urea/1% SDS by vortexing for 5 min at 4 °C in the presence of 150 μl of silica beads. The samples were then boiled for 5 min and the lysates cleared by centrifugation at 16,200*g* for 5 min at room temperature. SDS-PAGE was performed on 20 μg of protein separated on NuPAGE 10% w/v SDS-polyacrylamide gels (Invitrogen, Carlsbad, California, United States), and Western blots were visualized using SuperSignal West Dura Extended Duration ECL Substrate (Pierce Biotechnology, Rockford, Illinois, United States) according to the instructions of the manufacturer. Hac1p was detected using a polyclonal antibody raised against the carboxy terminus (see [Figure 4](#pbio-0020235-g004){ref-type="fig"}) or a monoclonal antibody raised against the HA epitope and directly coupled to horseradish peroxidase (see [Figure 5](#pbio-0020235-g005){ref-type="fig"}) (Molecular Probes, Eugene, Oregon, United States), and Pgk1p was detected using a commercially available polyclonal antibody (Molecular Probes). Protein abundance was quantified using the Epi Chemi II Darkroom GelDoc system (UVP). Parallel experiments using serial protein dilutions were performed to confirm that the detected protein levels were within the linear range of the system.
### Transcription shut-off {#s4a5}
The yeast strain JC218 ([@pbio-0020235-Sidrauski2]; *rbp1--1*) was grown in YPD at 23 °C to OD~600~ ≈ 0.5 and then shifted to a 37 °C water bath, shaking at 250 RPM. To induce the UPR, DTT was added to a final concentration of 6 mM. Cells were harvested and total RNA isolated, at 20 min intervals, as described above.
Supporting Information {#s5}
======================
Accession Numbers {#s5a1}
-----------------
The GenBank accession numbers of the sequences discussed in this paper are Hac1p (NP\_ 116622), Ire1p (NP\_011946), and tRNA ligase (NP\_012448).
Microarray data can be accessed at the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) database as platform number GPL999 and sample numbers GSM16978--GSM1984.
We thank Jason Brickner and other members of the Walter lab for helpful discussions and comments on the manuscript, Adam Carroll and Manuel Llinas for assistance with microarrays, Chris Patil and Hao Li for help with bioinformatic analyses, and Robert Fuller and Hillary Nelson for yeast strains. Also, we acknowledge Vladimir Denic for initial observations regarding *HAC1* mRNA transcriptional control. This work was supported by a University of California at San Francisco (UCSF) Chancellor\'s Fellowship to JL, by support from the UCSF Herbert H. Boyer Fund to SB, and by grants from the National Institutes of Health to PW. PW is an Investigator of the Howard Hughes Medical Institute.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** JHL and PW conceived and designed the experiments. JHL and SB performed the experiments. JHL and PW analyzed the data and wrote the paper.
Academic Editor: Steven McKnight, University of Texas Southwestern
Citation: Leber JH, Bernales S, Walter P (2004) *IRE1*-independent gain control of the unfolded protein response. PLoS Biol 2(8): e235.
DTT
: dithiothreitol
ER
: endoplasmic reticulum
GFP
: green fluorescent protein
I/T signal
: signal provided by inositol starvation or temperature shift
S-UPR
: Super-UPR
UMF
: unfolded protein response modulatory factor
UP signal
: signal provided by unfolded proteins in the ER
UPR
: unfolded protein response
UTR
: untranslated region
WT
: wild-type
| PubMed Central | 2024-06-05T03:55:46.758925 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509300/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e235",
"authors": [
{
"first": "Jess H",
"last": "Leber"
},
{
"first": "Sebastián",
"last": "Bernales"
},
{
"first": "Peter",
"last": "Walter"
}
]
} |
PMC509301 | Introduction {#s1}
============
Hematopoiesis involves the coordinated processes of cell proliferation and differentiation of a relatively small number of progenitor cells into billions of circulating red and white blood cells ([@pbio-0020237-Thisse1]). Hematopoiesis in vertebrates, from zebrafish to humans, is an evolutionarily conserved program that produces two waves of stem or progenitor cells that differ both in their embryonic origins and in the lineages of differentiated blood cells produced ([@pbio-0020237-Palis1]; [@pbio-0020237-Orkin1]; [@pbio-0020237-Galloway1]). The first, or primitive, wave of hematopoiesis originates from ventral mesoderm and gives rise to progenitor cells that differentiate in embryonic blood islands. The primitive wave of hematopoiesis produces a burst of embryonic erythrocytes and macrophages. The second, or definitive, wave of hematopoiesis arises from self-renewing stem cells that develop primarily in the intraembryonic aorta--gonad--mesonephros region. These definitive hematopoietic stem cells seed the later developing marrow spaces, to produce all lineages of adult blood cells, including definitive erythrocytes, myeloid cells, and lymphocytes.
We have undertaken a genetic approach to characterize genes that control hematopoiesis using the zebrafish as a model system ([@pbio-0020237-Thisse1]). As part of a large-scale forward genetic screen, we previously identified bloodless zebrafish mutants that failed to express the erythroid transcription factor *gata1* normally in embryonic hematopoietic precursors ([@pbio-0020237-Ransom2]). We named one of these zebrafish genes *moonshine (mon),* and another group named a noncomplementing allele *vampire* ([@pbio-0020237-Weinstein1]).
Here, we have determined that mutations in the *mon* gene cause a disruption in both primitive embryonic and definitive adult hematopoiesis, resulting in a severe loss of erythroid cells. Erythroid progenitor cells in *mon* mutants are initially present, but fail to express normal levels of hematopoietic transcription factors and undergo apoptosis.
Positional cloning identifies the *mon* gene as the zebrafish ortholog of mammalian *transcriptional intermediary factor 1γ (TIF1γ),* a member of the TIF1 family of transcriptional coactivators and corepressors ([@pbio-0020237-Le1]; [@pbio-0020237-Friedman1]; [@pbio-0020237-Kim1]; [@pbio-0020237-Venturini1]; [@pbio-0020237-Peng1]). The three members of the vertebrate TIF1 family (α, β, and γ) are large nuclear proteins that each contain an N-terminal RBCC or TRIM domain ([@pbio-0020237-Reymond1]) composed of a RING finger, two B-boxes, and a coiled-coil domain. TIF1 family members also contain a C-terminal plant homeodomain finger and bromodomain that are characteristic of chromatin remodeling factors. TIF1α has been shown to associate with a variety of ligand-bound nuclear hormone receptors ([@pbio-0020237-Le1]) and function as a coactivator for retinoic acid receptors ([@pbio-0020237-Zhong1]). TIF1β has been shown to act as a corepressor for the large family of Krüppel-associated box (KRAB) domain zinc-finger transcription factors ([@pbio-0020237-Friedman1]; [@pbio-0020237-Abrink1]). In contrast, TIF1γ does not associate directly with either nuclear receptors or KRAB domains that bind to the other TIF1 family members ([@pbio-0020237-Venturini1]; [@pbio-0020237-Abrink1]). Biochemical studies also demonstrate that TIF1γ forms both homo-oligomers and hetero-oligomers with TIF1α but not with TIF1β ([@pbio-0020237-Peng1]). The murine *Tif1α* and *Tif1γ* genes have not yet been subjected to gene targeting experiments, whereas analysis of mouse mutants demonstrates that *Tif1β* is required for postimplantation embryogenesis and mesoderm induction in particular ([@pbio-0020237-Cammas1]). Taken together, these studies suggest that a major function of TIF1 family members is to link DNA-binding proteins with other coactivators or corepressors during development.
Our studies establish that *tif1γ* functions as an essential regulator of embryonic and adult hematopoiesis in vertebrates. Cell transplantation studies demonstrate that *tif1γ* acts in a cell-autonomous manner during embryonic hematopoiesis. The *tif1γ* gene is expressed specifically in ventral mesoderm and hematopoietic progenitors, then downregulated as erythroid maturation occurs. Tif1γ protein localizes to a novel class of nuclear bodies in both primary mouse embryo fibroblasts and erythroleukemia cell lines. Taken together, our studies demonstrate that Tif1γ is required for normal erythroid cell development and survival.
Results {#s2}
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The Zebrafish *mon* Gene Is Essential for Both Primitive and Definitive Erythropoiesis {#s2a}
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In order to determine when the *mon* gene is required in development, we first examined hematopoietic gene expression and apoptosis in zebrafish homozygous *mon* mutant embryos. During embryogenesis, homozygous zebrafish *mon* mutants have no red blood cells (RBCs) visible in circulation ([@pbio-0020237-Ransom2]; [@pbio-0020237-Weinstein1]). The *mon* mutants initiate expression of *gata1* in hematopoietic cells around the five-somite stage, similar to wild-type embryos (data not shown); however, based on TUNEL staining, the differentiating erythroid cells undergo programmed cell death from the 12-somite stage to 22 h postfertilization (hpf) ([Figure 1](#pbio-0020237-g001){ref-type="fig"}A and [1](#pbio-0020237-g001){ref-type="fig"}B, arrows). At 12 somites, *gata1* expression is only slightly reduced. By 18--22 hpf, hematopoietic-specific markers such as *gata1, scl, gata2,* and *ikaros* are not detected in the embryonic blood island ([Figure 1](#pbio-0020237-g001){ref-type="fig"}A and [1](#pbio-0020237-g001){ref-type="fig"}B; unpublished data). The hematopoietic cells are thus correctly specified early during the development of *mon* mutant embryos, but these precursors undergo cell death. Based on expression of c-*myb* and *rag1* ([Figure 1](#pbio-0020237-g001){ref-type="fig"}B, arrows), *mon* mutants have normal myeloid and lymphoid development, respectively. In addition to the deficit of RBCs in *mon* mutants, there is a prominent loss of fin-fold and tail mesenchyme ([@pbio-0020237-Ransom2]). TUNEL staining of *mon* mutants demonstrates extensive apoptosis of mesenchymal cells in the trunk and tail bud regions ([Figure 1](#pbio-0020237-g001){ref-type="fig"}A and [1](#pbio-0020237-g001){ref-type="fig"}B, arrows). The *mon* gene is thus required for normal development and survival of both committed erythroid progenitor cells and posterior mesenchymal cells.
::: {#pbio-0020237-g001 .fig}
Figure 1
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###### Zebrafish *mon* Mutants Have Severe Defects in Primitive Hematopoiesis
\(A) Whole-mount TUNEL assays reveal that ventral-posterior mesodermal cells undergo apoptosis in homozygous *mon^tg234^* mutant embryos. Whole-mount in situ hybridization of *gata1* detected at the 12- and 18-somite stage in genotyped embryos. Posterior views, anterior to the left.
\(B) Extensive apoptosis is visible in the trunk and tail (arrowhead) and also in hematopoietic cells of the embryonic blood island at 22 h of development (arrow). Whole-mount in situ hybridization at 22 hpf including *scl, gata2, gata1, ikaros,* and *myb* in *mon^tg234^* mutants. Expression of *myb* is greatly reduced in the blood islands because of a loss of erythroid cells, but embryonic macrophages are still present (arrows). The expression of *rag1* in thymic T-cells appears normal in *mon* mutants at 5 d postfertilization (arrow heads). Lateral views of 22 hpf and 5-d-old embryos.
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We next examined definitive hematopoiesis in rare surviving homozygous adult zebrafish *mon* mutants. Mutations in *mon* are generally lethal by 10 to 14 d of development ([@pbio-0020237-Ransom2]), although rare *mon* homozygous mutants (approximately 1 in 500 bloodless embryos) of all tested alleles survive to adulthood. Adult *mon* mutants show cardiac hypertrophy, presumably due to the severe anemia leading to a high output state ([Figure 2](#pbio-0020237-g002){ref-type="fig"}). In wild-type zebrafish, the adult site of hematopoiesis is the kidney ([@pbio-0020237-Al1]), which contains erythroid, lymphoid, and myeloid populations at various stages of differentiation ([@pbio-0020237-Bennett1]). In *mon* homozygous mutants, there is a severe block in maturation at the proerythroblast stage ([Figure 2](#pbio-0020237-g002){ref-type="fig"}), whereas the differentiation of myeloid cells is normal (unpublished data). This demonstrates that the *mon* gene product acts during both primitive and definitive erythropoiesis.
::: {#pbio-0020237-g002 .fig}
Figure 2
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###### Zebrafish *mon* Mutants Also Have Severe Defects in Definitive Hematopoiesis
Adult phenotype of wild-type and *mon* mutants. A rare surviving *mon^tb222^* homozygous adult shows significant cardiomegaly in comparison to a wild-type age-matched control. Wright--Giemsa stained marrow of wild-type adult in comparison to a homozygous mutant. Note the dramatic reduction of terminally differentiated erythroid cells and the presence of abnormally large megaloblastic proerythroblasts in the *mon^tb222^* mutant marrow.
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Positional Cloning Identifies *mon* as the Zebrafish Ortholog of Mammalian TIF1γ {#s2b}
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We identified the *mon* gene by positional cloning using a panel of 2,200 diploid mutants collected from Tübingen background (TU)/WIK strain hybrid parents carrying the *mon^tg234^* allele. The *mon* mutant gene was positioned on Chromosome 8 between microsatellite markers z987 and z11001 ([Figure 3](#pbio-0020237-g003){ref-type="fig"}A) ([@pbio-0020237-Knapik1]). For positional cloning purposes, over 12,000 polymorphic markers were screened using amplified fragment length polymorphism (AFLP) ([@pbio-0020237-Ransom1]), and 36 markers within the interval were isolated. One of these, MA3, was found to be 0.3 cM from the gene ([Figure 3](#pbio-0020237-g003){ref-type="fig"}A) and was utilized as the starting point of a chromosomal walk. A critical P1 bacterial artificial chromosome clone (PAC), 107N19, was obtained that spanned the genetic interval. Two simple sequence conformation polymorphism (SSCP) markers found on this PAC clone flank the critical genetic interval. The marker 80M12-T7 maps two recombinants out of 4,400 meioses telomeric of the mutation, and the marker 157J23-T7 maps one recombinant centromic of the mutation ([Figure 3](#pbio-0020237-g003){ref-type="fig"}A). The end sequences and SSCP markers of PAC 107N19 are found in the zebrafish genomic sequence contig ctg23107 (<http://www.ensembl.org/Danio_rerio/>) containing a predicted zebrafish TIF1 family gene. This PAC was hybridized to a kidney cDNA library, resulting in the isolation of four clones that represented the same gene.
::: {#pbio-0020237-g003 .fig}
Figure 3
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###### Positional Cloning Identifies the *mon* Gene as Zebrafish *tif1γ*
\(A) Physical map of the *mon* locus on zebrafish Chromosome 8. Microsatellite markers z987 and z11001 were used to initially identify recombinants in a panel of 2,200 diploid *mon^tg234^* homozygous mutants. The AFLP marker MA3 was used to initiate a chromosomal walk in PAC libraries. The critical PACS that were isolated to encompass the *mon* locus are indicated by numbers above bar. The PAC 107N19 defines the critical interval for the *mon* gene. This PAC was used as a probe to screen cDNA libraries and to identify zebrafish *tif1γ* cDNAs. Numbers below the bar indicate the number of recombinants identified by SSCP analysis.
\(B) Clustal-W--generated phylogentic tree of zebrafish (Danio rerio \[Dr\]) Tif1γ and Tif1α peptide sequences in comparison to TIF1 family members: human (Hs) TIF1α, TIF1β, and TIF1γ; mouse (Mm) Tif1α, Tif1β, and Tif1γ;; and fly (Dm) bonus.
\(C) Diagrams illustrating the structure of the Tif1γ-predicted peptide and the three identified point mutants. RING finger (RING), B-boxes (B1 and B2), plant homeodomain finger (PHD) and bromodomain (BROMO). Numbers below the first diagram indicate the percent identity shared between each of these domains in zebrafish and human TIF1γ. The predicted truncated proteins are indicated.
\(D) DNA sequence chromatograms showing the three ENU-induced point mutants in comparison to wild-type control sequences
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The *mon* gene encodes a member of the TIF1 family of transcriptional cofactors ([Figure 3](#pbio-0020237-g003){ref-type="fig"}B and [3](#pbio-0020237-g003){ref-type="fig"}C). The coding sequence of *mon* is most similar to human TIFγ ([@pbio-0020237-Le1]; [@pbio-0020237-Friedman1]; [@pbio-0020237-Venturini1]), and the locations of exon boundaries are conserved between the zebrafish and human genes (unpublished data). The *mon* locus on zebrafish Chromosome 8 is also predicted to be syntenic to the region of human Chromosome 1p that contains the *TIF1γ* gene based on the conserved locations of 12 other orthologous gene pairs, including *NRAS,* mapped to these regions in human and zebrafish ([@pbio-0020237-Barbazuk1]). Therefore, based on sequence similarity and chromosomal location, the zebrafish *mon* gene is the likely ortholog of the human *TIF1γ* gene.
We have identified ethyl-nitrosourea (ENU)-induced point mutations in three alleles of *mon* ([Figure 3](#pbio-0020237-g003){ref-type="fig"}C and [3](#pbio-0020237-g003){ref-type="fig"}D), each of which generates a premature stop codon. The *mon^tb222b^* and *mon^tg234^* alleles have a severe phenotype with no circulating blood cells. In contrast, the *mon^m262^* allele has 10--100 circulating blood cells by 48 hpf, in comparison to the approximately 3,000 RBCs in the circulation of wild-type or heterozygous embryos at the same time point. The *mon^m262^* allele was found to encode a premature stop codon at position E40, which would encode a putative protein of only 40 amino acids. Although this mutation would be expected to lead to a complete loss of *mon* gene product, another methionine is found downstream at amino acid position 104. In vitro translation experiments in reticulocyte lysates demonstrate reinitiation of translation from this methionine (unpublished data). Therefore, the hypomorphic larval phenotype of the *mon^m262^* allele is likely due to partial loss of *mon* function or expression. The presence of mutations in each of the *mon* alleles indicates that defective Tif1γ function is the cause of the *mon* phenotype.
In order to determine whether *tif1*γ is expressed in hematopoietic mesoderm, we next examined zebrafish embryos by whole-mount in situ hybridization ([Figure 4](#pbio-0020237-g004){ref-type="fig"}A). *tif1γ* mRNA is expressed maternally and is found throughout the embryo during blastula stages. During gastrulation and epiboly stages, zygotic expression of *mon* is highest in the mesendoderm of the germ ring. At tail bud and early somite stages a high level of *tif1γ* expression delineates a horseshoe-shaped population of ventral/lateral mesoderm that will give rise to blood and also expresses *stem cell leukemia*hematopoietic transcription factor *(scl)* ([@pbio-0020237-Liao2]). This group of cells continues to express *tif1γ* and *scl* while it converges and forms the embryonic blood island ([@pbio-0020237-Detrich1]). The *tif1γ* gene is also highly expressed in the central nervous system as well as the mesenchyme of the trunk and tail. Homozygous *mon^tg234^* mutants have a greatly reduced amount of *tif1γ* mRNA in all tissues consistent with nonsense-mediated message decay. Thus, zebrafish *tif1γ* is specifically expressed in ventral mesoderm and putative hemangioblasts prior to and during the embryonic stages when hematopoietic progenitors are undergoing apoptosis in *mon* mutants. We also compared the expression of zebrafish *mon* to mouse *Tif1γ* ([Figure 4](#pbio-0020237-g004){ref-type="fig"}A and [4](#pbio-0020237-g004){ref-type="fig"}B). Mouse *Tif1*γ is highly expressed in erythroid blood islands of the yolk sac, and it is subsequently expressed in the fetal liver at a high level, and in other tissues, including the central nervous system. Taken together these results strongly suggest that zebrafish *mon* and mouse *Tif1γ* are orthologs that function during hematopoiesis.
::: {#pbio-0020237-g004 .fig}
Figure 4
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###### The *mon/tif1γ* Gene Is Highly Expressed in Hematopoietic Mesoderm
\(A) In situ hybridization of zebrafish embryos demonstrating the embryonic expression of *tif1γ. tif1γ* is initially expressed as a maternal mRNA. Increased expression of *tif1γ* in ventral-lateral mesoderm begins between the one- to three-somite stages and increases through early development. By five somites, *tif1γ* is strongly expressed in lateral stripes of mesoderm that also express *scl.* At 22 hpf *tif1γ* is expressed broadly in the brain, spinal cord, trunk, and tail mesenchyme, but is at much higher levels in hematopoietic cells of the blood island. Zebrafish *tif1α* is also broadly expressed but relatively more uniform in most tissues, in comparison to *tif1γ. Tif1α* is weakly expressed at early somite stages in hematopoietic mesoderm and uniformly expressed at 22 hpf, including expression in the blood islands. Expression of *scl* at five somites and 22 hpf highlights the embryonic blood island in comparison to *tif1γ* expression.
\(B) In situ hybridization of mouse embryos detects broad expression of *Tif1γ* at embryonic day 8.5 including the yolk sac blood islands (arrow). AT embryonic day 12.5, there is high level expression in the fetal liver (arrow) and broad expression in the embryonic brain, spinal chord, gut, and muscle.
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Given that mammalian TIF1γ has been shown to form hetero-oligomers with Tif1α ([@pbio-0020237-Peng1]), we searched for additional TIF1 family members in zebrafish to compare with *tif1γ.* Using zebrafish expressed sequence tag (EST) sequences, we designed primers to RT-PCR amplify a TIF1-related cDNA from embryonic 10-hpf and 24-hpf RNA. This cDNA encodes a predicted zebrafish ortholog of human TIF1α based on predicted amino acid sequences (see [Figure 3](#pbio-0020237-g003){ref-type="fig"}B). In addition, zebrafish *tif1α* ESTs map to LG4 in a region predicted to be syntenic to the region of human Chromosome 7 that contains the *TIF1α* gene based on the conserved locations of eight other orthologous gene pairs, including *SEMA3A,* mapped to these regions in human and zebrafish ([@pbio-0020237-Barbazuk1]). We next compared the embryonic expression pattern of *tif1α* mRNA to *tif1γ* by in situ hybridization. Like mammalian *TIF1α* ([@pbio-0020237-Le1]; [@pbio-0020237-Niederreither1]), the predicted zebrafish *tif1γ* gene is broadly expressed (see [Figure 4](#pbio-0020237-g004){ref-type="fig"}A). At five somites, zebrafish *tif1α* does not display the relatively high expression in the horseshoe-shaped region of hematopoietic mesoderm seen with *tif1γ.* At later stages, *tif1α* is evenly expressed throughout most of the embryo, including the developing blood islands. Therefore, *tif1α* is coexpressed in the same cells with *tif1γ* and may therefore be available to form hetero-oligomers in vivo.
Forced Expression of *tif1γ* Rescues Hematopoiesis in *mon* Mutants {#s2c}
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To further confirm that a mutation in the zebrafish *tif1γ* gene is responsible for the *mon* mutant phenotype we performed embryo rescue experiments ([Figure 5](#pbio-0020237-g005){ref-type="fig"}A; [Table 1](#pbio-0020237-t001){ref-type="table"}). Microinjection of synthetic wild-type *mon* mRNA at the one-cell stage rescues the formation of embryonic erythrocytes in genotyped mutant embryos without causing obvious defects in embryonic patterning or organogenesis. At 4 d of development, 70% (*n* = 10) of *mon^tg234^* mutants show significant (greater than 200 cells in comparison to a wild-type estimate of 3,000 cells) rescue of circulating hemoglobinized RBCs in comparison to control sibling mutants (*n* = 75). Based on the correction of the jagged fin-fold phenotype ([@pbio-0020237-Ransom2]), the mesenchymal cells are rescued to a similar extent as the anemia (unpublished data). Overexpression of *mon* did not result in expanded blood cell numbers in wild-type embryos and was not toxic at doses that rescue the phenotype of *mon* mutants (unpublished data). Since there were no expanded or ectopic blood populations in the embryos, these rescue experiments suggest that *mon* functions as a permissive factor required for hematopoiesis.
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Figure 5
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###### Overexpression of Wild-Type *tif1γ* mRNA or Marrow Transplantation Rescues Embryonic Hematopoiesis in *mon* Mutants
\(A) *mon^tg234^* mutants are rescued by injection of mRNA-encoding wild-type Tif1γ protein. At 4 d of development, large numbers of RBCs are visible in the circulation of wild-type zebrafish, shown here by o-dianisidine staining of hemoglobin. Uninjected *mon^ttg234^* homozygous mutants are completely bloodless. Injection of 100 pg of wild-type *tif1γ* mRNA rescues erythropoiesis in mutant embryos. o-dianisidine-stained larvae are shown in ventral views to highlight blood in vessels.
\(B) Transplantation of wild-type zebrafish marrow cells carrying a *gata1:GFP* transgene into 2-d-old embryos reconstitutes erythropoiesis, but not viability, in *mon^tg234^* homozygous mutants. Still frames from movies of live embryos at day 3 posttransplant highlight less than 100 GFP^+^ RBCs in circulation (top). Transplanted cells were observed to proliferate resulting in thousands of donor-derived erythrocytes 7 d later (bottom). Arrows indicate the hearts of control and transplanted zebrafish. See [Videos S1--S4](#s5){ref-type="sec"}.
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::: {#pbio-0020237-t001 .table-wrap}
Table 1
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###### Overexpression of *tif1γ* mRNA Rescues *mon* Mutants: Hematopoietic Phenotypes
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Synthetic *tif1γ* mRNA (100 pg) was injected at the one-cell stage into embryos of the indicated genotypes. For the *mon* embryos, circulating cells where counted each day through 4 d, when the embryos were fixed and stained with o-dianisidine to detect hemoglobin in mature RBCs. Normal embryos contain approximately 3,000 circulating cells at these time points. Results are given as number of embryos with the indicated phenotype. Numbers in parentheses represent percentage of total embryos analyzed
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Marrow Transplantation Rescues Erythropoiesis in *mon* Mutants {#s2d}
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The high levels of *tif1*γ expression in erythroid cells suggest that it functions as a cell-autonomous regulator of gene expression in hematopoietic cells. In order to test this hypothesis, we transplanted wild-type adult zebrafish kidney marrow cells carrying a *gata1:green fluorescent protein (GFP)* transgene into 48-hpf *mon* mutant embryos ([Figure 5](#pbio-0020237-g005){ref-type="fig"}B; [Table 2](#pbio-0020237-t002){ref-type="table"}). The *gata1:GFP* transgene makes use of the *gata1* promoter to drive GFP expression and can thus be used to mark donor-derived erythroid cells ([@pbio-0020237-Long1]). Untransplanted mutant embryos have no embryonic blood cells in circulation. Following transplantation, mutant host embryos were observed daily for 2 wk. Of 191 mutant embryos injected, 129 (68%) showed GFP^+^ cells in circulation 2 d later. Many recipients showed robust increases in donor-derived cells over the observation period. Of 81 recipients initially scored as having less than ten GFP^+^ cells at day 2 posttransplant, 13 (16%) of these demonstrated a marked increase in erythroid cells with 100--1,000 GFP^+^ cells in circulation 6 d later. By day 10, these transplanted embryos showed approximately 3,000 cells in circulation, similar to the number of blood cells in normal embryos. Despite robust reconstitution of blood cells, mutant recipients did not inflate their swim bladders and thus failed to survive longer than nontransplanted sibling controls, all dying by 3 wk of age. In contrast, 13/35 (37%) heterozygous *mon^tg234^* transplants survived to early adulthood. Similar transplants of wild-type cells can fully rescue *vlad tepes (gata1)* mutants ([@pbio-0020237-Traver1]). Therefore, the results of cell transplantations suggests that *tif1γ* plays a cell-autonomous role in erythroid cells, and its role in nonhematopoietic tissues, such as trunk mesenchyme or the nervous system, is also required for embryo survival.
::: {#pbio-0020237-t002 .table-wrap}
Table 2
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###### Marrow Transplantation Rescues Hematopoiesis But Not Survival in *mon* Mutants: Embryos with Transplanted Erythroid Cells
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Between 100 and 1,000 kidney marrow cells from adult *gata1:EGFP* transgenic donors were injected per zebrafish embryo at 48 hpf. Individual transplanted embryos were anesthetized and visualized for GFP^+^ erythroid cells. By 10 d posttransplantation the indicated number of embryos had an estimated 100 to 3,000 GFP^+^ cells in circulation. At 3 mo the indicated number of fish were alive. The relative percentage of embryos is shown in parentheses
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Tif1γ in Punctate Nuclear Foci Is Developmentally Regulated {#s2e}
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In order to examine the subcellular distribution of Tif1γ protein, we generated an affinity-purified rabbit polyclonal antiserum directed against the C-terminal 15 amino acids conserved in human TIF1γ and mouse Tif1γ. Immunofluorescence of mouse embryo fibroblast nuclei with the anti-Tif1γ antiserum demonstrates that Tif1γ is localized in small nuclear foci ([Figure 6](#pbio-0020237-g006){ref-type="fig"}A). The localization of Tif1γ protein appears different from the more diffuse nuclear patterns typically seen in studies of Tif1α ([@pbio-0020237-Remboutsika1]) or TIF1β ([@pbio-0020237-Cammas2]). A recent report demonstrates that TIF1β associates with heterochromatin-containing foci after retinoic acid treatment or serum starvation ([@pbio-0020237-Cammas2]). Thus, localization or expression of the TIF1 proteins may be regulated during distinct developmental processes or by environmental cues. The nuclear foci that contain Tif1γ do not colocalize with two markers of heterochromatin, HP1α protein and DAPI staining of DNA ([Figure 6](#pbio-0020237-g006){ref-type="fig"}A). Furthermore, Tif1γ does not colocalize with promyelocytic leukemia gene product (PML) nuclear bodies, DNA repair complexes that contain Mre11, or transcriptional complexes containing TFII-B (unpublished data). We next examined the expression of Tif1γ protein during the differentiation of G1E cells, a murine erythroleukemia cell line that can terminally differentiate into erythrocytes when a Gata1:estrogen receptor fusion protein is stabilized in response to estrogen exposure ([@pbio-0020237-Weiss1]). Western blot analysis demonstrated that Tif1γ protein expression decreases with terminal erythroid differentiation ([Figure 6](#pbio-0020237-g006){ref-type="fig"}B). Consistent with this finding, after 24 hpf, zebrafish *mon* mRNA expression falls during the terminal maturation of the primitive erythroid cells (unpublished data). In two different murine erythroleukemia cell lines (MEL and G1E), Tif1γ is also expressed in nuclear foci, and even though the overall Tif1γ protein level is reduced, this nuclear foci localization does not change with differentiation (unpublished data). This provides further support for the hypothesis that Tif1γ acts within novel nuclear foci, during erythroid differentiation.
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Figure 6
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###### Mammalian Tif1γ Protein Localizes to Nuclear Bodies Distinct from Heterochromatin
\(A) Deconvolved immunofluorescence images of a mouse embryonic fibroblast cell nucleus stained with an anti-Tif1γ antibody and stained with a monoclonal antibody directed against HP1α. This is also compared to DAPI staining. The merged images of the nucleus show that Tif1γ does not colocalize with the HP1α or DAPI staining of heterochromatin while HP1α and DAPI staining overlap.
\(B) G1ER mouse erythroleukemia cells express high levels of Tif1γ protein as detected by Western blot analysis. Expression of Tif1γ decreases during Gata1-dependent erythroid maturation induced by β-estradiol treatment to induce a Gata1--ER fusion protein.
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Discussion {#s3}
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The zebrafish is an excellent model system to elucidate the molecular machinery controlling gene expression during hematopoiesis ([@pbio-0020237-Thisse1]; [@pbio-0020237-Galloway1]). As part of a large-scale forward genetic screen, we originally identified a complementation group of independent mutant alleles in the zebrafish gene that we named *moonshine* ([@pbio-0020237-Ransom2]). Positional cloning was used to identify the *mon* gene, establishing a critical role for a transcriptional intermediary factor, Tif1γ, during hematopoietic development.
The *mon* Gene Encodes the Zebrafish Ortholog of Mammalian TIF1γ {#s3a}
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Our results strongly support the conclusion that we have positionally cloned the zebrafish *mon* gene correctly, and it is the ortholog of mammalian Tif1γ. Tif1γ is present in the critical genetic interval encompassing a single approximately 50-kb PAC clone defined by linkage analysis (see [Figure 3](#pbio-0020237-g003){ref-type="fig"}). Sequence analysis indicates that zebrafish *tif1γ* is most similar in predicted amino acid sequence and intron/exon structure compared to the predicted orthologous human and mouse genes. Zebrafish *tif1γ* is located in a region of zebrafish Chromosome 8 syntenic to the region of human Chromosome 1 containing *TIF1γ.* We identified point mutations in *tif1γ* from three different alleles of *mon* that each result in premature stop codons and mRNA decay. In addition, *tif1γ/Tif1γ* is highly expressed in hematopoietic cells throughout embryogenesis in both zebrafish and mouse (see [Figure 4](#pbio-0020237-g004){ref-type="fig"}). And as predicted, forced expression of wild-type *tif1γ* mRNA efficiently rescues hematopoiesis in *mon* mutants and does not perturb hematopoiesis in wild-type embryos (see [Figure 5](#pbio-0020237-g005){ref-type="fig"}). We have also cloned the predicted zebrafish ortholog of *tif1α,* which is more uniformly expressed in zebrafish embryos like mammalian *TIF1α* ([@pbio-0020237-Le1]; [@pbio-0020237-Niederreither1]) (see [Figures 3](#pbio-0020237-g003){ref-type="fig"}A and [4](#pbio-0020237-g004){ref-type="fig"}A) and may therefore be available to form hetero-oligomers with Tif1γ protein in developing hematopoietic cells. Comparing available zebrafish and mammalian TIF1-predicted amino acid sequences, it appears that the Tif1γ orthologs are the most highly conserved family members while the Tif1α sequences are relatively more divergent. We have not found a Tif1β ortholog, thus far, in the zebrafish or *fugu* genome or EST sequences. It is possible that Tif1β, like the KRAB domain transcription factors it binds to, may be present only in tetrapods ([@pbio-0020237-Urrutia1]). However, more complete genome sequences will be needed to confirm this hypothesis. Based on our analysis of zebrafish *mon* mutants, it is reasonable to predict that Tif1γ, the most evolutionarily conserved TIF1 family member, plays a similarly essential role in human and mouse hematopoiesis.
Mutations in *tif1γ* Cause Apoptosis of Erythroid Progenitors {#s3b}
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Our examination of hematopoietic gene expression, apoptosis, and marrow histology in *mon* mutants demonstrates that early erythroid progenitors are formed in homozygous mutants, but they fail to properly differentiate and instead undergo programmed cell death (see [Figure 1](#pbio-0020237-g001){ref-type="fig"}). The expression of *gata1* appears to initiate normally in the committed erythroid cells of *mon* mutants. However, the cells are abnormal prior to the complete loss of *gata1* expression. TUNEL-positive apoptotic cells are abundant by the 12-somite stage of development, and by 22 hpf all hematopoietic gene expression is extinguished. The expression of marker genes, including *scl* and *gata2,* characteristic of hematopoietic stem cells and primitive hematopoietic progenitors, are also not detected in the embryonic blood islands of mutants at 22 hpf. This indicates that the mutant hematopoietic cells are not blocked prior to commitment to the erythroid lineage, but instead develop as abnormal erythroid cells and undergo apoptosis, similar to *gata1-*deficient erythroid cells ([@pbio-0020237-Fujiwara1]; [@pbio-0020237-Lyons1]). Defective erythropoiesis and severe anemia were also observed in rare surviving homozygous mutant *mon* adults, demonstrating that *tif1γ* is also required in definitive hematopoiesis (see [Figure 2](#pbio-0020237-g002){ref-type="fig"}).
The zygotic phenotypes of *mon* mutants may not reveal the function of maternally inherited Tif1γ. Maternally expressed zebrafish Tif1γ may play roles in hematopoiesis or other aspects of organogenesis that are not detectable due to the presence of wild-type mRNA in eggs laid by heterozygous mothers. Analysis of the offspring of homozygous *mon* mutant female zebrafish will aid in defining the function of this maternal mRNA. The present analysis of zygotic *mon* mutants provides data that are consistent with the conclusion that *tif1γ* is essential for erythropoiesis but do not rule out essential functions in other hematopoietic lineages.
The hematopoietic phenotype of *mon* mutants resembles the loss of erythroid cells seen in both mouse *Gata1* knockout embryos and zebrafish *vlad tepes (gata1)* mutant embryos ([@pbio-0020237-Fujiwara1]; [@pbio-0020237-Lyons1]). In an effort to determine if there is a genetic relationship between *mon* and *gata1,* we tested their ability to rescue erythropoiesis. Both injection of *gata1* mRNA into *mon* homozygous mutant embryos and injection of *tif1γ* mRNA into *gata1* knock-down embryos failed to rescue hematopoiesis (unpublished data). We also tested for a direct interaction between Tif1γ and Gata1 proteins by coimmunoprecipitation and yeast two-hybrid assays and found no association (unpublished data). Although the mutations in each of these genes arrest cells at a similar stage of development, our results suggest that *gata1* and *tif1γ* act independently. This does not rule out the possibility that parallel genetic pathways involving *gata1* and *tif1γ,* operating together, regulate gene transcription within blood cells.
The Role of Tif1γ in Primitive and Definitive Erythropoiesis {#s3c}
------------------------------------------------------------
Taken together, our data suggest that *tif1γ* is required as a permissive cofactor for the erythroid lineage-specific control of hematopoietic gene expression. We reasonably predict that Tif1γ protein functions as a transcriptional intermediary factor in hematopoietic progenitor cells given that both TIF1α ([@pbio-0020237-Zhong1]) and TIF1β ([@pbio-0020237-Friedman1]; [@pbio-0020237-Abrink1]) have been shown to act as intermediary factors that positively or negatively regulate gene transcription. These studies indicate that TIF1α and TIF1β act as scaffolds that link different classes of DNA-binding proteins and chromatin-associated proteins into larger regulatory complexes. Tif1γ is detected within nuclear foci (see [Figure 6](#pbio-0020237-g006){ref-type="fig"}), which, based on our analysis, do not appear to correspond to several types of previously described nuclear bodies, including PML bodies. Localization of Tif1γ to these nuclear bodies may be regulated by posttranslational modification such as SUMO modification that is required for PML to form PML nuclear domains ([@pbio-0020237-Zhong2], [@pbio-0020237-Zhong3]; [@pbio-0020237-Best1]). These foci may serve as assembly points where Tif1γ forms multisubunit complexes with DNA-binding transcription factors and their other essential coactivators or corepressors, during the early stages of erythroid differentiation. It will be important to determine the identity of Tif1γ-interacting proteins in nuclear foci and establish how they function with Tif1γ to regulate blood cell development.
Materials and Methods {#s4}
=====================
{#s4a}
### Zebrafish and mouse strains and studies {#s4a1}
Zebrafish were maintained and staged as described ([@pbio-0020237-Westerfield1]). The alleles *mon^tb222b^* and *mon^tg234^* were generated in a large-scale screen for ENU-induced mutations ([@pbio-0020237-Ransom2]) on the TU, whereas the *mon^m262^* allele was derived on the AB strain and was originally called *vampire* ([@pbio-0020237-Weinstein1]). Mapping strains were constructed by mating to WIK or SJD polymorphic strains. Linkage analysis was performed on haploid or diploid embryos obtained from TU/SJD or TU/WIK hybrids. In situ hybridization and stainings of embryos were done as described ([@pbio-0020237-Thompson1]; [@pbio-0020237-Liao1]). In situ hybridization of mouse embryos was performed as described ([@pbio-0020237-Kingsley1]). Genomic DNA isolation, genotyping, AFLP analysis, and chromosomal walking were each performed as previously described ([@pbio-0020237-Brownlie1]; [@pbio-0020237-Ransom1]). A complete list of primers for genetic mapping, RT-PCR, and sequencing of *mon* are available on request.
### mRNA expression constructs, morpholinos, and microinjection {#s4a2}
The full-length *mon* cDNA was subcloned into EcoRI and XhoI sites in the pCS2^+^ vector. Synthetic mRNA was transcribed in vitro, and microinjection was performed essentially as described ([@pbio-0020237-Liao1]).
### Cell transplantation {#s4a3}
Whole kidney marrow cells were isolated from adult *gata1:EGFP* transgenic donors, resuspended in 0.9X phosphate-buffered saline + 5% fetal bovine serum, and injected into the sinus venosus of 2-d-old mon^tg234^ −/− and control embryos. Between 10^2^ and 10^3^ kidney marrow cells were injected per embryo. Individual transplanted embryos were anesthetized and visualized daily under an inverted fluorescent microscope (DM-IRE2; Leica, Wetzlar, Germany) for GFP^+^ cells over a span of 12 d. On day 13 posttransplant, all surviving larvae (12/129; 9%) were placed in tanks and monitored for survival.
### Antibodies, immunostaining, and immunoblots {#s4a4}
Antisera against the human C-terminal TIF1γ sequence RRKRLKSDERPVHIK was generated in rabbits (Genemed Synthesis, South San Francisco, California, United States) and affinity purified. Mouse embryonic fibroblasts grown on coverslips were immunostained with HP1α (Chemicon, Temecula, California, United States) and Tif1γ antisera simultaneously. In brief, cells were fixed in 4% paraformaldehyde for 5 min, washed with phosphate-buffered saline, and blocked with 5% serum (PBST) for 30 min. After incubation with the primary antibodies (PBST, 60 min) cells were washed three times with PBST and incubated with secondary antibodies (Jackson Laboratory, Bar Harbor, Maine, United States) followed by three washes in PBST. Cells were embedded with Vectashield/DAPI and analyzed using an Axioplan 2 microscope (Zeiss, Jena, Germany). Digital images were processed using the Volocity 1.0 software (Improvision, Lexington, Massachusetts, United States). G1E cell differentiation experiments were performed essentially as described ([@pbio-0020237-Weiss1]).
Supporting Information {#s5}
======================
Transplantation of wild-type zebrafish marrow cells carrying a *gata1:GFP* transgene into 2-d-old embryos reconstitutes erythropoiesis, but not viability, in mon^tg234^ homozygous mutants. Movies of live embryos at day 3 posttransplant highlight less than 100 GFP^+^ RBCs in circulation. Transplanted cells were observed to proliferate, resulting in thousands of donor-derived erythrocytes 7 d later. Movies present GFP-fluorescent images of live zebrafish larvae.
Video S1
::: {.caption}
###### Untransplanted Control *mon^tg234^* Homozygous Mutants Had No Fluorescent Cells in Circulation at 3 Days of Development
(13.7 MB MOV).
:::
::: {.caption}
######
Click here for additional data file.
:::
Video S2
::: {.caption}
###### One Day after Transplantation, Less Than 100 GFP^+^ Erythrocytes Were Visible in the Circulation of Three *mon^tg234^* Homozygous Mutants
(11.3 MB MOV).
:::
::: {.caption}
######
Click here for additional data file.
:::
Video S3
::: {.caption}
###### Untransplanted Control *mon^tg234^* Homozygous Mutants Had No Fluorescent Cells in Circulation at 9 Days of Development
(7.9 MB MOV).
:::
::: {.caption}
######
Click here for additional data file.
:::
Video S4
::: {.caption}
###### Seven Days after Transplantation, Thousands of Donor-Derived Erythrocytes Were Visible in the Circulation of a Representative *mon^tg234^* Homozygous Mutant
(11.2 MB MOV)
:::
::: {.caption}
######
Click here for additional data file.
:::
Accession Numbers {#s5a5}
-----------------
The GenBank (<http://www.ncbi.nlm.nih.gov/Genbank>) accession numbers for the genes and gene products discussed in this paper are fly bonus (AAF19646), human TIF1α (015164), human TIF1β (Q13263), human TIFγ (Q9UPN9), human TIF1γ (Q9UPN9), *mon* (AY59853), mouse Tif1α (Q64127), mouse Tif1β (AAH58391), and mouse Tif1γ (NP444400).
The cDNA sequences of zebrafish *mon/tif1γ* and *tif1α* have been deposited in GenBank under the accession numbers AY598453 and AY598454, respectively.
We thank A. Davidson, J. Amatruda, and J. Christian for critical review of this manuscript; J. Postlethwait and W. Talbot for helpful discussions and experimental advice; B. Weinstein for the gift of the *m262* allele of *mon;* and D. Giarla for administrative assistance. DGR was funded by the American Cancer Society and an award to Oregon Health and Science University by the Howard Hughes Medical Institute (HHMI) Biomedical Research Support Program for Medical Schools. LIZ and SHO are investigators of the HHMI. This work was supported by grants from the National Institutes of Health.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** DGR, NB, KN, DT, CB, NST, YZ, JP, SHO, and LIZ conceived and designed the experiments. DGR, NB, KN, DT, CB, NST, NPL, WJS, CAL, CH, BAB, and PDK performed the experiments. DGR, NB, KN, DT, CB, NST, NPL, YZ, JP, SHO, and LIZ analyzed the data. DGR, NB, KN, DT, NST, YZ, BAB, SL, and JP contributed reagents/materials/analysis tools. DGR, NB, KN, DT, and LIZ wrote the paper.
Academic Editor: William Talbot, Stanford University
¤Current address: Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, United States of America
Citation: Ransom DG, Bahary N, Niss K, Traver D, Burns C, et al. (2004) The zebrafish *moonshine* gene encodes transcriptional intermediary factor 1γ, an essential regulator of hematopoiesis. PLoS Biol 2(8): e237.
AFLP
: amplified fragment length polymorphism
ENU
: ethyl-nitrosourea
EST
: expressed sequence tag
GFP
: green fluorescent protein
hpf
: hours postfertilization
KRAB
: Krüppel-associated box
*mon*
: *moonshine;* PAC
PML
: promyelocytic leukemia gene product
RBCs
: red blood cells
*scl*
: *stem cell leukemia;* SSCP
TIF
: transcriptional intermediary factor
TU
: Tübingen background
| PubMed Central | 2024-06-05T03:55:46.763722 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509301/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e237",
"authors": [
{
"first": "David G",
"last": "Ransom"
},
{
"first": "Nathan",
"last": "Bahary"
},
{
"first": "Knut",
"last": "Niss"
},
{
"first": "David",
"last": "Traver"
},
{
"first": "Caroline",
"last": "Burns"
},
{
"first": "Nikolaus S",
"last": "Trede"
},
{
"first": "Noelle",
"last": "Paffett-Lugassy"
},
{
"first": "Walter J",
"last": "Saganic"
},
{
"first": "C. Anthoney",
"last": "Lim"
},
{
"first": "Candace",
"last": "Hersey"
},
{
"first": "Yi",
"last": "Zhou"
},
{
"first": "Bruce A",
"last": "Barut"
},
{
"first": "Shuo",
"last": "Lin"
},
{
"first": "Paul D",
"last": "Kingsley"
},
{
"first": "James",
"last": "Palis"
},
{
"first": "Stuart H",
"last": "Orkin"
},
{
"first": "Leonard I",
"last": "Zon"
}
]
} |
PMC509302 | Introduction {#s1}
============
Telomeres prevent the recognition of natural chromosome ends as double-stranded breaks (DSBs). When telomeres become dysfunctional due to shortening or loss of protective factors, chromosome ends activate a DNA damage response mediated (in part) by the ATM kinase ([@pbio-0020240-Karlseder1]; [@pbio-0020240-Takai1]). A major challenge in telomere biology is to define the mechanism by which functional telomeres prevent these events. Here we show that the human telomere-associated protein TRF2 is an inhibitor of the ATM kinase, suggesting a mechanism by which the telomeric protein complex prevents the activation of this DNA damage response transducer.
TRF2 is a small multimeric protein that binds to duplex telomeric (TTAGGG) repeats and recruits hRap1, ERCC1/XPF, WRN, and the Mre11/Rad50/Nbs1 complex to chromosome ends ([@pbio-0020240-Li1]; [@pbio-0020240-Zhu1], [@pbio-0020240-Zhu1] [@pbio-0020240-Zhu2]; [@pbio-0020240-Opresko1]; [@pbio-0020240-Machwe1]). TRF2 can be inhibited with a dominant-negative allele, TRF2^ΔBΔM^, which removes the endogenous TRF2 complex from telomeres ([@pbio-0020240-van1]). Upon expression of TRF2^ΔBΔM^, telomeres become uncapped and experience a series of deleterious events, including association with DNA damage response factors such as 53BP1, cleavage of the telomeric 3′ overhang by ERCC1/XPF, and telomere--telomere ligation by DNA ligase IV ([@pbio-0020240-van1]; [@pbio-0020240-de1]; [@pbio-0020240-Smogorzewska3]; [@pbio-0020240-Takai1]; [@pbio-0020240-Zhu2]). The DNA damage response to uncapped telomeres induces phosphorylation of DNA damage response proteins, including H2AX, SMC1, Rad17, CHK1, and CHK2, and upregulation of p53, p21, and p16, resulting in a G1 arrest ([@pbio-0020240-Karlseder1]; [@pbio-0020240-Smogorzewska1]; [@pbio-0020240-daAdda1]). Primary human cells with telomere damage undergo apoptosis or senescence ([@pbio-0020240-Karlseder1]; [@pbio-0020240-Smogorzewska1]).
An important transducer of the DNA damage signal is the ATM kinase (reviewed in [@pbio-0020240-Shiloh1]). ATM activation requires autophosphorylation on S1981 and concomitant dissociation into monomers, the presumed active form of the kinase ([@pbio-0020240-Bakkenist1]). DSBs and other genome stress lead to a rapid conversion of the ATM pool into active S1981--P monomers, which can phosphorylate regulators of the G1/S, intra-S, and G2/M cell cycle transitions ([@pbio-0020240-Bakkenist1]).
Activation of ATM also takes place in response to telomere damage. When telomeres become uncapped due to inhibition of TRF2, S1981-phosphorylated ATM associates with telomeres ([@pbio-0020240-Takai1]). Furthermore, ATM targets become phosphorylated in aging cells with shortened telomeres ([@pbio-0020240-daAdda1]). Genetic evidence for a role of ATM in the telomere damage pathway is provided by the diminished ability of ataxia telangiectasia (A-T) cells to mount a DNA damage response after telomere uncapping ([@pbio-0020240-Karlseder1]; [@pbio-0020240-Takai1]). However, several lines of evidence suggest that a second PIKK (phosphatidylinositol 3-kinase-like kinase), such as ATR or DNA-PKcs, can transduce the telomere damage signal in the absence of ATM ([@pbio-0020240-Takai1]; [@pbio-0020240-Wong1]).
One proposed mechanism of telomere protection is based on the ciliate telomere proteins, which envelop the single-stranded telomere terminus ([@pbio-0020240-Horvath1]). Such a protein cap, if sufficiently stable, could simply hide chromosome ends from the DNA damage surveillance machinery. Both budding and fission yeast also contain protective single-stranded telomere-binding proteins, but it is not known whether these proteins function similarly by forming a physical cap over the telomere terminus ([@pbio-0020240-Garvik1]; [@pbio-0020240-Baumann1]).
TRF2 must represent a different mechanism for telomere protection since it only binds to the duplex part of the telomere. TRF2 has been proposed to promote the formation of t-loops ([@pbio-0020240-Griffith1]; [@pbio-0020240-Stansel1]). In the t-loop configuration, the 3′ overhang of TTAGGG repeats is strand-invaded into the duplex part of the telomere. Although this could be an effective way to protect chromosome ends from nucleases and ligases, t-loops have several structural features resembling DNA lesions, including single strand to double strand transitions, 3′ and 5′ ends, and single-stranded DNA. Therefore, human telomeres may need additional mechanisms to circumvent checkpoint activation. The results presented here argue for a model in which TRF2 directly blocks activation of the ATM kinase.
Results {#s2}
=======
TRF2 was overexpressed in IMR90 primary fibroblasts using a retroviral vector. Under these conditions, TRF2 saturates its telomeric binding sites and is present in the nucleoplasm. While control IMR90 cells showed the expected reduction in mitotic index after ionizing radiation (IR), TRF2 overexpression partially abrogated this checkpoint response, increasing the percentage of cells entering mitosis from 0.3% to 8% ([Figure 1](#pbio-0020240-g001){ref-type="fig"}). The inappropriate entry into mitosis is indicative of a failure of the cell cycle checkpoints. Since cell cycle arrest after IR largely depends on ATM (reviewed in [@pbio-0020240-Shiloh1]), we asked whether TRF2 was acting on this kinase. Caffeine, an inhibitor of ATM and the related kinase ATR, suppressed IR-induced arrest to a similar extent as TRF2 ([Figure 1](#pbio-0020240-g001){ref-type="fig"}). Furthermore, caffeine had no additional effect on the ability of TRF2-overexpressing cells to bypass the DSB checkpoint, suggesting that TRF2 and caffeine target the same step in the response pathway.
::: {#pbio-0020240-g001 .fig}
Figure 1
::: {.caption}
###### TRF2 Inhibits the IR-Induced Cell Cycle Arrest
\(A) Retrovirally infected IMR90 cells were treated with 4 Gy IR (left and right) or treated with 4 Gy IR and exposed to caffeine (10 mM) directly after irradiation (middle). After 16 h, during which the cells were incubated in 1 μg/ml colcemide, the DNA was stained with DAPI and mitotic cells were identified by immunofluorescence with an antibody to phosphorylated histone H3.
\(B) Quantification of bypass of IR-induced cell cycle arrest. The mean percentage of phosphorylated histone H3-positive cells and SDs from three experiments are given. The low maximal incidence of phosphorylated H3-positive nuclei (approximately 18%) is due to loss of mitotic cells during processing; loss of mitotic cells occurred at the same level in control and experimental samples.
:::
:::
We then asked whether TRF2 overexpression inhibited other ATM-dependent readouts of the DNA damage response. ATM phosphorylates and stabilizes p53 in response to DNA damage (reviewed in [@pbio-0020240-Kastan1]). Quantitative immunoblotting showed that cells overexpressing TRF2 had a diminished ability to induce p53 after irradiation ([Figures 2](#pbio-0020240-g002){ref-type="fig"}A and [2](#pbio-0020240-g002){ref-type="fig"}B). Both the relative level of p53 protein and the induction of its downstream targets p21, Bax, and Hdm2 were dampened. By contrast, the p16/Rb pathway was not affected by TRF2 ([Figure 2](#pbio-0020240-g002){ref-type="fig"}A). We also examined the phosphorylation of Nbs1 on S343, a target of ATM ([@pbio-0020240-Gatei1]; [@pbio-0020240-Lim1]; [@pbio-0020240-Wu1]; [@pbio-0020240-Zhao1]). Phosphorylation of this residue causes a change in electrophoretic mobility shift and can also be detected using an antibody specific for the phosphorylated form of Nbs1. Extracts from irradiated control cells showed the previously reported retardation of Nbs1 and its reactivity with the S343--P-specific Nbs1 antibody ([Figure 2](#pbio-0020240-g002){ref-type="fig"}C). Both alterations could be reversed by phosphatase treatment of the Nbs1 immunoprecipitations (IPs). In contrast, irradiation did not appear to induce phosphorylation of Nbs1 in cells overexpressing TRF2, indicating that TRF2 diminished the ATM-dependent phosphorylation of Nbs1 ([Figure 2](#pbio-0020240-g002){ref-type="fig"}C).
::: {#pbio-0020240-g002 .fig}
Figure 2
::: {.caption}
###### Effect of TRF2 on Downstream Readouts of the IR-Induced ATM Response
\(A) Retrovirally infected IMR90 cells were exposed to 5 Gy IR and harvested after 0, 12, 24, and 36 h. Levels of p53, Bax, p21, Hdm2, p16, pRB, and γ-tubulin (loading control) were detected by immunoblotting of equal cell number equivalents.
\(B) Amount of p53 protein at the indicated timepoints (hours) was determined by densitometry of p53 immunoblots such as shown in (A). Amounts were normalized to the vector control at 0 h. Mean values from three experiments and standard deviations are shown.
\(C) Retrovirally infected IMR90 cells were exposed to 20 Gy IR and harvested after 45 min. Nbs1 was immunoprecipitated and subsequently detected by immunoblotting using a general Nbs1 antibody and a phosphospecific Nbs1 S343 antibody. IPs were treated with λ-phosphatase where indicated.
:::
:::
Because TRF2 overexpression blunted several cellular responses that depend (in part) on the ATM kinase, we determined TRF2\'s effect on the activation of ATM itself. Phosphorylation of ATM on S1981, an early and essential step in the activation of this kinase, can be detected rapidly after IR, even when a low level of DNA damage is induced ([@pbio-0020240-Bakkenist1]). To test whether TRF2 affected the autophosphorylation of ATM, the two proteins were expressed in 293T cells and ATM was activated with low doses of IR. TRF2 inhibited the activation of ATM as monitored by immunoblotting with an antibody specific for ATM S1981--P ([Figure 3](#pbio-0020240-g003){ref-type="fig"}A). The relative level of ATM S1981--P (normalized to total ATM protein) at 0.3 Gy was 49% of the vector control value (p = 0.002, Student\'s t test; n = 7). The TRF2 paralog TRF1, which also binds the duplex telomeric repeats, did not have a significant effect on ATM activation, demonstrating that the effect on ATM is specific to TRF2 ([Figure 3](#pbio-0020240-g003){ref-type="fig"}A). Overexpression of TRF2 also diminished the IR-induced ATM autophosphorylation of endogenous ATM in IMR90 fibroblasts to 55% of vector control value at 0.3 Gy and 60% of vector control value at 0.6 Gy ([Figure 3](#pbio-0020240-g003){ref-type="fig"}B).
::: {#pbio-0020240-g003 .fig}
Figure 3
::: {.caption}
###### Effect of TRF2 on IR-Induced ATM Phosphorylation
\(A) Overexpression of TRF2 inhibits IR-induced phosphorylation of transfected ATM in 293T cells. 293T cells co-transfected with ATM and either TRF2, TRF1, or vector were treated with the indicated doses of IR. After a 30-min recovery, cells were harvested and immunoblot analysis was performed on whole-cell lysates.
\(B) Overexpression of TRF2 inhibits IR-induced phosphorylation of endogenous ATM in primary fibroblasts. IMR90 primary fibroblasts infected with a retroviral construct expressing TRF2 or an empty virus were treated with the indicated doses of IR. After a 1 h recovery, cells were harvested and ATM was immunoprecipitated from whole-cell lysates. Immunoblot analysis was performed on immunoprecipitated ATM.
:::
:::
In order to understand the mechanism by which TRF2 inhibited ATM, we determined whether they interacted in vivo. IPs of the ATM kinase from primary human IMR90 fibroblasts resulted in recovery of a small fraction (approximately 1%) of endogenous TRF2 ([Figure 4](#pbio-0020240-g004){ref-type="fig"}A). This association was accentuated when TRF2 was overexpressed from a retroviral vector. TRF2 was not recovered in anti-ATM immunoprecipitates from A-T cells even when TRF2 was overexpressed ([Figure 4](#pbio-0020240-g004){ref-type="fig"}A), demonstrating that the recovery of TRF2 is dependent on the presence of functional ATM. The co-IP of TRF2 with ATM from IMR90 cells was resistant to the addition of ethidium bromide (data not shown), arguing that DNA tethering is not responsible for the association. A control IP with antibodies to the CycD1/Cdk4/Cdk6 kinase complex did not precipitate TRF2, and an irrelevant nuclear protein (Nova1), overexpressed in parallel in IMR90 cells, was not recovered in the ATM IP ([Figure 4](#pbio-0020240-g004){ref-type="fig"}A). The association of TRF2 with ATM was not dependent on the presence of DNA damage, since neither IR nor UV treatment enhanced the recovery of TRF2 in ATM IPs (data not shown).
::: {#pbio-0020240-g004 .fig}
Figure 4
::: {.caption}
###### TRF2 Interacts with the ATM Kinase In Vivo and In Vitro and TRF2 Does Not Localize to IRIF
\(A) Co-IP of TRF2 with ATM. Protein extracts from IMR90 cells and A-T cells (AG02496) infected with an empty virus or a TRF2-overexpressing virus were incubated with anti-ATM or anti-Cyclin D1 antibodies as indicated, and TRF2 was detected in the IP pellets by immunoblotting. The right panel represents IPs with anti-ATM antibodies from IMR90 cells infected with a retrovirus overexpressing Nova1 or the empty vector and detection of Nova1 by immunoblotting. For each extract 1% of the IP input (input) was processed for immunoblotting in parallel.
\(B) Bacterially expressed ATM--GST fusion proteins were purified on glutathione agarose beads and visualized by Western blotting with anti-GST antibody (Upstate Biotechnology \[Lake Placid, New York, United States\] \#06--332) (top). Unfused GST was run on a separate gel because of its low molecular weight. Equal amounts of fusion proteins and GST alone were incubated with purified baculoviral TRF2 (middle) or TRF1 (bottom), bound to glutathione beads, spun down, washed, and bound proteins were processed for immunoblotting with an anti-TRF1 or anti-TRF2 serum.
\(C) TRF2 does not localize to IRIFS. IMR90 primary fibroblasts infected with a retroviral construct expressing TRF2 or an empty virus were treated with 5 Gy IR. After a 90 min recovery, cells were fixed and processed for immunofluorescence with or without Triton X-100 extraction before fixation. Arrowheads denote foci of TRF2 signal previously demonstrated to represent telomeres. When overexpressed, some TRF2 is localized to nucleolus.
:::
:::
The nature of the association of TRF2 with ATM was explored further using in vitro pulldown experiments. GST-tagged fragments of ATM were tested for their ability to bind purified TRF2 protein expressed in a baculovirus system. In parallel, we used baculovirus-derived TRF1, which was previously shown to interact with ATM by co-IP ([@pbio-0020240-Kishi1]). TRF2 bound to two overlapping fragments of ATM spanning amino acids 1439 to 2138 ([Figure 4](#pbio-0020240-g004){ref-type="fig"}B). This region contains the FAT domain and S1981, the critical target of autophosphorylation ([@pbio-0020240-Bakkenist1]). TRF1 bound a different region of ATM ([Figure 4](#pbio-0020240-g004){ref-type="fig"}B), demonstrating the specificity of the observed interactions.
Blunting of the DNA damage response was observed when TRF2 was overexpressed throughout the nucleus. Because TRF2 is chiefly present at telomeres, the simplest interpretation is that the observed activity reflected a telomeric function. However, we also considered the possibility that TRF2 may have a heretofore clandestine role in the general DNA damage response. If this were true, TRF2 might be expected to localize to IR-induced foci (IRIF), where it would be in a position to modulate ATM. Previous data had shown that the endogenous TRF2 does not relocate from telomeres to IRIF ([@pbio-0020240-Zhu1]). Similarly, immunofluorescence analysis showed that overexpressed TRF2 did not form IRIF ([Figure 4](#pbio-0020240-g004){ref-type="fig"}C): the pattern of TRF2 localization was unchanged by IR, and there was no detectable colocalization with the known IRIF component 53BP1 ([@pbio-0020240-Schultz1]). This was also the case when TRF2 localization was examined in cells from which the nucleoplasmic proteins were extracted with a mild detergent ([Figure 4](#pbio-0020240-g004){ref-type="fig"}C). Thus, the inhibitory effect of TRF2 on ATM signaling does not reflect association of TRF2 with sites of DNA damage. Instead, we propose that the inhibition of ATM by TRF2 is an innate property of the protein, important at its natural location: telomeres. However, we cannot exclude the transient presence of TRF2 at DNA lesions and/or a role for TRF2 in the general DNA damage response.
Discussion {#s3}
==========
Natural chromosome ends require mechanisms to prevent the activation of the DNA damage response. Inhibition of the ATM kinase at human telomeres is particularly important since the telomeric complex contains the Mre11 complex, one of the DNA damage sensors of the ATM pathway ([@pbio-0020240-Carson1]; [@pbio-0020240-Petrini1]; [@pbio-0020240-Uziel1]). The telomeric protein TRF2 appears to play a central role in preventing telomeres from activating ATM. Removal of TRF2 from telomeres results in the localization of the active, phosphorylated form of ATM to unprotected chromosome ends ([@pbio-0020240-Takai1]) and induces ATM-dependent apoptosis ([@pbio-0020240-Karlseder1]). The data reported here are consistent with the hypothesis that TRF2 protects telomeres through a direct interaction with ATM that blocks its activation. As a result, TRF2 abrogates the downstream outcomes of the ATM-dependent DNA damage response, including phosphorylation of various ATM targets and cell cycle arrest.
We feel that the interaction of TRF2 with ATM is likely to be relevant to the mechanism by which TRF2 blocks ATM signaling. TRF2 binds ATM in a region surrounding S1981, which is functionally linked to the oligomerization state of ATM. Phosphorylation on S1981 occurs concomitant with the dissociation of ATM dimers (or oligomers), forming the monomeric, active form of the kinase ([@pbio-0020240-Bakkenist1]). TRF2 is also an oligomer of four to eight subunits, which are held together by the TRFH dimerization domain as well as other, yet to be defined, protein interactions ([@pbio-0020240-Broccoli1]; [@pbio-0020240-Fairall1]; [@pbio-0020240-Stansel1]). Owing to its oligomeric nature, TRF2 could potentially cross-link ATM monomers and thus hold the kinase in its inactive dimeric (or oligomeric) state. In this manner, TRF2 could abrogate the ATM pathway since it would block amplification of the ATM signal at an early step. In agreement with this idea, the in vitro GST pulldown experiments showed that TRF2 can interact with ATM when it is not phosphorylated on S1981. Because mutations in the TRF2 dimerization domain destabilize the protein, it has not been possible to test the contribution of TRF2 oligomerization on ATM repression directly. Alternatively, the interaction of TRF2 with the region surrounding S1981 may prevent ATM autophosphorylation or TRF2 binding could block a presumed interaction between ATM and a DNA damage sensor, such as the Mre11 complex. It is unlikely that TRF2 acts as an ATM target mimetic that titrates out genuine ATM targets since TRF2 is not a target of the ATM kinase (R. Drissi, M. B. Kastan, and J. Dome, unpublished data). Furthermore, TRF2 does not block ATM kinase activity in an in vitro assay (S. Kozlov, J. Karlseder, and M. F. Lavin, unpublished data). These findings are consistent with TRF2 acting at one of the earlier steps in the activation of the ATM kinase, including the interplay between ATM and DNA damage sensors, ATM autophosphorylation, or dissociation of ATM dimers.
In this study we have expressed TRF2 at high levels throughout the nucleus, whereas endogenous TRF2 is localized primarily to telomeres ([@pbio-0020240-van1]). Our estimates suggest that TRF2 is extremely abundant at telomeres. Human cells contain on the order of 1 million copies of TRF2 (X.-D. Zhu and T. de Lange, unpublished data), sufficient to position thousands of TRF2 molecules at each chromosome end. This number is consistent with the presence of thousands of TRF2-binding sites per telomere and the oligomerization potential of the protein. Thus, for every ATM kinase that could be activated at a chromosome end, there is a vast molar excess of its potential inhibitor, TRF2. Since TRF2 is specifically lodged at telomeres and remains there when DNA damage is induced, it is unlikely to interfere with activation of the ATM kinase at sites of DNA damage elsewhere in the genome. Hence, TRF2 could act as a telomere-specific inhibitor of ATM.
Previous studies have shown that overexpression of TRF2 can protect critically short telomeres generated by replicative aging ([@pbio-0020240-Karlseder2]). TRF2 reduced the incidence of end-to-end chromosome fusions in this setting and also delayed the onset of senescence. These findings suggested that senescence is induced by an altered telomere state, in which the telomere has become so short that the amount of TRF2 it can recruit is insufficient for the protection of the chromosome end. When TRF2 is overexpressed, this deficiency in TRF2 recruitment may be overcome. One possibility is that the altered state of critically short telomeres represents a situation in which telomeres have a diminished ability to form t-loops. The current findings raise the possibility that the altered state may also include a situation in which the telomere contains insufficient TRF2 to repress ATM. However, the ability of TRF2 to delay senescence was also observed in A-T cells ([@pbio-0020240-Karlseder2]), indicating that ATM repression is not the only pathway by which increased TRF2 loading can protect critically short telomeres.
As TRF2 can bind ATM, it has the inherent ability to recruit this protein to telomeres. ATM has not been observed at undamaged telomeres, but its abundance may be too low for detection. The idea that ATM could be recruited to telomeres by TRF2 is interesting considering that ATM-like kinases are necessary for telomere maintenance in Saccharomyces cerevisiae ([@pbio-0020240-Craven1]) and Schizosaccharomyces pombe ([@pbio-0020240-Matsuura1]). It is not excluded that human telomere maintenance similarly requires ATM. TRF2 could function to recruit ATM in an inactive form, perhaps allowing for highly regulated activation of ATM at appropriate times. Such regulation of DNA damage signaling and repair pathways at telomeres has been proposed previously in the context of the nonhomologous end-joining pathway and the role of the nucleotide excision repair endonuclease ERCC1/XPF ([@pbio-0020240-Smogorzewska3]; [@pbio-0020240-Zhu2]). In both cases, proteins with the potential to have detrimental effects on telomeres appear to be regulated such that their activities can be employed for telomere function.
Materials and Methods {#s4}
=====================
{#s4a}
### Cell culture and IR treatment {#s4a1}
IMR90 primary lung fibroblasts (ATCC, Manassas, Virginia, United States) and AG02496 and AG04405 primary A-T fibroblasts (Coriell Cell Repository, Camden, New Jersey, United States; PD 12 and PD 15) were grown and infected with retroviruses as described elsewhere ([@pbio-0020240-Karlseder2]). For γ-irradiation, 3 10^5^ cells were seeded in 5-cm culture dishes and exposed to a Ce~137~ source. Where indicated, the medium was replaced with medium containing 10 mM caffeine (Sigma, St. Louis, Missouri, United States) and 1 μg/ml colcemide (Sigma). Indirect immunofluorescence was performed as described ([@pbio-0020240-Smogorzewska2]; [@pbio-0020240-Takai1]).
### Co-transfection assay for S1981--P inhibition {#s4a2}
One day prior to transfection, approximately 5 10^6^ 293T cells were plated in 10-cm dishes. Cells were transfected with 1 μg of FLAG--ATM DNA ([@pbio-0020240-Canman1]) and 9 μg of N-terminally Myc-tagged TRF2 ([@pbio-0020240-Karlseder2]) or TRF1 in a pLPC vector backbone (gift of S. Lowe, Cold Spring Harbor Laboratory) or vector alone using CaPO~4~ coprecipitation. Two days after transfection, cells were harvested in media and divided into three equal fractions, which were exposed to 0, 0.3, or 0.6 Gy IR. Cells were allowed to recover for 30 min, washed with PBS, and resuspended in 250 μl of lysis buffer (50 mM Tris \[pH 7.4\], 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, with a complete mini-protease inhibitor tablet \[Roche, Basel, Switzerland\] per 10 ml). The NaCl concentration was raised to 400 mM, and the lysate was incubated on ice for 5 min. The NaCl concentration was reduced to 200 mM, cell debris was removed by centrifugation, and an equal volume of Laemmli buffer was added to the lysate.
### Immunoblotting {#s4a3}
For ATM immunoblots in the ATM S1981--P suppression assays: 40 μl of 293T cell lysate or ATM immunoprecipitated from approximately 5 10^6^ IMR90 fibroblasts were run on 7.5% precast polyacrylamide Bio-Rad (Hercules, California, UnitedStates) Ready Gels. PVDF Immobilon^TM^ Transfer Membrane (Millipore, Billerica, Massachusetts, United States) was prepared for protein transfer according to the manufacturer\'s instructions and the gel was transferred for 2 h at 90 V. Membranes were preincubated in 10% milk, 0.1% Tween-20 in PBS for 30 min at room temperature and subsequently incubated with primary antibodies: polyclonal rabbit ATM S1981--P ([@pbio-0020240-Bakkenist1]) and mouse monoclonal ATM antibody MAT3 (gift from Y. Shiloh) diluted in 0.1% milk, 0.1% Tween-20 in PBS overnight at 4 °C followed by three 10 min washes. Membranes were incubated for 45 min with HRP-conjugated secondary antibodies, washed, and developed using the ECL system (Amersham, Little Chalfort, United Kingdom). Immunoblots of Myc-tagged proteins (using Ab-1; Oncogene Research, Cambridge, Massachusetts, United States) in the ATM S1981--P suppression assays were performed as above, except that nitrocellulose (Schleicher and Schuell, Keene, New Hampshire, United States) filters were used. For all other immunoblots, cells were trypsinized, washed once with PBS, and subsequently lysed in Laemmli buffer at 10^4^ cells/μl. Lysates (10 μl) were separated on SDS-polyacrylamide gels (29:1 acrylamide: bisacrylamide, 8% for p53, TRF2, and γ-tubulin, 6% for pRB and Hdm2, 12% for Bax, p21, and p16) and transferred onto nitrocellulose membranes (Schleicher and Schuell) for 60 min at 90 V (Bio-Rad Mini-Protean II Cell). Membranes were preincubated in 10% nonfat dry milk, 0.1% Tween-20 in PBS for 30 min and subsequently incubated with primary antibodies: p53 D01 (Santa Cruz Biotechnology, Santa Cruz, California, United States); TRF2 serum 647 ([@pbio-0020240-Zhu1]); pRB \#554136 (PharMingen, Uppsalla, Sweden); Hdm2 \#3F3 (gift from, A. Levine); Bax \#sc-7480 (Santa Cruz Biotechnology); p21 sc-7480 (Santa Cruz Biotechnology); p16 \#15126E (PharMingen); γ-tubulin GTU88 (Sigma); Nova1 ([@pbio-0020240-Luque1]) in 5% dry milk, 0.1% Tween-20 in PBS overnight. Secondary antibody incubation and ECL were performed as described above. To quantify signals, band intensities were determined using an AlphaImager^TM^ 2200 using the SpotDenso function of AlphaEaseFC^TM^ Software Version 3.1.2 (Witec, Littau, Switzerland).
### IP {#s4a4}
For co-IP of ATM and TRF2, proteins were extracted from subconfluent cells by incubating trypsinized cells (approximately 10^7^ cells/0.1 ml buffer) in 20 mM HEPES (pH 7.9), 0.42 M KCl, 25% glycerol, 0.1 mM EDTA, 5 mM MgCl~2~, 0.2% NP40, 1 mM DTT, 0.5 mM PMSF, 1 μg/ml leupeptine, 1 μg/ml aprotinin, 10 μg/ml pepstatin on ice for 30 min. Debris was removed by centrifugation (14,000 rpm, 4 °C, 10 min). Protein concentration in the supernatant was determined using the Bradford assay and 400 μg of protein was diluted to 150 mM KCl and incubated for 20 min with 100 μl of protein G--Sepharose beads (Amersham), blocked with 1% fetal bovine serum in PBS. The beads were collected at 14,000 rpm for 1 min and the supernatant was incubated with 5 μg of anti-ATM antibody (AB3, Oncogene Research) or anti-Cyclin D1 antibody (\#sc-6281, Santa Cruz Biotechnology) for 1 h at 4 °C on a nutator. Protein G--Sepharose beads (30 μl) were added, and the mixture was incubated 1 h at 4 °C on a nutator. The beads were collected at 4,000 rpm at 4 °C, washed three times with wash buffer (150 mM NaCl, 1% NP40, 50 mM Tris \[pH 8.0\] with protease inhibitors as described above) by vortexing the suspension for 10 s. The beads were resuspended in Laemmli buffer and boiled 5 min, and proteins were separated by polyacrylamide gel electrophoresis. For IP of endogenous ATM from IMR90 fibroblasts for the phosphorylation assay, cells were resuspended in lysis buffer (50 mM Hepes \[pH 7.5\], 150 mM NaCl, 50 mM NaF, 1% Tween-20, 0.2% NP40, 1mM PMSF, with 1 complete mini-protease inhibitor tablet \[Roche, Basel, Switzerland\] ) and centrifuged at 13,000 rpm for 10 min. 400 μl of lysate was incubated with 30 μl of blocked protein G beads and 100 μl of D16.11 monoclonal supernatant ([@pbio-0020240-Alligood1]) for 1.5 h. Beads were washed once in lysis buffer and twice in RIPA buffer and resuspended in 60 μl of Laemmli buffer.
### Pulldown assays {#s4a5}
GST--ATM fusion plasmids ([@pbio-0020240-Khanna1]) were transformed into BL-21 cells. A 10-ml overnight culture was used to inoculate 500 ml of LB-Amp (50 μg/ml) and at OD~600~, 0.5--0.7, 0.3 mM IPTG (final) was added. After 3 h at 30 °C, cells were harvested, resuspended in 8 ml of lysis buffer (50 mM Tris \[pH 7.9\], 100 mM KCl, 1% Triton X-100, 2 mM DTT, 0.1 mM PMSF, 1 complete protease inhibitor tablet \[Roche\]) and sonicated three times for 30 s on ice. The lysate was cleared by centrifugation at 50,000 g at 4 °C and incubated with 600 μl of equilibrated glutathione beads for 2 h at 4 °C. The beads were washed three times for 10 min each (washes 1 and 3: PBS, 1% Triton X-100, 2 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 1 complete protease inhibitor tablet \[Roche\]; wash 2: 300 mM NaCl, 50 mM Tris \[pH 7.9\], 2 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 1 complete protease inhibitor tablet \[Roche\]) and a fourth time in wash 4 (50 mM Tris \[pH 7.9\], 100 mM KCl, 10% glycerol, 2 mM DTT, 0.1 mM PMSF). Fusion proteins were eluted in 500 μl of wash 4 containing 15 mM glutathione (reduced form). Two subsequent elutions were collected. Five micrograms of GST fusion proteins or GST alone were incubated with 2 μg of baculoviral TRF1 or TRF2 in binding buffer (150 mM NaCl, 100 mM KCl, 50 mM Tris \[pH 8.0\], 1% NP40, 0.1% SDS, 100 g/ml BSA) at 4 °C for 1 h. Glutathione beads (20 μl) were added and incubated for 1 h at 4 °C. Beads were collected by centrifugation at 5,000 rpm at 4 °C and washed three times for 10 min each with binding buffer, and bound protein was eluted by boiling the samples in Laemmli buffer. GST fusion proteins, TRF1, and TRF2 were detected by immunoblotting.
### Cell cycle arrest assay {#s4a6}
Cells were seeded on microscope coverslips and irradiated with 4 Gy of γ-irradiation as described above. Cells were incubated in growth medium containing 1 μg/ml colcemid for 16 h. Cells were fixed, and phosphorylated histone H3 was detected by indirect immunofluorescence using a phosphospecific antibody (6G3 monoclonal; Cell Signaling Technology, Beverly, Massachusetts, United States). Cells in mitosis were counted and expressed as a percentage of total cell number.
R. Darnell, Y. Shiloh, and M. Lavin are thanked for providing reagents. This work was funded by grants from the National Institutes of Health (NIH) to JHJP (GM59413 and GM56888), TdL (AG016642 and GM49046), and MK (CA71387 and CA21765) and by an Ellison Foundation Senior Scholar Award to TdL. JK was supported by fellowships from the Human Frontiers Science Program and the Charles H. Reversuson Foundation and by a grant from the L.Y. Mathers Charitable Foundation. KH was supported by NIH MSTP grant GM07739 to the Cornell/Rockefeller/Sloan--Kettering Tri-Institutional MD--PhD Program.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** JK, KMH, and TdL conceived and designed the experiments. JK, KMH, and OM performed the experiments. JK, KMH, JHJP, and TdL analyzed the data. JK, KMH, CB, MBK, JHJP, and TdL contributed reagents/materials/analysis tools. JK, KMH, JHJP, and TdL wrote the paper.
Academic Editor: Steve Elledge, Harvard Medical School
¤Current address: Regulatory Biology, The Salk Institute for Biological Studies, La Jolla, California, United States of America
Citation: Karlseder J, Hoke K, Mirzoeva OK, Bakkenist C, Kastan MB, Petrini JHJ, de Lange T (2004) The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response. PLoS Biol 2(8): e240.
A-T
: ataxia telangiectasia
DSB
: double-stranded break
IP
: immunoprecipitation
IR
: ionizing radiation
IRIF
: ionizing radiation-induced foci
| PubMed Central | 2024-06-05T03:55:46.766881 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509302/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e240",
"authors": [
{
"first": "Jan",
"last": "Karlseder"
},
{
"first": "Kristina",
"last": "Hoke"
},
{
"first": "Olga K",
"last": "Mirzoeva"
},
{
"first": "Christopher",
"last": "Bakkenist"
},
{
"first": "Michael B",
"last": "Kastan"
},
{
"first": "John H. J",
"last": "Petrini"
},
{
"first": "Titia",
"last": "de Lange"
}
]
} |
PMC509303 | Autoimmune diseases afflict a large segment of the population in Western countries. Many of them have been described, and rheumatoid arthritis, type I diabetes (also called insulin-dependent diabetes mellitus), multiple sclerosis, and systemic lupus erythematosus (SLE) are among the most common. Although tremendous progress has been made in disease management over the last decade, cures for these diseases have not yet been found. Consequently, a large research effort is sustained in this field. In addition, autoimmunity has intrigued basic immunologists since the early realization that the ability to discriminate self from non-self was at the core of the immune system\'s ability to protect an organism from pathogens while avoiding self-destruction. A failure of this mechanism results in autoimmune reactions that often lead to clinical disease. In spite of massive research efforts, the mechanisms by which autoimmune diseases develop are not clearly understood. Genetic predisposition as well as environmental triggers plays a role, but the identity of these factors has been largely elusive. The identification of the most common genetic and environmental factors that set off autoimmunity may lead to a better understanding of the ensuing pathogenesis, and offers the best hope for improved therapies, and ultimately, cures.
So far, animal models have proved the best way to probe the mechanisms of disease in general, and autoimmune diseases in particular. In the past few decades, the mouse has become the model of choice for experimental medicine, and the rat is following close behind. Starting in the early twentieth century at the Jackson Laboratory (Bar Harbor, Maine, United States), the production of inbred strains of mice and the systematic collection and characterization of naturally occurring mutants have created the building blocks on which much of the research using animal models is now based. Inbred strains are collections of genetically identical animals obtained through selective breeding. These strains have provided homogenous experimental groups, with interindividual variability reduced to environmental (and stochastic) factors. In addition, inbred strains have an assortment of distinct phenotypes that have then been exploited as models of human diseases.
Since the 1980s, techniques have been developed to manipulate the mouse genome. Specific genes now can be routinely over-expressed as transgenes, or eliminated by gene targeting, which creates a "knockout" ([@pbio-0020241-Smithies1]; [@pbio-0020241-Wassarman1]). This approach, known as reverse genetics, has shown itself to be a powerful tool with which to evaluate the role of individual genes in various biological processes. The use of genetically engineered mouse models will likely play a major role in deciphering the function of a multitude of new genes revealed in the recently completed sequence of the mouse genome ([@pbio-0020241-MGSC1]).
Interestingly, the percentage of mouse genes without any homolog currently detectable in the human genome (and vice versa) has been estimated to be less than 1%, a fact that strengthens the validity of mouse models of human diseases. However, one has to be careful in directly applying data obtained from animal models to human diseases. Most human autoimmune diseases show an extremely heterogeneous clinical presentation, which animal models present as simplified versions. A mouse model, as in any reductionist approach, is both inconvenient, because it provides only a partial representation of the real biological complexity underlying the human disease, and advantageous, because it is a more tractable tool with which to probe mechanistic issues. In addition, a number of differences exist between the human and rodent immune systems ([@pbio-0020241-Mestas1]). Since immune dysfunctions are at the root of autoimmune diseases, such differences may limit extrapolations from animal models to autoimmune patients.
Nonetheless, animal models are at the core of autoimmune research, and a large body of literature reflects the many advances brought by these models in terms of deciphering disease mechanisms. The relative lack of progress in certain human autoimmune diseases for which an animal model does not exist, such as neuropsychiatric lupus, corroborates the indispensable role played by animal models. Three basic types of animal models have been used in autoimmune research: spontaneous models, induced models, and genetically engineered models. The latter category has been further subdivided into transgenic and knockout strains ([Table 1](#pbio-0020241-t001){ref-type="table"}).
::: {#pbio-0020241-t001 .table-wrap}
Table 1
::: {.caption}
###### Examples of Common Mouse Models of Autoimmune Diseases
:::
:::
Spontaneous models were produced through fortuitous observations of clinical symptoms reminiscent of a given human autoimmune disease developing in a given mouse strain, or in crosses between mouse strains. This happened, for example, with the nonobese diabetic (NOD) mouse, which developed type 1 diabetes, and the hybrid between the New Zealand Black (NZB) and the New Zealand White (NZW) mouse, (NZB × NZW)F1, which developed a lupus-like disease.
Unfortunately, spontaneous models are not available for all human autoimmune diseases. Therefore, scientists have created induced models, often by exposing the animals to high doses of a suspected autoantigen at the same time as stimulating the immune system. Interestingly, marked differences exist between strains in their responses to these autoimmune inductions that reflect genetic variation associated with susceptibility to autoimmune diseases. For this reason, induced models have been limited to a small number of strains. For example, experimental autoimmune encephalomyelitis has been widely used as a model of multiple sclerosis. In this model, spinal cord homogenate or a protein derivative such as myelin basic protein is injected with a mixture of potent immunostimulants, most commonly in mice from the SJL strain. Another example of an induced model is collagen-induced arthritis, which has been used to study rheumatoid arthritis. In this model, type II collagen, a joint component, is injected also with immunostimulants, most commonly into mice from the DBA/1 strain.
Finally, the ability to turn specific genes on or off, in specific cell types and/or at specific times, has created a plethora of mouse models limited only by the immunologist\'s imagination. Genes suspected to play a role in the pathogenesis of various autoimmune diseases have been evaluated this way, and those studies have been very informative in mapping out functional pathways that are targeted in these diseases. However, these models have intrinsic problems that have become more apparent in the past few years, and require careful controls to avoid possible misinterpretation. These problems are a result of the fundamental way in which transgenic and knockout strains are produced. When a piece of DNA carrying the gene of interest is injected as a "transgene" into fertilized eggs, it integrates randomly into the genome, and in doing so, potentially modifies the expression of the gene it integrates into. Since all genetic studies have recognized that a large number of genes are involved in autoimmune disease susceptibility, the potential for a transgene to hit one of those susceptibility genes is not negligible. This potentially confounding factor is usually controlled by producing and comparing several independent transgenic lines.
More problematic is the interpretation of results obtained with knockout models. A gene is "knocked out" (KO) by homologous recombination of a disrupting piece of DNA within that gene. This genetic manipulation takes place in embryonic stem (ES) cells, which once mutated, are introduced into the inner cavity of a blastocyst (very early mouse embryo), creating chimeric embryos that are put back into female mice that carry the pregnancy to term. After multiple trials (and errors), ES cell lines from the 129 strain and blastocysts from the C57BL/6 (B6) strain have shown themselves to be superior in terms of efficiency and reliability. Consequently, this strain combination is at the origin of the overwhelming majority of knockout strains. Although the chimeric mice are usually backcrossed to B6 to dilute the contribution of the 129 genome, the knockout strains are always a mixture of the two genomes ([Figure 1](#pbio-0020241-g001){ref-type="fig"}). Most importantly, a large region flanking the KO gene remains of 129 origin, unless extreme measures are taken to select for recombination between tightly linked markers. There have been sporadic reports of phenotypes initially attributed to deficiency in the expression of a given gene that disappeared with additional backcrosses to B6. The only possible interpretation was that these phenotypes were in fact due to 129 alleles that were replaced by B6 alleles with further backcrossing.[](#pbio-0020241-g001){ref-type="fig"}
::: {#pbio-0020241-g001 .fig}
Figure 1
::: {.caption}
###### Breeding Strategy Usually Performed to Transfer a KO Allele from the 129 Genome to the C57BL/6 (B6) Genome
For clarity, only 4 of the 20 pairs of the mouse chromosomes are represented by black (B6) or red (129) bars. The KO allele is shown by a white box. Chimeric males are obtained from the integration of ES cells from the 129 strain that have been engineered to carry the KO allele to B6 blastocysts. These males are then bred to normal B6 females, resulting in an N1 progeny that is made up of 50% B6 and 50% 129 genome. N1 mice are subsequently "backcrossed" to B6, and their N2 progeny is selected for the presence of the KO allele. The contribution of 129 genome among the N2 progeny is normally distributed around a mean of 25%. This process can be repeated (shown here to N4), resulting in an average reduction of the 129 genome to one half of what it was in the previous generation. At any point in the process, homozygosity for the KO allele, which is necessary to prevent expression of that gene, can be obtained by intercrossing heterozygous mice, shown here at N4.
:::
:::
The production of autoantibodies and mild antibody-related renal pathology, highly relevant to autoimmune diseases, especially SLE, has been reported independently in four mouse models using the (129 × B6) genetic background ([@pbio-0020241-Obara1]; [@pbio-0020241-Botto1]; [@pbio-0020241-Bickerstaff1]; [@pbio-0020241-Santiago-Raber1]). It is generally accepted that genetic susceptibility to autoimmune diseases is conferred by multiple highly interactive genes that have small individual effects. In this context, the autoimmune phenotypes resulting from the combination of the 129 and B6 genomes may might therefore provide a primed background upon which the effects of deficiency in the target gene can be amplified. On the other hand, the autoimmune phenotype may be overwhelmingly contributed by the (129 × B6) genomic combination, with little if any effect of the deficiency of the KO gene.
In this issue of *PLoS Biology*, Marina Botto and her colleagues have tested this hypothesis by taking one of their knockout models for SLE, the serum amyloid P component deficient mouse (*Apcs* ^−/−^) ([@pbio-0020241-Bygrave1]). This group has published that *Apcs* ^−/−^ mice on a (129 × B6) genetic background develop a lupus-like disease, even after repeated backcrosses to B6 ([@pbio-0020241-Bickerstaff1]). Serum amyloid P component binds to debris generated from dying cells. Efficient removal of this debris has been shown to be critical to the prevention of the production of autoantibodies against intracellular material. *Apcs* was therefore an interesting candidate gene to evaluate. *Apcs*, however, is located in a region near the tip of mouse Chromosome 1 that is rich in SLE susceptibility loci and in genes that have been directly associated with SLE in humans ([@pbio-0020241-Wakeland1]). As mentioned above, the *Apcs* ^−/−^ mouse, although on a mostly B6 genomic background, has its entire *Apcs* flanking region replaced with 129 alleles. In a critical experiment, Botto and colleagues compared the autoimmune phenotypes of *Apcs* ^−/−^ mice to congenic mice (i.e., genetically identical, except for the gene of interest), carrying the same 129 region on Chromosome 1, but expressing *Apcs*. Amazingly, no difference was found between the two strains regarding the production of autoantibodies, clearly eliminating *Apcs* deficiency as a mechanism for this autoimmune process. *Apcs* deficiency was however associated with markedly increased renal damage, suggesting that this gene may be involved in preventing pathological consequences of autoantibody production.
It has been reported anecdotally that the most common outcome of a genetic knockout is a lupus-like disease. Botto and her colleagues may have identified the reason behind this somewhat surprising observation as a spurious consequence of the gene-targeting process. Gene targeting has been an invaluable tool in understanding the mechanisms of immunological diseases, and has still a very important role to play with increasingly sophisticated techniques of selective targeting. The immediate consequence of this work should be an increased scrutiny for appropriate controls, which may include congenic mice carrying the same 129 flanking region, but expressing the targeted gene ([Figure 2](#pbio-0020241-g002){ref-type="fig"}).[](#pbio-0020241-g001){ref-type="fig"}
::: {#pbio-0020241-g002 .fig}
Figure 2
::: {.caption}
###### Congenic Strains (Left and Right) of a Lupus-Prone Mouse (Middle). Image courtesy of Jessica Merritto
:::
:::
The past few years have shown that genetic susceptibility to autoimmune diseases involves a large number of genes with small individual contributions. In spite of this great complexity, advances have been made, and a small but growing number of susceptibility genes have been identified ([@pbio-0020241-Morahan1]). A common trait shared by successful studies has been the use of mouse models, either directly or indirectly. As the pace of genetic analysis increases in autoimmune diseases, and powerful tools have been created to navigate between the mouse and human genomes, the use of mouse models has been reaffirmed at multiple levels. Mouse models are used to discover new susceptibility genes that can then be assessed in patient populations, as well as to validate genes that have been directly identified in human genetic studies. Mouse models are also used to perform detailed functional and physiological analyses that cannot be conducted in humans. Finally, mouse models have been invaluable to screen disease-specific therapeutic agents.
Using mouse models has its pitfalls; many differences, both obvious and subtle, exist between mice and humans. Those differences are, however, outweighed by the power of the experimental system offered by the mouse. What the new study of Botto and colleagues reminds us is that the appropriate control is still crucial to meaningful data interpretation. Keeping that in mind, one can predict that many of the keys to human autoimmune diseases are still in the mouse room.
Laurence Morel is with the Department of Pathology, Immunology and Laboratory Medicine at the University of Florida College of Medicine, Gainesville, Florida, United States of America. E-mail: <morel@pathology.ufl.edu>
ES
: embryonic stem
KO
: knocked out
NOD
: non-obese diabetic
NZB
: New Zealand black
NZW
: New Zealand white
SLE
: systemic lupus erythematosus
| PubMed Central | 2024-06-05T03:55:46.769505 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509303/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e241",
"authors": [
{
"first": "Laurence",
"last": "Morel"
}
]
} |
PMC509304 | Introduction {#s1}
============
Tumor development and metastasis is a multistep process of somatic cell evolution that includes uncontrolled proliferation, impaired apoptosis, loss of differentiation, immortalization, neovascularization, invasion, and metastatic spread ([@pbio-0020242-Hanahan1]). This evolutionary transformation can be operationally divided into distinct stages, including initiation, promotion, progression, and metastasis ([@pbio-0020242-DiGiovanni1]). Mutations in both oncogenes and tumor suppressor genes are found in end-stage tumors, implying their causal role in tumor development. However, the association of mutations in specific genes with specific phenotypic states during tumor progression is poorly characterized for most human solid tumors. It is also largely unknown whether each mutation is an independent event or whether there is a preferred sequence or combination of mutations that is favored. The purpose of this study is to investigate the functional interactions between the mutational activation of the oncogene *Ras,* and two tumor suppressors, *p19 ^Arf^,* and *p53,* using a multistage epithelial tumor model.
*Ras* is among the most frequently mutated oncogenes in human cancer, with approximately 30% of tumors carrying an activating mutation in one of three family members, *Hras, Kras,* or *Nras* ([@pbio-0020242-Bos1]). Cancer-associated mutations in *Ras* result in constitutively active Ras protein. Ras is a nodal signaling molecule that regulates multiple signaling pathways, leading to profound changes in cellular proliferation, apoptosis, differentiation, senescence, cytoskeletal organization, adhesion, and migration ([@pbio-0020242-Campbell1]). Ras has also been shown to induce invasiveness and metastasis of cancer cells ([@pbio-0020242-Pozzatti1]; [@pbio-0020242-Webb1]; [@pbio-0020242-Varghese1]). These pleiotropic effects suggest Ras may influence multiple steps in tumor progression.
*p53* is the most frequently mutated tumor suppressor gene in human cancer, with more than 50% of tumors showing mutations ([@pbio-0020242-Hollstein1]). p53 is a nodal signaling protein that coordinates the cellular response to different types of stress, including oncogene activation, DNA damage, abnormal cell adhesion, altered ribonucleotide pools, hypoxia, and redox stress ([@pbio-0020242-Ko1]; [@pbio-0020242-Giaccia1]). These stress stimuli are thought to activate p53 by inducing posttranslational modifications that stabilize p53 and enhance its ability to act as a transcription factor ([@pbio-0020242-Siognov1]; [@pbio-0020242-Vousden1]). Loss of p53 function leads to loss of cell cycle checkpoints, impaired apoptosis, genomic instability, and tumor progression. However, a major unresolved issue is, of the many signals that have been shown to activate p53 using a variety of model systems, which one regulates p53 during autochthonous tumor progression.
A mechanistic connection between Ras signaling and activation of p53 that involves the tumor suppressor p19^Arf^ was recently established. p19^Arf^ (p14^Arf^ in humans; [@pbio-0020242-Stott1]) is encoded by the *p16^Ink4a^/p19^ Arf^* locus, but because it is transcribed in an alternative reading frame, the protein product is unrelated to the p16^Ink4a^ protein ([@pbio-0020242-Quelle1]; [@pbio-0020242-Kamijo1]). Deletions or mutations at the *p16^Ink4a^/p19^ Arf^* locus are frequently (more than 50% of cases) seen in human tumors ([@pbio-0020242-Ruas1]). p19^Arf^ was established as a bona fide tumor suppressor in studies showing that mice lacking p19^Arf^ are highly susceptible to spontaneous tumorigenesis ([@pbio-0020242-Kamijo1]). In vitro studies had shown that enforced expression of oncogenes such as *Ras, c-Myc,* and *E1A* activated p53 and induced growth arrest, senescence, or apoptosis depending on the cell type or oncogene used ([@pbio-0020242-Lowe1]; [@pbio-0020242-Hermeking1]; [@pbio-0020242-Serrano1]). These cellular responses were impaired in cells lacking p53, indicating that functional p53 was required. The involvement of p19^Arf^ was first suggested by experiments showing that enforced expression of Ras, Myc, and E1A in cells induced p19^Arf^, leading to G1 and G2 cell cycle arrest that was p53-dependent ([@pbio-0020242-Kamijo1]; [@pbio-0020242-Stott1]). Cells lacking p19^Arf^ showed impaired p53 induction in response to these oncogenes, and, like p53-deficient cells, escaped growth arrest and were immortalized ([@pbio-0020242-Zindy1]; [@pbio-0020242-de1]; [@pbio-0020242-Palmero1]; [@pbio-0020242-Lin1]; [@pbio-0020242-Ferbeyre1]). In vivo evidence linking oncogene signaling to p19^Arf^ and p53 was obtained in a lymphoma model. B-cell lymphomas from transgenic Eμ-myc mice also show a dependence on p19^Arf^ to activate p53, and Eμ-myc mice lacking either p19^Arf^ or p53 developed lymphomas much faster ([@pbio-0020242-Eischen1]; [@pbio-0020242-Schmitt1]). p19^Arf^ regulates p53 through mutual binding to the p53 regulator Mdm2. The levels of p53 in cells are normally kept low because of feedback regulation by the Mdm2 protein ([@pbio-0020242-Haupt1]; [@pbio-0020242-Kubbutat1]). Mdm2 binds to p53 and targets it for degradation by nuclear to cytoplasmic shuttling and through the E3 ubiquitin ligase activity of Mdm2 ([@pbio-0020242-Roth1]; [@pbio-0020242-Honda1]). p19^Arf^ sequesters Mdm2 from p53 and inhibits the ubiquitin ligase activity of Mdm2, resulting in increased stability and accumulation of p53 ([@pbio-0020242-Pomerantz1]; [@pbio-0020242-Tao1]; [@pbio-0020242-Weber1]; [@pbio-0020242-Zhang1]).
The importance of the Ras-p19^Arf^-p53 pathway in growth arrest was established in culture systems involving the ectopic overexpression of mutant Ras in both murine embryonic fibroblasts ([@pbio-0020242-Ferbeyre1]; [@pbio-0020242-Palmero1]) and primary epidermal keratinocytes ([@pbio-0020242-Lin1]). Both in vitro and in vivo models have established that the gene dosage of mutant *Ras* is critical for its oncogenic function. A single copy of mutant *Ras* is insufficient to transform cells; at least two mutant alleles are required ([@pbio-0020242-Finney1]). Duplication or even amplification of mutant *Ras* alleles is frequently observed in tumors ([@pbio-0020242-Quintanilla1]; [@pbio-0020242-Bremner1]). Ras activates multiple signaling pathways, and quantitative differences in Ras activity can lead to activation of different signals and qualitatively different cellular phenotypes ([@pbio-0020242-Shields1]). Cell culture conditions can add additional stress signals that are known to impinge on p19^Arf^ and p53, leading to cell cycle arrest ([@pbio-0020242-Sherr1]; [@pbio-0020242-Lowe2]), and cannot recapitulate the complex cellular ecology of tumor progression. Thus, to understand the interactions between Ras, p19^Arf^, and p53 that drive tumor progression, an autochthonous tumor model is required. Since more than 90% of human cancers are epithelial in origin, a carcinoma model system is favored.
Mouse skin carcinogenesis is perhaps the best-characterized in vivo model of epithelial neoplasia and was instrumental in establishing the concepts of initiation, promotion, and progression ([@pbio-0020242-DiGiovanni1]). The two-stage chemical protocol involves treatment of mice with a carcinogen, DMBA, followed by multiple applications of TPA. This treatment induces benign squamous cell papillomas, nearly 100% of which have sustained an AT mutation in codon 61 of *Hras* ([@pbio-0020242-Quintanilla1]). As this mutation results in constitutively active Ras protein, this protocol is ideal to study the biological consequence of Ras activation during the entire natural history of tumor progression. Papillomas consist of a series of folded epidermal or follicular hyperplasias that protrude from the skin surface. Papillomas have dysplastic characteristics including disturbed cell polarity, basal cell hyperplasia, disturbed maturational sequence, increased mitotic activity, and increased nuclear to cytoplasmic ratio ([@pbio-0020242-Yuspa1]). In most strains of mice, progression of these benign papillomas to malignant squamous cell carcinomas (SCCs) is a rare and late event. SCCs are usually endophytic tumors that present as plaques with an ulcerated surface. These tumors break through the basement membrane and progressively invade the underlying dermis and subcutaneous tissues, and rarely, can metastasize to regional and distant sites. SCCs are characterized by a disorderly proliferation of epithelial cells with increased cellular atypia and abnormal mitotic figures, and are classified into four grades: well-differentiated, moderately differentiated, poorly differentiated, and spindle cell carcinoma. The unique advantage of this skin tumor model is the ability to directly observe and quantify these evolutionary stages.
Loss of p53 function is strongly associated with the benign to malignant transition of chemically induced SCCs. Mutations in p53 are seen more frequently in carcinomas than in papillomas ([@pbio-0020242-Burns1]; [@pbio-0020242-Ruggeri1]). p53 knockout mice show accelerated malignant progression of SCCs ([@pbio-0020242-Kemp1]). The strongest association of p53 with malignant progression was revealed in *p53^+/−^* mice, in which loss of the remaining wild-type allele of *p53* was seen in carcinomas but not papillomas, indicating a strong selective pressure to completely inactivate p53 during this transition. Accelerated malignant progression seen in the absence of p53 was accompanied by extensive loss of differentiation and lymph node metastasis, indicating that p53 inhibits multiple steps involved in malignant tumor progression.
Here, we used the mouse skin tumor model to examine the role of p19^Arf^ in regulating the levels and tumor suppressor activity of p53. In addition, we addressed the biological and functional significance of alterations in p19^Arf^, p53, or both during tumor initiation, promotion, progression, and metastasis. Similar to *p53*-null mice, loss of *p19^ Arf^* resulted in increased malignant conversion, more aggressive tumors, and frequent and rapid metastasis. However, in contrast to *p53*-null mice, *p19 ^Arf^*-null mice had greater tumor numbers and tumor growth rates, indicating additional, p53-independent tumor suppressor functions for p19^Arf^.
Results {#s2}
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Increased Papilloma Number and Size in p19^Arf^-Deficient Mice {#s2a}
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Both in vitro and in vivo studies have demonstrated that p19^Arf^ is a tumor suppressor ([@pbio-0020242-Eischen1]; [@pbio-0020242-Kamijo2]; [@pbio-0020242-Schmitt1]; [@pbio-0020242-Lin1]). However, other than regulation of p53, little else is known about the role of p19^Arf^ in tumor suppression. To address this, groups of *p19^ Arf+/+^*, *p19^ Arf^* ^+/−^, and *p19^ Arf−^* ^/−^ littermates were treated with a single dose of DMBA followed by twice weekly application of TPA for 15 wk (see [Materials and Methods](#s4){ref-type="sec"}). Papillomas began to appear in all three genotypes after 9 wk of promotion. By 20 wk, *p19^ Arf+/−^* and *p19^ Arf−/−^* mice showed a significant increase in papilloma number and size compared to wild-type mice ([Figure 1](#pbio-0020242-g001){ref-type="fig"}A). Relative to wild-type littermates, *p19^ Arf−/−^* mice had an average of 2.97 more papillomas (95% CI (0.70, 5.24); *p* = 0.010) and *p19^ Arf+/−^* mice had an average of 2.60 more papillomas (95% CI (0.05, 5.14); *p* = 0.045) in weeks 18--30 after DMBA administration. Average papilloma size was also greater in both *p19^ Arf−/−^* and *p19^ Arf+/−^* mice compared to wild-type mice ([Figure 1](#pbio-0020242-g001){ref-type="fig"}B). This effect was seen as early as 12 wk and increased through time so that by 28 wk, 33% (47/141) of papillomas from *p19^ Arf−/−^* mice were greater than 8 mm in diameter versus 14% (38/267) from wild-type mice (*p* \< 0.0001). Papillomas from *p19^ Arf^*-deficient mice measured up to 16 mm in diameter while very few papillomas on wild-type mice measured more than 9 mm. Thus, *p19^ Arf^* deficiency resulted in faster growing papillomas, indicating a role for *p19^ Arf^* in regulating the early stages of benign tumor growth.
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Figure 1
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###### Skin Tumor Multiplicity, Size, and Progression in *p19^ Arf^*-Deficient Mice
\(A) Average number of papillomas (more than 2 mm in diameter) per mouse is plotted versus the number of weeks postinitiation. Both *p19^ Arf^ (Arf)^+/−^* and *p19^ Arf−/−^* mice show greater numbers of tumors than *p19^ Arf+/+^* mice.
\(B) Comparison of papilloma size (in mm) between *p19^ Arf+/+^, p19^ Arf+/−^,* and *p19^ Arf−/−^* mice through 28 wk postinitiation. An increase in the largest size class of tumors is seen in *p19^ Arf+/−^* and *p19^ Arf−/−^* mice but not *p19^ Arf+/+^* mice*.*
\(C) Percentage of mice bearing at least one carcinoma is plotted versus the number of weeks postinitiation*. p19^ Arf−/−^* mice show the shortest latency and greatest incidence of carcinoma conversion, with *p19^ Arf+/−^* mice showing an incidence between the *p19^ Arf−/−^* and *p19^ Arf+/+^* mice. Time of appearance of lymph node metastasis is noted above the graph as a vertical line for each mouse analyzed. Metastasis to lymph node occurred frequently and sooner in *p19^ Arf^*-deficient mice than in wild-type mice.
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Mutations in *Hras* are found in more than 95% of DMBA/TPA-induced skin tumors ([@pbio-0020242-Quintanilla1]; [@pbio-0020242-Kemp1])*.* In vitro studies showed that mutant Ras induces p19^Arf^, which, in turn, inhibits Ras-induced proliferation ([@pbio-0020242-Sherr2]). Thus, loss of p19^Arf^ might reduce or eliminate the need to mutate *Ras*. All papillomas from *p19^ Arf+/+^* (5/5), *p19^ Arf+/−^* (4/4), and *p19^ Arf−/−^* (5/5) mice contained the identical A→T transversion at codon 61 of *Hras,* resulting in an amino acid change from glutamine to leucine and a constitutively activated Ras protein ([@pbio-0020242-Quintanilla1]). Thus, mutation of *Ras* is very strongly selected for during epithelial carcinogenesis, with or without the presence of p19^Arf^, and loss of p19^Arf^ cooperates with activated Ras to accelerate tumor growth.
Increased Malignant Progression and Metastasis in p19^Arf^-Deficient Mice {#s2b}
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The rate of malignant conversion of papillomas to carcinomas is greatly increased in the absence of p53 function ([@pbio-0020242-Kemp1]). To determine if loss of p19^Arf^ had a similar effect, progression was quantified by visual inspection and confirmed by histologic analysis. The rate of conversion from papillomas to carcinomas was dramatically accelerated in p19^Arf^-deficient mice. Carcinomas developed in *p19^ Arf−/−^* mice as early as 14 wk after initiation, whereas papillomas from *p19^ Arf+/+^* mice began to convert much later, after 22 wk ([Figure 1](#pbio-0020242-g001){ref-type="fig"}C). By 28 wk, 100% of the *p19^ Arf−/−^* mice had at least one carcinoma, compared to only 25% of the wild-type mice. The *p19^ Arf+/−^* mice showed an intermediate conversion rate, with 60% of the mice bearing at least one carcinoma, indicating an p19^Arf^ gene dosage effect on malignant progression. In addition to reducing the latency, p19^Arf^ deficiency increased the frequency of malignant conversion. The odds of developing a carcinoma within 30 wk after DMBA administration was 8.10 times higher for *p19^ Arf−/−^* mice (95% CI (1.90, 34.56); *p* = 0.005) and 3.11 times higher for *p19^ Arf+/−^* mice (95% CI (0.90, 10.77); *p* = 0.073) compared to wild-type mice. The reduced carcinoma latency and increased conversion frequency in the *p19^ Arf^*-null mice implicate loss of p19^Arf^ as a critical rate-limiting step in malignant SCC progression.
Histologic analysis revealed that the carcinomas from control mice ranged in grade from well-differentiated to poorly differentiated SCCs. Carcinomas from *p19^ Arf+/−^* and *p19^ Arf−/−^* mice also showed a range of grades but a significant number (9/12) were characterized as spindle cell carcinomas. These were characterized by packed and spindle-shaped cells with elongated pleiomorphic nuclei and abundant abnormal mitotic figures. These cells grew in a homogenous pattern with very little evidence of the cellular organization typical of low-grade tumors. These tumors showed focal areas of squamous differentiation, indicating that they were derived from squamous epithelium.
*p19^ Arf^* deficiency also increased dissemination and establishment of metastatic SCCs. Carcinoma-bearing p19^Arf^-deficient mice frequently presented with enlarged lymph nodes, and in several cases tumors were noted on the lungs. ([Table 1](#pbio-0020242-t001){ref-type="table"}; [Figure 2](#pbio-0020242-g002){ref-type="fig"}). Histologic analysis revealed that these lymph nodes and lung tumors contained cells with features similar to the primary SCCs, including squamous differentiation, keratin pearls, high mitotic index, nuclear pleomorphism, and disturbed cell polarity ([Figure 2](#pbio-0020242-g002){ref-type="fig"}D and [2](#pbio-0020242-g002){ref-type="fig"}E). Immunostaining with a keratin-specific antibody showed that these cells were epithelial in origin, confirming that they were metastatic SCCs ([Figure 2](#pbio-0020242-g002){ref-type="fig"}F and [2](#pbio-0020242-g002){ref-type="fig"}G). 60% of local enlarged lymph nodes from carcinoma-bearing *p19^ Arf−/−^* mice contained such squamous carcinoma deposits, compared to 10% of those from wild-type mice ([Table 1](#pbio-0020242-t001){ref-type="table"}). Metastatic lesions from *p19^ Arf^*-deficient mice were seen as early as 16 wk after initiation and must have occurred very soon after or simultaneously with papilloma to carcinoma conversion (see top of [Figure 1](#pbio-0020242-g001){ref-type="fig"}C). In contrast, only one metastatic lesion was seen in one *p19^ Arf+/+^* mice through 36 wk of observation. *p19^ Arf+^* ^/−^ mice displayed an intermediate frequency of metastasis. Newly formed blood vessels, some measuring up to 2 mm in diameter, were seen on the underside of each tumor and appeared to lead directly to the inguinal or brachial lymph node ([Figure 2](#pbio-0020242-g002){ref-type="fig"}A and [2](#pbio-0020242-g002){ref-type="fig"}B). Several primary *p19^ Arf^*-deficient carcinomas showed clear evidence of penetration of tumor cells through blood vessel walls, with intravascular rafts of tumor cells seen ([Figure 2](#pbio-0020242-g002){ref-type="fig"}C), indicating a route by which tumor cells could migrate to distant organs through the circulation. Thus, in addition to increasing benign tumor growth, loss of p19^Arf^ accelerated both benign to malignant conversion and metastatic spread of epithelial tumors. Tumors lacking p19^Arf^ have a higher potential for metastatic spread.
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Figure 2
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###### Metastasis of Primary SCC to Lymph Nodes and Lungs in *p19^ Arf^*-Deficient Mice
\(A) Underside of skin from tumor-bearing mouse shows newly formed blood vessels surrounding tumor site (arrow) and leading to inguinal lymph node (arrowhead).
\(B) Enlarged inquinal lymph node (left) containing metastatic SCC and blood vessel formation (arrow) compared to normal lymph node (right).
\(C) H&E stain of carcinoma section with prominent blood vessel (bv). Carcinoma cells (ca) have penetrated blood vessel wall (arrow).
\(D) H&E stain of lymph node bearing infiltrating SCC cells (arrow) among normal lymphocytes (arrowhead).
\(E) H&E stain of lymph node bearing metastatic differentiated SCC.
\(F) Immunostain with pan-keratin antibody of papilloma.
\(G) Immunostain with pan-keratin antibody of lymph node with metastatic SCC.
(H and I) H&E stain of normal lung (arrowhead) with large metastatic SCC deposit (arrow).
\(J) H&E stain of lung metastasis with secondary site of infiltration (arrow).
(D--G, J): 20× magnification. Inserts in (E--G): 40× magnification.
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::: {#pbio-0020242-t001 .table-wrap}
Table 1
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###### Metastatic Frequency of SCC
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Denominator indicates number of tissues examined. Numerator indicates number of tissues bearing SCC
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Reduced p53 Expression in Papillomas from p19^Arf^-Deficient Mice {#s2c}
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The p53 expression in DMBA/TPA-induced papillomas is increased relative to adjacent normal skin ([@pbio-0020242-Kemp2]). As multiple signals can lead to the accumulation of p53, including activated oncogenes, DNA damage, and hypoxia, it was not clear which was operative in this setting. As nearly all carcinogen-induced papillomas carry mutations in *Hras,* we questioned whether increased p53 expression was due to signaling from Ras through p19^Arf^. Western blot analysis of nuclear lysates showed increased levels of both p19^Arf^ and p53 in wild-type papillomas compared to normal skin ([Figures 3](#pbio-0020242-g003){ref-type="fig"} and [4](#pbio-0020242-g004){ref-type="fig"}). In contrast, p53 expression was not detectable in papillomas from *p19^ Arf−^* ^/−^ mice and was intermediate and variable in papillomas from *p19^ Arf+/−^* mice. Immunostaining of paraffin-embedded sections confirmed the Western analysis, with nuclear staining of p53 detected in the epidermal cells of papillomas from wild-type mice, reduced numbers of p53-positive cells in the *p19^ Arf^* heterozygous papillomas, and undetectable p53 staining in *p19^ Arf^*-null papillomas ([Figure 3](#pbio-0020242-g003){ref-type="fig"}B). To determine if p53 could still be induced in the absence of p19^Arf^ by an alternative pathway, tumor-bearing mice were irradiated with 4Gy ionizing radiation and sacrificed 4 h later, and their tissues were examined for p53 expression. Both Western blot analysis and immunostaining revealed prominent induction of p53 in basal cells of normal skin and papillomas from both wild-type and *p19^ Arf^*-null mice ([Figure 3](#pbio-0020242-g003){ref-type="fig"}A and [3](#pbio-0020242-g003){ref-type="fig"}B). Thus, the induction of p53 seen in mutant Ras-containing tumors is due to signaling through p19^Arf^. These results provide in vivo confirmation of the model, largely derived from in vitro studies, that posits that signaling from mutant Ras acts through p19^Arf^ to induce p53. Other pathways to activate p53, such as those initiated by DNA damage, remain functional in the absence of p19^Arf^.
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Figure 3
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###### Reduced p53 Expression in Skin Tumors from *p19^Arf^*-Deficient Mice
\(A) Western blot analysis of nuclear lysates from skin tumors from *p19^ Arf^ (Arf)^+/+^, p19^ Arf+/−^,* and *p19^ Arf−/−^* mice using p53-specific antibody. PA, papilloma; skin IR, irradiated normal skin
\(B) p53 immunostain of paraffin-embedded skin tumor sections from *p19^ Arf+/+^, p19^ Arf+/−^,* and *p19^ Arf−/−^* mice (arrows indicate positive stained cells) (top). p53 immunostain of irradiated papillomas (IR) from *p19^ Arf+/+^* and *p19^ Arf−/−^* mice (bottom). p53 is not detected in normal skin or tumors from *p19^ Arf−/−^* mice, but is induced by irradiation in both normal and tumor cells from *p19^ Arf−/−^* mice.
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Figure 4
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###### LOH of Wild-Type *p19^Arf^* Allele in *p19^Arf+/−^* Tumors
\(A) LOH analysis by semiquantitative PCR of the wild-type *p19^Arf^* allele in *p19^Arf+/−^* papillomas and carcinomas. Gradient made from kidney DNA used for quantitation of wt/mu ratio (top row). wt, wild-type allele; mu, knockout allele; asterisk, loss or reduction of *p19^Arf^* wild-type band.
\(B) Western blot analysis of nuclear lysates from papillomas (PA) and carcinomas (CA) from *p19^Arf+/+^, p19^Arf+/−^,* and *p19^Arf−/−^* mice.
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Loss of the Wild-Type *p19^Arf^* Allele in Tumors from *p19^Arf+/−^* Mice Occurs During Benign to Malignant Conversion {#s2d}
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*p19^ Arf+/−^* mice displayed an intermediate rate of papilloma to carcinoma conversion (see [Figure 1](#pbio-0020242-g001){ref-type="fig"}C). Two genetic models could explain this heterozygous phenotype. *p19^ Arf^* could be haploinsufficient, in which case no mutation or loss of heterozygosity (LOH) should be seen in the remaining wild-type *p19^ Arf^* allele in carcinomas. Alternatively, *p19^ Arf^* could be recessive, in which case LOH or reduction to a homozygous null state would be expected. The fate of the wild-type allele of *p19^ Arf^* in tumors from heterozygous mice was assessed by semiquantitative PCR analysis of genomic DNA. Three of 15 (20%) papillomas examined showed evidence of loss of the wild-type *p19^ Arf^* allele, compared to ten of 15 (67%) carcinomas (*p* = 0.0027) ([Figure 4](#pbio-0020242-g004){ref-type="fig"}A), indicating LOH occurs primarily during malignant conversion. We next examined p19^Arf^ expression in tumor lysates by Western blot analysis with a p19^Arf^-specific antibody. In wild-type mice, p19^Arf^ protein was elevated in all papillomas and three of six carcinomas compared to normal skin ([Figure 4](#pbio-0020242-g004){ref-type="fig"}B). Increased expression of p19^Arf^ is consistent with activation of Ras in these tumors. p19^Arf^ expression was also increased in papillomas from *p19^ Arf+/−^* mice but not to the levels seen in wild-type mice, indicating that p19^Arf^ protein levels in tumors reflect *p19^ Arf^* gene dosage. p19^Arf^ protein was reduced or undetectable in four of six carcinomas from *p19^ Arf+/−^* mice, consistent with the LOH data. Collectively, these data indicate that p19^Arf^ expression is induced in tumors. Germline deletion of one *p19^ Arf^* allele provides a selective advantage during early tumor growth, and loss of the second allele confers an additional phenotype, destabilization of p53, and enhanced malignant progression.
Independent Contributions of p19^Arf^ and p53 to Tumorigenesis {#s2e}
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The observations that p19^Arf^ and p53 were upregulated in papillomas, that p53 expression was reduced in *p19^ Ar^* ^f^-null papillomas, and that loss of p19^Arf^ had a similar effect on tumor progression as that of loss of p53, provide strong in vivo support of the model whereby p19^Arf^ regulates p53 in response to mutational activation of *Hras*. However, enhanced tumor growth in *p19^ Arf^*-null mice, in contrast to reduced tumor growth in *p53*-null mice ([@pbio-0020242-Kemp1]), suggests additional tumor suppressor functions of p19^Arf^, independent of p53. To examine the effect of the combined loss of p53 and p19^Arf^ tumor suppressors, skin tumors were induced in *p19^ Arf^* and *p53* single and compound mutant littermates. Relative to wild-type mice, *p53*-null mice developed fewer tumors, averaging 4.05 fewer papillomas (95% CI (−6.10, −2.00); *p* = 0.0001) 10--16 wk after the DMBA administration, while *p19^ Arf^*-null mice averaged 2.68 more papillomas (95% CI (0.52, 4.84); *p* = 0.015) 18--40 wk after the DMBA administration ([Figure 5](#pbio-0020242-g005){ref-type="fig"}A). The *p19^Arf^ /p53* double-null mice showed a papilloma multiplicity similar to wild-type mice. *p53* ^−/−^ tumors were also smaller, while both *p19^Arf−/−^* and *p19^Arf−/−^p53^−/−^* tumors were larger compared to wild-type tumors. p19^Arf^ and p53 also affected tumor size and morphology. Wild-type papillomas were highly exophytic, while tumors from both *p19^ Arf^-* and *p53-*deficient mice grew in a flatter, endophytic pattern ([Figure 5](#pbio-0020242-g005){ref-type="fig"}B). Thus, loss of p19^Arf^ increased the number and size of both wild-type *p53* and *p53*-null tumors, demonstrating that p53 and p19^Arf^ contribute independently to the early stages of tumor development.
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Figure 5
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###### Tumor Multiplicity and Proliferative Index in *p19^ Arf^ /p53* Compound Mutant Mice
\(A) Average number of papillomas (more than 2 mm in diameter) per mouse is plotted against the number of weeks post-initiation.
\(B) Image of wild-type, *p19^ Arf^ (Arf)^−/−^, p53^−/−^,* and *p19^Arf−/−^p53^−/−^* mice with skin tumors at time of sacrifice. Wild-type mice show large exophytic tumors, while both *p19^ Arf^*- and p53-deficient mice have endophytic tumors. Note larger tumors in *p19^Arf^ /p53* compound mutant mice relative to *p53* single mutants.
\(C) BrdU-positive cells in papillomas from wild-type*, p53^−/−^, p19^ Arf−/−^,* and *p19^ Arf−/−^p53^−/−^* mice at 10 wk postinitiation. (Bars represent average counts ± standard deviation from ten fields and five mice). *p53^−/−^* tumors show significantly fewer BrdU-positive cells than either *p19^ Arf−/−^* or wild-type tumors (*p* \< 0.05, Wilcoxon one-sided t-test).
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To determine whether tumor growth in mice lacking p19^Arf^ or p53 is due to altered proliferation, additional cohorts of mice were treated with DMBA/TPA. Tumor-bearing mice 8--10 wk post DMBA treatment were injected with BrdU and sacrificed 1 h later. *p53*-null papillomas showed a reduced BrdU labeling index compared to wild-type mice, while the *p19^ Arf^*-null tumors showed a higher BrdU labeling index ([Figure 5](#pbio-0020242-g005){ref-type="fig"}C). The *p19^ Arf^ /p53* double-null papillomas showed a proliferative index similar to that of wild-type tumors. Thus, decreased proliferation contributes to the reduced tumor growth seen in the *p53*-null mice. Apoptotic cells were very rare in papillomas regardless of *p53* genotype or radiation exposure (apoptotic index over 40-fold less than proliferation index) (unpublished data). Thus, p53-regulated apoptosis does not appear to play a major role in SCC development, at least at the papilloma stage.
Tumor Progression in *p19^Arf^ /p53* Compound Mutant Mice {#s2f}
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To determine if p19^Arf^ and p53 cooperate during tumor progression, papilloma to carcinoma conversion was evaluated in *p19^Arf−/−^, p53^−/−^,* and *p19^Arf−/−^p53^−/−^* littermates. Tumor progression was accelerated in all *p19^ Arf^-* and *p53*-deficient genotypes compared to wild-type littermates ([Figure 6](#pbio-0020242-g006){ref-type="fig"}A). Carcinoma latency and multiplicity was almost identical for *p19^ Arf−/−^* mice regardless of *p53* genotype *(p53^+/+^, p53^+/−^,* or *p53^−/−^)* ([Figure 6](#pbio-0020242-g006){ref-type="fig"}A), indicating no cooperation between p19^Arf^ and p53 for malignant conversion per se. However, the size of carcinomas in both *p19^ Arf−/−^* and *p19^ Arf−/−^p53^−/−^* mice was considerably greater than that seen in *p53^−/−^* mice at comparable time points (see [Figure 5](#pbio-0020242-g005){ref-type="fig"}B). This confirms a significant impact of p19^Arf^ on suppressing tumor growth that does not require p53.
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Figure 6
::: {.caption}
###### Tumor Progression and *p53* LOH in *p19^Arf^ /p53* Compound Mutant Mice
\(A) Average number of carcinomas per mouse is plotted against the number of weeks postinitiation. Tumor progression was accelerated in all *p19^ Arf^ (Arf)-* and *p53*-deficient genotypes compared to wild-type littermates*.* Carcinoma latency and multiplicity was almost identical for *p19^ Arf−/−^* mice regardless of *p53* genotype (*p53^+/+^, p53^+/−^,* or *p53^−/−^*).
\(B) LOH of the wild-type *p53* allele by semiquantitative PCR in *p19^ Arf^ /p53* compound tumors. Gradient made from kidney DNA used for quantitation of wt/mu ratio (top row). wt, wild-type allele; mu, knockout allele; asterisk, tumors with reduction of wild-type *p53.*
:::
:::
In *p53^+/−^* mice there is strong selective pressure to lose the wild-type allele during conversion to malignancy ([@pbio-0020242-Kemp1]). As p19^Arf^ regulates p53, we next wished to determine if the selective pressure to lose p53 was reduced in the absence of p19^Arf^. Tumors from *p53^+/−^* mice of all three *p19^ Arf^* genotypes *( p19^ Arf+/+^, p19^ Arf+/−^,* and *p19^ Arf−/−^)* were assessed for LOH of *p53* by semiquantitative PCR analysis of genomic DNA. Two out of seven papillomas from *p19^ Arf+/+^p53^+/−^* mice show loss of the remaining *p53* allele, while all papillomas examined from *p19^ Arf+/−^p53^+/−^* (*n* = 8) and *p19^ Arf−/−^p53^+/−^* (*n* = 8) mice show retention of wild-type *p53* ([Figure 6](#pbio-0020242-g006){ref-type="fig"}B). Seven of 14 (50%) carcinomas from *p19^ Arf+/+^p53^+/−^,* and three of 14 (21%) from *p19 ^Arf+/−^p53^+/−^,* but only one of 12 (8%) from *p19^ Arf−/−^p53^+/−^* (*p* = 0.036) showed loss of *p53*. Thus, deletion of p19^Arf^ disrupts the activation of p53 and thereby reduces selection for mutations in *p53* during malignant progression.
Discussion {#s3}
==========
Using a multistage model of tumor progression, we have examined the functional interactions between the oncogene *Hras* and the tumor suppressors *p19^Arf^* and *p53*. Somatic mutation of *Ras* is an early and frequent event in this model of tumor development. Against this backdrop, p19^Arf^ has at least two distinct tumor suppressor properties, which act at different stages of tumor development and which show a range of gene dosage effects. Loss of one or both *p19^ Arf^* alleles leads to accelerated growth of benign tumors, indicating *p19^ Arf^* is partially haploinsufficient for suppression of this early growth phenotype. Although p19^Arf^ regulates p53 at this stage, suppression of tumor growth per se by p19^Arf^ does not appear to be mediated through p53. p19^Arf^ also inhibits the benign to malignant transition and subsequent tumor cell dissemination and metastasis, and this effect of p19^Arf^ is, in contrast, mediated through p53. LOH of *p19 ^Arf^* occurs preferentially in malignant tumors, indicating complete loss of p19^Arf^ is favored during progression. Thus, p19^Arf^ inhibits several stages in Ras-driven tumor progression. Furthermore, Ras is connected to both p19^Arf^ and p53 through a signaling pathway, indicating that selection for mutations in the p19^Arf^ / p53 pathway are a direct consequence of the initial *Ras* mutation.
Ras, p19^Arf^, p53, and Early Tumor Growth {#s3a}
------------------------------------------
The observation that *Hras* mutations are found at high frequency in papillomas from wild-type and both p19^Arf^- and p53-deficient mice indicates that squamous epithelial cells harboring *Hras* mutations have a strong selective advantage, with or without the presence of p19^Arf^ or p53. This permits analysis of the effects of Ras on defined genetic backgrounds in the natural setting of tumor cell evolution. The expression levels of both p19^Arf^ and p53 were increased in wild-type papillomas but not in *p19^ Arf−/−^* papillomas, indicating that p19^Arf^ regulates p53 in response to activated Ras in vivo. However, other signals to induce p53, such as those stemming from DNA damage, remain intact in the absence of p19^Arf^, as shown by the rapid increase in p53 in irradiated *p19^ Arf^*-null tumors. Thus, of the many stimuli that have been shown to activate p53 using a variety of experimental systems ([@pbio-0020242-Ko1]; [@pbio-0020242-Giaccia1]), the Ras-p19^Arf^ pathway appears to be the major signal that operates during SCC development. This indicates that p53 activation is an intrinsic consequence of the oncogenic pathway that drives tumor growth, and is not due to other microenvironmental factors (e.g., those induced by hypoxia or due to lack of survival factors) or exogenous stimuli (e.g., DNA damage inducers). However, it remains possible that these other modes of p53 activation might predominate in other tumor types or in other circumstances.
Despite the fact that both p19^Arf^ and p53 were induced in papillomas, loss of p19^Arf^ or p53 had opposite effects on early tumor growth. p19^Arf^ deficiency resulted in increased tumor cell proliferation and tumor growth while p53-deficient mice had reduced tumor cell proliferation, tumor numbers, and tumor size. The observation that tumors harboring mutant *Ras* grew faster in the presence of p53 than in the absence of p53 differs from in vitro studies, in which ectopically expressed Ras induces p53-dependent growth arrest or senescence ([@pbio-0020242-Palmero1]; [@pbio-0020242-Zindy1]; [@pbio-0020242-Lin1]). These different outcomes are likely due to different experimental conditions. Ex vivo culture per se induces stress, which can further induce p53 and accelerate senescence ([@pbio-0020242-Lowe1]; [@pbio-0020242-Serrano1]; [@pbio-0020242-Sherr1]). Moreover, the levels of active Ras protein likely differ; our autochthonous tumor model begins with a single cell that has undergone a mutation at the endogenous *Hras* locus, and subsequent tumor growth occurs in the context of surrounding normal cells. Locally produced growth factors or TPA treatment may attenuate the effect of Ras on p53, effectively dampening the response. Thus, the absolute levels of both Ras and p53, as well as cell type involved and the local cellular ecology, may dictate the outcome.
[@pbio-0020242-Greenhalgh1] also reported reduction in *Ras*-transgene-induced skin tumors in a *p53*-null background. We suggest that the acute effect of a constitutively active oncogene driving cellular proliferation, combined with lack of cell cycle checkpoints due to p53 deficiency, may generate excessive genetic instability, which may initially impair overall cellular fitness. This may be especially true at the early post-initiation stage where a small number of incipient tumor cells are competing with surrounding normal cells. This idea is consistent with the longstanding observation that p53-deficient mice ([@pbio-0020242-Donehower1]), and Li-Fraumeni patients who carry a germline *p53* mutation ([@pbio-0020242-Vogelstein1]), rarely develop multiple tumors, which would be expected if loss of p53 provided an early selective advantage. Also, in many human malignancies, mutations in *p53* are infrequent in early premalignant lesions and much more common in late-stage disease, indicating a long latency between oncogene activation and loss of p53. Further genetic or epigenetic changes may be required for a cell to adapt to the combined effect of a dominant oncogene and loss of p53 to gain a fitness advantage. Loss of p19^Arf^ appears to be one such change, as reduced tumor growth due to the absence of p53 was rescued by loss of p19^Arf^.
Although p19^Arf^ regulates p53, suppression of tumor growth per se by p19^Arf^ does not appear to be mediated through p53. Other reports have suggested that p19^Arf^ has tumor suppressor functions that are independent of p53. *p19^ Arf^*-null mice show a broader spectrum of spontaneous tumors compared to *p53*-null mice ([@pbio-0020242-Donehower1]; [@pbio-0020242-Kamijo2]). Mice lacking both *p19 ^Arf^* and *p53* showed a wider range of tumor types than animals lacking either gene alone, and many developed multiple primary tumors ([@pbio-0020242-Weber2]; [@pbio-0020242-Moore1]). Premalignant B-cells expressing oncogenic Eμ-myc and lacking both *p19^ Arf^* and *p53* proliferated at a faster rate than cells lacking either *p19^ Arf^* or *p53* alone ([@pbio-0020242-Weber2]). Indeed, both p19^Arf^ and p53 are lost in a wide spectrum of human cancers, both familial and sporadic, at very high frequency ([@pbio-0020242-Ruas1]; [@pbio-0020242-Vonlanthen1]). Microarray and GeneChip analysis of genes induced by a conditionally regulated p19^Arf^ has identified members of the B-cell translocation gene family whose induction is independent of p53 ([@pbio-0020242-Kuo1]). Expression of these genes inhibits cell proliferation and induces cell cycle arrest. p19^Arf^ can colocalize with the human replication protein A, suggesting a direct role for p19^Arf^in DNA synthesis ([@pbio-0020242-Yarbrough1]). p19^Arf^ has also been shown to inhibit ribosomal RNA processing ([@pbio-0020242-Sugimoto1]) and to repress NF-κB transactivation ([@pbio-0020242-Rocha1]). Finally, p19^Arf^ regulates vascular regression independent of p53, suggesting a role for p19^Arf^ in angiogenesis. ([@pbio-0020242-McKeller1]). The functional relevance of these phenotypes for tumor suppression by p19^Arf^ remains to be elucidated.
Ras, p19^Arf^, p53, and Malignant Progression {#s3b}
---------------------------------------------
In the DMBA/TPA model, conversion of papillomas to carcinomas is a relatively rare event and can take up to 6--12 mo. Phenotypes associated with conversion include loss of basement membrane integrity, invasion of epithelial tumor cells into the dermis, loss of differentiation, and increased cellular atypia. Even more rarely, these carcinoma cells can metastasize, which involves additional phenotypic changes including, extravasation, migration, attachment, and establishment of tumor growth in an ectopic tissue. Although benign tumor growth differed between p19^Arf^- and p53-deficient mice, both mutant mice showed dramatically accelerated progression to malignancy and rapid metastasis. Thus, benign tumors lacking either p19^Arf^ or p53 are at high risk for metastasis. In p19^Arf^-deficient mice, progression was similar with or without the presence of p53 and did not involve *p53* LOH, indicating that loss of p19^Arf^ decreased selection for *p53* mutations during progression and that p19^Arf^ acts through p53 at this stage. [@pbio-0020242-Schmitt1] also reported increased lymphoma dissemination in Eμ-myc *p53^−/−^* mice relative to Eμ-myc mice alone. From a clinical perspective, then, the most relevant effect of the p19^Arf^-p53 pathway may be to inhibit malignant conversion and metastasis. Loss of p19^Arf^ and/or p53 can increase progression and metastasis by several mechanisms. Deficiency in p19^Arf^ or p53 could indirectly affect progression via increased genetic instability, increased generation of mutants, and accelerated tumor evolution. This view postulates the existence of a distinct class of genes whose dysfunction increases progression and metastasis. It also requires a series of clonal evolutionary steps to select for cells carrying mutations in these genes. Alternatively, loss of p19^Arf^ or p53 can directly affect cellular phenotypes associated with progression through transcriptional regulation, or by relieving inhibition of Ras signaling.
Both p19^Arf^- and p53-deficient papillomas displayed several characteristics consistent with early malignancy. Conversion of papillomas to carcinomas is first characterized by a flattening, endophytic transition. p19^Arf^- and p53-deficient papillomas had this morphology from the outset, suggesting an early propensity for malignant conversion. More detailed histological and immunochemical characterization showed that p53-deficient papillomas were more dysplastic and had aberrant expression of differentiation markers such as E-cadherin, P-cadherin, and Keratin-13 ([@pbio-0020242-Cano1]). E-cadherin is a critical component of cell--cell adhesion and its down regulation is strongly associated with malignant progression ([@pbio-0020242-Birchmeier1]; [@pbio-0020242-Perl1]). That these "high risk" *p53*-null papillomas exhibited these early malignant features argues for a more direct effect of p53 on cellular phenotypes associated with progression. In addition, there was a lack of correlation between papilloma size and propensity to progress. *p53*-null papillomas were fewer and smaller, yet these showed the most rapid progression. Finally, metastasis of skin tumors in both p19^Arf^- and p53-deficient mice was observed within a matter of days after papilloma to carcinoma conversion, again, irrespective of precursor tumor size. Collectively, these data are inconsistent with a model in which loss of p19^Arf^ or p53 indirectly accelerates tumor progression by accelerating a series of independent genetic events, each followed by clonal selection, and instead favor a more direct model.
We suggest that Ras may be a major driving force for multiple steps in tumor progression, with loss of p19^Arf^ and p53 playing a facilitating role. In addition to the well-known effects of Ras on proliferation, consistent with an early role in neoplasia, Ras also contributes to a number of phenotypes that are involved in malignant progression, including metastasis. Ras induces cell motility, invasiveness ([@pbio-0020242-Lazarov1]; [@pbio-0020242-Dajee1]; [@pbio-0020242-Kim1]), epithelial to mesenchymal transition ([@pbio-0020242-Oft1]; [@pbio-0020242-Zondag1]), angiogenesis ([@pbio-0020242-Arbiser1]; [@pbio-0020242-Casanova1]), and metastasis ([@pbio-0020242-Pozzatti1]; [@pbio-0020242-Webb1]; [@pbio-0020242-Oft2]; [@pbio-0020242-Varghese1]). Ras activates a number of signaling cascades that drive these processes ([@pbio-0020242-Campbell1]). For example, Ras activation of the Raf-MAPK signaling cascade regulates the activities of nuclear transcription factors, including AP-1 ([@pbio-0020242-Campbell1]). In addition to regulating proliferation, AP-1 induces a motility/invasion program ([@pbio-0020242-Ozanne1]; [@pbio-0020242-Jochum1]; [@pbio-0020242-Young1]). Ras transgenic mice that lack Fos, a component of AP-1, develop benign skin tumors, but these fail to convert to carcinomas ([@pbio-0020242-Saez1]). MAPK activation also determines the ability of Ras-transformed fibroblasts to metastasize to the lung ([@pbio-0020242-Webb1]). Oncogenic Ras also works in concert with the Rho family of GTPases to regulate the intracellular actin cytoskeleton and promote cell motility and invasion leading to metastasis ([@pbio-0020242-Zohn1]). Sustained signaling by oncogenic Ras can permanently downregulate Rac activity and lead to an epithelial to mesenchymal transition. This transition is associated with changes in gene expression, loss of E-cadherin-mediated cell--cell adhesions, and increased invasiveness of tumor cells ([@pbio-0020242-Oft1], [@pbio-0020242-Oft2]; [@pbio-0020242-Zondag1]). This allows the cell to become mobile, invade surrounding tissue, and establish metastatic sites. An additional link between Ras and tumor progression was demonstrated in a genome-wide survey of Ras transformation targets that identified a significant increase in expression of genes triggering invasion and metastasis, such as the laminin receptor, collagenase (Mmp-1), stromolysin (Mmp-3), and the Cd44 glycoprotein ([@pbio-0020242-Zuber1]). Ras also repressed genes involved in anti-invasive or anti-angiogenic activity, such as syndecan-2, tissue inhibitor of metalloproteases-2 (Timp-2), and thrombospondin-1.
Further support for a continual involvement of Ras in tumor progression is indicated by the increase in copy number of mutant *Ras* alleles that is observed during tumor progression. In the DMBA/TPA skin tumor model, the mutant *Ras* allele is frequently duplicated in papillomas and amplified to multiple copies in carcinomas, especially in the most aggressive spindle cell tumors ([@pbio-0020242-Bremner1]; [@pbio-0020242-Buchmann1]). Increased expression of mutant *Ras* genes by gene amplification or other mechanisms is found in other tumor types ([@pbio-0020242-Schwab1]; [@pbio-0020242-Tanaka1]; [@pbio-0020242-Winter1]; [@pbio-0020242-Yokota1]) and has been shown to promote the growth of head and neck SCC and carcinoma of the cervix ([@pbio-0020242-Hoa1]; [@pbio-0020242-Soh1]). This indicates that there is a selective advantage to progressively increasing levels of Ras throughout tumor progression. Quantitative differences in Ras activity are known to differentially activate signaling pathways leading to different cellular outcomes ([@pbio-0020242-Shields1]). Loss of p53 may facilitate the increase in Ras levels, in that cells with inactivated p53 show greatly increased frequency of gene amplification ([@pbio-0020242-Yin1]).
Thus, mutation in *Ras* is much more than the initiating event: it directly contributes to many of the phenotypes associated with malignant progression ([Figure 7](#pbio-0020242-g007){ref-type="fig"}). As Ras induces both p19^Arf^ and p53, and both are antagonistic to Ras, we suggest that an important consequence of p19^Arf^ and p53 loss is that it permits increased Ras levels and signaling, fueling further tumor progression. In addition to counteracting Ras, p19^Arf^ and p53 likely contribute to tumor suppression through additional pathways. For example, loss of p19^Arf^ increases tumor growth, and loss of p53 confers resistance to apoptosis and loss of cell cycle checkpoints, leading to genetic instability. The observation that oncogene mutations are linked to tumor suppressor gene activation through mechanistic signaling pathways indicates that selective pressure in favor of tumor suppressor gene mutations originates from the initial oncogenic lesion and is thus intrinsic to the tumor.
::: {#pbio-0020242-g007 .fig}
Figure 7
::: {.caption}
###### An Integrated Model of SCC Progression
At the genetic level, treatment of mouse skin with DMBA induces mutations in *Hras* resulting in initiated cells that express constitutively active Ras protein (grey rectangles). TPA treatment is required for clonal expansion of these *Hras* mutant cells to form papillomas. Frequent duplication of the mutant *Ras* allele in papillomas indicates increased Ras signaling is favored. Mutation of *p53,* as well as additional *Ras* gene amplification, is seen in carcinomas, particularly in the most aggressive tumors (black rectangles). At the signaling level, mutant Ras upregulates p19^Arf^ (Arf), which in turn activates p53. p19^Arf^, in turn, inhibits growth of Hras-driven tumors in a p53-independent manner. p19^Arf^, acting through p53, also inhibits malignant progression and metastasis. As Ras signals through p19^Arf^ and p53, selection for subsequent mutations in *p19^ Arf^* or *p53* is a direct consequence of the initial *Ras* mutation. In this model, Ras drives tumor progression through direct signaling effects and by dictating the evolution pathway of the tumor.
:::
:::
Materials and Methods {#s4}
=====================
{#s4a}
### Mice {#s4a1}
p19^Arf^-deficient mice (C57BL/6 × 129SvJ) were provided by Martine Roussel and Charles Sherr ([@pbio-0020242-Kamijo1]). To increase their susceptibility to skin tumor development, *p19^ Arf−/−^* mice were backcrossed to the susceptible NIH/Ola strain (Harlan Olac, Oxfordshire, United Kingdom), and carcinogenesis studies were performed on the F~3~ littermates of this cross. 20 mice of each genotype, *p19^ Arf−/−^, p19^ Arf+/−^,* and *p19^ Arf+/+^* were treated. The backs of 8-wk-old male and female mice were shaved and treated with a single application of DMBA (25 μg in 200 μl of acetone; Sigma, St. Louis, Missouri, United States) followed a week later by twice weekly applications of TPA (200 μl of 10^−4^ M solution in acetone) for 15 wk. The number and size of papillomas on each mouse were recorded every 2 wk. Mice were sacrificed if moribund or following detection of carcinomas. Tumors were frozen for DNA extraction and/or fixed in formalin to be processed and stained with hematoxylin/eosin (H&E) for histological examination.
Mice deficient for *p53* ([@pbio-0020242-Donehower1]) (F~7~ backcross to NIH) were crossed to *p19^ Arf^*-deficient (F~4~ backcross to NIH) mice to generate *p19^ Arf+/−^ p53^+/−^* mice. These mice were intercrossed to generate all nine possible *p19^ Arf^ /p53* genotypes*.* Some 20--30 mice of each genotype were subjected to the same DMBA/TPA protocol and monitored as described above.
### Immunoblotting {#s4a2}
In order to remove abundant keratin present in papillomas and carcinomas, nuclear extracts were prepared as described ([@pbio-0020242-Schreiber1]) with modifications. Pieces of skin or tumor were ground in liquid nitrogen with a mortar and pestle, and the resulting powder was dissolved in buffer A and further homogenized for 1 min on ice (PowerGen 125, Fischer Scientific, Pittsburgh, Pennsylvania, United States). Buffers A and C both contained 1 mM DTT, 0.4 mg/ml Pefablock, 25 mg/ml Aprotinin, 10 mg/ml Pepstatin, and 10 mg/ml leupeptin (Roche, Alameda, California, Unites States) to inhibit proteases. Protein concentrations were standardized using the Bradford assay (Bio-Rad, Hercules, California, United States) and equal loading (50 μg/lane) was confirmed by Ponceau S staining of the nylon membrane after blotting. Western blot analysis was performed using specific antibodies against p19^Arf^ (Novus Biologicals, Littleton, Colorado, United States) and p53 (Novocastra Laboratories, Newcastle-upon-Tyne, United Kingdom).
### *Hras* sequencing {#s4a3}
Genomic DNA was prepared from tumor and normal tissue by QIAamp DNA Mini Kit (Qiagen, Valencia, California, United States). A 400-bp PCR fragment containing exon 2 of *Hras* was amplified with standard PCR (3.0 mM MgCl~2~ andannealing for 2 min at 37 °C with 40 cycles), with 5′-GACTCCTACCGGAAACAGGT-3′ and 5′-CTGTACTGATGGATGTCCTC-3′ primers. We used the internal primer 5′-TGGTCATTGATGGGGAGACA-3′ to sequence exon 2, using PE Biosystems (Applied Biosystems, Foster City, Calfornia, United States) Dye-Terminator and Big-Dye cycle sequencing.
### Histological analysis {#s4a4}
Sections of normal skin, papillomas, carcinomas, and other organs were removed and fixed in 10% normal buffered formalin for 4 h. After fixation, tissues were processed and then embedded in paraffin. From the tissues, 4-μm sections were cut and stained for either p53 (CM5, Novocastra) or pan-keratin (AE1/AE3, Novocastra) using high-temperature antigen retrieval in 10 mM citrate buffer (pH 6), or for BrdU (Dako, Glostrup, Denmark) after treating with 2N HCl followed by 0.1% trypsin. After staining with the primary antibody, the sections were stained with a biotin-conjugated secondary (Vector Laboratories, Burlingame, California, United States) followed by StreptABComplex/HRP (Dako). Slides were developed with DAB/NiCl and counterstained with methyl green. Control sections with no primary antibody were run concurrently. Other sections were cut and stained using a standard H&E method. Proliferation index was determined by counting the number of BrdU-stained cells per 40× field. The apoptotic index was determined by counting the H&E slides for the number of apoptotic figures per 40× field. All counts were done on a Nikon (Tokyo, Japan) Labophot-2 microscope.
### Semiquantitative PCR analysis {#s4a5}
Genomic DNA was prepared from tumor tissue or normal kidney by QIAamp DNA Mini Kit (Qiagen). To measure LOH of the *p19^Arf^* wild-type allele in *p19^Arf+^* ^/−^ tumors, wild-type and knockout alleles were amplified by PCR separately then combined for electrophoresis. Primers 5′-AGTACAGCAGCGGGAGCATGG-3′ *(Arf1),* 5′-TTGAGGAGGACCGTGAAGCCG-3′ *(Arf2),* and 5′-ACCACACTGCTCGACATTGGG-3′ *(ArfN)* were used to amplify wild-type *(Arf1* and *Arf2)* and knockout alleles *(Arf2* and *ArfN)* from 100 ng of genomic DNA using 68 °C for annealing and extension at 90 s for 30 cycles. Equal amounts of each PCR product were then combined for electrophoresis on a 2% TAE agarose gel. Wild-type and knockout alleles of *p53* ^+/−^ tumors were amplified in a separate reaction as described ([@pbio-0020242-Timme1]) for 30 cycles. PCR products were electrophoresed on a 2% TAE agarose gel. Comparison gradients for *p19^Arf^* and *p53* were established by combining wild-type and knockout genomic DNA in quantified ratios, then amplifying as described above.
### Statistical methods {#s4a6}
In order to assess the impact of *p19^Arf^* (or *p19^Arf^ /p53*) genotypes on the development of papillomas, longitudinal profiles of papilloma counts were analyzed using the generalized estimating equation (GEE) approach ([@pbio-0020242-Zeger1]). GEE is an established statistical approach to the regression analysis of longitudinal data. Our analysis used papilloma counts of each mouse measured every 2 wk as the outcome variable and incorporated within-mouse correlations of papilloma counts over time in making statistical inference. Using GEE, we estimated average differences of papilloma counts across genotypes after DMBA administration. Since the development of papillomas depends on the time since the DMBA administration and may differ by the sex of the mice, the effects of the weeks since the DMBA administration and sex were controlled for in the GEE analysis as covariates. A working correlation structure of the GEE analysis was specified as the first-order autoregressive structure over the time since the DMBA administration; however, GEE is robust against the misspecification of the correlation structure. In contrast to papillomas, the overall number of carcinomas developed during the experiment was relatively small. Thus, we analyzed differences by genotype in the probability of developing a carcinoma after DMBA administration. A logistic regression model ([@pbio-0020242-Clayton1]) was used to assess the odds of developing a carcinoma during the experimental period and to compare it across *p19^Arf^* (or *p19^Arf^ /p53*) genotypes. Estimates of relative odds were adjusted for sex effects. Fisher\'s exact test was used for comparing two proportions such as comparing LOH proportions between papillomas and carcinomas. All statistical tests were two-sided.
We thank S. Lawrence Bailey for excellent technical assistance, Martine Roussel and Charles Sherr for generously providing p19^Arf^ knockout mice, and Denny Liggett for assistance with histopathological evaluation of tumor samples. We also thank numerous colleagues at the Fred Hutchinson Cancer Research Center for critical reading of the manuscript. This work was supported by a National Institutes of Health T32 CA8046 Interdisciplinary Training Grant in Cancer Research to KSK-S and NCI R01 (CA099517) and NIEHS U01 (ES11045) grants to CJK.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** KSK-S and CJK conceived and designed the experiments. KSK-S and KEG performed the experiments. KSK-S, YY, and CJK analyzed the data. KSK-S and KEG contributed reagents/materials/analysis tools. KSK-S and CJK wrote the paper.
Academic Editor: Nicholas Hastie, Western General Hospital
Citation: Kelly-Spratt KS, Gurley KE, Yasui Y, Kemp CJ (2004) p19^Arf^ suppresses growth, progression, and metastasis of Hras-driven carcinomas through p53-dependent and -independent pathways. PLoS Biol 2(8): e242.
Arf
: p19^Arf^
GEE
: generalized estimating equation
H&E
: hematoxylin/eosin
LOH
: loss of heterozygosity
SCC
: squamous cell carcinoma
| PubMed Central | 2024-06-05T03:55:46.771436 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509304/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e242",
"authors": [
{
"first": "Karen S",
"last": "Kelly-Spratt"
},
{
"first": "Kay E",
"last": "Gurley"
},
{
"first": "Yutaka",
"last": "Yasui"
},
{
"first": "Christopher J",
"last": "Kemp"
}
]
} |
PMC509305 | Introduction {#s1}
============
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by the production of autoantibodies (auto-Abs) against a wide spectrum of self-antigens, mainly from subcellular compartments, especially the cell nucleus. Genetic predisposition is an important contributor to susceptibility to SLE in both humans and animals ([@pbio-0020243-Vyse1]; [@pbio-0020243-Harley1]; [@pbio-0020243-Theofilopoulos2]; [@pbio-0020243-Wakeland1]). Genes in multiple pathways participate in mediating disease pathogenesis, and epistatic interactions amongst these genes influence the expression of disease. In this context, both genetic linkage studies in spontaneous lupus-prone models and synthetic murine models of autoimmunity generated by targeted disruption of specific genes modulating the immune system have widely been used to investigate the complexity of SLE.
The best-studied strains of mice that spontaneously develop a lupus-like pathology are the New Zealand Black/New Zealand White hybrid strain (NZB/WF1); the MRL/Mp *lpr/lpr* strain, which carries the *lpr* mutation of the FAS receptor gene; and the BXSB strain, which carries the Y chromosome autoimmune accelerator (*Yaa*) gene ([@pbio-0020243-Theofilopoulos1]). Extensive genetic mapping studies in all three strains have identified multiple intervals associated with disease susceptibility. Interestingly, the majority of the intervals detected are strain-specific, confirming the genetic complexity of the disease and indicating the presence of extensive heterogeneity in the genes contributing to the pathogenesis of the disease. However, some susceptibility loci have been mapped to similar chromosome locations in different strains, suggesting that at least some susceptibility may be shared amongst lupus-prone strains. Amongst these shared susceptibility loci, the most striking are loci on distal Chromosome 1, for which important contributing genes have been found in New Zealand and BXSB models ([@pbio-0020243-Theofilopoulos2]; [@pbio-0020243-Wakeland1]).
Although considerable efforts have been made to identify the genes responsible for the development of the disease, with the exception of the *lpr* mutation, none of the genetic contributions to disease in the three well-documented murine SLE strains have yet been fully resolved at the molecular or protein level. Thus, targeted genetic disruption of candidate genes encoding proteins of the immune system has been extensively used to examine their role in immune regulation. However, the most surprising result of this powerful approach has been the high frequency with which such mutations have been associated with an autoimmune phenotype. In this regard, it is of note that hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, are spontaneously predisposed to development of humoral autoimmunity with low levels of glomerulonephritis ([@pbio-0020243-Obata1]; [@pbio-0020243-Botto2]; [@pbio-0020243-Bickerstaff1]; [@pbio-0020243-Santiago-Raber1]). Furthermore, the genetic background markedly influences the autoimmune phenotype in gene-targeted mice ([@pbio-0020243-Bolland1]; [@pbio-0020243-Santiago-Raber1]; [@pbio-0020243-Mitchell1]). These observations led to the hypothesis that the autoimmune phenotype described in some gene-targeted mice might be due primarily to combinations of as-yet-uncharacterised background genes, originating from 129 and C57BL/6 mice strains, interacting or not with the mutated allele. To test this, we conducted a genome-wide scan analysis of two large cohorts of (129 × C57BL/6)F2 mice, one of which carried a mutation in the serum amyloid P component gene (*Apcs*). The *Apcs*-deficient mice (*Apcs* ^−/−^) were chosen as an example of a gene-targeted strain previously reported to develop a lupus-like disease on the hybrid genetic background (129 × C57BL/6); autoimmunity in *Apcs* ^−/−^ mice persists even after backcrossing the mutated gene onto C57BL/6 ([@pbio-0020243-Bickerstaff1]). We chose this targeted gene in particular to study since the *Apcs* gene is located on Chromosome 1, approximately 94 cM from the centromere, within a region where several lupus-susceptibility loci, designated *Sle1* ([@pbio-0020243-Morel2]), *Nba2* ([@pbio-0020243-Drake1]; [@pbio-0020243-Vyse2]), and *Bxs3* ([@pbio-0020243-Hogarth1]; [@pbio-0020243-Haywood1]), have been mapped in NZW, NZB, and BXSB lupus-prone strains, respectively. This region contains several genes, including those encoding FcγRII, the complement receptor CR1/2 (CD35/CD21), and the decay-accelerating factor CD55 ([@pbio-0020243-Prodeus1]; [@pbio-0020243-Bolland1]; [@pbio-0020243-Miwa1]; [@pbio-0020243-Wu1]), which have each been implicated in the causation of SLE when inactivated by gene-targeting in 129 embryonic stem cells.
Here we show that there are multiple genetic loci, derived from both 129 and C57BL/6 mice, contributing to autoimmunity. Furthermore, a 129-derived interval on distal Chromosome 1, when transferred onto the C57BL/6 genome, a combination commonly created by backcrossing onto C57BL/6 a gene that has been inactivated in 129 embryonic stem cells, was sufficient to cause humoral autoimmunity in its own right, irrespective of the presence of the mutated *Apcs* gene. These results demonstrate important epistatic interactions between genes from 129 and C57BL/6 genomes on the development of autoimmunity and illustrate the important effects of background genes in the analysis and interpretation of autoimmune phenotypes associated with targeted genetic disruptions.
Results {#s2}
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Disease Traits in (129 × C57BL/6)F2 and (129 × C57BL/6)F2.*Apcs* ^−/−^ Mice {#s2a}
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To investigate the genetic basis of the lupus-like disease observed in the (129 × C57BL/6) hybrid mice, we generated two large cohorts of (129 × C57BL/6)F2 animals, one carrying a mutation in the *Apcs* gene, and monitored them for 1 y under identical environmental conditions. Since female mice in the original reports showed a higher penetrance of disease, the present study was conducted only on female mice. The results of the phenotypic analysis at 1 y of age are summarised in [Tables 1](#pbio-0020243-t001){ref-type="table"} and [2](#pbio-0020243-t002){ref-type="table"}. As previously reported ([@pbio-0020243-Botto2]), the wild-type (129 × C57BL/6)F2 mice developed lupus traits with elevated levels of auto-Abs, starting from 6 mo of age (data not shown), and histological evidence of proliferative glomerulonephritis. In agreement with our previous observations ([@pbio-0020243-Bickerstaff1]), the titres of anti-nuclear Abs (ANAs) and anti-chromatin Ab were considerably greater in the (129 × C57BL/6)F2.*Apcs* ^−/−^ mice compared with the strain-matched controls. However, in contrast to our original findings, the levels of the other two disease serological markers analysed (anti-single-stranded DNA \[ssDNA\] and anti-double-stranded DNA \[dsDNA\] Abs) and the severity of the renal pathology were not different between the two experimental groups. In view of the possibility of an association between the fixed 129-derived segment flanking the mutated *Apcs* gene and the autoimmune traits observed, the genome-wide linkage analysis of the two experimental cohorts was carried out separately.
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Table 1
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###### Spontaneous Auto-Abs in *Apcs* ^−/−^ and Wild-Type (129 × C57BL/6)F2 Female Mice
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Significant differences between *Apcs* ^−/−^ and wild-type controls by Mann--Whitney U test. The numbers tested for different phenotypes are not equal due to the limited amount of serum or death of the mice before the end of the experiment. NS, not significant
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Table 2
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###### Histological Assessment of Kidney Sections in *Apcs* ^−/−^ and Wild-Type (129 × C57BL/6)F2 Female Mice
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The renal sections were scored on a 0--3 scale on the intensity and extent of the histopathological changes as described in Materials and Methods. The difference between the two experimental groups was not significant
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Mapping of Loci Predisposing to Lupus in the Hybrid Strain (129 × C57BL/6) {#s2b}
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Mice were genotyped with 143 microsatellite markers distributed throughout the autosomes such that 98% of the genes were within 20 cM of an informative marker. A summary of the genome-wide linkage analysis for each of the disease traits measured is shown in [Table 3](#pbio-0020243-t003){ref-type="table"}. The areas of linkage were defined according to the parental origin, 129 or C57BL/6. Only linkages identified in both experimental groups are reported in [Table 3](#pbio-0020243-t003){ref-type="table"}, with the exception of the Chromosome 1 distal segment, where the linkage analysis could not be applied to the (129 × C57BL/6)F2.*Apcs* ^−/−^ mice as this region was of fixed 129 origin. Chromosomes where linkages were present only in one of the two cohorts are shown in [Figures 1](#pbio-0020243-g001){ref-type="fig"}--[3](#pbio-0020243-g003){ref-type="fig"}.
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Figure 1
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###### Linkage of Chromosome 2 with ANA and Anti-Chromatin Abs in (129 × C57BL/6)F2*.Apcs^−/−^* Mice
These associations were not detected in (129 × C57BL/6)F2 animals. Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from <http://www.informatics.jax.org>. Dotted lines and the dashed line indicate the threshold over which linkages were considered suggestive or significant, respectively, as defined in Materials and Methods.
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::: {#pbio-0020243-g003 .fig}
Figure 3
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###### Linkage of Chromosome 4 with Anti-dsDNA Abs
The estimated peak in (129 × C57BL/6)F2 mice was at position 51.3 cM, whilst in the (129 × C57BL/6)F2*.Apcs^−/−^*animals it was was at position 71 cM, indicating that most likely these were two independent loci. Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from <http://www.informatics.jax.org>. Dotted lines the indicate threshold over which linkage was considered suggestive, as defined in Materials and Methods**.**
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::: {#pbio-0020243-t003 .table-wrap}
Table 3
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###### Summary of Genome-Wide Linkage Analysis in *Apcs* ^−/−^ and Wild-Type (129 × C57BL/6)F2 Female Mice
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^a^ Suggestive linkage
^b^ Significant linkage
^c^ Highly significant linkage as defined in the material and method section
Chr, Chromosome; Est. Peak, Estimated Peak; LOD, logarithm of odds; N/A, not applicable
Chromosomes where linkages were not present in both experimental groups are not illustrated. See [Materials and Methods](#s4){ref-type="sec"} for details. The oligonucleotide sequences and approximate positions of the microsatellite markers used were taken from the Mouse Genome Database, Mouse Genome Informatics, Jackson Laboratory, Bar Harbor, Maine, United States (<http://www.informatics.jax.org>)
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The quantitative trait linkage (QTL) analysis identified several intervals on Chromosome 1 with linkage to disease serological markers, and these regions were all derived from the 129 mouse strain (see [Table 3](#pbio-0020243-t003){ref-type="table"}; [Figures 4](#pbio-0020243-g004){ref-type="fig"} and [5](#pbio-0020243-g005){ref-type="fig"}). Interestingly, whilst ANA and anti-chromatin Ab levels showed suggestive or significant linkages only to the telomeric region of Chromosome 1, with an estimated peak occurring at a position approximately 90 cM near the *Apcs* gene, anti-dsDNA or anti-ssDNA Ab production was also linked to other segments on Chromosome 1, indicating a more complex genetic contribution from the 129 mouse strain.
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Figure 4
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###### Interval Mapping Scans Showing QTL on Chromosome 1 with Anti-dsDNA and Anti-ssDNA Abs
Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from <http://www.informatics.jax.org>. Dotted lines indicate the threshold over which linkage was considered suggestive, dashed lines indicate the threshold over which linkage was considered significant, and dotted/dashed lines indicate highly significant linkage, as defined in Materials and Methods. See [Table 3](#pbio-0020243-t003){ref-type="table"} for additional details.
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Figure 5
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###### Interval Mapping Scans Showing QTL on Chromosome 1 with ANA and Anti-Chromatin Abs
Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from <http://www.informatics.jax.org>. Dotted lines indicate the threshold over which linkage was considered suggestive, and dashed lines indicate the threshold over which linkage was considered significant, as defined in Materials and Methods. See [Table 3](#pbio-0020243-t003){ref-type="table"} for additional details.
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Guided by these observations, we investigated whether the increased levels of ANA and anti-chromatin Ab observed in the *Apcs* ^−/−^ mice were caused by a gene(s) within the fixed 129 region surrounding the mutated *Apcs* gene, rather than caused by the mutated *Apcs* gene itself. We compared the levels of these auto-Abs between all (129 × C57BL/6)F2.*Apcs* ^−/−^ mice and a group of 33 wild-type mice that were selected for being homozygous 129 in the region of Chromosome 1 between microsatellites D1Mit105 and D1Mit 223 (80--106 cM) ([Figure 6](#pbio-0020243-g006){ref-type="fig"}A--[6](#pbio-0020243-g006){ref-type="fig"}D). In contrast to the results reported in [Table 1](#pbio-0020243-t001){ref-type="table"}, this comparison showed no significant differences between the two experimental groups. This result demonstrates that, most likely, the 129-derived region and not the lack of *Apcs* was mediating the production of ANA and anti-chromatin Ab. Consistent with this explanation, we found that the 129 mice have significantly higher levels of *Apcs* in circulation compared with the C57BL/6 mice (median, 83 mg/l; range, 25--208; *n* = 16 versus median, 5 mg/l; range, 4--9; *n* = 10, respectively; *p* \< 0.0001). The C57BL/6 strain has previously been reported to be one of the murine strains defined as low *Apcs* producers ([@pbio-0020243-Pepys3]; [@pbio-0020243-Baltz1]). In addition, sequence analysis of the entire *Apcs* coding region in both strains failed to identify any coding sequence polymorphisms in the *Apcs* gene (data not shown), indicating that a structural variant of the protein is unlikely to be the explanation for our findings. This is consistent with a previous report by [@pbio-0020243-Drake2] that showed no *Apcs* coding sequence differences amongst several autoimmune and nonautoimmune murine strains.
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Figure 6
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###### Auto-Ab Profiles
\(A) ANA titres in the (129 × C57BL/6)F2.*Apcs* ^−/−^ mice and (129 × C57BL/6)F2 at 1 y of age. A small circle represents one mouse; a large circle, a variable number of animals, as indicated in parentheses. Serum samples were titrated to endpoint.
\(B) ANA titres in the (129 × C57BL/6)F2.*Apcs* ^−/−^ mice and a selected number of wild-type (129 × C57BL/6)F2 animals carrying the Chromosome 1 region between D1Mit105 and D1Mit223 (80--106 cM) of 129 origin. The symbols are as in (A).
(C and D) Anti-chromatin Ab levels expressed in AEUs related to a standard positive sample, which was assigned a value of 100 AEU. The comparison is between the same groups of mice as in (A) and (B), respectively. The symbols are as in (A).
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In addition to the 129-derived segments, in both cohorts the C57BL/6 strain contributed to the autoimmune traits with one major susceptibility locus on Chromosome 3. A genomic region between D3Mit40 and D3Mit13, with an estimated peak at position approximately 51 cM, showed a significant linkage to ANA production and weaker linkages to anti-ssDNA and anti-chromatin production (see [Table 3](#pbio-0020243-t003){ref-type="table"}; [Figure 7](#pbio-0020243-g007){ref-type="fig"}). The high frequency of autoimmune phenotype in the (129 × C57BL/6) hybrid genetic background and its absence in either of the inbred parental strains imply that there are essential interactions between 129- and C57BL/6-derived alleles for the expression of autoimmunity. We investigated further the effects of genes from the C57BL/6 background by repeating the linkage analysis in (129 × C57BL/6)F2 mice, whilst controlling for the very strong 129 effect on distal Chromosome 1, as previously described ([@pbio-0020243-Zeng1]). The results of this analysis showed that the statistical support for the linkage of the C57BL/6 locus on Chromosome 3 for ANA increased from logarithm of odds (LOD) 5.4 to LOD 6.4.
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Figure 7
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###### Interval Mapping Scans Showing QTL on Chromosome 3 with ANA, Anti-Chromatin, and Anti-ssDNA Abs
See [Table 3](#pbio-0020243-t003){ref-type="table"} for additional details. Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from <http://www.informatics.jax.org>. Dotted lines indicate the threshold over which linkage was considered suggestive, the dashed line indicate the threshold over which linkage was considered significant, and dotted/dashed lines indicate highly significant linkage, as defined in Materials and Methods.
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In contrast to these strong associations with disease serological markers, the QTL analysis identified only two potential linkages to glomerulonephritis: one in the wild-type mice on Chromosome 7 across a 10 cM region between D7Mit246 (15 cM) and D7Mit145 (26.5 cM) of 129 origin (LOD 2.86, *p* = 0.0013), and one on Chromosome 17 between D17Mit100 (11.7 cM) and D17Mit216 (29.4 cM) from the C57BL/6 strain (LOD 1.3, *p* = 0.049 and LOD 1.67, *p* = 0.021 in the wild-type and *Apcs* ^−/−^ mice, respectively). Histological evidence of glomerulonephritis was only found in approximately 20% of the mice in each cohort, which reduces the power of the QTL analysis for this disease trait.
Production of a C57BL/6.129 Chromosome 1 Congenic Line and Its Phenotypic Analysis {#s2c}
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We generated a C57BL/6 congenic line carrying the telomeric region of Chromosome 1 from the 129 mouse strain, in order to dissect the complex polygenic disease phenotype of the (C57BL/6 × 129/Sv)F2 hybrid strain into its individual genetic components. The 129 interval was backcrossed seven times onto C57BL/6, and at each generation the presence or absence of the Chromosome 1 interval was determined with several microsatellite markers. Each backcrossed generation was screened with more than three markers per chromosome to facilitate the removal of unselected 129 genomic regions. At the end of the backcrossing, the 129-derived Chromosome 1 interval in the congenic mice extended from microsatellite marker D1Mit105 to D1Mit223 (80--106 cM), which encompasses the most important 129 interval identified by the linkage studies in the (C57BL/6 × 129/Sv)F2 mice.
Female Chromosome 1 congenic mice (C57BL/6.129\[D1Mit105--223\]), together with sex-matched *Apcs* ^−/−^ mice backcrossed onto C57BL/6 for ten generations (C57BL/6.*Apcs* ^−/−^) and C57BL/6 controls, were monitored for the presence of lupus. In the C57BL/6.*Apcs* ^−/−^ mice, the 129 genome around the *Apcs* locus was mapped as a stretch of approximately 17 cM, positioned from 87.9 cM (D1Mit15) to 105 cM (D1Mit17). Thus, the congenic line carried a similar 129 region (80--106 cM) to the one present in the C57BL/6.*Apcs* ^−/−^ mice (87.9--105 cM). At 1 y of age, all animals were sacrificed, the auto-Abs assessed, and the renal histology examined. The results of this analysis are shown in [Figure 8](#pbio-0020243-g008){ref-type="fig"}. As previously reported ([@pbio-0020243-Bickerstaff1]), the levels of auto-Abs were markedly increased in the C57BL/6.*Apcs* ^−/−^ compared to the wild-type C57BL/6 controls. However, the C57BL/6.129(D1Mit105--223) animals also expressed high levels of auto-Abs, and these titres were not different from those detected in the matched congenic mice containing a null mutation of the *Apcs* gene. These results clearly demonstrated that epistatic interactions between 129 loci on Chromosome 1 and C57BL/6 genes were sufficient to mediate the loss of tolerance to nuclear autoantigens. However, in contrast to the serological data, the histological assessment of the kidneys showed evidence of markedly increased glomerulonephritis in the C57BL/6.*Apcs* ^−/−^ compared to both control groups ([Figure 9](#pbio-0020243-g009){ref-type="fig"}), suggesting that the lack of *Apcs*, when combined with other C57BL/6 susceptibility alleles, can induce the development of severe renal damage.
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Figure 8
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###### Auto-Ab Profiles
\(A) ANA titres in C57BL/6 mice, C57BL/6.*Apcs* ^−/−^ mice, and C57BL/6.129(D1Mit105--223) congenic mice at 1 y of age. Small symbols represent one mouse; large symbols, a variable number of animals as indicated in parentheses. Serum sample were titrated to endpoint.
(B and C) Anti-ssDNA (B) and anti-chromatin (C) Ab levels in the same cohorts of mice as in (A). The Ab levels are expressed in AEUs related to a standard positive sample, which was assigned a value of 100 AEU.
\(D) Anti-dsDNA Ab levels. Serum samples were screened at 1:20. Samples that were positive were titrated to endpoint. The symbols are as in (A).
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::: {#pbio-0020243-g009 .fig}
Figure 9
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###### Renal Histological Assessment
C57BL/6 mice, C57BL/6.*Apcs* ^−/−^ mice, and C57BL/6.129(D1Mit105--223) congenic mice were sacrificed at 1 y of age to obtain age-matched autopsy specimens. Bouin\'s fixed kidney sections were scored blinded for glomerulonephritis**.** Glomerulonephritis was graded on a 0--3 scale (see [Materials and Methods](#s4){ref-type="sec"} for details).
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Discussion {#s3}
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There is accumulating evidence that background genes may influence the expression of autoimmunity in gene-targeted mice. Here we report what is to our knowledge the first systematic study that has examined this in the 129 and C57BL/6 mouse strains, widely used for gene targeting. Our results demonstrate interacting loci between 129 and C57BL/6 mice that can cause the expression of a powerful autoimmune phenotype in these animals, in the absence of any gene-targeted mutations. We also developed a congenic mouse strain bearing a portion of 129 Chromosome 1 on a C57BL/6 background and showed that this wild-type congenic line expressed striking anti-nuclear autoimmunity. By comparing this Chromosome 1 congenic strain with matched congenic mice lacking the *Apcs* gene, we demonstrated that serum amyloid P component deficiency influences the severity of glomerulonephritis, but is not the prime mover in the induction of anti-nuclear autoimmunity, contrary to our own original interpretation of our data ([@pbio-0020243-Bickerstaff1]). The same consideration applies to other genes located in the same Chromosome 1 region that have been implicated in the development of SLE when inactivated by gene-targeting in 129 embryonic stem cells and then backcrossed onto a pure genetic background ([@pbio-0020243-Bolland1]; [@pbio-0020243-Miwa1]; [@pbio-0020243-Wu1]). For each, there has to be a question as to whether the anti-nuclear autoimmunity is due to the gene-targeted mutant gene or to the normal 129 genes expressed in the same region as the targeted gene.
The influence of background genes on the development of spontaneous autoimmune disease is well known, especially with respect to the *lpr* and *Yaa* disease-susceptibility genes. In MRL/Mp mice, the presence of the *lpr* gene accelerates the development of high level and broad-spectrum auto-Ab production and lethal glomerulonephritis, in addition to marked lymphoproliferative disease. In contrast, homozygosity of the *lpr* gene in other strains such as C57BL/6, AKR, LG/J, and C3H leads only to auto-Ab production ([@pbio-0020243-Izui1]). Similarly, the Y-chromosome-linked *Yaa* gene in BXSB and MRL/Mp males enhances the rapid development of auto-Abs and glomerulonephritis ([@pbio-0020243-Izui2]; [@pbio-0020243-Merino1]). However, in the C57BL/6 background, the *Yaa* gene does not lead to an autoimmune phenotype ([@pbio-0020243-Izui2]). Not surprisingly, important effects of the genetic background on the expression of autoimmunity have also been reported in gene-targeted mice ([@pbio-0020243-Bolland1]; [@pbio-0020243-Santiago-Raber1]; [@pbio-0020243-Mitchell1]). Thus, SLE exists as a complex-trait disorder in which specific combinations of susceptibility alleles are required for the expression of the full phenotype.
Through the use of microsatellite marker maps, the identification of murine SLE susceptibility intervals in experimental crosses has been made possible. These mapping studies have shown that the disease expression in relation to the inheritance of the different alleles followed a threshold liability pattern in which a positive phenotype depended upon the presence of multiple discrete susceptibility loci with no single locus being a prerequisite factor. We have employed the same approach to analyse the genetic basis of disease inheritance in the (129 × C57BL/6) hybrid strain, the most common genetic background in gene-targeted mice. Although spontaneous autoimmunity has not been documented in either of the pure 129 or C57BL/6 strains, a spontaneous lupus-like phenotype has been described in (129 × C57BL/6) hybrid strains ([@pbio-0020243-Obata1]; [@pbio-0020243-Botto2]; [@pbio-0020243-Bickerstaff1]; [@pbio-0020243-Santiago-Raber1]), suggesting that the predisposition in these hybrid mice may arise as a result of the interaction between specific combinations of alleles inherited from both the 129 and C57BL/6 parental strains.
This was confirmed by the mapping study reported here. We showed that there are multiple genetic loci contributing to the disease and these are derived from both 129 and C57BL/6 mice. We demonstrated that a 129-derived segment of Chromosome 1 was strongly linked to the expression of auto-Abs. This region is probably capable of causing the initiation of a humoral autoimmune response to nuclear antigens; however, this response does not occur in the absence of C57BL/6 genes. In support of this, we identified a C57BL/6 segment on Chromosome 3, which may interact with the 129 genes on Chromosome 1 to mediate the loss of tolerance. Interestingly, although the C57BL/6 SLE-susceptibility region on Chromosome 3 is novel, disease-modifying alleles derived from C57BL/10 and C57BL/6 strains have been mapped to a portion of Chromosome 3 close to the region identified in this study ([@pbio-0020243-Morel1]; [@pbio-0020243-Haywood1]). Furthermore, the region on Chromosome 7 associated with the development of lupus nephritis has been linked to the same trait in other murine models of SLE ([@pbio-0020243-Santiago1]; [@pbio-0020243-Morel1]; [@pbio-0020243-Xie1]), suggesting the possibility of shared susceptibility loci.
Taken together our results suggest a complex genetic contribution from the (129 × C57BL/6) hybrid background genome, with both enhancing as well as inhibitory loci from the 129 mouse, in addition to genes promoting autoimmunity from the C57BL/6 mice. The impact that these interacting loci may have on the lupus-like disease present in several gene-targeted animals was further assessed by comparing *Apcs* ^−/−^ mice with Chromosome 1 genetically matched controls.
In the context of SLE susceptibility, one of the most consistently mapped non-MHC regions of the mouse genome is the telomeric Chromosome 1 segment, where several disease loci, designated *Sle1* ([@pbio-0020243-Morel2]), *Nba2* ([@pbio-0020243-Drake1]; [@pbio-0020243-Rozzo1]; [@pbio-0020243-Vyse2]), and *Bxs3* ([@pbio-0020243-Hogarth1]), have been mapped in lupus-prone strains. Moreover, this region of mouse Chromosome 1 is orthologous to a region on human Chromosome 1q22--1q25, which has also been linked with human SLE ([@pbio-0020243-Moser1]).
The *Apcs* gene is one of the candidate genes known to lie within this region. The human serum amyloid P component binds avidly to DNA, chromatin, and apoptotic cells in physiological conditions in vitro ([@pbio-0020243-Pepys1]; [@pbio-0020243-Pepys2]; [@pbio-0020243-Butler1]) and also to exposed chromatin and apoptotic cells in vivo ([@pbio-0020243-Hintner1]; [@pbio-0020243-Breathnach1]; [@pbio-0020243-Familian1]). We have previously reported that (129 × C57BL/6).*Apcs* ^−/−^ mice spontaneously produce a wide range of ANAs and develop significant immune complex glomerulonephritis ([@pbio-0020243-Bickerstaff1]). On the basis of these observations, it was postulated that *Apcs*, by altering the clearance of chromatin, contributes to the pathogenesis of SLE. However, in this study we found that only ANA and anti-chromatin Ab levels were significantly increased in the (129 × C57BL/6)F2.*Apcs* ^−/−^ mice. A possible explanation for this discrepancy may lie in the fact that the *Apcs* ^−/−^ mice analysed in the original study were generated from a limited number of founders and that this may have caused a nonrandom inheritance of the loci from the parental strains. Furthermore, the whole-genome analysis identified the 129 region surrounding the *Apcs* gene as the main locus contributing to the development of ANA and anti-chromatin Ab. In agreement with this, when we carried out a selective comparison between the (129 × C57BL/6)F2.*Apcs* ^−/−^ mice and Chromosome 1 genetically matched controls, we failed to detect any significant difference in the levels of these two auto-Abs. These findings, taken together, indicated that the phenotype associated with *Apcs* deficiency was caused by the presence of unaltered 129 genes from the telomeric region of Chromosome 1 operating in the C57BL/6 genomic background. Strong supportive evidence for this was provided by the analysis of the C57BL/6 mice congenic for this 129 region.
The generation and analysis of congenic strains have successfully been used to dissect the contribution of individual susceptibility alleles to a multigenic trait such as SLE. We adopted the same strategy to investigate the relative contribution of the 129 Chromosome 1 segment and the *Apcs* gene to each disease trait. Using this approach, we demonstrated that the 129 interval on distal Chromosome 1, when transferred onto the C57BL/6 genome, a combination commonly created by backcrossing onto C57BL/6 a mutated gene located in that chromosomal region, was sufficient to mediate the production of auto-Abs. In this context, it is of note that several strains of mice with targeted mutations of genes encoded in this region have been reported to express a lupus-like illness, including mice lacking FcγRIIB ([@pbio-0020243-Bolland1]), complement receptors (CR1/2) ([@pbio-0020243-Prodeus1]; [@pbio-0020243-Wu1]), and decay-accelerating factor (CD55) ([@pbio-0020243-Miwa1]). In each case, the autoimmune phenotype was described in mice in which the null mutation was generated in 129 embryonic stem cells and then backcrossed to the C57BL/6 or another genetic background. Thus, in view of our findings, one may postulate that in each of these murine models of SLE, the effects of the targeted null gene were irrelevant. Similar conclusions may apply to other gene-targeted animals carrying mutations of genes mapped in the 129-derived susceptibility allele on Chromosome 7 ([@pbio-0020243-OaKeefe1], [@pbio-0020243-OaKeefe2]).
The expression of anti-nuclear autoimmunity was identical comparing the congenic with the *Apcs^−/−^* mice. The only difference in phenotype between these mice was in the expression of glomerulonephritis, which was more pronounced in the *Apcs^−/−^* mice compared with the congenic mice. Although these findings demonstrate that *Apcs* is not implicated in the processing of autoantigens, as it had previously been suggested, they indicate that *Apcs* might still play an important protective role in lupus nephritis. In support of this, the expression of the human C-reactive protein, an acute-phase protein closely related to *Apcs*, has been shown to delay the onset and severity of lupus nephritis in the NZB/W strain by preventing the deposition of immune complexes in the renal cortex ([@pbio-0020243-Szalai1]). Consistent with this, a polymorphism associated with reduced basal level of C-reactive protein has been reported to be linked to SLE in humans ([@pbio-0020243-Russell1]). However, as the congenic mice and the *Apcs^−/−^* mice carried similar but not identical 129 regions on Chromosome 1, an alternative explanation for our findings may still lay in the numerous and complex synergistic and counteractive interactions between 129 and C57BL/6 genes involved in self-tolerance and end organ damage. Thus, whilst the lack of lupus nephritis in the congenic mice is consistent with the need for multiple susceptibility genes for the full expression of lupus, further studies will be required to fully elucidate the role of *Apc*s in the pathogenesis of renal damage.
In summary, our findings demonstrate the impact of epistatic interactions between 129 and C57BL/6 genomes on the development of SLE and illustrate how these background gene effects may lead to incorrect interpretations when analysing the autoimmune phenotype of specific genetic disruptions.
Materials and Methods {#s4}
=====================
{#s4a}
### Mice. {#s4a1}
All the mice were females. Wild-type C57BL/6 and 129/Sv (129S6, according to the revised nomenclature) were bred and maintained in the animal care facility at Imperial College, London, United Kingdom. (129 × C57BL/6)F1 mice were generated by intercrossing the two wild-type strains and (129 × C57BL/6)F2 mice by interbreeding the (129 × C57BL/6)F1 mice. The *Apcs* ^−/−^mice were generated as previously reported ([@pbio-0020243-Botto1]), and the (129 × C57BL/6)F2.*Apcs* ^−/−^ mice were generated by intercrossing *Apcs* ^−/−^ mice on the 129 genetic background with *Apcs* ^−/−^ animals backcrossed onto C57BL/6 for ten generations. A total of 141 (129 × C57BL/6)F2 and 158 (129 × C57BL/6)F2.*Apcs* ^−/−^ female mice were produced and monitored for 1 y. Wild-type congenic C57BL/6.129(D1Mit105--223) mice were generated by backcrossing the 129 interval between microsatellites D1Mit105 and D1Mit223 (80 cM to 106 cM) onto the C57BL/6 strain. Inherited 129 regions were mapped with microsatellite markers polymorphic between 129 and C57BL/6 mice (see below). After seven generations of backcrossing, siblings were intercrossed to generate C57BL/6.129(D1Mit105--223) congenic mice homozygous for the 129 Chromosome 1 interval. Inside 129 markers at positions 81.6 cM (D1Mit159) and 105 cM (D1Mit17), respectively, and an outside C57BL/6 marker at position 74.3cM (D1Mit159) were used to further define the interval. In the C57BL/6.*Apcs* ^−/−^ mice (backcrossed onto C57BL/6 for ten generations), the 129 genome around the *Apcs* locus was mapped as a segment from 87.9 cM (D1Mit15) to 105 cM (D1Mit17). In this analysis, the inside 129 markers were at positions 93 cM (D1Mit36) and 99.7 cM (D1Mit115) and the outside C57BL/6 markers at positions 81.6 cM (D1Mit159) and 106 cM (D1Mit223). Along with 28 C57BL/6.*Apcs* ^−/−^mice and 30 C57BL/6 wild-type animals, 26 C57BL/6.129(D1Mit105--223) female mice ^−/−^were followed up to 1 y of age. Animals were maintained in specific pathogen-free conditions. All animal procedures were in accordance with institutional guidelines.
### Serological analyses. {#s4a2}
Sera, collected at 6 and 12 mo of age, were assayed for the presence of auto-Abs. Levels of IgG ANA were sought by indirect immunofluorescence using Hep-2 cells, and anti-dsDNA Abs were detected by indirect immunofluorescence on Crithidia luciliae as previously described ([@pbio-0020243-Mitchell1]). Serum samples were screened at a 1:80 (ANA) or 1:20 (anti-dsDNA) dilution and the positive samples titrated to endpoint. Abs to ssDNA and anti-chromatin were measured by ELISA, as previously described ([@pbio-0020243-Mitchell1]). Samples were screened at a 1:100 dilution, and the results were expressed in arbitrary ELISA units (AEUs) relative to a standard positive sample (derived from an MRL/Mp.*lpr/lpr* mouse), which was assigned a value of 100. For interplate comparison, serial dilutions of a positive control serum sample were included on each plate. Apcs levels were assessed by ELISA using sheep anti-mouse Apcs and rabbit anti-mouse Apcs Abs (Calbiochem, Nottigham, United Kingdom). Samples were screened at a 1:3,000 dilution, and the results were expressed in milligrams per liters, referring to a standard curve derived from an acute-phase serum with a known concentration of Apcs (Calbiochem). *Apcs* ^−/−^ mouse serum was included as a negative control.
### Histological analysis. {#s4a3}
All the mice, except the few that died before the end of the experiment, were sacrificed at 1 y of age, and kidney portions were fixed in Bouin\'s solution and paraffin embedded, and sections were stained with periodic acid--Schiff reagent. Glomerular histology was graded in a blinded fashion as follows: grade 0, normal; grade 1, hypercellularity involving greater than 50% of the glomerular tuft in 25%--50% of glomeruli; grade 2, hypercellularity involving greater than 50% of the glomerular tuft in 50%--75% of glomeruli; grade 3, glomerular hypercellularity in greater than 75% of glomeruli or crescents in greater than 25% of glomeruli.
### Statistical analysis {#s4a4}
Non-parametric data are expressed as median with range of values in parentheses. All statistics were calculated using GraphPad Prism^TM^ version 3.0 for Windows (GraphPad Software, San Diego, California, United States). Non-parametric tests were applied throughout, with differences being considered significant for *p* values \< 0.05. The Mann--Whitney test was used for comparison of two groups, whilst for analysis of three groups the Kruskal--Wallis test with Dunn\'s multiple comparison test was used.
### Genotypic analysis {#s4a5}
Genotyping was carried out by PCR of genomic DNA using 143 polymorphic markers (list available on request) distributed throughout all 19 autosomes. PCRs were performed using standard reagents containing 1.5 mM MgCl~2~ and 0.4 μM primers. Microsatellite markers were screened for size polymorphisms between 129 and C57BL/6 mice. Only primers with differences detectable on ethidium bromide-stained agarose gels or on SDS-polyacrylamide gels were used.
### Linkage analysis {#s4a6}
The QTL program MAPMANAGER.QTL (<ftp://mcbio.med.buffalo.edu/pub/MapMgr/>) was used, and the two experimental groups were examined independently. Only data from mice at 12 mo of age were analysed. Log transformations of auto-Abs levels resulted in more normalised distribution and were used in QTL mapping. LOD thresholds for suggestive and significant linkages were determined by using a cohort- and trait-specific permutation test (1,000 permutations). The average threshold for suggestive, significant, and highly significant linkages were LOD ≥ 2.1 (*p* ≤ 7.8 × 10^−3^), LOD ≥ 3.6 (*p* ≤ 2.4 × 10^−4^), and LOD ≥ 5 (*p* ≤ 1 × 10^−5^), respectively ([@pbio-0020243-Manly1]).
Supporting Information {#s5}
======================
Accession Numbers {#s5a1}
-----------------
The LocusLink (<http://www.ncbi.nlm.nih.gov/LocusLink/>) ID numbers for the genes and gene products discussed in this paper are *Apcs* (LocusLink ID 20219), CD35/CD21 (LocusLink ID 12902), CD55 (LocusLink ID 13136), the FAS receptor gene (LocusLink ID 14102), and FcγRII (LocusLink ID 14130).
::: {#pbio-0020243-g002 .fig}
Figure 2
::: {.caption}
###### Linkage of Chromosome 8 with Anti-Chromatin and Anti-dsDNA Abs in (129 × C57BL/6)F2 Mice
These linkages were not detected in (129 × C57BL/6)F2*.Apcs^−/−^* animals. Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from <http://www.informatics.jax.org>. Dotted lines indicate the threshold over which linkage was considered suggestive, as defined in Materials and Methods.
:::
:::
We are grateful to M. Lewis for the processing of the samples for histology and to F. Reid and D. Mitchell for their help. We thank all of the staff in the animal facility for their technical assistance. This work was supported by the Wellcome Trust (grant number 061438). JCH was a recipient of a fellowship from the National Institute of Health, Spain (BEFI 99/9212).
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** MJW, TJV, and MB conceived and designed the experiments. AEB, KLR, JW, JC-H, and RJR performed the experiments. AEB, KLR, RJR, and HTC analyzed the data. MB wrote the paper.
Academic Editor: David Nemazee, Scripps Research Institute
¤ Current address: The Wellcome Trust, London, United Kingdom
Citation: Bygrave AE, Rose KL, Cortes-Hernandez J, Warren J, Rigby RJ, et al. (2004) Spontaneous autoimmunity in 129 and C57BL/6 mice---Implications for autoimmunity described in gene-targeted mice. PLoS Biol 2(8): e243.
Ab
: antibody
ANA
: anti-nuclear antibody
AEU
: arbitrary ELISA unit
*Apcs*^−/−^
: Apcs-deficient mice
*Apcs*
: serum amyloid P component gene
dsDNA
: double-stranded DNA
LOD
: logarithm of odds
QTL
: quantitative trait linkage
SLE
: systemic lupus erythematosus
ssDNA
: anti-single-stranded DNA
| PubMed Central | 2024-06-05T03:55:46.775425 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509305/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e243",
"authors": [
{
"first": "Anne E",
"last": "Bygrave"
},
{
"first": "Kirsten L",
"last": "Rose"
},
{
"first": "Josefina",
"last": "Cortes-Hernandez"
},
{
"first": "Joanna",
"last": "Warren"
},
{
"first": "Robert J",
"last": "Rigby"
},
{
"first": "H. Terence",
"last": "Cook"
},
{
"first": "Mark J",
"last": "Walport"
},
{
"first": "Timothy J",
"last": "Vyse"
},
{
"first": "Marina",
"last": "Botto"
}
]
} |
PMC509306 | Introduction {#s1}
============
The vast majority of all cellular secretory and membrane proteins are folded and modified in the endoplasmic reticulum (ER), from which they are transported to their final destination in the secretory pathway. When the protein folding capacity of the ER is exceeded or experimentally impaired, unfolded proteins accumulate in the ER and activate the unfolded protein response (UPR). The UPR allows the ER to communicate with the nucleus ([@pbio-0020246-Patil1]), where a comprehensive gene expression program is induced to adjust the protein folding capacity of the cell according to need.
In the yeast *S. cerevisiae,* unfolded ER proteins stimulate the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1p ([@pbio-0020246-Cox2]; [@pbio-0020246-Mori2]; [@pbio-0020246-Sidrauski1]). When activated, Ire1p excises a 252-nucleotide intron from the mRNA encoding Hac1p, a bZIP transcription factor required for induction of all UPR target genes ([@pbio-0020246-Cox1]; [@pbio-0020246-Mori3]; [@pbio-0020246-Sidrauski1]). Removal of the *HAC1* intron and subsequent ligation of the two liberated exons by tRNA ligase ([@pbio-0020246-Sidrauski2]) produces a spliced mRNA that is efficiently translated ([@pbio-0020246-Kawahara1]). In the absence of splicing, the intron blocks translation of the mRNA ([@pbio-0020246-Ruegsegger1]). Splicing is therefore a prerequisite for Hac1p production and thus serves as the key regulatory step in the UPR. When it is produced, Hac1p binds an upstream activating sequence (UAS), the unfolded protein response element (UPRE), found in the promoters of UPR target genes ([@pbio-0020246-Mori1]; [@pbio-0020246-Kohno1]), thereby stimulating the transcriptional response to protein unfolding.
Several salient features of the UPR are conserved between yeast and metazoans. In metazoans, Ire1p orthologs Ire1-α and Ire1-β remove a short intron from the *XBP-1* mRNA, which encodes a bZIP transcription factor analogous to Hac1p ([@pbio-0020246-Wang1]; [@pbio-0020246-Miyoshi1]; [@pbio-0020246-Urano1]; [@pbio-0020246-Calfon1]). The metazoan UPR, however, is implemented by at least two additional ER-resident sensors, which are thought to act in parallel and induce multiple downstream transcriptional activators not known to exist in yeast. A second branch of ER-to-nucleus signaling is mediated by ATF-6, a bZIP transcription factor that is synthesized as an integral ER transmembrane protein ([@pbio-0020246-Haze1]). Upon UPR induction, ATF-6 is proteolytically cleaved, liberating a soluble fragment that moves to the nucleus to induce transcription in association with XBP-1 ([@pbio-0020246-Wang2]; [@pbio-0020246-Ye1]; [@pbio-0020246-Steiner1]; [@pbio-0020246-Yoshida1]; [@pbio-0020246-Lee1]). A third branch of the metazoan UPR provides translational control by the ER transmembrane kinase PERK ([@pbio-0020246-Harding1]; [@pbio-0020246-Liu1]). When activated in response to protein misfolding in the ER, PERK phosphorylates the translation initiation factor eIF-2α, thereby down-tuning translation of many mRNAs (and decreasing the translocational load on the ER) ([@pbio-0020246-Harding2], [@pbio-0020246-Harding3]). Under conditions of limiting eIF-2α activity, however, some mRNAs containing short upstream open reading frames (ORFs) in their 5′ UTR are preferentially translated. One of these mRNAs encodes a third bZIP transcription factor, ATF-4, which collaborates with XBP-1 and other cellular stress signaling factors to activate UPR targets ([@pbio-0020246-Harding2], [@pbio-0020246-Harding4]; [@pbio-0020246-Ma1]).
The UPR target genes of yeast have been comprehensively defined by microarray expression profiling, where they comprise a significant fraction of the yeast genome (381 genes, more than 5% of the ORFs) ([@pbio-0020246-Travers1]). The UPR target genes encode many proteins that play critical roles in the ER, the Golgi apparatus, and throughout the secretory pathway. Hence, the UPR can be thought of as a means of homeostatic control, serving to remodel the secretory pathway according to the cell\'s need.
The set of 381 genes was defined by microarray hybridization expression profiling, using a stringent quantitative "filter" that required the expression profile of each target gene to closely match that of previously known and well-characterized UPR target genes. In particular, the filter demanded that the expression profile of a target gene closely correlate to that of canonical UPR targets over a time course of UPR induction, and that induction be significantly greater in wild-type (WT) than in either Δ*ire1* or Δ*hac1* cells.
The identification of this vast set of target genes poses an enigma in light of the previously characterized UPRE. The UPRE was originally defined as a 22-bp sequence element of the *KAR2*/BiP promoter ([@pbio-0020246-Mori1]) and subsequently carefully refined to nucleotide precision as a semipalindromic seven-nucleotide consensus, CAGNGTG ([@pbio-0020246-Mori4]). Point mutations in any one of the six conserved nucleotides or deletion of the central nucleotide was shown to have severely detrimental effects on the ability of the element to function as an autonomous UAS when placed into an otherwise silent promoter. Yet, inspection of the 381 promoter sequences of the experimentally defined set of target genes failed to reveal a recognizable UPRE in most of them. This observation is particularly surprising given that the UPRE is thought to be the Hac1p binding site, and *HAC1* has been shown to be required for activation of all UPR target genes. One possible resolution to this paradox is that additional, heretofore unrecognized UPREs exist that are required for the activation of the genes lacking the "classical" UPRE. A requirement for new *cis*-activating sequences in the promoters of UPR target genes raises the possibility that such sequences could be bound by other *trans*-acting factors, alone or in combination with Hac1p, and thus contribute to the transcriptional complexity of the UPR.
Results {#s2}
=======
Computational Identification of Target Motifs {#s2a}
---------------------------------------------
To identify sequence motifs shared by the set of UPR target genes, we employed a bioinformatics approach to build a "dictionary" of putative regulatory elements from the promoters of these genes. In this approach, DNA sequence is considered as a "text" (a long string of nucleotides), which is modeled as having been composed by concatenating "words" (short oligonucleotides) drawn from a probabilistic "dictionary" according to their frequencies. To infer the dictionary from the observed text, we employed the previously developed computational algorithm, MobyDick, which was developed based on a probabilistic segmentation model ([@pbio-0020246-Bussemaker1], [@pbio-0020246-Bussemaker2]). MobyDick has been used previously to identify regulatory sites in large sets of promoters activated during sporulation or by specific cell-cycle stage.
We first constructed a dictionary from the UPR target gene promoters. To this end, we compiled a text from the promoters of all 381 UPR target genes as previously defined ([@pbio-0020246-Travers1]). We defined the promoter region for each ORF as the 600 nucleotides upstream of the initiation codon. Probabilistic segmentation analysis using the MobyDick algorithm indicated that the target gene promoters are best modeled by a dictionary of about 300 words of eight nucleotides or less (for details of this and subsequent calculations, see [Materials and Methods](#s4){ref-type="sec"}; a complete report of the dictionary with associated statistics appears in [Table S1](#st001){ref-type="supplementary-material"}). These words represent the sequences that are most frequent in the target gene promoters.
Because words with similar sequences are likely to possess similar biological activity, we considered groups of related words as units in our subsequent analysis. We grouped the dictionary into motifs by performing every possible pairwise alignment between all words, and then clustering words with high mutual alignment scores. A motif may contain two or more words, or just a single word. For a multiword motif, the words defining the motif are similar to one another and share common core sequences ([Figure 1](#pbio-0020246-g001){ref-type="fig"}B; [Table S2](#st002){ref-type="supplementary-material"}). The clustering procedure yielded about 100 motifs, about half of which contain multiple words.
::: {#pbio-0020246-g001 .fig}
Figure 1
::: {.caption}
###### Computational Selection of Candidate Regulatory Motifs
\(A) Candidate regulatory motifs are overrepresented in UPR target promoters. Sequence motifs were ranked in order of overrepresentation, i.e., on the number of observed appearances in target promoters relative to the expectation from the total appearances in all promoters. −log~10~ *P,* a metric of overrepresentation, is plotted against rank (circles). Eight motifs were chosen for experimental characterization (open circles).
\(B) Best words grouped into eight candidate motifs. The eight most overrepresented motifs from [Fig. 1](#pbio-0020246-g001){ref-type="fig"}A, aligned to illustrate common core sequences. The example of each motif chosen for experimental characterization is underlined.
:::
:::
We reasoned that motifs that are likeliest to represent bona fide regulatory elements will be nonrandomly distributed in the genome and appear more often in the UPR target gene promoters than expected by chance. Therefore, we counted the number of times each motif (i.e., a sequence match to any of the words the motif comprises) appeared in the approximately 6,000 promoters in the genome, and computed from this figure the frequency with which each motif would be expected to appear in a promoter if it were distributed randomly throughout the genome. We then counted the number of times the motif was actually found in the 381 target promoters and calculated the probability *P* of this many or more appearances occurring by chance. A small *P* value (high −log~10~ *P*) indicates that the motif is overrepresented relative to the expectation. [Figure 1](#pbio-0020246-g001){ref-type="fig"}A shows the motifs ranked in order of decreasing overrepresentation, with −log~10~ *P* for each motif plotted against this rank. We chose the eight highest-ranking motifs (open circles) as candidates for experimental testing ([Figure 1](#pbio-0020246-g001){ref-type="fig"}B), analyzing a single example of each (underlined sequences).
Experimental Verification of Novel UPREs {#s2b}
----------------------------------------
To determine whether any of the eight candidate motifs would function as bona fide UPREs, we introduced three tandem repeats of a single representative sequence of each motif into a *lacZ* reporter construct that contains a crippled version of the *CYC1* promoter that is transcriptionally silent in the absence of a UAS ([@pbio-0020246-Guarente1]). Analogous constructs containing the "classical," *KAR2*-derived UPRE inserted upstream of the core promoter have been shown to drive transcription of this reporter gene under ER stress ([@pbio-0020246-Mori1]; [@pbio-0020246-Cox2]). As a positive control for UPR-dependent gene expression, we used a construct containing a triple repeat of the *KAR2*-derived UPRE ([@pbio-0020246-Cox1]). We transformed the resulting plasmids into yeast and assayed for β-galactosidase activity in response to ER stress.
Of the eight reporter constructs, the two containing Motif 1 and Motif 8 were transcriptionally activated when cells were treated with tunicamycin (Tm) ([Figure 2](#pbio-0020246-g002){ref-type="fig"}A), or dithiothreitol (DTT) (unpublished data), both inducers of the UPR. The other six motifs showed no activity above baseline (unpublished data). Neither Motif 1 nor Motif 8 showed any activity in the absence of ER stress, and no activation was observed upon UPR induction in either Δ*ire1* or Δ*hac1* strains. Hence, as with the "classical" UPRE, these two motifs are sufficient to confer transcriptional activation upon a promoter in an *IRE1*-*, HAC1*-*,* and ER stress-dependent manner. We therefore conclude that the bioinformatics analysis has identified two novel UPREs present in target gene promoters; hereafter, we refer to Motif 1 and Motif 8 as UPRE-2 and UPRE-3, respectively. Correspondingly, we shall refer to the classical, *KAR2*-derived UPRE as UPRE-1.
::: {#pbio-0020246-g002 .fig}
Figure 2
::: {.caption}
###### Identification of Two Novel Sequence Motifs Necessary and Sufficient for UPR Activation
\(A) Motif 1 and Motif 8 are sufficient to confer UPR-responsive transcription on an artificial promoter*.* Single representative sequences of the *KAR2-*derived UPRE and candidate regulatory motifs Motif 1 and Motif 8 were cloned into a crippled promoter driving *lacZ,* transformed into yeast (WT, Δ*ire1,* and Δ*hac1*), and β-galactosidase activity monitored in response to Tm treatment.
\(B) UPRE-2 (Motif 1) is necessary for UPR-dependent activation of the *ERO1* promoter. lacZ was placed under the control of the WT *ERO1* promoter (+ UPRE-2) or a mutant (− UPRE-2), and β-galactosidase activity monitored in response to DTT treatment.
\(C) UPRE-3 (Motif 8) is necessary for UPR-dependent activation of the *DHH1* promoter. As in (B), except using the *DHH1* promoter, in which UPRE-3 appears once.
\(D) Novel motifs explain a greater fraction of UPR target gene activation. Sets of genes whose promoters contain UPR-responsive UASs UPRE-1, UPRE-2, UPRE-3, or a combination, are here depicted in Venn diagram format as subsets of the 381-gene UPR target set.
:::
:::
To test whether these motifs are also necessary for transcriptional activation, we designed *lacZ* reporter constructs derived from two native promoters in which the motifs appear. We chose for UPRE-2 the promoter of *ERO1,* encoding an ER resident redox protein, and for UPRE-3 the promoter of *DHH1,* encoding an RNA helicase. Both genes are robust targets of the UPR ([@pbio-0020246-Travers1]) and lack a recognizable UPRE-1. First, we verified that the reporters responded to ER stress in a UPR-dependent manner. WT but not Δ*ire1* or Δ*hac1* cells bearing the UPRE-2-containing *ERO1*-promoter-driven reporter expressed higher levels of β-galactosidase after treatment with DTT ([Figure 2](#pbio-0020246-g002){ref-type="fig"}B, "+ UPRE-2" columns). In a mutant version of this reporter construct, in which the UPRE-2 was ablated and replaced by an unrelated sequence of identical length, inducibility of the *ERO1* promoter was decreased by approximately 4-fold ("− UPRE-2" columns). Similarly, WT but not Δ*ire1* or Δ*hac1* cells bearing the UPRE-3-containing *DHH1*-promoter-driven reporter expressed higher levels of β-galactosidase after treatment with DTT ([Figure 2](#pbio-0020246-g002){ref-type="fig"}C, "+ UPRE-3" columns); ablation of UPRE-3 from the *DHH1* promoter entirely eliminated induction by ER stress ( "− UPRE-3" columns).
Taken together, the data presented so far indicate that, as with the classical UPRE-1, UPRE-2 and UPRE-3 are both sufficient ([Figure 2](#pbio-0020246-g002){ref-type="fig"}A) and necessary ([Figure 2](#pbio-0020246-g002){ref-type="fig"}B and [2](#pbio-0020246-g002){ref-type="fig"}C) to confer UPR inducibility on a target promoter. The addition of UPRE-2 and UPRE-3 to the repertoire of UPREs triples the number of genes in the UPR target set whose induction we can explain by invoking the presence of a well-defined UAS ([Figure 2](#pbio-0020246-g002){ref-type="fig"}D).
Identification of High-Copy Activators of UPRE-2 {#s2c}
------------------------------------------------
The existence of functional *cis*-regulatory elements that differ in sequence from the canonical UPRE-1 suggests that *trans*-activating factors other than Hac1p may bind these elements. Alternatively, Hac1p, alone or accompanied by another factor or factors, may be able to recognize multiple sequences. To distinguish between these possibilities and potentially reveal novel regulatory factors, we attempted to identify genes which, when overexpressed, activate transcription of the UPRE-2 reporter plasmid in the absence of an ER stress signal. The design of this screen recapitulates the approach which identified *HAC1* as a high-copy activator of the UPRE-1 ([@pbio-0020246-Cox1]).
We transformed a strain bearing the UPRE-2-*lacZ* reporter with a 2-μm-plasmid-derived ( high-copy) genomic DNA library ([@pbio-0020246-Miller1]). A Δ*ire1* strain was used in order to focus the screen on genes acting downstream of *IRE1.* Use of the Δ*ire1* strain also avoided a background of false positives resulting from library plasmids encoding secretory proteins whose overexpression might activate Ire1p. Transformants were plated on synthetic defined media and, after appearance of colonies, overlaid with soft agar containing the β-galactosidase substrate X-gal. Colonies that turned significantly more blue than control (untransformed) colonies were recovered and rescreened by the same assay. Plasmids from positively rescreened clones were retransformed into the Δ*ire1* UPRE-2-*lacZ* strain to verify plasmid linkage of the activator phenotype.
We screened a total of 112,000 transformants, representing a predicted genomic coverage of approximately 50x. Thirty-eight positive transformants passed through repetition and plasmid linkage tests, and 18 of these stably maintained the activator phenotype over many generations. Positive plasmids fell into two classes, as defined by the minimal region of overlap of their insert sequences. One class of inserts (ten plasmids) shared the *IRE1* locus and surrounding sequences; *IRE1* has been previously shown to be activated by overexpression and is a high-copy activator of UPRE-1 ([@pbio-0020246-Cox2]). Recovery of this locus demonstrates that the screen was able to capture genes of physiological relevance to the pathway.
The second class of positive inserts (eight plasmids) shared the *GCN4* locus. *GCN4* encodes a bZIP transcription factor, which has been well-characterized as a component of the cellular response to amino acid starvation and other stresses ([@pbio-0020246-Natarajan1]; reviewed in [@pbio-0020246-Hinnebusch2]) but has not been previously demonstrated to play a role in the UPR. We constructed a 2-μm plasmid bearing only *GCN4,* transformed it into WT, Δ*ire1,* and Δ*hac1* strains carrying UPRE-1-*lacZ,* UPRE-2-*lacZ,* and UPRE-3-*lacZ* reporters, and assayed for β-galactosidase activity ([Figure 3](#pbio-0020246-g003){ref-type="fig"}A). *GCN4* overexpression stimulated UPRE-2-driven reporter activity in all three genotypes ( "+ GCN4 2μ" columns), indicating that overexpression of *GCN4* is sufficient to stimulate transcription from the UPRE-2-driven reporter gene in the absence of ER stress, Ire1p activity, or Hac1p production. We also starved cells for histidine by administering 3-aminotriazole (3-AT), which induces translation of Gcn4p ([@pbio-0020246-Albrecht1]). As when cells expressed high levels of *GCN4,* amino acid starved cells exhibited a significant increase of UPRE-2 transcription in the absence of ER stress ( "+3-AT, −Tm" columns). *GCN4* overexpression alone did not activate transcription from either UPRE-1 or UPRE-3 reporter genes, emphasizing that these motifs are not synonymous with UPRE-2.
::: {#pbio-0020246-g003 .fig}
Figure 3
::: {.caption}
###### *GCN4* Encodes a Novel Transcription Factor in the UPR
\(A) Overexpression of *GCN4* is sufficient for activation of UPRE-2, but not UPRE-1 or UPRE-3. UPRE-driven transcriptional activity as a function of Gcn4p levels, elevated either as a result of overexpression (+ *GCN4--2μ*) or amino acid starvation (+ 3-AT), in the presence or absence of ER stress (Tm).
\(B) *GCN4* and *GCN2* are necessary for ER stress-dependent activation of UPRE-1 and UPRE-2. UPRE-driven transcriptional activity as a function of *GCN4* pathway genes (WT, Δ*gcn4,* and Δ*gcn2*) in the presence or absence of ER stress (Tm).
\(C) *GCN4* and *GCN2* are required for UPR-dependent transcriptional activation of a subset of target genes. Fold changes in mRNA levels were determined by microarray for DTT-treated vs. -untreated WT, Δ*ire1,* Δ*gcn4,* and Δ*gcn2* strains (columns). Histograms show distribution of log~2~-fold changes for non-UPR target genes (light bars) and for UPR target genes (dark bars), which contain UPRE-1, UPRE-2, UPRE-3, or still unidentified UPREs (rows) in their promoters.
\(D) Target gene regulation differs significantly in WT and *Δgcn4/Δgcn2* mutants. Means *(μ)* and standard deviations *(σ)* for log~2~-fold change in gene expression for non-UPR target genes, and for genes that fall inside the UPR target gene set and contain UPRE-1, UPRE-2, or UPRE-3 in their promoters. Z statistic *(z)* and *P* value *(P)*: higher *z* reflects a greater difference between the distribution for UPRE-containing target genes and nontarget genes; lower *P* indicates a more highly significant difference. For detailed calculations, see [Materials and Methods](#s4){ref-type="sec"}.
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*GCN4* and *GCN2* Are Required for Activation of All Three UPREs {#s2d}
----------------------------------------------------------------
Having demonstrated that *GCN4* overexpression is sufficient to activate transcription from a UPRE-2 reporter, we next asked whether *GCN4* is also necessary to activate transcription in response to ER stress. We deleted *GCN4* from strains bearing UPRE-1, UPRE-2, and UPRE-3 reporter constructs and assayed β-galactosidase activity in response to UPR activation. Upon UPR induction, *HAC1* mRNA was spliced normally, and Hac1p was produced at WT levels in Δ*gcn4* mutants (unpublished data). However, Δ*gcn4* cells failed to induce transcription, not only of the UPRE-2-driven reporter but also of the UPRE-1- and UPRE-3-driven reporters ([Figure 3](#pbio-0020246-g003){ref-type="fig"}B). Hence we conclude that *GCN4* is required for ER stress responsiveness of all three UPREs.
Consistent with the genetic requirement for *GCN4,* high levels of Gcn4p potentiate transcription from all UPREs when the UPR is activated. *GCN4* overexpression increases the level of reporter activation in WT cells when the UPR is induced ([Figure 3](#pbio-0020246-g003){ref-type="fig"}A, compare "*GCN4* +Tm" to "*GCN4 −*Tm" data), suggesting that *GCN4* activity is limiting for UPR-dependent transcription from all three UPREs. Similarly, stimulation of Gcn4p production by amino acid starvation also increases the magnitude of the transcriptional response ([Figure 3](#pbio-0020246-g003){ref-type="fig"}A, "+3-AT, +Tm" data).
In its role in the transcriptional response to amino acid starvation, *GCN4* is activated at the translational level. Uncharged tRNAs are detected by the kinase Gcn2p, which phosphorylates initiation factor 2α (eIF-2α); when eIF-2α is phosphorylated, scanning ribosomes fail to initiate at upstream ORFs encoded by the *GCN4* 5′ UTR and are able to initiate translation at the *GCN4* ORF itself ([@pbio-0020246-Hinnebusch2]). We therefore asked whether *GCN2* is also required for *GCN4* activity in the context of the UPR. As with Δ*gcn4* cells, Δ*gcn2* strains were also unable to mount a transcriptional response from any of the reporter constructs ([Figure 3](#pbio-0020246-g003){ref-type="fig"}B).
Given that *GCN4* and *GCN2* are necessary for ER stress-dependent transcription in an artificial promoter context, we next asked whether these genes are required for upregulation of the target genes of the UPR. To this end, we measured steady-state mRNA levels by microarray hybridization, comparing WT, Δ*ire1,* Δ*gcn4,* and Δ*gcn2* cells treated with DTT for 30 min (by which time the UPR is qualitatively complete; [@pbio-0020246-Travers1]) to untreated samples of the same genotype. WT cells were taken as a positive control for UPR induction, and Δ*ire1* cells as a negative control. Fold change in expression of a given gene was computed as the ratio of mRNA level in the treated sample to the level in an untreated sample of the same genotype.
In our analysis, we considered five subsets of genes: the sets of UPR target genes containing a UPRE-1, UPRE-2, or UPRE-3 in their promoter, the set of UPR target genes without an identified UPRE in their promoters ("no UPRE"), and the set of genes previously identified as UPR-independent ("nontargets") ([@pbio-0020246-Travers1]). The distributions of the log~2~-fold changes for each subset of genes in each genotype relative to the set of nontarget genes are illustrated in [Figure 3](#pbio-0020246-g003){ref-type="fig"}C. For each gene set in each genotype, we determined the difference between the distributions of log~2~-fold changes in UPRE target genes and those in nontarget genes. The statistical significance of these differences is represented by the *z* scores and *P* values enumerated in [Figure 3](#pbio-0020246-g003){ref-type="fig"}D; higher *z* and lower *P* indicate a greater difference between distributions and higher significance (for details see [Materials and Methods](#s4){ref-type="sec"}).
The majority of the genes in the nontarget set ([Figure 3](#pbio-0020246-g003){ref-type="fig"}C, all histograms, light bars) are not differentially regulated by ER stress in the WT and mutant strains. As previously shown, however, genes of the UPR target set are significantly more upregulated in the WT than in Δ*ire1* cells ([Figure 3](#pbio-0020246-g003){ref-type="fig"}C, compare dark bars versus light bars between histograms *a* and *b, e* and *f, i* and *j,* and *m* and *n*). This is the case both for target genes bearing any UPRE in the promoter ([Figure 3](#pbio-0020246-g003){ref-type="fig"}C, histograms *a--l*) as well as the remainder of the target set for which a UPRE has not been identified ([Figure 3](#pbio-0020246-g003){ref-type="fig"}C, histograms *m--p*). For those genes with an identified UPRE in their promoters, expression patterns in both Δ*gcn4* ([Figure 3](#pbio-0020246-g003){ref-type="fig"}C, histograms *c, g,* and *k*) and Δ*gcn2* mutants ([Figure 3](#pbio-0020246-g003){ref-type="fig"}C, histograms *d, h,* and *l*) show trends similar to those in Δ*ire1*. In both mutants, the sets of genes whose promoters contain a UPRE are significantly less upregulated relative to their induction in the WT. Some UPR target genes exhibit residual upregulation in Δ*gcn4* and Δ*gcn2,* suggesting that these promoters have only a partial requirement for *GCN4*/*GCN2*. This effect is most prominent for genes containing UPRE-1 in the Δ*gcn2* mutant ([Figure 3](#pbio-0020246-g003){ref-type="fig"}C, histogram *j*), where the residual induction crosses the threshold into marginal statistical significance ([Figure 3](#pbio-0020246-g003){ref-type="fig"}D, "Δ*gcn2,* UPRE-1"; *p* = 3.4 × 10^−4^); it is possible that the residual levels of Gcn4p present in a Δ*gcn2* mutant are sufficient to allow UPR transcription from these promoters, or alternatively that UPRE-1 promoters are relatively less sensitive to Gcn4p levels (and concomitantly, relatively more reliant on Hac1p) for induction (see Discussion). In contrast, induction of the "no UPRE" genes is quite high in Δ*gcn4* and Δ*gcn2* cells ([Figure 3](#pbio-0020246-g003){ref-type="fig"}C, histograms *o* and *p* versus *m*). As a population, these genes are not significantly less upregulated in the mutants than in the WT. It would appear that the UPREs identified to date define a special subset of UPR target genes that are responsive not only to *IRE1* and *HAC1* but that are particularly sensitive to the *GCN4*/*GCN2* branch of the pathway.
Overall, in both Δ*gcn4* and Δ*gcn2* mutants, the pattern of gene regulation during the UPR is similar to that in the Δ*ire1* mutant: Mean fold changes of UPRE-containing target genes are lower in these mutants than in the WT. We conclude that *GCN4* and *GCN2* play a broad role in the UPR, contributing significantly to the upregulation of a large subset of UPR target genes.
Gcn4p Is Upregulated in Response to ER Stress {#s2e}
---------------------------------------------
Given the requirement for *GCN4* in UPR-dependent transcription, and in particular the observation that Gcn4p appears to be limiting for the magnitude of the transcriptional response ([Figure 3](#pbio-0020246-g003){ref-type="fig"}A), we asked next whether Gcn4p levels would be subject to posttranscriptional regulation under conditions of ER stress. We discounted the possibility that *GCN4* would be regulated at the transcriptional level, as our previous studies showed that *GCN4* mRNA levels are unchanged over the course of the UPR ([@pbio-0020246-Travers1]).
We constructed strains expressing a C-terminally myc-epitope-tagged allele of Gcn4p, which complements the slow growth phenotype of a Δ*gcn4* mutant and is inducible by amino acid starvation resulting from 3-AT treatment ([Figure 4](#pbio-0020246-g004){ref-type="fig"}A, "Gcn4p" lanes, compare "wt, +3-AT" to "wt, 0 min"). Over a time course of UPR induction, Gcn4p-myc levels exhibited a transient increase of 2.5-fold, peaking after 15 min and gradually decaying to uninduced levels after 60--120 min ([Figure 4](#pbio-0020246-g004){ref-type="fig"}A, "WT" lanes; quantitated in [Figure 4](#pbio-0020246-g004){ref-type="fig"}B). This temporary increase in Gcn4p was not observed in UPR-deficient mutants: neither Δ*ire1* nor Δ*hac1* mutants exhibited increased levels of Gcn4p over the time course of UPR induction.
::: {#pbio-0020246-g004 .fig}
Figure 4
::: {.caption}
###### Gcn4p Protein Levels Are Upregulated during the UPR
\(A) Gcn4p levels, but not eIF-2α phosphorylation, rise under ER stress in a UPR-dependent manner. Cells bearing a C-terminally *myc*-tagged allele of *GCN4* were treated with Tm for 0, 15, 30, 60, or 120 min. Western blots probed with anti-myc recognizing Gcn4p-myc (top panels) or phospho-specific anti-eIF-2α antibody (bottom panel). Gcn4p blot for the Δ*gcn2* mutant is 5x overexposed so that the bands are visible.
\(B) Quantitation of the Gcn4p-myc protein levels in [Figure 4](#pbio-0020246-g004){ref-type="fig"}A. Data reflect an average of four experiments, normalized against Gcn4p levels in the WT *t* = 0 samples.
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In the context of other stress responses (e.g., amino acid starvation), Gcn4p levels are regulated via phosphorylation of eIF-2α by Gcn2p ([@pbio-0020246-Dever1]; [@pbio-0020246-Hinnebusch1]; [@pbio-0020246-Diallinas1]). Because *GCN2* is required for induction of UPR-dependent transcription, we asked whether *GCN2* was required for the rise in Gcn4p levels we observed during Tm treatment. Basal levels of Gcn4p are low in a Δ*gcn2* strain (less than 10% of WT), as previously reported ([@pbio-0020246-Hinnebusch1]; [@pbio-0020246-Tavernarakis1]). We observed no increase in Gcn4p levels during the time course in this mutant ([Figure 4](#pbio-0020246-g004){ref-type="fig"}B).
These data are consistent with two possibilities: first, that Gcn2p is responsible for both basal levels of Gcn4p and its induction upon ER stress; or second, that Gcn2p is responsible only for maintaining basal levels of Gcn4p, while another pathway mediated by Ire1p/Hac1p further elevates Gcn4p levels during the UPR. If Gcn2p is responsible for upregulation of Gcn4p during the UPR, we should observe a concomitant increase in the level of eIF-2α phosphorylation. We did not observe such an increase ([Figure 4](#pbio-0020246-g004){ref-type="fig"}A, "eIF-2α-P" lanes), which is consistent with the idea that Gcn2p\'s role in the UPR is primarily to maintain basal levels of Gcn4p, not to upregulate Gcn4p via increased eIF-2α phosphorylation. Other workers have observed a transient increase in phospho-eIF-2α under Tm treatment ([@pbio-0020246-Cherkasova1]). It is possible that strain differences or the significantly greater doses of Tm used in the previous study (4 and 20 μg/ml versus our 1 μg/ml) explain this disparity. Consistent with our findings, [@pbio-0020246-Cherkasova1] predict derepression of *GCN4* by ER stress mediated by increased phospho-eIF-2α. Here, we observe increased Gcn4p levels under ER stress conditions even when phospho-eIF-2α levels are not detectably altered.
Epistasis of *HAC1* and *GCN4* {#s2f}
------------------------------
*GCN4* plays an essential role in the UPR, with a knockout phenotype closely resembling that of Δ*ire1* and Δ*hac1:* the absence of any of these genes prevents transcriptional activation by ER stress. This observation could be a consequence of one of several different mechanisms: Gcn4p might act upstream or downstream of Hac1p in the same linear pathway, or act in a parallel pathway that converges at target promoters. Two lines of evidence from data already introduced argue that Gcn4p does not act upstream of Hac1p. First, *GCN4* overexpression is sufficient to activate transcription from UPRE-2 in a Δ*hac1* mutant (see [Figure 3](#pbio-0020246-g003){ref-type="fig"}A), indicating that Gcn4p\'s influence on target promoters can occur by a Hac1p-independent mechanism. Second, the transient upregulation of Gcn4p levels observed under ER stress is absent in the Δ*hac1* mutant (see [Figure 4](#pbio-0020246-g004){ref-type="fig"}A), indicating that Hac1p levels determine Gcn4p levels.
Further evidence that Gcn4p does not act upstream of Hac1p is provided by the observation that expression of Hac1p cannot activate transcription in a Δ*gcn4* mutant ([Figure 5](#pbio-0020246-g005){ref-type="fig"}). In a WT cell, expression of Hac1p produced from a *HAC1* gene lacking the intron is sufficient to activate transcription from the UPRE-1 ([@pbio-0020246-Cox1]; [Figure 5](#pbio-0020246-g005){ref-type="fig"}, "UPRE-1" columns). Constitutive expression of Hac1p is also sufficient to activate UPRE-2, and to a lesser extent UPRE-3, in the absence of ER stress ([Figure 5](#pbio-0020246-g005){ref-type="fig"}, "WT, +Hac1p" columns) and in the absence of Ire1p ([Figure 5](#pbio-0020246-g005){ref-type="fig"}, "Δ*ire1*, +Hac1p" columns). In the absence of *GCN4,* however, the constitutive expression of Hac1p does not activate transcription from any of the three reporter constructs ([Figure 5](#pbio-0020246-g005){ref-type="fig"}, "Δ*gcn4,* +Hac1p" columns), suggesting that Hac1p\'s function at promoters containing any one of the three UPREs requires the presence of Gcn4p. Thus, Gcn4p must act at the same point as or downstream of Hac1p. Following the same line of reasoning, for UPRE-1 and UPRE-3, *GCN4* overexpression alone is insufficient to activate transcription in the absence of *HAC1* (e.g., see [Figure 3](#pbio-0020246-g003){ref-type="fig"}A, Δ*hac1* mutants), indicating that at UPRE-containing promoters Hac1p must act at the same point as or downstream of Gcn4p. Thus, the observations enumerated here are consistent with the interpretation that Gcn4p and Hac1p act together at target gene promoters.
::: {#pbio-0020246-g005 .fig}
Figure 5
::: {.caption}
###### *GCN4* Acts with or Downstream of *HAC1*
UPRE reporter activity as a function of Hac1p expression and UPR pathway genes. To express Hac1p in the absence of ER stress, we used an intron-less allele of *HAC1,* which is constitutively translated.
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A Gcn4p/Hac1p Complex Binds Both the UPRE-1 and UPRE-2 {#s2g}
------------------------------------------------------
To explore this possibility directly, we performed gel-retardation assays with the UPRE-1-containing segment of the *KAR2* promoter (oligo 1), used in previous experiments demonstrating direct binding of Hac1p to UPRE-1 ([@pbio-0020246-Cox1]), and the UPRE-2-containing segment of the *ERO1* promoter (oligo 2). ^32^P-labeled oligonucleotides were incubated with cell extracts and subjected to native (nondenaturing) polyacrylamide gel electrophoresis, and visualized by autoradiography ([Figure 6](#pbio-0020246-g006){ref-type="fig"}).
::: {#pbio-0020246-g006 .fig}
Figure 6
::: {.caption}
###### Hac1p and Gcn4p Directly Interact with UPRE-1 and UPRE-2
^32^P-labeled oligos bearing either UPRE-1 or UPRE-2 promoter were incubated with crude cell extracts, and subjected to nondenaturing polyacrylamide gel electrophoresis.
\(A) Extract: Samples were of the WT, or bore deletions in *IRE1*Δ*ire1), GCN4 (*Δ*gcn4),* or *GCN2 (*Δ*gcn2),* and were treated with Tm (+) or mock treated (−). Labeled oligos contained either UPRE-1 (1) or UPRE-2 (2). Binding reactions were incubated with no unlabeled competitor (−) or with 100x excess of unlabeled WT UPRE-1 (1), an inactive mutant version of UPRE-1 (1\*), UPRE-2 (2), or an inactive mutant version of UPRE-2 (2\*).
\(B) Extract: Samples from a strain overexpressing *GCN4* (2μ-*GCN4;* lanes 1 and 2) or from a strain expressing myc-tagged Gcn4p and HA-tagged Hac1p *(GCN4-myc* and *HA-HAC1).* Binding reactions were incubated with no antibody (−), anti-myc recognizing Gcn4p-myc (myc), anti-HA recognizing HA-Hac1p (HA), or both antibodies simultaneously (myc/HA). Bands represent the following: *a,* Gcn4p + Hac1p + anti-myc + anti-HA; *b,* Gcn4p + Hac1p + anti-HA; *c,* Gcn4p + Hac1p + anti-myc; *d,* Gcn4p + Hac1p; *e,* Gcn4p. \*, an unidentified band that appears only when extracts include both Gcn4-myc and HA-Hac1p and when both antibodies are included in the binding reaction.
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As previously observed, oligo 1\'s mobility was retarded when incubated with crude extracts from UPR-induced cells, but not extracts from untreated cells ([Figure 6](#pbio-0020246-g006){ref-type="fig"}A; compare lane 2 to lane 1). Likewise, oligo 2 was specifically shifted by extracts from UPR-induced cells (compare lane 6 to lane 5). The binding activity is specific: for both oligos, the mobility shift was competed out by 100-fold excess of an unlabeled identical sequence (lanes 3 and 7) but not by a transcriptionally inactive point mutant of the same sequence (lanes 4 and 8). The binding activity is dependent on an intact UPR. No gel retardation was observed for either sequence in an Δ*ire1* mutant (lanes 9 and 12), in which Hac1p cannot be synthesized. Likewise, in both Δ*gcn4* and Δ*gcn2* mutants, the binding activity observed in WT cells was absent. In both Δ*gcn4* and Δ*gcn2* mutants, however, a faster migrating complex appeared, which likely represents Hac1p alone binding the oligos (lanes 10, 11, 13, and 14).
To demonstrate Gcn4p and Hac1p binding conclusively, we performed supershift analyses of the WT complex by addition of antibodies to either protein. We constructed a strain expressing both HA-epitope-tagged Hac1p and myc-tagged Gcn4p. Extracts from Tm-treated cells were incubated with antibodies against either or both tagged proteins. Antibodies recognizing either the tagged Gcn4p-myc ([Figure 6](#pbio-0020246-g006){ref-type="fig"}B, lanes 5 and 6) or HA-Hac1p (lanes 7 and 8) supershifted the bound complex to different extents (compare lanes 7 and 8 to lanes 3 and 4). Hence, both Gcn4p and Hac1p can bind to sequences containing UPRE-1 and UPRE-2. Addition of both antibodies to the same binding reaction resulted in an ultrashifted band, migrating more slowly than the bands in either of the single antibody reactions (lanes 9 and 10). If Hac1p and Gcn4p bound DNA in distinct, separate complexes, we would expect to see two bands of identical mobility to those seen in lanes 5--8. We conclude that the mobility-shifted complex observed in UPR-induced WT cells therefore must contain both transcription factors, since no ultrashift would occur if the proteins were bound to separate complexes, and that Hac1p and Gcn4p act together at the same location to activate transcription upon UPR induction. (Similar gel-shift experiments performed with an oligonucleotide representative of UPRE-3 failed, indicating that transcription factor binding may be of reduced affinity at this sequence. This interpretation is consistent with the overall lower activity of the UPRE-3 reporter constructs (see [Figure 2](#pbio-0020246-g002){ref-type="fig"}A).
Further evidence that Gcn4p can bind UPRE-2 is provided by the observation that overexpression of *GCN4* alone in an otherwise WT cell, in the absence of ER stress, resulted in a mobility shift for oligo 2 ([Figure 6](#pbio-0020246-g006){ref-type="fig"}B, lane 2). This complex migrated faster than the WT complex (e.g., [Figure 6](#pbio-0020246-g006){ref-type="fig"}B, lane 4). Because the extract was made from untreated cells, no Hac1p was present, indicating that the complex contains Gcn4p alone. The *GCN4*-dependent shift is not observed for oligo 1, consistent with observations above that Gcn4p overproduction is sufficient to activate transcription of a UPRE-2 reporter but not a UPRE-1 reporter (see [Figure 3](#pbio-0020246-g003){ref-type="fig"}A). Reciprocally, Hac1p is present in the Δ*gcn4* and Δ*gcn2* mutants, but Gcn4p is absent; it therefore seems likely that the faster migrating bands in Δ*gcn4*/Δ*gcn2* mutants ([Figure 6](#pbio-0020246-g006){ref-type="fig"}A, lanes 10, 11, 13, and 14) represent oligonucleotides bound to Hac1p alone.
Discussion {#s3}
==========
Identification of Novel UASs {#s3a}
----------------------------
Beginning only with the set of genes induced by the UPR and the promoter sequences of all genes in the genome, we computationally identified candidate motifs that obeyed the statistical properties we would expect of regulatory sequences, i.e., high frequency in UPR target promoters, and enrichment in the target promoters relative to the rest of the promoters in the genome. Two of these motifs, UPRE-2 and UPRE-3, are both necessary and sufficient to confer ER stress responsiveness in an *IRE1*- and *HAC1*-dependent manner on promoters which contain them. These novel sequences are activated under the same conditions as UPRE-1. Functional non-synonymy of these sequences, however, is illustrated by the activation of UPRE-2 by *GCN4* overexpression alone, a condition under which UPRE-1 and UPRE-3 are silent, and by the quantitative difference with which the motifs respond to UPR activation (UPRE-2 \> UPRE-1 \> UPRE-3). Although the two new UPRE sequences look at first glance entirely different from the well-characterized UPRE-1, one of them may share "half-site" similarity: UPRE-2 has a three base identity with UPRE-1 at the 3′ end (TACGTG versus CAGNGTG); whether these bases make equivalent contacts with the bound transcription factors remains to be determined. Taken together, the sequence diversity of the motifs conferring similar transcriptional control upon binding of the same transcriptional activators illustrates the difficulty of predicting biological regulation from promoter sequences alone, even if binding sites in one context are well defined experimentally.
The identification of these novel sequences allows a greater proportion of UPR target gene regulation to be explained within the paradigm of modular transcriptional control, i.e., in which a "portable" sequence module (a UAS) located within a promoter confers pathway responsiveness on the gene in question. The two novel motifs described triple the number of target genes whose regulation can be described in terms of a modular control mechanism, thus adding significantly to the repertoire of *cis*-acting elements known to act in the UPR.
And yet, the resulting description of UPR transcription remains incomplete, as approximately 50% of the target genes still lack a recognizable UPREs. It may be that more biologically active motifs exist among the 109 motifs that emerged from the overrepresentation analysis, as many of the untested motifs are overrepresented relative to chance in the UPR target set by many orders of magnitude. For the eight motifs tested, we tested whether a motif was necessary for promoter induction only if it had already been shown to be sufficient in the artificial promoter system. Because of this experimental approach, it remains possible that some motifs not found to be sufficient are dependent for their activity on some contextual parameter (e.g., particular nearby flanking sequences). Thus it may be that some UPREs are not generally portable to other contexts, but are nonetheless necessary for UPR responsiveness of the native promoters in which they reside. Also, particularly rare motifs would have been omitted from the dictionary; thus, it is possible that complementary computational approaches might allow detection of uncommon motifs that this analysis missed. Finally, some UASs may remain ultimately undiscoverable within the paradigm of modular regulation. Motifs that are particularly sensitive to chromatin structure or position relative to the transcription initiation site would not be detected by an approach that neglected these parameters.
It might be argued that the approach here enjoys no relative advantage over testing random oligonucleotides from UPR promoters. If every sequence from each target promoter were to be tested for activity, it is possible that additional elements not revealed by the bioinformatic approach would be discovered. For example, the residual upregulation of *ERO1* after removal of UPRE-2 (see [Figure 2](#pbio-0020246-g002){ref-type="fig"}B) suggests that at least one cryptic element exists in that promoter. On the other hand, the *DHH1* promoter shows no residual upregulation after removal of UPRE-3 (see [Figure 2](#pbio-0020246-g002){ref-type="fig"}C). If the average number of sites (candidate plus cryptic) per promoter is similar (1--2) throughout the target gene set, our computational approach represents a highly efficient means of identifying a subset of regulatory motifs. On the other hand, if the average is significantly higher, it is possible that testing random subsequences of target promoters would also be efficient. From the small number of promoters we studied in depth, it is not possible to calculate a meaningful upper bound for the average number of undiscovered regulatory sites per promoter. Nonetheless, within the sample size of our study, the yield of active regulatory sites per candidate tested (two of eight) is much higher than any reasonable a priori estimate of the density of regulatory elements in the UPR target promoters.
One indication of a possible shortcoming of our computational approach is the finding that the probabilistic segmentation did not return the classical UPRE-1 as a significant "word," i.e., the approach failed to generate a comprehensive list of all known active UPREs. The absence of UPRE-1 from the dictionary indicates that no sequence matching the experimentally defined degenerate consensus CAGNGTG is intrinsically overrepresented in the target promoters, i.e., this motif does not occur in the "text" of target gene promoters with a higher frequency than that with which its component subsequences would appear together by chance. Neither is this sequence overrepresented in the target promoter set relative to the promoters of the nontarget genes. The motif CAGNGTG has an overrepresentation score −log~10~ *P* of 0.37, far beneath the enrichment of any of the 109 motifs assembled from dictionary words (see [Figure 1](#pbio-0020246-g001){ref-type="fig"}A). Hence, among genes that possess a UPRE-1 in their promoters, there are more instances of unresponsiveness to the UPR than instances of regulation, even though UPRE-1 has been experimentally demonstrated to be necessary and sufficient for upregulation in response to ER stress.
A plausible resolution to this paradox may be that the UPRE-1 is heavily dependent on context. The experiments that defined the key core nucleotides proceeded by single point mutation at each position while holding constant the identity of all other nucleotides from the source 22-bp stretch of the *KAR2* promoter; thus the seven-nucleotide "core sequence" may only specify those bases which are necessary for activity, but not define a module which is generally functional outside its original context of flanking sequence. If this were the case, we would not expect to recover UPRE-1 in a bioinformatic analysis of all target genes. Indeed, alignment of the *KAR2* promoter from S. cerevisiae and three related budding yeasts reveals that UPRE-1 lies in the middle of a highly conserved 21-bp sequence which is 100% identical across three of the species ([Figure 7](#pbio-0020246-g007){ref-type="fig"}A). This conserved stretch may represent a context that is essential for the transcriptional function of the core sequence. We speculate that recognition of the extended context may be performed by Hac1p without the collaboration of Gcn4p, as suggested by the observation that promoters which contain a UPRE are more dependent on *GCN4*/*GCN2* than are those genes in which a short modular UAS has not been identified (see [Figure 3](#pbio-0020246-g003){ref-type="fig"}C, histograms *o* and *p*).
::: {#pbio-0020246-g007 .fig}
Figure 7
::: {.caption}
###### Multiple Alignment of UPRE-1 and UPRE-2 from Three Budding Yeasts
Alignment of partial promoter sequences from S. cerevisiae and homologous sequences in related yeasts. Numerical coordinates reflect the distance from the first nucleotide of the initiation codon in the S. cerevisiae promoter.
\(A) A segment of the *KAR2/YJL034W* promoter and homologs. The core sequence of UPRE-1 is indicated.
\(B) A segment of the *ERO1*/*YML130C* promoter and homologs. The core sequence of UPRE-2 is indicated (above). The consensus binding site of Gcn4p is aligned (below).
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Despite these qualifications, the approach has successfully uncovered novel information about how the UPR is regulated. The appealing aspect of the strategy described here is that such studies are not limited to the UPR but can be generally employed in the study of any transcriptional response in any organism for which promoter sequences for all genes are known and in which the comprehensive genomic output of the response can be measured by expression profiling. The sole requirement of the probabilistic segmentation/overrepresentation computations is that a partition of the genome (into "target genes" and "nontarget genes") be made on the basis of some meaningful difference in expression levels under the conditions of interest; the analysis thereafter proceeds by comparing the distribution of candidate motifs in the target gene set and the remainder of the genome. Further refinement of the mathematical tools therefore promises to be of invaluable help in our quest for a comprehensive understanding of the logic and complex interactions of transcriptional programs in eukaryotic cells.
*GCN4* Is an Essential Transcription Factor of the UPR {#s3b}
------------------------------------------------------
The overexpression screen for activators of UPRE-2 revealed a role for the transcription factor Gcn4p, which we show to be required not only for activity of UPRE-2 but for all three known UPREs. Gcn4p and its upstream activator Gcn2p thus join Ire1p, Hac1p, and Rlg1p in the list of essential players in the yeast UPR.
*GCN4* encodes a well-characterized transcription factor acting in several distinct stress responses including amino acid starvation, glucose limitation, and ultraviolet irradiation ([@pbio-0020246-Hinnebusch2]; [@pbio-0020246-Yang1]; [@pbio-0020246-Natarajan1]; [@pbio-0020246-Stitzel1]), but has not previously been demonstrated to play any role in the UPR. Here, we demonstrate that *GCN4* is required for normal induction of UPR transcription, both in the context of artificial promoters containing any of the known UPREs and in the context of the native promoters of most target genes. *GCN2,* a gene implicated in regulating *GCN4* in other stress responses, is similarly required for a normal UPR, perhaps because *GCN2* function is required to maintain the basal level of Gcn4p in a cell even under normal growth conditions.
Our gel-mobility shift studies demonstrate a direct physical association between Hac1p and Gcn4p and the sequence motifs UPRE-1 and UPRE-2. Gcn4p and Hac1p are bZIP proteins, a family whose members bind DNA as dimers ([@pbio-0020246-Ransone1]; [@pbio-0020246-Hsu1]). It therefore seems likely that Gcn4p and Hac1p stimulate transcription by binding promoter DNA as a heterodimer, although we cannot rule out higher order complexes.
The promoter sequences UPRE-1 and UPRE-2 have identical genetic requirements for activation, but their behavior in response to genetic perturbations is not strictly identical. UPRE-2 can be activated by high levels of *GCN4* alone (see [Figure 3](#pbio-0020246-g003){ref-type="fig"}A), but UPRE-1 cannot. This can be explained by the binding studies, which demonstrate that UPRE-2 (but not UPRE-1) can bind Gcn4p in the absence of Hac1p (see [Figure 6](#pbio-0020246-g006){ref-type="fig"}B, lanes 1 and 2); indeed, Gcn4p is known to bind DNA as a monomer as well as a dimer ([@pbio-0020246-Cranz1]) and can bind DNA sequences containing even a consensus half-site ([@pbio-0020246-Hollenbeck1]).
The basis for this differential affinity for Gcn4p is strongly suggested by a refined consensus sequence for UPRE-2, and is illustrated by multiple species alignment of the *ERO1* promoter ([Figure 7](#pbio-0020246-g007){ref-type="fig"}B). We searched for examples of UPRE-2 core sequences that were conserved in UPR target genes across five yeast species, and extracted core and flanking sequences to derive a generalized consensus (see [Materials and Methods](#s4){ref-type="sec"}). The resulting consensus was revealed to be T(C/T)ACGTGT(C/T)(A/C), which differs from the experimentally established UPRE-1 consensus by two nucleotides essential for activity in the *KAR2* promoter context. The conserved extended context of UPRE-2 in this promoter aligns with a consensus binding site for Gcn4p defined by computational analysis of the set of promoters that bind Gcn4p in a genome-wide chromatin immunoprecipitation assay (analysis by W. Wang and H. Li, unpublished data; chromatin immunoprecipitations in [@pbio-0020246-Lee2]). Comparison of multiple alignments of the extended contexts of UPRE-1 and UPRE-2 in the *KAR2* and *ERO1* promoters (compare [Figure 7](#pbio-0020246-g007){ref-type="fig"}A and [7](#pbio-0020246-g007){ref-type="fig"}B) reveals that the two sequence contexts share a six-nucleotide segment, CGTGTC. The match between UPRE-2 and the Gcn4p consensus is imperfect (five of seven positions), suggesting that the association with Gcn4p and UPR promoters is not identical to the binding of Gcn4p to its "classical" amino acid starvation targets. Rather, these observations suggest that the proposed Gcn4p/Hac1p heterodimeric complex binds to a composite site, of which UPRE-1 and UPRE-2 represent different forms with stronger relative affinities to Hac1p and Gcn4p, respectively. Such a model would explain the residual upregulation of UPRE-1-containing genes in a Δ*gcn2* mutant (see [Figure 3](#pbio-0020246-g003){ref-type="fig"}C, histogram *j*), which retains some expression of Gcn4p. In the absence of Hac1p but in the presence of high concentrations of Gcn4p (e.g., when *GCN4* is overexpressed), Gcn4p can bind the UPRE-2 on its own, either as a homodimer or a monomer.
Upregulation of Gcn4p by ER Stress {#s3c}
----------------------------------
The transient upregulation of Gcn4p levels, which we observe upon UPR induction, may therefore serve to increase the transcriptional output of the response, especially early in the response. Most UPR target genes are robustly induced after 15 min of ER stress ([@pbio-0020246-Travers1]); hence, the increase in Gcn4p levels occurs at a time suggestive of a role in the initial response.
Gcn4p itself mediates a broad transcriptional program in response to a diverse set of cellular conditions and stresses ([@pbio-0020246-Natarajan1]). The recruitment of Gcn4p therefore provides an opportunity for crosstalk between regulatory pathways and fine-tuning of the magnitude of the UPR. For example, under amino acid starvation, Gcn4p levels are high relative to the baseline of normal growth. In this state, cells with accumulated unfolded ER protein might wish to upregulate ER-associated protein degradation (one output of the UPR; [@pbio-0020246-Casagrande1]; [@pbio-0020246-Friedlander1]; [@pbio-0020246-Travers1]) beyond the level normally provided by the UPR. Such a mechanism might provide for an additional source of amino acids through protein catabolism. Elevated Gcn4p levels and the concomitant increased induction of UPR target genes would serve this need. This view raises the possibility that those genes that most stringently require *GCN4* for normal UPR induction are those that are most urgently required by the cell under specific conditions, under which UPR is induced *and* Gcn4p levels are high for reasons unrelated to ER stress. The relationship between the cellular stress responses that regulate Gcn4p and the potentiation of UPR transcription will therefore be an important subject for future study.
The mechanism by which *IRE1* and *HAC1* mediate the transient increase in Gcn4p remains to be elucidated. Given that Hac1p and Gcn4p are observed in the same complex with DNA, one intriguing possibility is that association with Hac1p serves to stabilize Gcn4p.
*GCN4* and the Super-UPR: Two Ways to Modulate the UPR {#s3d}
------------------------------------------------------
We propose a model of UPR transcriptional activation that is illustrated in [Figure 8](#pbio-0020246-g008){ref-type="fig"}. According to the circuit diagram in [Figure 8](#pbio-0020246-g008){ref-type="fig"}A, *HAC1* mRNA splicing retains its role as the "switch" that turns the UPR on or off. Gcn4p, whose levels appear to be limiting for the extent of gene regulation, would therefore play a role in setting the "gain" or "volume" of the response, perhaps allowing communication from other stress response pathways in the cell. Such a gain control could serve as an adjunct to the "Super-UPR" (S-UPR) gain control described in the accompanying paper ([@pbio-0020246-Leber1]), whereby an *IRE1-*independent ER surveillance mechanism regulates the transcription of the *HAC1* mRNA in response to compound stresses on the secretory pathway. S-UPR induction proceeds unimpaired in Δ*gcn4* cells, indicating that the S-UPR is mechanistically distinct from the regulation described here ([@pbio-0020246-Leber1]). Whereas the S-UPR monitors conditions of the ER, the *GCN4* branch would integrate information gleaned from the cytosol. Both of these gain controls have the potential to act not only as modulators of the magnitude of the response but also as a tuning dial: UPR targets respond differentially to increased level of *HAC1* during the S-UPR (see the Class 1, 2, and 3 genes in [Figure 6](#pbio-0020246-g006){ref-type="fig"} of [@pbio-0020246-Leber1]). Likewise, different UPR targets exhibit differential dependence on Gcn4p, as is apparent from the variable upregulation of UPR targets in Δ*gcn4* and Δ*gcn2* mutants (see [Figure 3](#pbio-0020246-g003){ref-type="fig"}C). The observations suggest that increased levels of Gcn4p might serve to differentially upregulate a subset of target genes.
::: {#pbio-0020246-g008 .fig}
Figure 8
::: {.caption}
###### Model of Gcn4p/Hac1p Action in the UPR
\(A) The expanded circuitry of the UPR*.* The classical UPR (red), the S-UPR (blue), and regulated Gcn4p levels (green) are integrated at target promoters. Transcriptional regulation of *HAC1* mRNA levels, providing one level of gain control, is depicted as a rheostat under supervision of a logical AND gate informed by multiple inputs from the ER. Splicing of *HAC1* mRNA by Ire1p, providing a binary on/off control, is depicted by a switch. Regulation of Gcn4p levels by Gcn2p under changing cellular conditions adds an additional layer of gain control. Together, activity levels of Hac1p, Gcn4p, and the proposed UPR modulatory factor ([@pbio-0020246-Leber1]) collaborate to determine the magnitude of the transcriptional output signal.
\(B) Mechanism of Gcn4p/Hac1p action at target promoters. In the absence of Hac1p, Gcn4p is present in the cell as a consequence of baseline activity of Gcn2p. At normal concentrations, Gcn4p is unable to bind or activate a target UPRE, but it may bind when Gcn4p levels are elevated. Upon induction of the UPR, Ire1p is activated and Hac1 is synthesized. Hac1p can bind, but not activate, target UPREs. Binding of target DNA by a Gcn4p/Hac1p heterodimer results in a transcriptionally active complex. Gcn4p levels are upregulated under UPR induction, perhaps as a consequence of stabilization by interaction with Hac1p.
:::
:::
From a mechanistic standpoint, ER stress activates Ire1p, which, through nonconventional splicing, induces Hac1p production ([Figure 8](#pbio-0020246-g008){ref-type="fig"}B). Hac1p can bind to the known UPREs, but by itself forms a protein--DNA complex that is not competent to upregulate transcription. Gcn4p, which is present at a basal level in cells under normal growth conditions as a result of baseline Gcn2p activity, is unable to bind UPREs in the absence of Hac1p. Gcn4p may bind some UPRE sequences, providing a weak bypass of Hac1p, when it is present at physiologically elevated levels. When Hac1p is produced, Gcn4p is recruited to the UPRE, presumably forming a more stable ternary complex containing promoter DNA, Gcn4p, and Hac1p, and transcription is induced. This ternary complex could be established serially, in which case an inactive Hac1p/UPRE complex would be recognized by Gcn4p, or by recognition of the UPRE by a preformed heterodimer of Gcn4p and Hac1p.
Conservation between Yeast and Mammalian UPR {#s3e}
--------------------------------------------
Advances in the understanding of the metazoan UPR system has been richly informed by the study of yeast. The elucidation of a role for Gcn4p in the yeast UPR allows us to draw even stronger parallels between the yeast and metazoan systems. In higher eukaryotes, the ER-resident transmembrane kinase PERK is activated by protein unfolding. PERK\'s cytosolic domain is homologous to Gcn2p and likewise phosphorylates eIF-2α, thereby downregulating general translation but also promoting the selective translation of mRNAs containing upstream ORFs in their 5′ UTR sequences. One of these mRNAs encodes ATF-4, a bZIP transcription factor that represents the metazoan ortholog of Gcn4p. Intriguingly, and in strict analogy to the joint action of Gcn4p and Hac1p proposed here, ATF-4 in metazoan cells collaborates with the Hac1p ortholog XBP-1 to stimulate UPR target gene transcription.
The analogies between the roles of Gcn4p/Hac1p/Gcn2p and ATF-4/XBP-1/PERK suggest that the function of these proteins has been amazingly conserved in the UPR, although the nature of the connections between pathway components may have been adapted over evolutionary time: Yeast does not have an identified PERK ortholog that feeds ER-derived information into the *GCN4* branch of the network. Another parallel concerns S-UPR regulation. In the accompanying paper, [@pbio-0020246-Leber1] demonstrate that compound secretory stress upregulates *HAC1* mRNA. The mode of modulation of the UPR by the superimposed control of the S-UPR bears a resemblance to the known function of another metazoan transcription factor, ATF-6, which is activated by regulated proteolysis in response to ER stress and in turn upregulates XBP-1 transcription.
In comparison to the metazoan UPR, where multiple ER-resident proteins communicate in a seemingly parallel way with multiple downstream transcription factors, Ire1p and Hac1p remain the central players in the yeast UPR. *GCN4* and the S-UPR provide modulatory functions. Nonetheless, the addition to the repertoire of the yeast UPR effectors of an additional transcription factor (Gcn4p) and of a mechanism for transcriptional regulation of Hac1p (S-UPR; [@pbio-0020246-Leber1]) suggests that the UPR functions as a regulatory network, with its opportunities for crosstalk with other pathways and regulation by cellular state. But most importantly, both the central players and the connectivity of the circuits involved appear to be conserved among eukaryotes and evolutionarily ancient.
Materials and Methods {#s4}
=====================
{#s4a}
### Computational and quantitative methods {#s4a1}
To build the dictionary of putative regulatory elements for UPR target genes, we first extracted the 600-bp upstream regions of all UPR target genes. To get rid of simple repeats unlikely to be regulatory elements (such as AT-rich repeats and transposable elements), we removed exact repeats of lengths 15 bp or longer, and kept the remaining fragments of lengths longer than 50 bp. What remained was the input sequence for the dictionary construction. We used the MobyDick algorithm based on probabilistic segmentation ([@pbio-0020246-Bussemaker2]) to build a dictionary of putative regulatory elements. MobyDick builds the dictionary by iterating through fitting and testing steps. Starting with the frequencies of single bases, the algorithm finds overrepresented two-nucleotide pairs (testing step), adds them to the dictionary, determines their probabilities by maximizing the likelihood of observing the sequence data (fitting step), and continues to build larger fragments iteratively. Adjustable parameters were as follows: *L,* the maximum word length, was set to 8, and *MaxP,* the probability of seeing at least one false positive at each testing step when all words of length up to *L* are tested, was set to 0.999 (relaxed cutoff). MobyDick generated a dictionary of 328 words. We filtered out words that were too short, appeared in too many copies (such as AT-rich short repeats), or were of low quality (the algorithm calculates a quality factor for each word describing how likely it is that the word can be made by chance from shorter words). With the filters *number\_of\_copies* \< 200, *length* \> 4, and *quality\_factor* \> 0.2, we obtained 201 words.
Using the filtered dictionary, we grouped similar words into motifs using the clustering algorithm CAST ([@pbio-0020246-Ben-Dor1]), as follows: We first computed pairwise alignment scores for all the words in the dictionary, using gapless alignment with a scoring scheme derived from a simple mutation model. The model assumes that a base *x* mutates to any other given base *y* with probability *p*/3, and remains the same base with a probability (1 − *p*). The score for a pair *x--y* is given by the log-odds-ratio of observing the pair under the mutation model versus observing the pair at random. With the choice of *p* = 0.5 (the result is insensitive to the actual *p* value chosen as long as *p* is much smaller than 0.75), a matching pair scores ln(2), and a mismatch scores ln(2/3). We normalized the scores to fall between 0 and 1 by the largest score. We then used the CAST algorithm to group words into clusters, with the threshold parameter set at 0.7 (the lower bound of the normalized score averaged over all pairs in a cluster). This procedure generated 109 motifs.
To test which motifs are significantly overrepresented in the promoters of UPR target genes, we counted for each motif the total number of occurrences in all promoters, and calculated the expected number of occurrences *N~exp~* in the UPR target gene promoters based on the genome-wide frequencies. We then counted the observed number of occurrences *N~obs~* of the motif in the promoters of UPR target genes. We used Poisson statistics to calculate the probability *P* of observing a number of occurrences equal to or greater than *N~obs~* by chance, based on *N~exp~*. The test based on Poisson statistics is a very good approximation of the more rigorous test based on the binomial distribution, where the probability *P* is the probability of seeing a specific instance of the motif in the UPR gene set and the total number of trials *N~t~* is the total number of copies of the motif in the genome. Since *P* is small (0.059) and *N~t~* is large (ranging from approximately ten to approximately 1000) but the product *N~exp~* is finite, the resulting distribution is well approximated by a Poisson distribution with mean = *N~exp~*.
To derive a general consensus for UPRE-2 that includes context information beyond the core motif, we took the five-nucleotide core ACGTG from the Motif 1 alignment (see [Figure 1](#pbio-0020246-g001){ref-type="fig"}B) and searched the promoters of UPR target genes for the occurrences of this core motif that are conserved across five yeast species. We first took the sequence data for *S. cerevisiae, S. bayanus, S. mikatae, S. paradoxus,* and S. kudriavevii ([@pbio-0020246-Cliften1]; [@pbio-0020246-Kellis1]) and performed multiple sequence alignment on all the orthologous promoters. We then searched for conserved blocks on both strands where ACGTG occurs in all species and is correctly aligned. We found 60 instances of conserved blocks in UPR target gene promoters for which multiple sequence alignment data were available. We then extracted ACGTG plus 10-bp flanking sequences on both side in S. cerevisiae and performed a multiple local sequence alignment of the S. cerevisiae sequences from each of the 60 conserved blocks using the Consensus algorithm ([@pbio-0020246-Hertz1]), setting *matrix\_width* to 15. The result of the alignment was a position-specific frequency matrix. We derived a consensus sequence from the matrix using the convention by [@pbio-0020246-Cavener1]. The alignment matrix and raw sequence data are available in [Table S3](#st003){ref-type="supplementary-material"}.
### Plasmids and recombinant DNA {#s4a2}
DNA manipulations, cloning, and yeast culture were performed as previously described ([@pbio-0020246-Sherman1]; [@pbio-0020246-Ausubel1]; [@pbio-0020246-Guthrie1]) unless otherwise noted.
UPRE reporter constructs (used in [Figures 2](#pbio-0020246-g002){ref-type="fig"}A, [3](#pbio-0020246-g003){ref-type="fig"}A, [3](#pbio-0020246-g003){ref-type="fig"}B, and [5](#pbio-0020246-g005){ref-type="fig"}) were based on the plasmid pPW344/pJC104 ([@pbio-0020246-Cox2]), which contains a triple repeat of the *KAR2*-derived UPRE; this plasmid was used as the UPRE-1 reporter in all experiments. To construct UPRE reporters used to test Motifs 1--8, we removed the UPRE-1 repeat from pPW344 by digestion with BglII and XhoI, and replaced it with a triple repeat of a 15-nucleotide sequence encompassing the motif in question and the flanking sequence context. Source sequences were chosen from promoters that exhibited robust induction by the UPR ([@pbio-0020246-Travers1]) and, if possible, did not contain a match to the canonical (*KAR2*-derived) UPRE. Intact promoter reporter constructs (pPW668--pPW671) used in [Figure 2](#pbio-0020246-g002){ref-type="fig"}B and [2](#pbio-0020246-g002){ref-type="fig"}C were also based on plasmid pPW344. Here the promoter of pPW344 (BamHI/BglII fragment) was replaced by a single PCR fragment spanning the approximately 600 nucleotides immediately upstream of either the *ERO1* or *DHH1* initiation codon, or by two fragments spanning the same sequence but with the UPRE motif replaced by a restriction site. The high-copy *GCN4* plasmid (pPW672) used in [Figure 3](#pbio-0020246-g003){ref-type="fig"}A consists of the region plus1000 nucleotides one either side of the *GCN4* ORF. Source sequence contexts, olignucleotide sequences, and select PCR primers are compiled in [Table S4](#st004){ref-type="supplementary-material"}. The plasmids expressing the activated allele of *HAC1* used in [Figure 5](#pbio-0020246-g005){ref-type="fig"} (pPW322/pRC43) and the N-terminally HA-tagged allele of *HAC1* (pPW353/pJC316) used in [Figure 6](#pbio-0020246-g006){ref-type="fig"}B were as previously described ([@pbio-0020246-Cox1]).
Knockouts of *GCN4* and *GCN2* and the integrated *GCN4-myc* were constructed by PCR cassette/generic primer mutagenesis ([@pbio-0020246-Longtine1]).
### Yeast strains {#s4a3}
All base strains used in this study are enumerated in [Table 1](#pbio-0020246-t001){ref-type="table"}. As appropriate, these strains were transformed with plasmids from [Table 2](#pbio-0020246-t002){ref-type="table"} for use in experiments.
::: {#pbio-0020246-t001 .table-wrap}
Table 1
::: {.caption}
###### Yeast Strains
:::
:::
::: {#pbio-0020246-t002 .table-wrap}
Table 2
::: {.caption}
###### Yeast Plasmids
:::
:::
### Cell culture and growth conditions {#s4a4}
For all experiments, samples were diluted from saturated overnight cultures and regrown to midlog phase (OD600 = 0.5) prior to addition of drug.
DTT (Sigma, St. Louis, Missouri, United States) was added to cultures to a final concentration of 2 mM. Tm (Boehringer Mannheim, Indianapolis, Indiana, United States) was added to cultures to a final concentration of 1 μg/ml. 3-AT (Sigma) was added to cultures to a final concentration of 10 mM. All 3-AT treatments were performed on strains WT for the *HIS3* gene; for histidine-deprived cultures, overnight cultures were washed three times in SD-histidine, then diluted to low density in SD-histidine and grown to midlog phase before the addition of the drug.
To assay β-galactosidase activity on solid growth media, we overlaid plates with buffered soft agar containing X-gal (Sigma) as described previously ([@pbio-0020246-Cox1]). For liquid cultures, we used a colorimetric ONPG assay ([@pbio-0020246-Holley1]).
### Gene expression profiling. {#s4a5}
Strains were grown in YPD (pH 5.4) as in [@pbio-0020246-Travers1] to midlog phase (OD = 0.5) and then either treated with 2 mM DTT or left untreated. RNA was extracted as described by [@pbio-0020246-Ruegsegger1], and mRNA was purified with a PolyATtract kit (Promega, Madison, Wisconsin, United States).
Microarray analysis used yeast spotted-cDNA ORF arrays printed at the University of California, San Francisco, Core Center for Genomics and Proteomics (<http://derisilab.ucsf.edu/more>) and was performed as described previously ([@pbio-0020246-Carroll1]). Measurements reported are the average of three independent experiments.
We tested the statistical significance of the induction for the three gene sets (UPRE-1, UPRE-2, and UPRE-3 genes) in four different strains (WT, Δ*ire1,*Δ*gcn4,* and Δ*gcn2*) using a *z*-score scheme. For a given gene set and a given strain, we calculated the average fold induction for genes in the set and compared it to the value for the genome overall. The null hypothesis was that the selected gene set was no different from a randomly selected set (same total number) from the genome overall. Under this hypothesis, the average *μ* has a distribution well approximated by a normal distribution (due to the central limit theorem) with mean *μ~genome~* and standard deviation σ/√(*Nset*), where *N~set~* is the total number of genes in the test set. We computed a *z*-score, *z*=√(*Nset*)(μ−μ*genome*)/σ, which should have a standard normal distribution (zero mean and unit variance) under the null hypothesis. The *P* value was calculated by integrating the standard normal curve from *z* to infinity.
### Isolation and detection of protein. {#s4a6}
Protein preparation, electrophoresis, and Western blotting proceeded as described in the accompanying paper ([@pbio-0020246-Leber1]). Gcn4p-myc (see [Figure 5](#pbio-0020246-g005){ref-type="fig"}A) was detected using a mouse anti-myc monoclonal antibody (Molecular Probes, Eugene, Oregon, United States); eIF-2α-phosphate was detected by a commercial phospho-specific mouse polyclonal (Upstate Biotechnology, Lake Placid, New York, United States).
### Gel retardation analysis. {#s4a7}
Gel shifts were performed as previously described ([@pbio-0020246-Cox1]) except that we found it important to elevate the acrylamide concentration to 5% and lower the in-gel glycerol concentration to 4%. UPRE-1 oligo and UPRE-1 mutant are based on sequences previously described ([@pbio-0020246-Cox1]). UPRE-2 oligo is a fragment of the *ERO1* promoter centered around the UAS. UPRE-2 mutant is a point mutation that does not support transcription in an artificial promoter context (unpublished data). For sequences, see [Table S4](#st004){ref-type="supplementary-material"}. Competition experiments used a 100-fold excess of unlabeled oligonucleotide.
Supporting Information {#s5}
======================
Table S1
::: {.caption}
###### Dictionary of "Words" Compiled by MobyDick
This table contains an alphabetical list of the dictionary "words" compiled by the MobyDick algorithm from the "text" comprising the promoters of UPRE target genes. Associated statistics for each word are as follows: *N,* the average number of times the string is delimited as a word among all segmentations of the data; *Xi,* the number of matches of the word anywhere in the text; *p,* the frequency of drawing the word from the dictionary, optimized over all words to give the maximum likelihood of observing the text; *Z* = *p* + *p~s~*, where *p~s~* is the probability with which the word can be made by combining shorter words from the dictionary; *sig* = significance = *Np*/sqrt(*N*\[Z − *p*\]).
(10 KB TXT).
:::
::: {.caption}
######
Click here for additional data file.
:::
Table S2
::: {.caption}
###### Ranked Listing of the Motifs Assembled by Clustering from the Dictionary Words
N~tot~ is the number of times a given motif appeared in the promoters of the genome overall; N~exp~ is the number of times one would expect a given motif to appear in the 381 promoters of UPR target genes if the motif were distributed randomly throughout all promoters; N~obs~ is the number of times a given motif actually appears in the target gene promoters; and −log~10~ *P* is a measure of overrepresentation based on Poisson statistics (*P* is the likelihood that a given observed distribution would occur by chance).
(3 KB TXT).
:::
::: {.caption}
######
Click here for additional data file.
:::
Table S3
::: {.caption}
###### UPRE-Containing Promoter Alignments
This table contains CLUSTALW alignments for the *KAR2* and *ERO1* promoters, derived from S. cerevisiae and related budding yeasts. Asterisk indicates 100% conserved residues. Scer, *S. cerevisiae;* Skud, *S. kudriavevii;* Spar, *S. paradoxus;* Smik, *S. mikatae;* Sbay, S. bayanus.
(8 KB TXT).
:::
::: {.caption}
######
Click here for additional data file.
:::
Table S4
::: {.caption}
###### Oligonucleotide Sequences and Cloning Schemes
This table contains the sequences of primers and olignonucleotide sequences used in construction of plasmids for this study, as well as oligonucleotide sequences used for probes in the gel-shift analysis.
(38 KB DOC).
:::
::: {.caption}
######
Click here for additional data file.
:::
Accession Numbers {#s5a5}
-----------------
The GenBank accession numbers of the gene products discussed in this paper are Dhh1p (NP\_010121), Ero1p (NP\_013576), Gcn4p (NP\_010907), Gcn2p (NP\_010569), Hac1p (NP\_011946), and Ire1p (NP\_116622).
Microarray data can be accessed at the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) database as platform number GPL1001 and sample numbers GSM16985--GSM1988.
The authors wish to thank Jason Bricker, Isabella Halama, Jess Leber, and Feroz Papa for enthusiastic technical assistance with all experiments; Adam Carroll for particular assistance with microarray hybridization experiments; Sebastian Bernáles, Jason Brickner, Jess Leber, and Feroz Papa for close readings of the manuscript; and Jeffery Cox, Steven McCarroll, Aaron McGee, Jonathan Weissman, and past and present members of the Walter lab for expert advice and valuable discussion throughout the course of the project.
This work was supported by an Howard Hughes Medical Institute predoctoral fellowship and a Burroughs Welcome Fund/Program in Quantitative Biology predoctoral fellowship for CP; by a Sandler\'s opportunity award and a Packard Science and Engineering Fellowship to HL; and by grants from the National Institutes of Health to HL and PW. PW is an Investigator of the Howard Hughes Medical Institute.
**Conflicts of interest.** The authors declare that no conflicts of interest exist.
**Author contributions.** CP and PW conceived and designed the experiments. CP performed the experiments. HL analyzed the data. CP, HL, and PW wrote the manuscript.
Academic Editor: Steven McKnight, University of Texas Southwestern
Citation: Patil CK, Li H, Walter P (2004) Gcn4p and novel upstream activating sequences regulate targets of the unfolded protein response. PLoS Biol 2(8): e246.
3-AT
: 3-aminotriazole
DTT
: dithiothreitol
ER
: endoplasmic reticulum
ORF
: open reading frame
S-UPR
: Super-UPR
Tm
: tunicamycin
UAS
: upstream activating sequence
UPR
: unfolded protein response
UPRE
: unfolded protein response element
WT
: wild-type.
| PubMed Central | 2024-06-05T03:55:46.778877 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509306/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e246",
"authors": [
{
"first": "Christopher K",
"last": "Patil"
},
{
"first": "Hao",
"last": "Li"
},
{
"first": "Peter",
"last": "Walter"
}
]
} |
PMC509307 | Sooner or later, a gardener looking for "nontoxic" ways to control the inevitable attack on a favorite plant will discover the nicotine remedy. Steep a cup of loose tobacco in a gallon of water, let it sit overnight, strain, and spray away. Caterpillars, aphids, and a diverse array of insects predisposed to devouring plants will soon abandon your vegetables and flowers in search of less disagreeable forage.
The ultimate sitting duck, plants rely on an arsenal of chemical metabolites to fend off predators. Many of these chemicals harbor anti-herbivore properties, which have been exploited for commercial use. Nicotine, it turns out, is so toxic that it was one of the first chemicals used in agricultural insecticides. It\'s not clear, though, whether these toxic metabolites are really defending plants against hungry herbivores in their natural environment, especially since many insects can tolerate various plant chemicals and sometimes even incorporate them into their own defenses. Though scientists have cataloged a long list of these presumed resistance traits, there\'s no evidence that they offer plants a competitive advantage against their leaf-covetous foes in nature.[](#pbio-0020250-g001){ref-type="fig"}
::: {#pbio-0020250-g001 .fig}
::: {.caption}
###### Spodoptera exigua larva feeding on Nicotiana attenuata
:::
:::
With plant and plant-eater engaged in an ever-escalating battle of evolutionary one-upmanship and with plants capable of producing an array of defensive responses, teasing out the predator-resistant effects of individual plant metabolites has proved challenging. Theoretically, one could track down a resistance gene by breeding plants that are genetically identical save for the gene that controls expression of a particular resistance trait. In practice, however, traditional breeding techniques aren\'t that precise and tend to generate additional variations in genomic regions that are linked to the target gene and that might affect resistance as well.
The tools of genetic engineering have largely overcome such limitations, allowing scientists far greater control and specificity. Following this approach, Ian Baldwin and colleagues use transgenic silencing (which introduces gene "constructs" into an organism to inactivate a gene of interest) to investigate a single resistance trait, nicotine production. Even though nicotine is one of the best-studied putative resistance traits, its specific role has been unclear.
To isolate the resistance effects of nicotine from possible confounding factors, Baldwin and colleagues blocked nicotine production in the Nicotiana attenuata tobacco plant. Focusing on an enzyme, called putrescine methyl transferase (PMT), central to nicotine biosynthesis, the authors used two techniques that interfere with PMT production by silencing the gene, *pmt*, that encodes the enzyme. One of the techniques (which adds genetic sequences called "inverted repeats" to gene fragments) proved far more effective at silencing *pmt*, producing 29 out of 34 plant lines with only 3%--4% of normal nicotine levels.
With suitably nicotine-deprived plants, Baldwin and colleagues could directly test nicotine\'s role in tobacco fitness. They transplanted the transgenic plants, along with nonmutant cultivated plants, in southwestern Utah, N. attenuata\'s native habitat. A subset of the plants was also treated with a chemical known to increase both nicotine content and resistance to herbivore attack. Predictably, several of the plant\'s natural insect enemies made an appearance. Untreated nicotine-deficient transgenic plants fared the worst, losing twice as much foliage to herbivores as nonmutant plants. Transgenic plants treated with the chemical boost performed much better, showing about the same amount of damage as the nonmutants. Interestingly, tobacco hornworms---which, as their name implies, feed primarily on tobacco---preferred nicotine-free plants when given the choice. Though the worms have evolved strategies for coping with nicotine\'s deleterious effects, these adaptations come at a price: worms feeding on nicotine-deficient tobacco grew bigger and faster than those feeding on plants with normal nicotine levels.
These results clearly show that nicotine protects tobacco plants in their native habitat, the authors conclude, and that tobacco-chewing insects "prefer low nicotine diets." Removing nicotine from the equation reveals the relentless pressure that plants face from herbivores. Without such defenses, plants would be unceremoniously eliminated posthaste, leaving a world without greenery, not to mention oxygen. But these experiments also demonstrate the unprecedented power of transgenic tools to peel back the obfuscating layers inherent in ecological interactions to reveal the fundamental properties of those interactions. And that scientists working to unravel the tangled web of ecological interactions would do well to take advantage of the longest running experiment on earth---the natural environment.
| PubMed Central | 2024-06-05T03:55:46.785259 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509307/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e250",
"authors": []
} |
PMC509308 | As the presumed "seat of consciousness," the cerebral cortex mediates the higher-level cognitive processing---such as abstract thought---that humans like to think distinguishes them from other animals. The cerebral cortex is, in fact, significantly larger in the human brain and has far more "columns" than it does in other mammals, particularly compared to the rat, a traditional model for brain study. Neurons in these cortical columns have similar response properties and form fundamental units of brain processing. It is the larger brain surface area, accommodating a greater number of cortical columns, that gives humans the computational edge.
By studying the genetic and molecular agents of cortex development, scientists hope to understand the nature and extent of cortical cognitive function and identify effective therapies to repair brain injury and disease. Analyses of mutant mice have provided insights into the mechanisms controlling cerebral cortex development, but many details of cortical development remain to be revealed. Identifying individual molecules and genes involved in discrete brain processes is particularly difficult given the complexity of brain structure and function. Geneticists have traditionally linked genes to specific biological pathways by first screening large numbers of individuals of a species for unusual physical traits (phenotypes) and then determining the genetic makeup of these mutants to home in on the faulty gene. This approach, called forward genetics, typically requires large numbers of individuals to find unusual phenotypes and so has traditionally focused on fast-breeding organisms like zebrafish and fruitflies. But zebrafish and fruitflies are unlikely to reveal the secrets of higher consciousness.
Now Andrew Peterson and colleagues have updated the forward genetic screen and added a new resource to the neuroscientist\'s "brain dissection" toolkit. Their approach, which labels specific populations of neurons with protein "reporters," offers a novel way to find mutations in developing neurons and to identify mutations that interfere with cerebral cortex development. The reporter used here highlights mutations that disrupt interneuron migration into the cortex as well as those that affect cortex growth and morphology. (Interneurons are one of the primary types of cortical neurons.)[](#pbio-0020254-g001){ref-type="fig"}
::: {#pbio-0020254-g001 .fig}
::: {.caption}
###### Distribution of GABAergic interneurons in wild-type (top) and mutant (bottom) cortex
:::
:::
Like most forward genetic approaches, the researchers started with a genetically well-characterized breed, then used a chemical mutagen to damage the organism\'s DNA. After two or three rounds of breeding, the researchers looked for cortical-related defects in the developing embryos. In this case, however, the region of the mouse brain that gives rise to developing interneurons was labeled in the original mice. Thus, when the researchers screened for mutants with defects associated with forebrain development and interneuron migration, they could easily find the cells and genes involved.
The screen identified thirteen mutations affecting cortical development and interneuron migration. (Developing neurons travel along the fibers of other brain cells before reaching their ultimate destination in the brain.) Three mutations are variants of genes known to play a role in cortical development; nine mutations were in genes that had not been linked to cortical development before.
The screen described here takes advantage of a chemical mutagen (called ENU) that induces mutations at a single base pair, or nucleotide, in DNA. These mutations tend to have quite selective effects on protein function---by changing the composition of a single domain---which can provide information on how the protein should normally function and highlight its role in a particular process. The selective nature of ENU, the authors argue, offers new information about how the mutated genes identified here function in cortical development and how these putative roles might be tested.
Altogether, the results suggest that this type of focused screening, so long a resource in fly genetics, can be a powerful tool in mammalian biology as well. That the strategy outlined here could identify novel mutations in a process as complicated as cerebral cortex development suggests that it could do the same for a broad range of biological processes. If the success of other model systems moving in this direction is any indication, this new strategy in the mouse offers researchers a powerful resource for identifying the genetic underpinnings of living systems.
| PubMed Central | 2024-06-05T03:55:46.785931 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509308/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e254",
"authors": []
} |
PMC509309 | For a three-millimeter invertebrate, the Drosophila fruitfly has a remarkably sophisticated immune system. Granted, it can\'t customize an immune response by grooming cells to "remember" and target specific pathogens. But it can rally the less specialized tools of innate immunity to fight disease and infection, and in so doing draws on several aspects of blood cell development (called hematopoiesis)---the foundation of the cellular immune response---also found in vertebrates.
In fruitflies, as in vertebrates, hematopoiesis occurs in distinct stages and locations, with nascent cell populations migrating to establish new hematopoietic frontiers. A population of progenitor cells generates all the blood cell types in the organism. These cells arise in two distinct waves and in two distinct locations, with their progeny differentiating into the specialized tissues and organs of the hematopoietic system. In mammals, these organs include the liver and bone marrow, an ongoing source of blood cells after embryogenesis. In fruitflies, the definitive hematopoietic organ is the lymph gland, which churns out three types of blood cells: plasmatocytes and crystal cells, which are also produced by embryonic hematopoietic precursors, and lamellocytes. Plasmatocytes account for up to 95% of circulating fruitfly blood cells and act much like their mammalian counterpart, the macrophage, engulfing substances deemed foreign and dangerous. Crystal cells account for most of the rest and are involved in melanization reactions, which trigger mechanisms involved in containing and killing invading microbes. Unlike plasmatocytes and crystal cells, lamellocytes appear en masse and only under certain conditions, such as the unwelcome appearance of parasitic wasp eggs. Lamellocytes encapsulate and neutralize the invader.[](#pbio-0020255-g001){ref-type="fig"}
::: {#pbio-0020255-g001 .fig}
::: {.caption}
###### Cellular immune response to parasitization in Drosophila requires the EBF ortholog Collier
:::
:::
Molecular factors involved in determining the fate of hematopoietic cells have been identified for plasmatocytes and crystal cells but not for lamellocytes---until now. In a search for genes that might precipitate lamellocyte differentiation, Marie Meister, Alain Vincent, and colleagues homed in on a protein, called Collier (Col), that is expressed in lymph glands at the end of embryogenesis. Col is quite similar to a mammalian protein, called Early B-cell Factor (EBF), that controls B-cell development in mice. Both proteins are transcription factors, exerting control by initiating gene transcription.
To investigate Col\'s part in lamellocyte development, the researchers had to get around the fact that mutations that render Col nonfunctional eventually kill the embryo. Using tricks of the genetics trade, Meister and colleagues generated fly larvae that survive loss-of-function mutations in the gene that encodes Col, allowing them to study its role in hematopoiesis.
The mutants had normal amounts of circulating plasmatocytes and crystal cells, but when exposed to parasitic wasp eggs, could not muster the requisite response: lamellocyte production. With no lamellocytes, fly larvae had no means of protection against encroaching wasp eggs, which, uncontested, developed into parasitic larvae. The flies with normal Col levels had no such problem, producing considerable numbers of lamellocytes. In these flies, Col expression was restricted to a lymph region called the posterior signaling center (PSC). Col\'s influence on lamellocyte fate was strong enough that forcing Col expression in precursor blood cells induced lamellocyte differentiation even in the absence of wasp infestation. Based on these findings, Meister and colleagues propose a model for Col-mediated lamellocyte differentiation in which wasp infestation activates Col-expressing cells in the PSC, which then instructs immature blood cells in the lymph gland to become lamellocytes and dispatch the gathering threat.
Col\'s role in fruitfly hematopoiesis closely parallels that of its mammalian ortholog in white blood cell development, EBF. Both are required to generate specialized populations of cells in response to a particular immune threat, and both confer an extra line of defense when faced with special circumstances---key features of vertebrate adaptive immunity. Could it be that building blocks of adaptive immunity were already in place some 550 million years ago, when flies and vertebrates parted ways? Researchers will have to investigate the molecular agents of immunity in intervening species to find out.
| PubMed Central | 2024-06-05T03:55:46.787004 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509309/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e255",
"authors": []
} |
PMC509310 | My graduate adviser, Sydney Brenner, used to exclaim 'Revenons à nos mutants!' as he sat down at the bench to search for yet more genetic variants of C. elegans. Those mutants won him a Nobel Prize, some thirty years later. Armand Leroi is another aficionado of C. elegans mutants, but he decided to write a book---and then to make a television series---on mutants of humanity, not of worms. He says at the beginning and end of the series: "We are all mutants, but some of us are more mutant than others." This is a good slogan, and very proper for embracing humanity as a whole. He backs it up with an aphorism from Etienne Geoffroy Saint-Hilaire, pioneer of teratology, who proclaimed: "There are no monsters and Nature is one." What Geoffroy meant was that abnormalities provide clues to normal processes, and hence are invaluable to science, if they can be properly understood. But monsters and mutants are, undeniably, fascinating in their own right.[](#pbio-0020262-g001){ref-type="fig"}
::: {#pbio-0020262-g001 .fig}
:::
*Mutants*, the book, is excellent: impressively researched and illustrated and extremely well written. The resulting television series on Channel 4 in the United Kingdom has a distinctly different impact. The series covers only a few of the subjects dealt with in the book, and handles the material in a different way. Inevitably, television can\'t include much science or scholarly detail, but it compensates with human images that are wholly gripping---both the preserved specimens and the living subjects who talk about the strange conditions that they live with. It\'s a freak show, but a freak show with thoughtful scientific commentary.
Each of the three programmes in the series has a particular theme. The first, 'The Mystery of Growth', is devoted mainly to skeletal disorders. We meet Carole Ozel, who copes with extraordinary courage with a terrible disease called fibrodysplasia ossificans progressiva (FOP), in which bony tissue forms throughout the body, gradually immobilizing the body in a second skeleton. Later we encounter a crew of charming and articulate dwarfs taking time out from a disco at the Reno convention of the Little People of America. They are happy to be called dwarfs or little people, but midget is no longer an acceptable term. Being a dwarf, in fact, is sometimes a ticket to fame and fortune, as in the case of Joseph Boruwlawski, last of the court dwarfs, who enchanted European royalty, married a beautiful woman, and lived happily to the age of 98.
The second part, 'The Dangerous Womb', is about birth defects, conjoined twins, and basic embryology. The makers of the piece went to the trouble of getting the developmental biologist Eddie de Robertis to reconstruct the classic experiment of Mangold and Spemann that revealed the underlying basis of some of these defects. The programme goes into some detail about what is now known about the molecular basis of normal and abnormal development, and how we can begin to explain such extraordinary forms as that of the Parodi twins, who had distinct heads and shoulders, but merged into a single torso and a single pair of legs.
The third programme, 'The Meaning of Beauty', deals with lesser but still striking abnormalities such as albinism and hypertrichosis (excess hair). It also moves into contentious areas, in a frank discussion of race genetics. Leroi makes the essential point loudly and clearly: there is much more genetic variation within any village on earth than there is between different human populations. We are extraordinarily unified, from a genetic standpoint. But, as he notes, the idea of race persists, and about 7% of global genetic variation in human DNA includes AIMs, ancestry informative markers. Some of these distinguish, for example, Africans and Europeans and provide objective information about the ancestry of different human populations.
This being television, the series closes with a discussion of beauty, which Leroi proposes to be simply the absence of visible mutant defects, using Saira Mohan, *Newsweek*\'s idea of human facial perfection, as an exemplar. Yes, she looks nice, but nice can be boring. Beauty, above all other human attributes, is profoundly influenced by culture, and it is hard to take this interpretation of beauty as an adequate explanation, rather than just a pretty way to finish the series.
For the most part, Leroi makes an agreeable and humane commentator, though he is not immune to the slight self-satisfaction that seems to overcome all scientists on television. The camera also spends an excessive amount of time dwelling on him, to a point where it becomes irritating to see him walking---frequently in slow motion--- into yet another museum or laboratory. Sometimes the focus on the presenter pays off, as when we see the six-foot scientist looking like a small child beside Chris Greener, the tallest man in Britain, or witness Leroi\'s faint chagrin at discovering that his DNA is mostly European, despite his cosmopolitan family history. More questionable are the bits where he paddles casually through a tub of preserved viscera from some long-gone sufferer from situs inversus (mirror-reversed organs) and succumbs to laughter at the sight of Ditto, the amazing two-faced pig. This may be an honest attempt at portraying the conflicted reactions we all have to abnormality, but it seems bound to cause trouble.
Arty camerawork is a running feature of the programmes, and there is a great deal of smoke and mirrors about the whole production. Perhaps this is deliberate, reminding us that it is hard to look directly at extreme deformity, but there is an air of 'I wants to make your flesh creep' about the many sidelong shots of mutant babies in bottles in the Vrolik Museum, to which we return again and again throughout the series. Viewing the already distorted fetuses through further distorting camera angles and under green lighting doesn\'t really achieve anything. The treatment begins to resemble the first *Alien* movie, in which the audience was never allowed to see the monster directly.
In the end, what is most memorable about these programmes is the living people themselves, and how they have coped with their various genetic disorders. It is very touching to see the home movies of Tiffany York, born with mermaid syndrome, or sirenomelia (in which the legs are fused together), taking her first tottery steps after corrective surgery, and to listen to her talking philosophically about her life as she floats in a Florida swimming pool. It is similarly cheering to hear from the dwarfs and albinos, or from Chuy Aceves, who has hypertrichosis and looks like the original Hollywood wolfman but suffers no ill effects and is proud of his rare condition. For these sections alone, let alone the serious and well-explained scientific background, the series is well worth seeing. It makes one feel surprisingly good about the human race and the human spirit.
Jonathan Hodgkin is in the Genetics Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom. E-mail: <jonathan.hodgkin@bioch.ox.ac.uk>
| PubMed Central | 2024-06-05T03:55:46.787597 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509310/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e262",
"authors": [
{
"first": "Jonathan",
"last": "Hodgkin"
}
]
} |
PMC509311 | Cell and molecular biologists have a good start at understanding the adult salamander\'s enviable ability to completely regrow a lost limb or jaw. (Salamanders can even regenerate portions of their eyes and heart.) Happily, mammals share many of the required cellular skills---though in an untapped form. In response to injury, fully differentiated salamander muscle cells can produce less specialized cells that are capable of multiplying and recreating lost tissue. In this month\'s *PLoS Biology*, biochemist Jeremy Brockes and his colleagues at University College London tie this muscle cell de-differentiation to expression of a single gene, *Msx1*, a player in limb development and regeneration across species.[](#pbio-0020266-g001){ref-type="fig"}
::: {#pbio-0020266-g001 .fig}
::: {.caption}
###### An adult salamander
:::
:::
The protein encoded by *Msx1* appears at the borders of maturing tissues in salamanders, chickens, and mice, and represses differentiation of muscle cells (called myofibers) during development. *Msx1* promotes amphibian limb and tail regeneration, zebrafish fin repair, and regrowth of the tips of digits in mice. Here, Anoop Kumar et al. clarify the gene\'s role in muscle repair by demonstrating that *Msx1* expression is required for de-differentiation of salamander myofibers---mature muscle tissue without the ability to produce new cells---into cells with single nuclei (mononucleate cells) that are able to multiply.
Large, elongated cells, myofibers are distinguished by a striped pattern of actin and myosin, the proteins that produce muscle contractions. They develop from precursor cells, which proliferate and fuse together into multinucleate intermediates called myotubes. As myotubes bulk up on actin and myosin, pushing their many nuclei to the cell periphery, they mature into myofibers. Once differentiated, myofibers are committed to the life of a muscle workhorse. They cannot divide; mature muscle in mammals adds strength and repairs injury by accumulating more actin and myosin, and by fusing single-nucleus "satellite" cells into new or existing myofibers.
Unlike their mammalian counterparts, salamander and newt myofibers respond to injury by splitting into several multinucleated fragments, or by budding off several of their nuclei to create individual cells. These mononucleate progeny multiply and develop to replace lost tissue. Kumar et al. found that more than half of salamander myofibers spontaneously fragment and bud when dissected and placed in cell culture. Prior study has shown that mouse myofibers don\'t normally behave this way, although cultured mouse myotubes can imitate amphibian myofibers if given a push, in the form of *Msx1*. A fraction of mouse myotubes made to express *Msx1* break off mononucleate cells that can be induced to express markers for bone, cartilage, fat, or muscle.
In the salamander cells, *Msx1* RNA and protein appeared in actively fragmenting and budding myofibers, especially in and around their nuclei. When Kumar et al. blocked the translation of *Msx1* mRNA, the myofibers did not generate new cells.
The results confirm *Msx1* as a pivotal regulator of muscle cell re-entry into the cycle of cell division and tissue growth. In this context, it doesn\'t drive cell division---on the contrary, the authors showed that *Msx1* induced cell splitting without DNA replication. The gene drove committed muscle cells to donate nuclei, creating a pool of new cells to divide, differentiate, and repair damage. Given what it can do for mouse cells in culture, the question remains whether *Msx1* might help awaken a latent capacity for regeneration in living mammals.
| PubMed Central | 2024-06-05T03:55:46.788410 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509311/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e266",
"authors": []
} |
PMC509312 | At first glance, the preschool classroom on the other side of the two-way mirror looks like any other---brightly colored rugs, scattered toys, and tiny chairs. But almost immediately an observer notices differences in the Team Toddle students here at the Neuropsychiatric Institute of the University of California at Los Angeles (UCLA) (Los Angeles, California, United States).
A therapist instructs a toddler on his colors, flashing a rapid sequence of blocks at him. When the toddler starts rocking in his chair and repeatedly touching his forehead, the therapist physically restrains his hands, placing them back on the tabletop until he stops the repetitive behaviors and focuses once again on her face and the blocks. During playtime, a two-year-old girl sits by herself in the corner, fixated on some picture cards, oblivious to a group of other children playing with a racetrack and to the therapist who tries to draw her out to join the group.
These children lack some of the key social skills that normal toddlers pick up naturally---looking to others for reassurance or cues, focusing on faces, and playing together. Social and communication impairment is a hallmark of autism and can show up as early as 12--18 months of age. But with an unknown cause, and genetic linkages still hazy, there is little consensus among researchers on how the disorder develops in children and how it causes a broad spectrum of social, language, and behavioral deficits.
Following one line of research, David Amaral\'s laboratory at the M.I.N.D. Institute at the University of California at Davis Medical Center in Sacramento (California, United States) has recorded, in autistic brains, a brain volume increase in a specific structure, the amygdala, which is thought to be important for social behavior. A similar study at the University of Washington in Seattle (UW) (Seattle, Washington, United States) has reached the same conclusion. "There are so few facts about autism, to have two labs come up with the same data is phenomenal," says Amaral. "We feel confident this is a real finding, but what does it mean to these kids?"
On another research track, using functional imaging, Ralph-Axel Müller, a cognitive neuroscientist at San Diego State University (San Diego, California, United States) sees a scattering of brain activation in autistic brains that he views as an indication of a more general brain development problem underlying the disorder ([Figure 1](#pbio-0020267-g001){ref-type="fig"}). He has hypothesized that the early-developing basic functions may require more brain area in autism, pushing out and disturbing the later specialization for more complex functions. "I\'m sure this is wrong," he says, "but it will allow us to look in a more hypothesis-driven way at animal studies of how the cerebral cortex develops specialization." Animal models may, in turn, yield clues about normal and abnormal brain development in humans.
::: {#pbio-0020267-g001 .fig}
Figure 1
::: {.caption}
###### Brain Activation Scattering in Autism
Autistic individuals show less activity, during a movement task, in areas that are normally activated (premotor and superior parietal cortex; blue areas), but unusually increased activity around these normal sites of activation (red areas). Images courtesy of Ralph-Axel Müller.
:::
:::
"Since there is no major hypothesis as to cause \[of autism\], there are many plausible ideas," says Amaral. "If we go after all of them, we will waste all of our resources. \[We have to\] come to some consensus about which are most plausible." At least two levels of pursuit exist for tracing brain problems associated with autism---the exploration of the general developmental disruptions that result in an autistic brain, and the examination of more specific problems in particular brain structures that produce symptoms. Although scientists still debate how autism evolves in a patient, the field has begun in the last decade to replicate findings and make science-based arguments for interventions. Progress has come in small steps, with advances in neuroimaging and more rigorous experimental designs.
Research focuses have shifted from "curing" autism to finding better diagnostics for early intervention, improving behavioral therapies, and gaining insight into the development and function of the autistic brain. Both advocacy groups and government programs have started to bring together neuroscience and genetics experts, clinicians, and families to sharpen the focus of studies and ensure progress in what has often been a messy field.
A World Apart {#s2}
=============
Autism spectrum disorder strikes between one and six out of every 1,000 children around the world, but diagnosis and treatment are currently limited to developed countries. Autism is four times more prevalent in boys than girls, but makes no racial, ethnic, or socioeconomic distinctions. It is characterized by three main symptoms: impaired language, social and communicative deficits, and repetitive and stereotyped behaviors, such as hand flapping, rocking, and unusual responses to sensory stimuli. Autism spectrum disorders can be broken down into other categories, such as low-functioning autism (IQ below 70), high-functioning autism (IQ above 70), and Asperger syndrome (similar to high-functioning autism but with no language deficit).
Researchers suspect that there are even more distinct subsets of autism patients. For example, some patients also have epilepsy, and it has been suggested that there is a regressive form of autism---children who, at two or three years of age, appear to regress and lose developmental milestones they had already achieved. Researchers say that sorting out these different profiles---or phenotypes---of autism will be especially important in sorting out which genes or which brain abnormalities are implicated for particular deficits. This sorting should also help clarify the mounds of contradictory data that have dogged the field, by tamping down the experimental "noise" in studies. Boosting the number of children studied and following them from early infancy through adolescence and beyond will also be key components of future studies.
"There is not going to be rapid progress in autism research unless we subtype," Amaral says. He predicts that "brain differences in kids with a regressive form of autism will be different than those of kids with the more congenital type of autism." He and others are teaming up in an autism phenotyping project that will characterize 600 children into categories of autism (comparing them to 600 children with mental retardation and 600 controls). Splitting autism into subtypes will boost both neurobiology and genetics studies ([Box 1](#box1){ref-type="boxed-text"}) to find real effects related to specific traits.
Facing Up to Autism {#s3}
===================
A key area of research explores the brain\'s response to human faces at a young age. Studies at the UW Autism Center have shown that unlike typically developing three-year-olds, autistic children do not show a differential brain response to their mother\'s face compared to that of a stranger. While dysfunctional face recognition may be one of the more devastating symptoms for caregivers, it is also one of the most promising avenues for research to determine how autistic brains process their world differently.
Sara Webb, a child psychologist at UW, has followed about 70 autistic children since the age of three for a longitudinal study that will test many parameters until they reach age nine. Her work has already shown that autistic three-year-olds process seeing a strange toy differently from seeing a favorite toy, in the same way a normal child does. But activity in their brains---measured through a network of electrodes placed on the scalp---is similar whether the face is familiar (for example, mom) or strange. This, Webb says, led to two hypotheses: either the brain area for face processing is not set up correctly in autistic children, or the way these children incorporate experiences from their environment is so different that the brain area develops improperly.
"We think the latter is a more likely explanation at this point," says Webb. "By the time they are adolescents or adults, they are showing the \[proper\] response for familiar faces." Indeed, a functional MRI (fMRI) study by UW neuroimaging researcher Elizabeth Aylward showed that the brains of high-functioning adolescents and adults did activate the face-recognition center, the fusiform gyrus, when shown a very familiar face. However, the same subjects did not activate the center when viewing strange faces. This points to the possibility that greater experience seeing the familiar face (i.e., on a daily basis for many years) can eventually influence the appropriate brain areas.
"You need the biological wiring set up properly, but you also need experience for it to function normally," says Aylward. "We\'re guessing what is missing is the experience." To test that idea, one of her graduate students will "train" half of the autistic patients in face recognition---something most children pick up on their own---by having them study, manipulate, and match faces using computer games. Then fMRI scans will be done again to see if the fusiform gyrus might now be activated when viewing strange faces, as it is in control subjects. Intense training of a similar type for reading has already been shown to effect change in brain activation in as little as three weeks for children with dyslexia.
In their model, it is as if "all the parts are there, ready to go, but somehow they haven\'t gotten the ignition turned on," says Aylward. At the 2004 annual meeting of the American Association for the Advancement of Science (Washington DC, United States), the UW center director Geraldine Dawson explained that this tackling of specific deficits will help researchers attach them to particular "mind modules" in the brain and will ultimately lead to the genes that control the development or function of those modules. That modular view, however, is not shared by many of her colleagues elsewhere, who argue that autistic behaviors are the result of a system-wide perturbation of early brain development and connectivity.
Structural Support {#s4}
==================
For example, Müller points to structural studies that seem to uphold his theory of overall disorganization of the brain\'s cortex. Work by Manuel Casanova and colleagues at the University of Louisville (Louisville, Kentucky, United States) shows that the "minicolumns" of neurons that make up the cortex are narrower and more numerous in autistic brains. Normally, these organized bundles appear very early in the developing fetal brain. In postmortem studies of autistic brains, Casanova found that the minicolumns had the same number of neurons, but smaller margins between the bundles. The margins, Casanova says, may act like "a shower curtain of inhibition that prevents information from flooding adjacent minicolumns."
Reducing those margins, he hypothesizes, could mean that an autistic brain has too much positive feedback, acting like a noisy amplifier. "For an autistic individual who is trying to piece together too much information from a face, maybe it\'s like looking at the sun," he says.
More general studies of adult autistic neuroanatomy have given conflicting results---most likely from diversity in the study populations---that make functional inferences difficult, if not impossible. But recent studies that focus on developing autistic brains earlier in life have revealed intriguing differences from normally developing children.
Several studies have shown that from ages two to four, autistic children have larger overall brain volumes (and correspondingly larger head circumferences) than normal children, but that the difference had disappeared by about age six or seven. Since autism is usually diagnosed around age two or three, when the brain is already abnormally large, Eric Courchesne and colleagues at University of California, San Diego (San Diego, California, United States) hypothesized that brain overgrowth must occur earlier, before signs of autism appear.
In an elegant retrospective study, the team analyzed head circumference and brain volume measurements of autistic children that started at birth and continued until 14 months of age. The study revealed that at birth, autistic children\'s head size is much smaller than healthy children, in the 25th percentile, but by 6--14 months, their head size had increased to the 84th percentile, an excessive growth rate. The increase correlated with increased brain volumes of both gray and white matter regions measured by structural imaging between ages two to five.
The Courchesne study strongly suggests that with autism, significant unregulated brain growth occurs in the first year of life. The team also found an association between greater increases in brain size in infancy and a later age for first word, worse repetitive behavior, and a trend toward more severe autistic symptoms later, at diagnosis. The rapid growth of autistic brains may produce too many connections too quickly, without the opportunity to be shaped by the experience and input that a typically developing child accumulates over many years. At age six or later, when the growth slows, the already derailed connections may no longer be able to incorporate experiences. "By that time," write Courchesne et al., "the period of plasticity that allows the exquisite and graceful complexity of the human brain to emerge will have passed."
Playing Well with Others {#s5}
========================
This idea that autistic brains are developing at warp speed, to their detriment, fits intriguingly well with what is known about treatment of autism---the earlier and more intense behavioral therapy an autistic child receives, the better the outcome will be. That\'s why the toddlers at UCLA get one-on-one training by therapists, who fire rapid questions and physically repeat tasks until they sink in.
Stephanny Freeman, co-director of the Early Childhood Partial Hospitalization program at UCLA (Los Angeles, California, United States), says these methods would be alien to, and lost on, typically developing two-year-olds, who would be bewildered by such a highly structured environment. Her colleague and co-director, Tanya Paparella, chimes in, "It as if we are opening a window or door to the autistic brain." Keeping that door open as long as possible in very young autistic patients seems to give them a better prognosis than older children, who are more difficult to treat.
But while most agree that early and intense therapy is good for autistic children, until recently, little research on intervention methods existed. Connie Kasari, an educational psychologist at UCLA, along with Freeman and Paparella, has run one of the first randomized, controlled trials on therapies designed to teach autistic kids social skills. The group tested two skills in particular---sharing attention with others and pretend playing ([Figure 2](#pbio-0020267-g002){ref-type="fig"}). The team hypothesizes that these skills, which normal children pick up easily and early, lay important groundwork for language development.
::: {#pbio-0020267-g002 .fig}
Figure 2
::: {.caption}
###### Pointing as an Example of Joint Attention
A child with autism (three years old) pointing to the fish in an aquarium. Photo courtesy of Connie Kasari.
:::
:::
The team\'s results show that autistic children can learn these skills from intense training. At least anecdotally, some of these children have gone on to function in normal school classrooms, even making a few friends, although they are still a bit socially awkward. Whether or not improvements in those skills will correlate with language improvements will require further testing. But Kasari notes that this work is not universally accepted in the autism therapy community, and that many more controlled studies will have to be published before a system-wide change in autism preschool education can occur.
Funding the Search {#s6}
==================
In the last decade, National Institutes of Health funding for autism research has increased from \$10 million to \$80 million, and much of that has been funneled into large, multidisciplinary research projects. Advocacy groups such as Cure Autism Now (Los Angeles, California, United States) and the National Alliance for Autism Research (Princeton, New Jersey, United States) greatly influence which autism research projects get funded, both through their own grant programs and also by lobbying Congress for increased federal grants. Some question whether it is wise to let emotions and the desire to find a cure drive research agendas. In the past, tensions between government programs and advocacy programs have run high.
Casanova, for one, criticizes the disproportionate flow of money to what he calls imaging and genetic "fishing expeditions" and says more should go to neuropathology studies. He points out that only about 40 postmortem, mostly adult, autistic brains have been studied so far, a tiny fraction compared to those studied in other neuropathological disorders like Alzheimer\'s disease or schizophrenia.
But Daniel Geschwind, a neurogeneticist at UCLA, defends this approach, saying that a well-planned fishing expedition that uses the right technology and looks in the appropriate places can result in a "freezer full of fish." He also says that parent organizations keep the field honest by "constantly reminding us to keep an eye on the ball and don\'t get distracted." Geschwind, Amaral, and other top experts have recently been recruited by advocacy groups or by friends with autistic children to shift some of their research questions to examining autism.
As more researchers in genetics and neuroscience have become involved, Amaral says, the tensions between the parent groups and the National Institutes of Health have eased. "The parents communicated to the scientists the tremendous need for research and the scientists convey back to them which \[research projects\] make sense to fund," he says. He adds that advocacy groups have been indispensable to research, setting up large genetic and brain tissue banks and enlisting families to participate in those efforts.
So, researchers say, the goals of the National Institutes of Health programs and the advocacy programs have started to come together to focus on well-executed studies that might lead to better diagnostics and earlier, proven interventions. The work of Courchesne et al. suggests that children at risk for autism might easily be diagnosed by head circumference measurements as early as the first few months of life. Imaging studies combined with training programs, such as the work at UW on face recognition, may one day be able to verify that behavioral interventions are effective at activating target brain areas. As researchers work to untangle the causes and effects of brain dysfunctions in autism, Aylward notes, there is good reason to be hopeful: "Although this is a genetic disorder, we know there is plasticity in the young brain."
::: {.caption}
###### Box 1. Genetic Power-Up
:::
Evidence abounds that autism results from multiple gene mutations. Identical twins share an autism diagnosis 60%--95% of the time, and a younger sibling of an autistic child is 50 times more likely to have autism. There are also four times as many autistic males as females, indicating a possible sex chromosome difference in inheritance. Genetics researchers estimate that autism is the result of mutations in anywhere from 2 to 20 genes.
By studying the commonly inherited pieces of chromosomes in autistic siblings, geneticists have identified a handful of chromosome hotspots. However, each region contains hundreds of individual genes, and narrowing down to specific mutations will require studies that either involve thousands of families or tackle specific phenotypes. Daniel Geschwind, a neurogeneticist at UCLA, has already completed such a study. It reveals a linkage---the probability that a region contains a gene or genes linked to the disorder---between language deficits and a hotspot region on Chromosome 7. His team looked at a more homogenous group of autistic patients, all of whom had a similar language delay measured quantitatively by time to first spoken word.
"Endophenotypes measure something that underlies the disorder in a significant way and \[therefore probably\] also underlies a genetic component," says Geschwind. "We\'re trying to identify characteristics that really underlie the genetic peaks of interest." Another such study, by Margaret Pericak-Vance and colleagues at Duke University Medical Center (Durham, North Carolina, United States), used the characteristic of "insistence on sameness"---a subset of stereotyped behaviors such as resisting change in routine or environment, and compulsions. By running a genetic analysis on a group of patients with the highest "insistence on sameness" scores from diagnostic tests, the Duke team increased the linkage score and further narrowed the hotspot region on Chromosome 15.
Kendall Powell is a freelance journalist from Broomfield, Colorado, United States of America. E-mail: <kendall2@nasw.org>
fMRI
: functional MRI
UCLA
: University of California at Los Angeles
UW
: University of Washington in Seattle
| PubMed Central | 2024-06-05T03:55:46.788912 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509312/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e267",
"authors": [
{
"first": "Kendall",
"last": "Powell"
}
]
} |
PMC509313 | Machinery within the endoplasmic reticulum (ER) of eukaryotic cells modifies, folds, and assembles proteins as needed to suit their functions at or past the cell membrane. When this system is hampered or overtaxed, a buildup of unfolded or misfolded proteins within the ER triggers the "unfolded protein response," which alerts the nucleus to boost production of protein-processing machinery that helps proteins fold. This system for adjusting manufacturing capacity is similar in organisms from yeast to human. If the unfolded protein response cannot be turned on when needed, cells die. Prior study suggested that, in yeast cells, the response to unfolded protein buildup is binary: either off or on. In this month\'s *PLoS Biology*, biochemist Peter Walter and his colleagues from the University of California at San Francisco demonstrate two new signaling mechanisms that appear to give the yeast unfolded protein response the means for amplitude adjustment.
In yeast, a transcription regulator called Hac1p activates the genes required for the unfolded protein response. A cytoplasmic pool of *HAC1* messenger RNA waits in readiness for ER emergencies, each molecule locked against translation into protein by intronic RNA sequences that interrupt mRNA translation. Accumulation of unfolded or misfolded proteins in the ER releases this translational block, triggering production of Hac1p and activating the unfolded protein response. Previously, this binary *HAC1* signal was the only known regulator of the unfolded protein response in yeast. In two complementary papers, Walter and colleagues now present evidence that the repertoire includes new factors and regulators that amplify the unfolded protein response under conditions of ER stress.[](#pbio-0020269-g001){ref-type="fig"}
::: {#pbio-0020269-g001 .fig}
::: {.caption}
###### Wiring the unfolded protein response
:::
:::
Leber et al. stressed yeast cells by exposing them to substances that cause protein misfolding or buildup in the ER. In response, the cells ratcheted up transcription levels of *HAC1* severalfold. Primed with high levels of *HAC1* mRNA, the cells were ready to produce a bumper crop of Hac1p and to induce a supercharged unfolded protein response. In the accompanying paper by Patil et al., the authors show that Hac1p is not working alone. A second regulator of transcription called Gcn4p is required to activate most of the genes associated with the unfolded protein response. The regulatory elements of these genes now appear far more diverse than previously appreciated. The authors propose that cells adjust the levels of Hac1p and Gcn4p to drive a continuum of transcriptional programs equipped to deal with incoming challenges. Together, these two papers demonstrate that the control of the unfolded protein response is far from a simple on/off mechanism, but exhibits complex fine-tuning through a network of signaling pathways that interpret and respond to the cell\'s needs.[](#pbio-0020269-g002){ref-type="fig"}
::: {#pbio-0020269-g002 .fig}
::: {.caption}
###### Control elements of UPR target genes
:::
:::
| PubMed Central | 2024-06-05T03:55:46.791217 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509313/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e269",
"authors": []
} |
PMC509314 | To maintain the integrity of their genetic content, cells closely monitor the state of their chromosomal DNA. Any break or pairing anomaly in the double helix is perceived as damage that must be repaired. Mechanisms that sense DNA damage enlist the help of cell cycle checkpoint proteins, which stall cell division until the damage has been repaired. The exposed ends of linear chromosomes of eukaryotic cells (cells with nuclei) resemble the double-strand breaks of damaged DNA, which should make them vulnerable to unnecessary manipulations by repair enzymes. But chromosomes have special DNA sequences at their tips, called telomeres, that are coated with telomere-binding proteins that appear to protect ends from the unwanted attentions of repair enzymes. For instance, removing a telomere-specific DNA-binding protein, called TRF2, from telomeres leads to a rapid attack on chromosome tips, associated with the activation of the DNA damage pathway. The biochemical basis for TRF2\'s shielding effects remains obscure.
In this issue of *PLoS Biology*, Jan Karlseder et al. propose that TRF2 is a direct inhibitor of an early mediator of the DNA damage signal. Their observations offer new insights into how telomeres resist the inappropriate interventions of the DNA repair machinery.
Ataxia telangiectasia mutated (ATM) kinase is an enzyme that induces the activation of DNA repair enzymes as well as regulators of the cell cycle and apoptosis. Its enzymatic function is triggered by DNA damage sensors. Several observations suggest an antagonism between TRF2 and ATM: ATM is activated in aging cells with shortened telomeres and participates in ushering the cell into replicative senescence. (Cells divide only so many times during their lives before forgoing the process altogether.) In contrast, TRF2 overexpression protects shortened telomeres from decay and delays cell entry into senescence.
Here the authors examine the effect of TRF2 overexpression in cells subjected to radiation-induced DNA damage. This treatment is expected to lead to cell cycle arrest, an outcome mediated for the most part by ATM activation. Irradiated cells that overexpress TRF2, however, continue to enter cell division. Known targets of ATM\'s enzymatic activity are activated to a lesser degree in these cells, which harbor only about half the amount of active ATM detected in controls. This suggests that TRF2 may interfere with the DNA damage pathway early on, when ATM is activated.
Do these findings reflect a direct interaction between ATM and TRF2? Karlseder et al. suggest they might. Though only a small fraction of TRF2 associates with ATM in normal cells, ATM and TRF2 proteins can form a complex in a test tube. The region of the ATM protein that interacts with TRF2 is the very region required for ATM\'s activation by DNA damage sensors. The authors propose that TRF2 binds to inactive ATM, and in so doing, prevents ATM\'s transition to an active state.
The authors integrate their results in an elegant proposal that relates a cell\'s health to the length of its telomeres. They have previously shown that TRF2 binds another protein called Mre11, a DNA damage sensor known to activate ATM. Thus, by inhibiting ATM, TRF2 may nip in the bud any misguided attempts by Mre11 to "repair" DNA breaks in telomeres. But in aging cells, whose shortened telomeres no longer retain large amounts of TRF2, ATM activation, no longer muffled, eventually allows the onset of senescence. While TRF2 is abundant in normal cells, the authors note that its strict association with telomeres should locally limit its effect on ATM, leaving the DNA damage pathway intact at other chromosomal locations.
| PubMed Central | 2024-06-05T03:55:46.791700 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509314/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e270",
"authors": []
} |
PMC509315 | There is an old Chinese curse: 'May you live in interesting times.' According to those who know about such things, we live in a momentous time, the time of the Sixth Mass Extinction! But most of us do not feel at all cursed. Because, in fact, the Sixth is quite different to the previous Big Five---no-one would notice this one if we were not repeatedly reminded of it by ecologists. Previous mass extinctions were not so bashful, so discreet. The fossil record reveals the disappearance of pollen during previous ones, replaced by an abundance of fungus spores, telling us of a world of devastated forests rotting away. The earliest sediments after the mass extinction that did away with the dinosaurs are barren of fossils: so it is not just that species were going extinct, conditions for life itself were bad. Not only did species diversity drop, the abundance of life did as well.
But conditions for life itself have never been better than today. In the history of the planet, there has never been anything as productive of life as a wheat field in Kansas. It may not have a large diversity of species, but that is a different matter. In fact, one of the reasons for the ongoing loss of plant diversity from grasslands is the very reason the wheat field is so productive---fertilisation. We are pouring nitrogen fertiliser into the environment and, through the wellstudied 'paradox of enrichment', this reduces species diversity while increasing actual biomass. Now, there is no question that if current trends of habitat alteration and climate change continue then we will ultimately lose large numbers of species---diversity will drop---but this does not necessarily translate into a loss of abundance of life, and that is a big difference between now and previous mass extinctions. Looking at specific groups of organisms tells the same story. So, for example, many island bird species are threatened, like the kagu of New Caledonia, but British seabird populations, like puffins, are booming. Worldwide amphibian diversity is threatened, but cane toads are a pest in Australia. Introduced species pose a threat to diversity---the 'McDonald-isation' of nature---precisely because they achieve enormous abundances.
Actually, all six mass extinctions may have one very important thing in common: from the point of view of the vast bulk of life on the planet they are probably not mass extinctions at all. By any criterion---number of individuals or total biomass---the vast majority of life on earth is invisible---microbial. So, for example, at least 10% of the living biomass on earth consists of bacteria living deep in the oceans\' sediments: it would take more than an asteroid impact to disturb them. And microbial life is extraordinarily robust: microbes can be found living happily in pressurised water hotter than your boiling kettle, in concentrated acid, and in rock, and their spores can survive for years in the rigours of outer space.
In talks and lectures, the renowned oceanographer and paleontologist Jeremy Jackson paints a vivid picture of what is currently happening to coastal ecosystems, talking about a wall of slime emanating from populated areas and growing outwards inexorably towards the open oceans, replacing beloved ecologies like coral reef systems. What he means is that the visible life that we find attractive and useful---pretty fish, turtles, and so on---is being replaced by microbes in splendid profusion. It is taken completely for granted that this is disastrous. From a utilitarian point of view indeed it is disastrous, since we like eating fish and turtles, and don\'t like snorkling in slime. But from the point of view of life per se, again things have never been better. Life is so abundant that in some places all the oxygen in the water is completely used up. These are called 'dead zones', but they are no more 'dead' than the Dead Sea, which is actually teeming with life---just not fish. But, nonetheless, we consider what is occurring to be a disaster not just from a utilitarian point of view, but at some deeper level giving us an emotional reaction to the word 'slime'---somehow it is just plain wrong.
But this reflects nothing other than our evolutionary origins. Evolution has programmed us to be positively interested in plants and animals, our food, and to be repelled by slimes and oozes, teeming with potentially harmful microbes. These emotional responses colour our view of ecology, for example, in a way that has no parallel in other sciences: physicists do not just study particles that they find pretty. No ecologist wants to study the rich ecosystem that each of us carries around inside our gut, because evolution has programmed our brains to find bottom-related matters disgusting. I think it likely that naturalists from a different planet, silicon entities evolved under very different circumstances, would find tropical forests uninteresting (mainly primary producers with some herbivory and mutualisms) and animal guts fascinating, with their complex metabolic networks in which each node is manned by different species with wildly varying means of energy production.
Our guts should be an ecological scientist\'s dream come true, ecological theatres that are replicated billions of times, which operate on a fast time scale and are easy to get to! (If aliens are ecologists, this would explain why they always 'probe' their abductees.) Many natural experiments are going on all the time as antibiotics and probiotics are administered and people find all sorts of different ways, voluntary or otherwise, to establish migration links between their gut ecologies. Microbiologists are increasingly interested in our guts from an ecological point of view but, unlike ecologists, they are used to faeces from their work in sewage plants.
Two thousand years ago the Roman senator Cicero noted the creation of barren desert-like land in North Africa after the forests were felled for their timber, providing the earliest record of an ecosystem 'service' provided by forests---the stabilisation of soils. Other services provided by biodiversity readily come to mind, like pollination and carrion clean-up, and there may be many more. But perhaps the clearest example of an ecosystem service provided by biodiversity comes from our gut. Throughout our history, until very recently, we all had worms. In rich countries we have quite happily eradicated them from our inner ecosystems with none of the handwringing we expend on rhinos. But it is increasingly believed that the loss of worms from our internal ecology is responsible for the upsurge in inflammatory bowel disorders such as Crohn\'s disease and colitis. In fact, there are clinical trials underway in the United States testing the efficacy of worms as treatment for these diseases. The mechanism is clear: worms trigger one arm of the immune system which down-regulates another, inflammatory arm. Our immune system has evolved to expect a certain constellation of species in our gut: in that context, worms provide an ecosystem service of balancing the immune system.
Our perception of our impact on the planet as equivalent to a mass extinction simply reflects the evolutionary prism through which we view life. Of course, we may yet live up to our own publicity and pull off something apocalyptic like a runaway greenhouse that sterilises the Earth. But it is at least as likely that the microbial world, resentful at being either ignored or exterminated, will come up with something to consign us to a footnote in the history of life when it is ultimately written by the silicon entities. The Spanish flu, SARS, and HIV have just been early experiments.
Sean Nee is at Ashworth Laboratories, University of Edinburgh, Edinburgh, Scotland, United Kingdom. E-mail: <sean.nee@ed.ac.uk>
| PubMed Central | 2024-06-05T03:55:46.792328 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509315/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e272",
"authors": [
{
"first": "Sean",
"last": "Nee"
}
]
} |
PMC509316 | Organisms of vastly differing morphologies, ecologies, and behaviors---such as fruit flies and humans---are now known to share a multitude of molecular, cellular, and developmental processes. Not only is there extensive similarity in the sequences of fly and human genes, but in addition, almost all of the proteins and major signal transduction pathways that control cell division and differentiation in mammals are also found in the fruitfly Drosophila melanogaster ([@pbio-0020276-Rubin1]; <http://flybase.bio.indiana.edu/>). Components in these pathways perform the same biochemical functions and act in the same order in both fruitfly and mammalian cells.
Evolutionary conservation is of considerable practical and theoretical importance to biologists. First, it provides a valuable source of data for the reconstruction of phylogeny ([@pbio-0020276-Salemi1]). Evolutionary connections between organisms that were once hidden by morphology have now been exposed in genomic analyses. Second, the conservation of evolutionary processes or traits is a prime area of investigation in theoretical evolutionary biology ([@pbio-0020276-Gould1]). What can, and cannot, be changed evolutionarily? In a constantly evolving world, how can any biological system or trait survive unchanged ([@pbio-0020276-VanValen1])? Finally, conservation provides fundamental insights into how complex biological systems, such as immunity, are assembled, maintained, and altered in evolution.
Elements of Immunity {#s2}
====================
"Innate" immunity refers to the variety of physical, cellular, and molecular features that provide the first lines of defense against infections. The relatively quick innate immune responses operate along with slower but more targeted adaptive immune responses that generate antigen-specific mechanisms that eventually lead to the destruction and elimination of the pathogen.
In mammals, the skin and the epithelial lining of the mucosal tissues act as the primary nonspecific barriers, impeding infectious agents from entering the body. The mucous membrane barrier traps microorganisms, and the cilia present on the epithelial cells assist in sweeping the microbes towards the external openings of the respiratory and gastrointestinal tracts. If infectious agents gain entry into the body, internal innate immune responses become activated and rapidly eliminate the infection. Internal innate immune agents and responses include (amongst others) low pH of the stomach and vagina, proteolytic enzymes and bile in the small intestine, and phagocytosis.
Phagocytosis is a fundamental innate immune mechanism carried out by a number of different cell types, including macrophages. Specific macrophage subpopulations are associated with different tissues (alveolar macrophages in the lung, microglial cells in the central nervous system, etc.). Their main function is to consume microorganisms, other foreign substances, and old, dying cells.
Innate immunity is present from birth, and the information for innate immune responses is inherited. Cells in the mammalian innate immune system (e.g., macrophages) detect "microbial nonself" by recognizing pathogen-associated molecular patterns (PAMPs; [@pbio-0020276-Janeway1]). PAMPs are products of microbial metabolism that are conserved over evolution, distributed in a wide variety of pathogens, and not found in host cells. Lipopolysaccharide is an example of a PAMP and is found in bacteria, viruses, and fungi. Receptors, called pattern recognition receptors, are present on surfaces of host cells and recognize PAMPs. When activated, pattern recognition receptors induce intracellular signaling via the transcription factor NF-κB, resulting in the activation of genes involved in host defense.
Adaptive immunity is characterized by greater specificity than innate immunity, as the adaptive immune response can not only distinguish foreign cells from self, but can also distinguish one foreign antigen from another. Another hallmark of adaptive immunity is memory, which enables the body to remember specific adaptive responses in response to specific antigens. Immunological memory allows the body to make a greater and more rapid second response when the body is reinfected by the same pathogen. Immunological memory underlies both immunization and resistance to reinfection, conferring a tremendous evolutionary advantage to vertebrates. The adaptive immune response has nearly infinite flexibility: the T and B lymphocytes of the acquired immune system can rearrange the elements of their immunoglobulin and T-cell receptor genes to create billions of clones with distinct antigen receptors. In organisms where both innate and acquired immune systems are present, there is a clear interdependence between the two systems. For a fully functional immune system, these components must act in synergy.
Innate Immunity in Drosophila {#s3}
=============================
Because it lacks an adaptive immune response, Drosophila melanogaster serves as a wonderful model for studying aspects of the innate immune system that might otherwise be obscured by the actions of the adaptive immune response. Insects defend themselves against parasites and pathogens by invoking a multitude of innate immune responses ([Figure 1](#pbio-0020276-g001){ref-type="fig"}; for more details, see recent reviews by [@pbio-0020276-Hoffmann1], [@pbio-0020276-Hultmark1], [@pbio-0020276-Brennan1], [@pbio-0020276-Meister1], and [@pbio-0020276-Theopold1]). Like humans, Drosophila protects itself against microbes and parasites via epithelial barriers: for example, epithelial cells of the trachea, gut, genital tract, and Malpighian tubules produce antimicrobial peptides (local response).
::: {#pbio-0020276-g001 .fig}
Figure 1
::: {.caption}
###### Innate Immune Responses of Drosophila
\(A) Posterior region of a third instar larva showing the cuticle and the trachea. These structures provide a physical barrier against infections. Cellular immune reactions consist of phagocytosis, encapsulation, and melanization.
\(B) A dead and melanized crystal cell phagocytosed by a plasmatocyte.
\(C) Encapsulation of an egg of a Drosophila parasite. The parasite is a wasp that normally infects larvae. Cells surrounding the egg are lamellocytes. The cells and the egg are stained with a fluorescent nuclear stain.
\(D) Clot formation occurs during wound healing.
\(E) Crystal cells in contact with the larval cuticle. The contents of the crystal cells are melanized. Melanization occurs in response to intruding pathogens or parasites and is also observed during wound healing.
\(F) Humoral immune reaction. The expression of antimicrobial peptides in the larval fat body is induced by microbes. Cells of the fat body appear green due to the presence of a transgene encoding the green fluorescent protein, under the control of the *drosomycin* promoter. The *drosomycin* promoter is activated in response to fungal infections and is under the control of the Toll pathway (see [Figure 2](#pbio-0020276-g002){ref-type="fig"}). Antimicrobial peptides are released from the fat body into the hemolymph. This response is therefore systemic. A similar antimicrobial gene activation response can occur locally in specific body parts such as the trachea or the gut (not shown).
:::
:::
Once within the body cavity, microbes may be consumed by the phagocytic blood cells called plasmatocytes ([Figure 1](#pbio-0020276-g001){ref-type="fig"}). Larger pathogens (such as eggs of parasitic wasps) are inactivated by encapsulation, an immune response carried out by specialized cells called lamellocytes ([Figure 1](#pbio-0020276-g001){ref-type="fig"}). Lamellocytes differentiate in response to macroscopic pathogens, and their precursors are thought to reside in the larval lymph gland. The transcription factors (GATA, Friend-of-GATA, and Runx family proteins) and signal transduction pathways (Toll/NF-κB, Serrate/Notch, and JAK/STAT) that are required for specification and proliferation of blood cells during normal hematopoiesis, as well as during the hematopoietic proliferation that accompanies immune challenge, are conserved ([@pbio-0020276-Evans1]; [@pbio-0020276-Meister1]). In this issue of *PLoS Biology*, [@pbio-0020276-Crozatier1] identify the transcription factor Collier as being critical for the differentiation of lamellocytes in Drosophila. The mammalian ortholog of Collier (Early B-cell Factor) is involved in B-cell differentiation in mice.
In addition to triggering cellular immune responses, invading pathogens also activate humoral reactions. Microbes induce the rapid secretion of antimicrobial peptides from the cells of the fat body into the larval or adult body cavity (systemic response; [Figure 1](#pbio-0020276-g001){ref-type="fig"}). A microbial infection initiates a zymogen cascade that plays a crucial role in the activation of the antimicrobial genes in the fat body. Infection or wounding also triggers a protein-cleaving cascade that results in the production of toxic intermediates and melanin around microbes or wound sites. This proteolytic cascade is similar to the vertebrate clotting cascade. Drosophila hemolymph also coagulates and participates in host defense and wound healing ([Figure 1](#pbio-0020276-g001){ref-type="fig"}; [@pbio-0020276-Theopold1]). Given the evolutionary success of insects, this combination of defense mechanisms has proven to be extremely effective, allowing insects to thrive in septic environments.
NF-κB Activation: The Toll and Imd Pathways of Drosophila {#s4}
=========================================================
The Drosophila genome encodes several members of the multifunctional Toll family of receptors ([@pbio-0020276-Beutler1]). Mutations in the Drosophila Toll gene (as well as in other components in the pathway) make the fly susceptible to fungal or gram-positive bacterial infections. However, Toll does not act as a pattern recognition receptor in the fly; instead its activation depends on the presence of the processed (active) form of the growth-factor-like polypeptide Spätzle. Processing of Spätzle depends on a serpin-controlled proteolytic cascade ([Figure 2](#pbio-0020276-g002){ref-type="fig"}).
::: {#pbio-0020276-g002 .fig}
Figure 2
::: {.caption}
###### Molecular Components of the Toll and Imd Pathways Involved in Drosophila Immunity
Toll is activated by the processed Spätzle (left). Toll activation leads to intracellular signaling via cytoplasmic proteins Tube and Pelle, leading to the degradation of Cactus and nuclear localization of NF-κB proteins Dorsal and Dif. These transcription factors bind to promoters of target genes, such as *drosomycin*, activating their transcription. The NF-κB protein for the Imd pathway, Relish, activates *diptericin* transcription. The signaling events resulting in Dorsal/Dif/Relish activation in the fly are "recycled" in mammals in the activation of mammalian NF-κB. See reviews and [@pbio-0020276-De1] for more details.
:::
:::
While components of the Drosophila Toll pathway were identified in earlier genetic screens for developmental mutants, those in the Imd pathway have been the focus of more recent studies, mainly in the context of Drosophila immunity ([@pbio-0020276-Hoffmann1]; [@pbio-0020276-Hultmark1]). The effector NF-κB transcription factor of the Imd pathway is Relish, which upon immune activation is cleaved by the Dredd caspase ([Figure 2](#pbio-0020276-g002){ref-type="fig"}). Using a combination of the RNA interference approach of silencing gene function and a high-throughput cell culture assay, [@pbio-0020276-Foley1] report the identification of two new conserved members of this Imd pathway: Sickie is a novel protein required for Relish activation, and Defense repressor 1 is a novel inhibitor of the Dredd caspase.
The impressive progress in our understanding of innate immunity in Drosophila is now guiding scientists to explore the immune system of other insects such as the mosquito, Anopheles gambiae, that spreads human malaria. Immune responses in this mosquito are linked to the elimination of the malarial parasites ([@pbio-0020276-Osta1]). A comparison of the immunity-related genes in Anopheles and Drosophila reveals the presence of the Toll signaling pathway in the mosquito genome, even though there are some differences in genes encoding pathogen recognition and signal transduction molecules ([@pbio-0020276-Christophides1]). A detailed and comparative view of the genetic mechanisms underlying their host defense will contribute to the identification of new targets for insecticide development, and provide opportunities for controlling the transmission of pathogens.
Concluding Remarks {#s5}
==================
The homologs of many genes involved in innate immune responses in flies and humans have also been found in mice, sharks, nematodes, and plants (e.g., [@pbio-0020276-Pujol1]; [@pbio-0020276-Nurnberger1]). In species studied to date, host defense appears to be mediated by homologous proteins. Taken together, these findings suggest that the regulatory mechanisms of host defense may be hard-wired in the genome much as DNA replication and cell division are. Protein motifs, domains, and signaling elements have, for millions of years, not only retained their ancestral biochemical features but have also continued to participate in similar physiological responses. It is crucial that our evolving knowledge of "genomic recycling" be used to enhance our understanding of the evolution of humans, not only in the context of "descendants of ancient apes," but in the larger context of our fundamental unity and shared genetic history with all other species. This simple but fundamental idea has yet to be adopted by the majority of our students and teachers. Unless we do more to overcome resistance to the idea that humans share deep evolutionary connections with all animal life, students will become increasingly isolated from an understanding of, and participation in, the genomics and bioinformatics revolution that is transforming the biological and biomedical sciences.
Shubha Govind and Ross H. Nehm are in the Department of Biology and The Graduate Center, City College of New York, New York, New York, United States of America. E-mail: <sgovind@ccny.cuny.edu> (SG)
PAMP
: pathogen-associated molecular pattern
| PubMed Central | 2024-06-05T03:55:46.793366 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509316/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e276",
"authors": [
{
"first": "Shubha",
"last": "Govind"
},
{
"first": "Ross H",
"last": "Nehm"
}
]
} |
PMC509317 | When gene therapy was introduced nearly fifteen years ago, it was widely hailed as a panacea. Since many diseases have a genetic component, the hope was that gene therapy could replace compromised genes with healthy versions to treat everything from inherited disorders like cystic fibrosis to cancer and HIV. That great promise was quashed when a teenager suffering from a rare hereditary liver disorder died after participating in an experimental gene therapy trial in 1999: four days after being injected with millions of viruses engineered to deliver healthy genes to his liver, Jesse Gelsinger died. It seems the virus, derived from an adenovirus, targeted his immune cells rather than his liver cells, which triggered an immune response against the virus, resulting in massive organ failure.
In another case, two young boys who received gene therapy for the severe immunodeficiency disorder known as "bubble boy disease" developed leukemia-like symptoms 30 months after treatment. In this case, the viral vector inserted itself near a promoter region---a site that initiates gene transcription---of a proto-oncogene, a gene that can initiate cancer. Since viral vectors can integrate at various genomic locations, the safety and effectiveness of gene therapy ultimately depends on being able to predict a virus\'s particular bias. Comparing retroviral vectors derived from three viruses, including two common gene therapy vectors, Rick Mitchell et al. report 3,127 sites where these viruses typically integrate into the human genome. The different vectors, they found, show different target preferences.
Retroviruses use viral enzymes to copy their own genome, which is stored in an RNA transcript, into DNA. Now recognizable by the host\'s genome, the virus can integrate into one of the host\'s chromosomes. In this study, Mitchell et al. studied vectors derived from the human immunodeficiency virus (HIV), avian sarcoma-leukosis virus (ASLV), and murine leukemia virus (MLV). Introducing the viral vectors into human cells, the authors analyzed the gene expression profiles of the cells to determine where vectors integrate into human chromosomes and which, if any, genes they activate. Mitchell et al. then compared the integration sites with the transcription profiles.
Each retrovirus, they discovered, showed distinct preferences for genome integration. HIV vectors tend to integrate into sites of active transcription, favoring chromosomal regions rich in expressed genes. MLV vectors tend to integrate near transcription initiation sites, confirming the results of a previous study, with a weak bias toward active genes. In contrast, the authors report, the ASLV vector "does not favor integration near transcription sites, nor does it strongly favor active genes."
Early efforts to understand how chromosomes may influence where viruses insinuate themselves into a chromosome focused on factors governing accessibility. Viruses are more likely to be integrated into chromosomal regions that are more accessible, which tend to be transcriptionally active sites. But since each of the three viruses studied here routinely targeted different sequences, the authors note, accessibility is probably just one factor. Specific chromosomal proteins, for example, might interact with the viral integration machinery and facilitate integration at nearby sites. Another possibility, the authors propose, is that DNA-binding proteins that bind to specific DNA sequences assist integration of one virus while impeding another.
This could explain why ASLV behaved as it did in the human cells studied here. The virus might have more refined integration preferences during normal infection of chicken cells, the authors note, but its integration machinery can\'t interact properly with human cells. The leukemia-like effects of the bubble boy gene therapy stemmed from integration of a mammalian retrovirus---the MLV vector---near an oncogene promoter region. Since ASLV tends to avoid both transcription initiation sites and active gene sites, it could be a more promising candidate for human gene therapy. With the draft chicken genome sequence now complete, researchers can investigate whether that proves true. But for now, Mitchell et al. make the case that scientists can gain more control over where viral vectors integrate into the human genome by selecting different retroviral integration systems. Only time will tell whether more control translates into safer gene therapy protocols.
| PubMed Central | 2024-06-05T03:55:46.794907 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509317/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e281",
"authors": []
} |
PMC509318 | Every multicellular organism depends on the coordinated actions of a multitude of cell types, each designed to carry out specific jobs. In vertebrates, for example, the task of ferrying oxygen to organs, tissues, and cells throughout the body is shouldered by red blood cells. These cells must develop early in the embryo to nourish the body and must be maintained at the right levels throughout adulthood to keep the organism healthy.
Specialized red blood cells arise from undifferentiated stem cells in a developmental process called hematopoiesis. All vertebrates have the same basic plan for hematopoiesis during their development, with one group of stem cells producing embryonic blood cells and, later, another group in a different part of the body making adult blood cells. Such developmental programs are tightly orchestrated by suites of transcription factors---proteins that turn genes on and off.
But which transcription factors act---and how and where they act---is still largely unknown. Scientists and doctors alike want to better understand differentiation in order to grasp not only how it proceeds normally but also how it can go wrong and lead to diseases, such as leukemia, in which white blood cells become cancerous, and aplastic anemia, in which bone marrow stem cells make too few red blood cells.
To discover the genes involved in hematopoiesis, Leonard Zon\'s group at Children\'s Hospital, Boston, and their collaborators exposed zebrafish to mutagens and then studied the individuals that developed relevant disorders. In 1996, the group produced three zebrafish lines with embryos lacking fully differentiated red blood cells; since these fish also had especially shimmery tails, the lines were named moonshine.
Now Zon and his colleagues have identified the mutant gene responsible for the moonshine zebrafish lines\' peculiar traits. The *moonshine (mon)* gene, it turns out, encodes a transcription factor with wide effects on the embryo\'s mesoderm, the set of cells that eventually form the circulatory system, muscles, and skeleton. The researchers found that all three lines of mutant zebrafish had mutations in the *mon* gene.
In the mutant fish, stem cells that produce red blood cells were present initially, but the defective moonshine protein was unable to keep the blood cells alive. These blood cells underwent apoptosis, or programmed cell death, leaving the fish unable to make red blood cells, and they died at about two weeks old.[](#pbio-0020282-g001){ref-type="fig"}
::: {#pbio-0020282-g001 .fig}
::: {.caption}
###### Hemoglobin staining shows that hematopoiesis is defective in a *moonshine* mutant (bottom) compared to a wild-type zebrafish embryo (top)
:::
:::
The researchers found that the protein encoded by the *mon* gene is most similar to the human and mouse Tif1-gamma, one of a family of proteins known to link DNAbinding proteins with other factors that activate or repress gene activity. Using a DNA analysis technique in mouse cell cultures, the researchers found Tif1-gamma in nuclear bodies, multi-protein complexes in cell nuclei that help regulate gene expression. But the Tif1-gamma nuclear bodies didn\'t fit into any known class of such complexes, so it\'s an open question how Tif1-gamma acts and what other proteins help regulate it. Finding a transcription factor such as Tif1-gamma involved in early cell specialization opens a door to a suite of studies on the targets of the transcription factor, as well as other genes that act along with *mon* to affect red blood cell development.
| PubMed Central | 2024-06-05T03:55:46.795466 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509318/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e282",
"authors": []
} |
PMC509319 | In the 2002 thriller *Memento*, the protagonist, Lenny, is plagued by a crippling neurological disorder that renders him incapable of storing memories for longer than fifteen minutes. He wants nothing more than to avenge the murder of his wife, but as another character in the film tells him, "Even if you get revenge, you\'re not going to remember it. You\'re not even going to know it happened." Lenny has anterograde amnesia, a condition typically caused by stroke or other illness. (In the film, it\'s caused by a blow to the head.) Most forms of amnesia---including the more common retrograde amnesia, which involves the loss of long-term memory---are caused by some type of brain injury--- particularly to the hippocampus.
Retrograde amnesia can arise from brain damage that interferes with memory storage, retrieval, or consolidation. And it is by studying retrograde amnesia that scientists have developed theories explaining where the brain stores memory traces and how it consolidates them into long-term memories. What ultimately causes amnesia---a failure to store memories or a failure to retrieve them---is not clear.
A major challenge in resolving this question experimentally is being able to determine whether an animal has truly recovered a lost memory or has simply re-learned the task at hand. By refining a classic behavioral neuroscience experiment to test spatial learning and memory, Livia de Hoz, Stephen Martin, and Richard Morris have developed a novel protocol that distinguishes retrieval from new learning. The experiment, developed by Morris over fifteen years ago, takes advantage of the fact that rats are good swimmers but prefer life out of water. Rats are placed in a pool and trained to remember the location of an escape platform, hidden just under the surface. (The rats can\'t see it because the water is cloudy.)
Here, de Hoz et al. trained rats to a particular platform location---their escape route---then gave them hippocampal lesions. One group received "sham" lesions, another partial lesions, and a third complete lesions. Partial lesions, the authors explain, should damage only a subset of the stored memory traces and thus weaken the rats\' memory rather than completely disrupting it.
The rats\' postoperative memory was tested by first placing them in the pool with the platform hidden: as expected, the lesioned rats couldn\'t remember where it was. After a minute, the platform was raised above the water, to remind them that it existed and that they could escape by climbing onto it. The platform was revealed in the original location for half the animals and in a novel location---which could be construed as misleading information if you\'re a rat---for the other half. If raising the platform was facilitating re-learning, then animals capable of learning would look for the platform in the new location. But if raising the platform functioned as a reminder, then animals should gravitate toward the place they were trained to, regardless of whether the platform reminder was in the original location or a new one. And that\'s what happened to the rats with partial lesions.
De Hoz et al. then repeated the experiments in a new environment. If the rats managed to escape in this situation, the authors explain, then the reminder treatment could be causing new learning rather than triggering recall of the original training experience. Not surprisingly, the rats with total lesions failed to learn in this new environment (or to be reminded in the previous experiment), while the control rats adapted to the new location. The group with partial lesions failed to learn.
Since partially lesioned rats responded to the reminder treatment by recalling the original platform location, their initial memory failure couldn\'t have resulted from a storage failure. And since they did not realize they could escape from the new location, the reminder didn\'t induce new learning. Among the many questions these results raise is what role the hippocampus plays in memory storage and retrieval. It could be, as the authors propose, that essential components of spatial memory traces are either stored in the hippocampus or reactivated there, since only the partially lesioned rats responded to reminders. Whether that proves true, de Hoz et al. have contributed a much needed resource for investigating the neural basis of memory loss.[](#pbio-0020283-g001){ref-type="fig"}
::: {#pbio-0020283-g001 .fig}
::: {.caption}
###### Rats given partial hippocampal lesions after training in a watermaze are initially unable to remember the location of the escape platform
:::
:::
| PubMed Central | 2024-06-05T03:55:46.795965 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509319/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e283",
"authors": []
} |
PMC509320 | The essential difference between cancer cells and normal cells is that cancer cells evolve. Most cancers arise from a single cell through a sequential evolutionary process of mutation and selection. Cancer cells harbor mutations in a number of critical genes that, at various stages during the evolution of the tumor, provide those cells with a selective advantage. Many of the phenotypes, or physical outcomes, conferred by these mutant genes are subverted from a normal cell\'s repertoire, including proliferation, invasion, migration, loss of differentiation, and loss of apoptosis (programmed cell death); other phenotypes, such as immortalization, are novel. Tumor evolution is thought to adhere to Darwinian principles, with mutations arising randomly within an individual cell, followed by selection for mutant clones with favorable traits.
Support for this idea stems from the observation that end-stage tumors have mutations in a number of genes. But linking mutations in particular genes with defined stages is difficult for most human cancers and especially so for the last and deadliest stage: when cancer disseminates throughout the body during metastasis. It\'s unclear, for example, whether there are mutations in particular genes or sets of genes that enhance metastasis. That is, do metastatic lesions develop through a continuation of Darwinian evolution, or is metastasis an intrinsic property of the primary tumor, meaning that further genetic evolution is not required? It is also unclear whether there is a "preferred" sequence of mutations, such that selective pressure for particular mutations depends on preexisting mutations.[](#pbio-0020284-g001){ref-type="fig"}
::: {#pbio-0020284-g001 .fig}
::: {.caption}
###### Squamous cell carcinoma invading muscle layer from *ARF*-null mouse
:::
:::
Since the earliest days of research on oncogenes---genes that can cause a cell to become cancerous---it has been known that certain oncogenic mutations cooperate to transform normal cells into cancer cells. For example, an "activating" mutation in the oncogene *Ras* and the loss of the tumor suppressor *p53* cooperate to transform cells.
The paper by Christopher Kemp and his colleagues at the Fred Hutchinson Cancer Research Center sheds light on some of these questions, and many of the issues center around the most notorious oncogene: *Ras*. Using a well characterized mouse model of squamous cell carcinogenesis, which generates a form of skin cancer, the authors examine both the functional and evolutionary relationships between three cancer genes that play major roles in most human cancers: *Ras* and the tumor suppressors *Arf* and *p53*.
Two seminal early observations set the stage: mutational activation of *Ras* is the initiating genetic event in this cancer model, while mutation of *p53* occurs later, during the benign to malignant transition; and expression of mutant *Ras* in cells activates *p53* via signaling through the protein encoded by *Arf*.
Kemp et al. confirm that this pathway is active in "autochthonous" tumors---which grow and develop where they are initiated---by showing that *p53* expression in tumors with *Ras* mutations is dependent on the presence of *Arf*. Thus, during the early benign stages of tumor growth, *Ras* activates *Arf*, which in turn activates *p53*, thereby inhibiting tumor progression. This provides strong selective pressure in favor of cells with mutations in either *Arf* or *p53*, and these mutations are indeed observed as the tumors progress to malignancy. That *Arf* and *p53* function as tumor suppressors was confirmed by demonstrated accelerated tumor progression in mice lacking either *Arf* or *p53*. This answers a longstanding question concerning the nature of the signal that activates *p53* during autochthonous tumor development: Mutation of *Ras* not only initiates tumor development but, through its intracellular signaling through *Arf* and *p53*, directly influences the subsequent evolutionary trajectory of the tumors. In this view, secondary evolutionary events are determined by the preexisting genetic lesion, as a result of direct signaling interactions.
The authors go on to show that tumors lacking *Arf* or *p53* show accelerated metastatic dissemination, a phenomenon rarely seen in mouse squamous cell cancer models. Thus both benign and malignant tumors lacking these tumor suppressors are at high risk for metastasis. As *Ras* is well known to confer many phenotypes required for the metastatic process, it appears that *Ras*, together with loss of its inhibitors, *Arf* and *p53*, may be sufficient to drive this process. More direct evidence that metastasis does or does not require further genetic evolution awaits.
| PubMed Central | 2024-06-05T03:55:46.796605 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509320/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e284",
"authors": []
} |
PMC509321 | DNA mutates, and it\'s a good thing it does. If it didn\'t, there could only be one kind of life, not the millions there are today, and species could not adapt to new challenges. This is because mutations in genes---the coding portion of DNA---are the raw material for evolution. However, genes make up a surprisingly small fraction of our DNA. If the genome were a cookbook, its 30,000-odd genetic recipes would be scattered among millions of pages of apparently meaningless nonsense.
Mutations affect all DNA, not just the genes, and this provides population geneticists with a veritable toolbox of methods useful, for example, in DNA profiling. Importantly, all these methods rely on the idea of a "molecular clock," the notion that mutations rain down on noncoding DNA like a fine drizzle, so constantly that genetic similarity is a good measure of evolutionary time. Thus, if orangutans diverged from humans twice as long ago as did chimpanzees, on any given piece of DNA we would find twice as many differences between the orangutan sequence and the human sequence as between humans and chimps. The mutations are marking time.
If the molecular clock works, scientists can do wonderful things like estimating how long ago it was that the common ancestor of all humans lived, or when birds evolved from dinosaurs. The clock assumes that mutations occur independently of each other and at a constant rate. By analyzing thousands of noncoding DNA sequences scattered throughout the human genome, Edward Vowles and William Amos have found that the clock is anything but constant. Instead, a mutation in one spot in the genome affects the chance of getting another mutation nearby.[](#pbio-0020287-g001){ref-type="fig"}
::: {#pbio-0020287-g001 .fig}
::: {.caption}
###### Non-random base frequencies around microsatellites
:::
:::
Not all noncoding DNA is made up of benign tracts of random letters. Some sequences appear to be more difficult to copy than others, and these trouble spots can give rise to alphabetic stuttering. DNA is made up of four component chemical units, called nucleotides, which are often referred to by their initial letters: A, C, G, and T. Stuttering occurs when the same pairs or triplets of letters occur together, for example ACACAC. Such regions are called microsatellites, and instead of mutating by swapping one letter for another, as most nucleotides do, these sequences evolve mainly by gaining and losing triplets or pairs like "AC."
In this study, Vowles and Amos used the published sequence of the human genome to track down and compare thousands upon thousands of microsatellites. If the molecular clock ran smoothly, they would expect to find no similarity at all between the DNA sequences surrounding any pair of unrelated microsatellites. To their surprise, they found the complete reverse, with entirely unrelated microsatellites showing widespread and obvious similarities in their flanking DNA. This meant that mutations near microsatellites were not random, but favored certain letters in certain positions. Just as a new shipwreck will attract its own special community of marine life, so microsatellites appear gradually to change the surrounding DNA towards a common pattern. The result is convergent evolution, an unusual state of affairs where, as time goes by, DNA sequences become more similar, not less.
As yet, the exact mechanisms remain unclear, though it probably has something to do with how comfortably different combinations of letters sit next to each other. In English, "U" always follows "Q" and "B" never follows "V." Similar rules may apply to DNA, albeit on a much subtler level. For example, if a microsatellite contains alternating As and Cs, the flanking regions also tend to have As at alternate positions, in phase with the As in the microsatellite. It is as if the DNA prefers the pattern in the microsatellite to extend into the flanking DNA, rather than abruptly stopping at the end of the microsatellite.
These findings suggest that it may be wise to take the notion of a molecular clock at face value. With a perfect clock, two or three identical mutations would be highly unlikely, but we now know that this may be possible near microsatellites. Vowles and Amos estimate that as much as 30% of the genome may show evidence of convergent evolution, simply because microsatellites are so common. These mutation biases probably exist to a lesser extent in most sequences. Once scientists understand more fully how and where these biases operate, they may be able to estimate more accurately the risk of any given mutation occurring, be it one that causes human disease or makes a virus more virulent. These findings represent yet another windfall from the Human Genome Project, and act as a powerful reminder that unexpected results always lurk around the corner as we delve deeper into the secret world of the genome.
| PubMed Central | 2024-06-05T03:55:46.797516 | 2004-8-17 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509321/",
"journal": "PLoS Biol. 2004 Aug 17; 2(8):e287",
"authors": []
} |
PMC509406 | Introduction {#s1}
============
Emery-Dreifuss muscular dystrophy (EDMD) is inherited through mutations in either of two different genes: *LMNA,* encoding A-type lamins, and *STA,* which encodes a nuclear membrane protein named emerin ([@pbio-0020231-Nagano1]; [@pbio-0020231-Emery1]; [@pbio-0020231-Bengtsson1]). Lamin filaments and emerin interact at the nuclear inner membrane ([@pbio-0020231-Burke1]; [@pbio-0020231-Holaska1]). Together, emerin and lamin A form stable tertiary complexes with other binding partners in vitro ([@pbio-0020231-Holaska2]), suggesting that emerin and lamins together provide a structural foundation for oligomeric protein complexes. Mutations in emerin cause the X-linked recessive form of EDMD ([@pbio-0020231-Bione1]; [@pbio-0020231-Bonne1]). Both emerin and lamin A are expressed in most cells, but EDMD disease strikes specific tissues: skeletal muscles, major tendons, and the cardiac conduction system. To explain the tissue specificity of EDMD, it was proposed that emerin might have tissue-specific binding partners such as transcription factors and signaling molecules that regulate gene expression ([@pbio-0020231-Wilson1]; [@pbio-0020231-Bonne1]; [@pbio-0020231-Ostlund1]). There is growing evidence to support "gene expression" models for emerin, as discussed further below. However, a second model, not mutually exclusive, proposes that emerin helps maintain the structural integrity of the nuclear envelope. According to structural models, loss of emerin selectively disrupts tissues under high mechanical stress, such as skeletal muscle and tendons ([@pbio-0020231-Bonne1]; [@pbio-0020231-Ostlund1]). Although this model fails to explain the cardiac conduction phenotype of EDMD, it is consistent with structural defects (aberrant shape and nuclear envelope herniations) seen in nuclei from EDMD patients ([@pbio-0020231-Fidzianska1]) and in a subset of patients with other diseases linked to mutations in *LMNA* ("laminopathies"; [@pbio-0020231-Holaska1]; [@pbio-0020231-Ostlund1]). Whereas structural and mechanical roles are expected for lamins, which form nuclear intermediate filaments, mechanical roles for emerin have not been investigated.
Emerin is detected in most human cells tested, except the nonmyocytes of the heart ([@pbio-0020231-Manilal1]). In Caenorhabditis elegans emerin is expressed ubiquitously ([@pbio-0020231-Lee2]; [@pbio-0020231-Gruenbaum1]). Emerin belongs to the LEM-domain family of proteins, which are defined by an approximately 40-residue folded domain known as the LEM domain. In vertebrates, other family members include LAP2β and MAN1 at the nuclear inner membrane ([@pbio-0020231-Dechat1]; [@pbio-0020231-Lin1]; [@pbio-0020231-Cohen1]), LAP2α in the nuclear interior ([@pbio-0020231-Dechat1]), and at least three additional uncharacterized LEM-domain proteins ([@pbio-0020231-Lee1]). A major shared function of all characterized LEM-domain proteins is their binding (via the LEM domain) to a small protein named barrier-to-autointegration factor (BAF; reviewed by [@pbio-0020231-Segura-Totten1]). BAF is a highly conserved chromatin protein essential for cell viability ([@pbio-0020231-Zheng1]), with direct roles in higher order chromatin structure and nuclear assembly ([@pbio-0020231-Haraguchi1]; [@pbio-0020231-Segura-Totten2]), and gene regulation ([@pbio-0020231-Wang1]; [@pbio-0020231-Holaska2]).
Supporting gene regulation models for EDMD, emerin binds directly to BAF and two other transcription repressors, germ cell-less (GCL; [@pbio-0020231-Holaska2]) and Btf ([@pbio-0020231-Haraguchi2]) as well as an mRNA-splicing factor named YT521-B ([@pbio-0020231-Wilkinson1]). Interestingly, GCL and LAP2β, a LEM-domain protein closely related to emerin, comediate transcription repression in vivo ([@pbio-0020231-Nili1]). On the other hand, emerin also has a growing number of structural or anchoring partners, including a spectrin-repeat (SR) membrane protein named nesprin-1α ([@pbio-0020231-Mislow1], [@pbio-0020231-Mislow2]), lamins A and C ([@pbio-0020231-Clements1]; [@pbio-0020231-Lee3]; [@pbio-0020231-Sakaki1]), and lamin B ([@pbio-0020231-Dreger1]). Lamins form type-V intermediate filaments that are critical for the integrity of the nucleus and confer unique elasticity and incompressibility properties to the nuclear envelope ([@pbio-0020231-Dahl1]). Nuclear morphology and lamina architecture are disrupted in a fraction of cells that express disease-causing missense mutations in A-type lamins ([@pbio-0020231-Ostlund1]). However, the line between gene expression and mechanical models for disease has become blurred. For example, the subnuclear localization of Rb, a transcription and cell-fate regulator, depends on both LAP2α and lamin A, since Rb is mislocalized in cells that either lack A-type lamins ([@pbio-0020231-Johnson1]) or express disease-causing lamin-A mutations ([@pbio-0020231-Markiewicz1]). A second putative anchoring partner for emerin is nesprin-1α, an integral nuclear inner-membrane protein with seven SR domains ([@pbio-0020231-Zhang1]; [@pbio-0020231-Mislow2]). Each SR domain consists of approximately 100 residues and folds into a tightly packed triple α-helical structure ([@pbio-0020231-Bennett1]). Tandem SR domains, as seen in nesprin-1α, form a rigid, elongated tertiary structure ([@pbio-0020231-Djinovic-Carugo1]). More important, SR domains provide binding sites for other proteins, the specificity of which is determined by exposed residues ([@pbio-0020231-Bennett1]). SR domains 1--7 (and particularly domains 1--5) of nesprin-1α mediate high-affinity binding to emerin ([@pbio-0020231-Mislow1]). Interestingly, SR domains 5--7 of nesprin-1α bind directly to lamin A in vitro, suggesting that nesprin-1α, lamin A, and emerin might form stable tertiary complexes. Such complexes have the potential to stabilize lamin filaments at the nuclear envelope, in addition to anchoring and spacing emerin.
Notably, both emerin and lamin A also bind G-actin in vitro ([@pbio-0020231-Sasseville1]; [@pbio-0020231-Fairley1]). Actin binds two regions in the lamin-A tail ([@pbio-0020231-Zastrow1]). Both α- and β-actin bind emerin in vitro, and emerin coimmunoprecipitates with actin from cell lysates ([@pbio-0020231-Fairley1]; [@pbio-0020231-Lattanzi1]). The significance of these findings was unclear, in part because nuclear actin has been both documented and debated for over 35 y ([@pbio-0020231-Pederson1]). However there is a growing consensus that nuclear actin is no artifact ([@pbio-0020231-Pederson1]; [@pbio-0020231-Bettinger1]). Both α- and β-actin have been shown, definitively, to reside in the nucleus ([@pbio-0020231-Scheer1]; [@pbio-0020231-Gonsior1]; [@pbio-0020231-Olave1]; [@pbio-0020231-Lattanzi1]) and to form short filaments in the nucleus ([@pbio-0020231-Clark1]). Actin and actin-related proteins (Arps) are required for chromatin remodeling and transcription ([@pbio-0020231-Olave1]; [@pbio-0020231-Percipalle1]). Also interesting is that polymerase II--dependent mRNA transcription requires a nuclear-specific myosin I motor (nuclear myosin I; [@pbio-0020231-Nowak1]; [@pbio-0020231-Pestic-Dragovich1]). Thus, actin probably has a variety of roles in the nucleus.
To test the hypothesis that emerin forms multiprotein complexes in vivo, we affinity-purified emerin-binding proteins from nuclear extracts of HeLa cells. We identified actin itself plus several actin-binding proteins as bona fide emerin-associated proteins, and we further discovered that emerin stimulates actin polymerization in vitro by binding and stabilizing the pointed end of growing filaments. These results suggest that emerin contributes to the formation of an actin cortical network at the nuclear inner membrane.
Results {#s2}
=======
We used affinity chromatography to purify emerin-binding complexes from HeLa nuclear extract; as the negative control, beads were conjugated to bovine serum albumin (BSA). Mass spectrometry (data not shown) and Western blotting identified β-actin as a major emerin-binding protein ([Figure 1](#pbio-0020231-g001){ref-type="fig"}A). Six other proteins, including nuclear-enriched αII-spectrin, were also identified and will be reported in full elsewhere (J. M. H. and K. L. W., unpublished data). Consistent with previous reports ([@pbio-0020231-Fairley1]; [@pbio-0020231-Lattanzi1]), antibodies specific for emerin coprecipitated actin from HeLa cell nuclear lysates, as shown by Western blotting with immune serum ([Figure 1](#pbio-0020231-g001){ref-type="fig"}B, Im). Only background levels of actin were precipitated by preimmune sera ([Figure 1](#pbio-0020231-g001){ref-type="fig"}B, PI). These findings led us to hypothesize that emerin might bind filamentous actin (F-actin).
::: {#pbio-0020231-g001 .fig}
Figure 1
::: {.caption}
###### Affinity Purification of Emerin-Associated Proteins
\(A) Immunoblot of HeLa nuclear lysate proteins (L), or proteins affinity-purified using either BSA beads or emerin beads (see [Materials and Methods](#s4){ref-type="sec"}), probed with antibody against actin.
\(B) HeLa nuclear lysate proteins (L) were immunoprecipitated using either immune (Im) or preimmune (PI) serum 2999 against emerin, resolved by SDS-PAGE, and Western blotted using antibodies specific for actin (upper panel) or emerin (lower panel), in succession.
\(C) Cosedimentation assays using F-actin and purified, recombinant wild-type emerin (residues 1--222). G-actin (2 μM) was polymerized and then incubated in the absence or presence of 2 μM emerin. Emerin was incubated alone in polymerization buffer as a negative control. After 30 min samples were centrifuged 1 h at 100,000*g,* resolved by SDS-PAGE, and stained with Coomassie blue. L, load (100%); S, supernatant (100%); P, pellet (100%).
\(D) F-actin column was used to determine the affinity of F-actin for emerin. The K~d~ was 480 nM for the experiment shown; range was 300--500 nM, *n* = 8.
\(E) Binding of wild-type (WT) or mutant emerin protein to F-actin beads. Recombinant emerin proteins were incubated with F-actin beads, and bound emerins were eluted with SDS-PAGE sample buffer, resolved by SDS-PAGE, blotted, and probed with antibodies against emerin ("bound"; all emerin mutants are recognized by this antibody; [@pbio-0020231-Lee3]; [@pbio-0020231-Holaska2]). The input amounts (10%) of each emerin mutant ("load") were visualized either by immunoblotting (top row, top panel) or Coomassie staining (top row, bottom panel).
\(F) Domains in emerin required for binding to BAF, lamin A, transcription repressor GCL, or actin ([@pbio-0020231-Lee3]; [@pbio-0020231-Holaska2]; present study). Asterisks indicate EDMD disease-causing mutations.
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:::
Emerin Binds F-Actin with High Affinity {#s2a}
---------------------------------------
Emerin was first tested for binding to F-actin in a cosedimentation assay. Actin filaments were incubated 30 min in the presence or absence of recombinant emerin (4 μM) and then pelleted at 100,000*g.* Approximately 75% of input emerin pelleted in the presence of F-actin, compared to 15% in the absence of F-actin ([Figure 1](#pbio-0020231-g001){ref-type="fig"}C), demonstrating that emerin binds polymerized actin in vitro. The stoichiometry of this interaction was one emerin molecule per approximately 300 actin monomers, demonstrating that emerin binds actin filaments with an average length of 0.9 μm (data not shown). Emerin binds F-actin with high affinity, as determined by binding to an F-actin column (K~d~ = 480 nM, range = 300--500 nM, *n* = 8; [Figure 1](#pbio-0020231-g001){ref-type="fig"}D). The F-actin columns were also used to screen selected emerin mutants for binding to F-actin. Wild-type emerin and EDMD disease-causing mutants S54F and P183H, and alanine substitution mutant m196 (Ser^196^Ser^197^ to Ala^196^Ala^197^; [@pbio-0020231-Holaska2]), bound efficiently to an F-actin column, whereas 12 other tested mutants, including EDMD disease-causing mutant Q133H, showed no significant binding to F-actin ([Figure 1](#pbio-0020231-g001){ref-type="fig"}E). Similar results were seen in coimmunoprecipitation assays using antibodies against actin to immunoprecipitate F-actin in the presence of recombinant wild-type or mutant emerin proteins (data not shown). Fifteen additional emerin missense mutants, including LEM-domain mutants, were tested in blot overlay assays; mutants m11, m24, m30, m40, m207, m214, and m217 bound detectably to actin, whereas no significant binding was detected for mutants m45A, m45E, m61, m141, m145, m161, and m206 (data not shown; see [@pbio-0020231-Holaska2] for details of mutations). Thus, three independent assays all showed that emerin binds F-actin. Furthermore, based on the positions of missense mutations that blocked binding to F-actin, this recognition involved almost the entire nucleoplasmic domain of emerin, with the notable exception of the LEM-domain ([Figure 1](#pbio-0020231-g001){ref-type="fig"}F). The putative actin-binding domain in emerin overlaps with both the lamin- and repressor-binding (GCL) domains described previously ([@pbio-0020231-Holaska2]).
Emerin Regulates Actin Polymerization {#s2b}
-------------------------------------
The above results suggested that in vivo emerin might (a) use actin filaments as anchors, (b) stabilize F-actin networks, or (c) actively influence actin dynamics. To test these models, we first used reactions containing 5% pyrene-labeled actin (final actin concentration, 2 μM) to determine if emerin influenced actin polymerization in vitro. Results were graphed as the rate of emerin-induced polymerization (R) divided by the control rate (cR; rate of actin polymerization in the absence of emerin). At concentrations ranging from 0.1 to 4.4 μM, emerin increased the rate of pyrene--actin polymerization 4-fold (mean = 6.2 ± 2.2, *n* = 32; one experiment is shown in [Figure 2](#pbio-0020231-g002){ref-type="fig"}A). These experiments also yielded an equilibrium affinity of 420 nM ([Figure 2](#pbio-0020231-g002){ref-type="fig"}A), consistent with our previous results (480 nM; see [Figure 1](#pbio-0020231-g001){ref-type="fig"}D).
::: {#pbio-0020231-g002 .fig}
Figure 2
::: {.caption}
###### Emerin Stimulates Actin Polymerization
\(A) Graph of a representative experiment (*n* = 32) showing that emerin increases the rate of actin polymerization 4-fold. R, rate of polymerization in the presence of emerin; cR, control rate of polymerization (actin alone). These data also yielded an equilibrium binding affinity of emerin for actin of 420 nM.
\(B) Representative graph (*n* = 17) in which each recombinant emerin mutant protein (1.0 μM) was added to 2.0 μM G-actin, and polymerization rates were calculated. R, rate of polymerization in the presence of emerin; cR, control rate of polymerization (actin alone). Stars indicate EDMD disease-causing mutations.
\(C) Critical concentration assays were performed in the presence or absence of 625 nM emerin. Actin was polymerized in the absence (barbed-end growth) or presence of 5 nM gelsolin--actin seeds (pointed-end growth) for 16 h at room temperature. Barbed-end growth with (□) or without (▪) emerin. Pointed-end growth with (○) or without (•) emerin. Actin monomers (Δ).
:::
:::
We next tested eight emerin mutants (1 μM) in the pyrene--actin polymerization assay ([Figure 2](#pbio-0020231-g002){ref-type="fig"}B). Five mutants (Q133H, m151, m164, m192, and m198) that failed to bind actin in vitro did not stimulate actin polymerization; instead, they reduced the rate of actin polymerization slightly, by 5%--40% ([Figure 2](#pbio-0020231-g002){ref-type="fig"}B). Mutant 196, which had wild-type binding to F- and G-actin in coimmunoprecipitation assays, stimulated actin polymerization approximately 50% as well as wild-type emerin ([Figure 2](#pbio-0020231-g002){ref-type="fig"}B). The two disease-causing mutants with apparently normal binding to F-actin, S54F and P183H, enhanced the rate of actin polymerization at least as well as wild-type emerin ([Figure 2](#pbio-0020231-g002){ref-type="fig"}B). Critical concentration assays were done to determine if emerin acted on the barbed or pointed end of growing actin filaments. Pointed-end growth was examined by capping filaments with gelsolin. Emerin had no significant effect on the critical concentration of barbed-end growth ([Figure 2](#pbio-0020231-g002){ref-type="fig"}C, + emr/no emr), but it increased the critical concentration for pointed-end growth by 2.3- to 2.7-fold ([Figure 2](#pbio-0020231-g002){ref-type="fig"}C, + gelsolin no emr/+ gelsolin + emr). We therefore hypothesized that emerin, like tropomodulin ([@pbio-0020231-Fowler2]), might stabilize growing filaments by capping the pointed end.
Emerin Binds F-Actin at the Pointed End {#s2c}
---------------------------------------
The vast majority of actin-binding proteins that influence subunit addition do so by binding the barbed end ([@pbio-0020231-dos1]). However, because emerin failed to influence the critical concentration for barbed-end growth ([Figure 2](#pbio-0020231-g002){ref-type="fig"}C), we used gelsolin--actin seeds to test the hypothesis that emerin binds the pointed end. Gelsolin binds and caps the barbed end of actin filaments ([@pbio-0020231-Burtnick1]), thereby restricting subunit addition to the pointed end only. We measured the extension of gelsolin--actin dimers (10 nM) in the presence of 2 μM actin plus 0 to 2 μM wild-type emerin ([Figure 3](#pbio-0020231-g003){ref-type="fig"}A). Emerin blocked actin polymerization in a concentration-dependent manner, supporting our model that emerin binds and caps the pointed end of actin filaments. Based on this assay, the affinity (K~d~) of emerin for F-actin was 430 nM (range, 300--500 nM, *n* = 12; [Figure 3](#pbio-0020231-g003){ref-type="fig"}A). Interestingly, the affinity determined by either direct binding (see [Figure 1](#pbio-0020231-g001){ref-type="fig"}D) or activity measurements (see [Figures 2](#pbio-0020231-g002){ref-type="fig"}A and [3](#pbio-0020231-g003){ref-type="fig"}A) differed by a maximum of 2-fold. We conclude that emerin binds F-actin with an affinity of 300--500 nM.
::: {#pbio-0020231-g003 .fig}
Figure 3
::: {.caption}
###### Emerin Binds the Pointed End of Actin Filaments
\(A) Gelsolin--actin seeds were incubated with increasing concentrations of wild-type emerin residues 1--222. Emerin significantly reduced the rate of subunit addition at the pointed end, with an apparent K~d~ of 430 nM (range, 300--500 nM, *n* = 12). R, rate of polymerization in the presence of emerin; cR, control rate of polymerization (actin alone).
\(B) Emerin inhibits depolymerization of actin filaments (2 μM) preformed from gelsolin--actin seeds, with an apparent K~d~ of 380 nM (range, 350--450 nM, *n* = 6). R, rate of depolymerization in the presence of emerin; cR, control rate of depolymerization (actin alone).
\(C) Rhodamine--phalloidin-stabilized actin filaments were formed from 2 μM actin, then capped at the barbed end by the addition of 100 nM capping protein, and finally diluted 2-fold in the presence of buffer or 1 μM emerin, GST, or tropomodulin (Tmod). Samples were then incubated with actin (3.2 μM) and Alexa-488 phalloidin (3.2 μM) for 2 min, diluted 1:500, placed on polylysine-coated coverslips, and viewed by fluorescence microscopy. Bar is 1 μm and applies to all panels.
\(D) Actin (2 μM) was incubated with gelsolin--actin seeds (500 nM) in the presence of rhodamine--phalloidin (2 μM). These red filaments were diluted 10-fold and incubated with buffer alone or with 1 μM emerin or tropomodulin (Tmod) for 10 min, followed by incubation with actin (2 μM) and Alexa-488-labeled (green) phalloidin (2 μM) for 2 min. Samples were diluted 1:500, placed on polylysine-coated coverslips, and viewed by fluorescence microscopy. Bar is 1 μm and applies to all panels.
(E--H) Alexa-488-labeled emerin (green) was incubated 30 min with actin filaments stabilized by Alexa-546 phalloidin (red) and centrifuged at 100,000*g* to recover filaments, which were diluted 1:500 for viewing.
:::
:::
To independently confirm that emerin binds the pointed end, we tested the effect of emerin on actin depolymerization in two separate assays. First, gelsolin--actin seeds were incubated with 2 μM actin and grown in the absence of emerin. The resulting filaments were then diluted to 0.2 μM actin in the presence of increasing concentrations of emerin (0--2 μM), and assayed immediately. Preformed actin filaments depolymerized rapidly in the absence of emerin ([Figure 3](#pbio-0020231-g003){ref-type="fig"}B, arrow), as expected. Supporting our model, depolymerization was slowed up to 8-fold by emerin ([Figure 3](#pbio-0020231-g003){ref-type="fig"}B). To independently confirm that emerin blocked subunit addition at the pointed end, preformed actin filaments were capped at the barbed end with capping protein (CapZ), a high-affinity barbed-end binding protein ([@pbio-0020231-Cooper1]), then incubated in the presence or absence of emerin, and diluted into 0.2 μM actin; emerin slowed the rate of depolymerization by 10-fold (data not shown). Based on these three assays, we conclude that emerin binds and protects the pointed end of actin filaments in vitro, thereby stabilizing actin filaments.
Fluorescent actin polymerization assays were done to demonstrate visually that emerin blocks pointed-end growth of single actin filaments. Rhodamine--phalloidin-stabilized actin filaments (red) were preformed from 2 μM actin, then capped on the barbed end with capping protein, and diluted 2-fold into a final concentration of 1 μM emerin. Pointed-end growth was then initiated by increasing actin to 3.2 μM in the presence of 3.2 μM Alexa-488 phalloidin (green) for 2 min. In the absence of emerin ([Figure 3](#pbio-0020231-g003){ref-type="fig"}C, buffer or GST), actin filaments containing both red and green segments are seen ([Figure 3](#pbio-0020231-g003){ref-type="fig"}C), demonstrating pointed-end growth. The average lengths of the growing filament segments (green) as measured for buffer and GST were 1.45 ± 0.3 μm and 1.47 ± 0.3 μm, respectively (*n* = 30). However, in the presence of either emerin or tropomodulin, a pointed-end binding protein, most red filaments lacked green segments ([Figure 3](#pbio-0020231-g003){ref-type="fig"}C), consistent with capped pointed ends. In the presence of emerin or tropomodulin, the lengths of the green segments were 0.05 ± 0.09 μm (*n* = 60) and 0.09 ± 0.13 μm (*n* = 50), respectively. Single filament assays were also done using small red filaments formed from gelsolin--actin seeds ([Figure 3](#pbio-0020231-g003){ref-type="fig"}D). Here, actin (2 μM) was incubated with gelsolin--actin seeds (500 nM) and rhodamine--phalloidin (2 μM). The resulting red filaments were diluted 10-fold and incubated with 1 μM emerin for 10 min, then incubated 2 min with actin (2 μM) and Alexa-488-labeled (green) phalloidin (2 μM). In the absence of emerin, single filaments contained both short red (gelsolin--actin seeds) and longer green (pointed-end growth) segments ([Figure 3](#pbio-0020231-g003){ref-type="fig"}D). The average length of these growing filament segments was 2.1 ± 0.6 μm (*n* = 40). However, when emerin was present, the preformed filaments remained predominantly short and red, demonstrating that emerin blocks pointed-end growth ([Figure 3](#pbio-0020231-g003){ref-type="fig"}D). Similar results were obtained in control reactions containing tropomodulin, the pointed-end binding protein ([Figure 3](#pbio-0020231-g003){ref-type="fig"}D, Tmod). The average lengths of growing filaments incubated with emerin or tropomodulin were 0.1 ± 0.1 μm (*n* = 40) and 0.1 ± 0.11 μm (*n* = 40), respectively. These experiments also show that emerin does not stimulate branching ([Figure 3](#pbio-0020231-g003){ref-type="fig"}D). To directly visualize emerin bound to actin filaments, green (Alexa-488-labeled) emerin was incubated with red (Alexa-546 phalloidin) actin filaments ([Figure 3](#pbio-0020231-g003){ref-type="fig"}E--[3](#pbio-0020231-g003){ref-type="fig"}H). Under these conditions 85% of labeled emerin molecules were bound to actin filaments; of these, 92% were localized at a filament end (*n* = 306). Interestingly, 10% of actin-associated emerin proteins localized to "aster-like" structures ([Figure 3](#pbio-0020231-g003){ref-type="fig"}G and [3](#pbio-0020231-g003){ref-type="fig"}H), presumably due to the aggregation of emerin proteins on different actin filaments.
Discussion {#s3}
==========
This work shows for the first time that a nuclear membrane protein, emerin, is a pointed-end F-actin-binding protein. Similar to the activity of tropomodulin ([@pbio-0020231-Fowler1]; [@pbio-0020231-Cooper1]; [@pbio-0020231-Fowler2]), emerin caps the pointed end, thereby stabilizing the growing filament. Only three other pointed-end binding proteins have been reported: the Arp2/3 complex ([@pbio-0020231-Mullins1]), tropomodulin, and mSWI/SNF, a component of a nuclear complex that remodels chromatin structure ([@pbio-0020231-Rando1]). The Arp2/3 complex initiates filament branching at the cell surface ([@pbio-0020231-Mullins1]; [@pbio-0020231-Mullins2]). We have no evidence that emerin initiates branching. Instead, emerin behaves most like tropomodulin, which binds the pointed end of F-actin with high affinity (K~d~ = 110 nM) and stimulates actin polymerization by stabilizing the actin filament ([@pbio-0020231-Fowler2]).
Our analysis of 15 emerin missense mutants suggested that the actin-binding region in emerin overlaps with regions required for binding to lamin A ([@pbio-0020231-Lee3]), transcription factors GCL and YT521-B ([@pbio-0020231-Holaska2]; [@pbio-0020231-Wilkinson1]), and nesprin-1α (J. M. H. and K. L. W., unpublished data). However this overlap does not necessarily imply that actin competes with these other proteins. Indeed, despite similar overlap, GCL and lamin A can form stable ternary complexes with emerin in vitro ([@pbio-0020231-Holaska2]). Further work is needed to determine if F-actin cobinds or competes with lamin A, nesprin-1α, or other emerin-binding proteins.
A Proposed Actin Network at the Nuclear Envelope {#s3a}
------------------------------------------------
We propose that emerin stabilizes and promotes the formation of a nuclear actin cortical network, analogous to the actin cortical network at the plasma membrane ([Figure 4](#pbio-0020231-g004){ref-type="fig"}). Another LEM-domain protein, LAP2β, also an integral nuclear inner-membrane protein, was 20-fold less active than emerin in actin polymerization assays (data not shown), suggesting that LAP2β binds actin with an affinity 20-fold lower than that of emerin. Other LEM-domain proteins have not yet been tested for binding to actin. Whether emerin has specialized roles involving actin, or shares this function with other nuclear membrane proteins, are both interesting possibilities. An actin-based cortical network could help anchor emerin and possibly other nuclear membrane proteins and lamin filaments, contributing significantly to the structural integrity of the nuclear envelope and potentially reinforcing sites of chromatin attachment ([Figure 4](#pbio-0020231-g004){ref-type="fig"}).
::: {#pbio-0020231-g004 .fig}
Figure 4
::: {.caption}
###### Model in Which Emerin Binding to the Pointed End of F-Actin Stabilizes an Actin Cortical Network at the Nuclear Inner Membrane
Our model is based on the actin cortical network at the cell surface of erythrocytes, except that lamin filaments also anchor to emerin-based junctional complexes. Spectrin heterodimers bind short actin filaments at the erythrocyte membrane; we therefore speculate that nuclear isoforms of αII-spectrin (J. M. H. and K. L. W., unpublished data) act similarly. Direct binding of emerin and αII-spectrin has not yet been tested. Nuclear isoforms of protein 4.1, which are essential for nuclear assembly ([@pbio-0020231-Krauss1]), have the potential to cross-link short actin filaments and spectrin filaments at the inner membrane (IM). Further work is necessary to test our model and identify other components of this proposed nuclear actin cortical network. OM, nuclear outer membrane.
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:::
Since lamin A also binds G-actin in vitro ([@pbio-0020231-Sasseville1]), we are currently testing the actin-binding properties of lamin A. Because emerin forms stable complexes with lamin A in vitro ([@pbio-0020231-Clements1]; [@pbio-0020231-Lee3]), and because the nuclear envelope localization of emerin depends on lamins, we speculate that emerin might interlink multiple filament networks (actin, spectrin, and lamins) at the nuclear envelope. This model will be tested in future experiments by determining whether lamin A and actin compete for binding to emerin, or form trimeric complexes. Such complexes could significantly reinforce the mechanical properties of the nuclear envelope. Our nuclear actin cortical network model is further supported by the properties of the nesprin family of nuclear membrane proteins, which includes nesprin-1α (see [Introduction](#s1){ref-type="sec"}) and NUANCE. Nesprin-1α binds directly to both emerin (K~d~ = 4 nM) and lamin A (affinity undetermined; [@pbio-0020231-Mislow1]). NUANCE is a large (796 kd), alternatively spliced isoform of nesprin that localizes to the nuclear envelope and nucleoplasm and binds F-actin ([@pbio-0020231-Zhen1]).
The organization of the membrane skeleton in erythrocytes (red blood cells) includes integral membrane proteins (e.g., Band 3), anchoring proteins (ankyrin), spectrin filaments, and "junctional complexes" (short actin filaments, protein 4.1, adducin, tropomodulin, and tropomyosin; [@pbio-0020231-Delaunay1]). Tropomodulin and tropomyosin stabilize the junctional complex. Spectrin filaments (α/β-spectrin heterodimers) attach to junctional complexes through direct binding to protein 4.1, adducin, and actin. At the inner nuclear membrane, our working model is that emerin stabilizes junctional complexes ([Figure 4](#pbio-0020231-g004){ref-type="fig"}), consisting of short actin filaments, nuclear-specific αII-spectrin ([@pbio-0020231-McMahon1]), and nuclear isoforms of protein 4.1 ([@pbio-0020231-Krauss1]). This model is the first step toward understanding the structural function of nuclear actin.
Materials and Methods {#s4}
=====================
{#s4a}
### Antibodies and proteins {#s4a1}
A pan-actin antibody (Sigma, St. Louis, Missouri, United States; catalog \#A-5060) was used at 1:1,000 for immunoblotting. An antibody specific for β-actin (Sigma; catalog \#A-5316) was used at 1:10,000 for immunoblotting and 1:1,000 for immunoprecipitation. Our rabbit polyclonal emerin antibody (serum 2999), described previously ([@pbio-0020231-Lee3]), was used at 1:20,000 for immunoblotting and 1:2,000 for immunoprecipitation. Purified chicken actin was a kind gift of Doug Robinson (Johns Hopkins Medical School). Purified rabbit actin was purchased from Cytoskeleton. (Denver, Colorado, United States; catalog \#AKL95 and \#AKL99). Alexa-488 actin (\#A12373), Alexa-594 actin (\#A34050), Alexa-488 phalloidin (\#A-12379), rhodamine--phalloidin (\#R-415), and Alexa-546 phalloidin (\#A-22283) were purchased from Molecular Probes (Eugene, Oregon, United States). Purified CapZ (capping protein) was a kind gift from John Cooper (Washington University, St. Louis). The full nucleoplasmic domain of wild-type emerin (residues 1--222) and corresponding mutants (detailed in [@pbio-0020231-Holaska2]) were expressed in bacteria and purified as described ([@pbio-0020231-Lee3]; [@pbio-0020231-Holaska2]). Emerin protein was labeled with Alexa-488 (Molecular Probes, catalog \#A20000) per manufacturer\'s instructions.
### Affinity purification using emerin-conjugated beads {#s4a2}
Wild-type emerin residues 1--222 (comprising the entire nucleoplasmic domain of emerin and lacking the transmembrane domain) or BSA (as a negative control) were coupled to Affigel-15 beads (Bio-Rad, Hercules, California, United States) per manufacturer\'s instructions. Nuclear extracts were prepared by hypotonic lysis ([@pbio-0020231-Offterdinger1]) from 10^10^ HeLa-S3 cells, obtained as frozen cell pellets from the National Cell Culture Center. For each affinity purification, we incubated 50 mg of nuclear lysate proteins with 2 ml of either emerin beads (0.5 mg/ml) or BSA beads in binding buffer (50 mM HEPES, 250 mM NaCl, 0.1% Triton X-100) for 4 h at 4 °C. Beads were collected by centrifugation at 500*g,* washed five times with binding buffer, and eluted with SDS-PAGE sample buffer. Dr. Robert Cole at the Johns Hopkins Mass Spectrometry Facility performed MALDI-TOF. Actin and αII-spectrin were two of seven emerin-associated proteins identified unambiguously in this work, which will be reported separately (J. M. H. and K. L. W., unpublished data).
### F-actin-binding assays {#s4a3}
F-actin columns were assembled as described ([@pbio-0020231-Forero1]). Equal amounts of purified recombinant wild-type and mutant emerin proteins (residues 1--222) were incubated with each column in PBS containing 0.1% Triton X-100 (PBST) for 1 h at 22 °C. After washing beads five times with PBST, bound proteins were eluted and resolved by SDS-PAGE, and detected either by Coomassie blue staining, or by immunoblotting with rabbit serum 2999 against emerin.
Coimmunoprecipitation assays were performed as described ([@pbio-0020231-Lee3]). Briefly, equal masses (5 μg) of actin and either wild-type or mutant emerin were incubated 2 h, then incubated 4 h with protein-A Sepharose-coupled antibodies against emerin or actin. The beads were washed five times with buffer, and bound proteins were eluted with SDS-sample buffer, resolved by SDS-PAGE, then blotted and probed with antibodies specific for either actin or emerin.
To measure emerin binding to single actin filaments, actin was polymerized by the addition of KMEI buffer (2 mM MgCl, 50 mM KCl, 10 mM imidazole \[pH 7. 0\], and 2 mM EGTA). After 30 min, the indicated form of emerin (4 μM, recombinant wild-type or mutant emerin residues 1--222, with or without conjugation to Alexa-488) was added to the filaments and incubated 30 min; we lastly added Alexa-546 phalloidin (final concentration, 0. 33 μM). These red F-actin polymers were then pelleted at 100,000*g* for 60 min. For experiments with unlabeled emerin, corresponding load, supernatant, and pellet fractions were resolved by SDS-PAGE and stained with Coomassie blue. For experiments with phalloidin-546-labeled F-actin, filaments were viewed using a Zeiss Axiovert 200 fluorescent microscope (Zeiss, Oberkochen, Germany) and images captured using a Quantix CCD camera (Photometrics, Huntington Beach, California, United States) attached to an Apple G4 computer using IPLab (version 3.6; Scanalytics, Fairfax, Virginia, United States) software.
### Actin polymerization and depolymerization assays {#s4a4}
Actin polymerization assays were performed per manufacturer\'s instructions (Cytoskeleton). Rabbit actin (2 μM; Cytoskeleton, catalog \#AKL95) plus pyrene--actin (0.1 μM; Cytoskeleton, catalog \#AP05) were used in all assays, unless otherwise stated. Actin polymerization was measured in a fluorimeter (Fluoromax 2; SPEX, Edison, New Jersey, United States), with excitation wavelength 365 nm and emission wavelength 407 nm, and plotted using DataMax-Std (version 2.2; SPEX, Edison, New Jersey, United States). Graphs were refined using Cricketgraph III (version 1.0, Computer Associates, Smithfield, Rhode Island, United States) and Kaleidagraph (version 3.5.1, Synergy Software, Reading, Pennsylvania, United States). Pyrene--actin was always present at 5% of total actin. Increasing concentrations of recombinant emerin were added just prior to initiating actin polymerization. The actin depolymerization assays were performed exactly as described ([@pbio-0020231-Mullins1]) with increasing amounts of recombinant emerin protein. Briefly, F-actin was polymerized by adding 2 μM G-actin to gelsolin--actin seeds (100 nM), then diluted 10-fold in the absence or presence of increasing concentrations of recombinant emerin (residues 1--222). Both gelsolin and actin were obtained from Cytoskeleton (catalog \#HPG5 and \#AKL95, respectively). Gelsolin--actin seeds were made exactly as described ([@pbio-0020231-Blanchoin1]). Alternatively, 2 μM G-actin was polymerized in the absence of gelsolin for 2 h. Polymerized filaments were then incubated with or without 100 nM CapZ. Subsequent polymerization and depolymerization assays were assayed as described above.
Actin polymerization in the presence or absence of emerin was monitored by fluorescent microscopy, as described ([@pbio-0020231-Blanchoin1]; [@pbio-0020231-Amann1]). Samples were diluted 1:500--1:1000, viewed on a Nikon Eclipse E600W microscope (Nikon, Tokyo, Japan), and images were captured with a Q-imaging Retiga Exi CCD camera (Q Imaging, Burnaby, British Columbia, Canada) using IPLab (version 3.9.2) attached to an Apple G5 computer. Images were converted to TIFF images and lengths of filaments were measured in Photoshop version 7.0 (Adobe Systems, San Jose, California, United States).
These studies were funded by the National Institutes of Health (F32 GM067397 and T32 HL07227 to JMH and R01 GM48646 to KLW) and the American Heart Association Scott B. Deutschman Memorial Research Award (KLW). We are grateful to Dyche Mullins, Tom Pollard, and Doug Robinson for advice. We thank the National Cell Culture Center for providing the HeLa cell pellets. Protein identification was done by the AB Mass Spectrometry/Proteomics Facility at Johns Hopkins School of Medicine (<http://www.hopkinsmedicine.org/msf>) with support from National Center for Research Resources shared instrumentation grant 1S10-RR14702, the Johns Hopkins Fund for Medical Discovery, and the Institute for Cell Engineering.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** JMH conceived and designed the experiments. JMH performed the experiments and analyzed the data. AKK and KLW contributed reagents/materials/analysis tools. JMH and KLW wrote the paper. KLW was the principal investigator.
Academic Editor: Marc W. Kirschner, Harvard University
Citation: Holaska JM, Kowalski AK, Wilson KL (2004) Emerin caps the pointed end of actin filaments: Evidence for an actin cortical network at the nuclear inner membrane. PLoS Biol 2(9): e231.
Arp
: actin-related protein
BAF
: barrier-to-autointegration factor
BSA
: bovine serum albumin
EDMD
: Emery-Dreifuss muscular dystrophy
F-actin
: filamentous actin
GCL
: germ cell-less
SR
: spectrin-repeat
| PubMed Central | 2024-06-05T03:55:46.798202 | 2004-8-24 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509406/",
"journal": "PLoS Biol. 2004 Sep 24; 2(9):e231",
"authors": [
{
"first": "James M",
"last": "Holaska"
},
{
"first": "Amy K",
"last": "Kowalski"
},
{
"first": "Katherine L",
"last": "Wilson"
}
]
} |
PMC509407 | Introduction {#s1}
============
The continuous shaping of plant form is a marvel of signal integration. In early seedling development this is particularly clear, as environmental cues, such as light, profoundly alter the innate morphogenetic program. How diverse pathways merge to determine a discrete cellular growth response is largely unknown. Auxin, the first plant hormone identified, has been implicated in patterning or growth of virtually every plant tissue from earliest embryo to developing fruit ([@pbio-0020258-Liscum1]). Brassinosteroids (BRs), the polyhydroxylated steroid hormones of plants, have been linked to many of these same processes, including photomorphogenesis ([@pbio-0020258-Clouse1]). The nature of the relationship between these hormones has remained largely undefined.
Many factors in the signal transduction pathways operating downstream from BRs and auxin have been identified. Brassinosteroid Insensitive-1 (BRI1), a plasma-membrane-localized receptor serine/threonine kinase, is essential for BR perception and accounts for most BR-binding activity in *Arabidopsis* ([@pbio-0020258-Wang1]). A Shaggy/GSK3-type kinase, Brassinosteroid Insensitive-2 (BIN2), acts as a negative regulator of the pathway downstream of BRI1 action ([@pbio-0020258-Li1]). When BR levels are low, proteins in the BES1/BZR1 family are hyperphosphorylated by BIN2 and targeted for degradation by the proteasome ([@pbio-0020258-He1]; [@pbio-0020258-Yin1]). Upon BR perception, BIN2 is inactivated by an unknown mechanism which allows hypophosphorylated BES1/BZR1 proteins to accumulate in the nucleus, where they presumably provoke changes in gene expression ([@pbio-0020258-He1]; [@pbio-0020258-Yin1]).
In contrast to BRs, no auxin receptor has been identified. However, exposure to auxin is known to promote rapid turnover of nuclear Aux/IAA proteins by ubiquitin-mediated targeting to the 26S proteasome ([@pbio-0020258-Gray2]). Aux/IAAs are direct negative regulators of the Auxin Response Factor (ARF) family of transcription factors and contain four highly conserved domains numbered I to IV ([@pbio-0020258-Abel1]). Domains III and IV are also found in most ARFs and facilitate dimerization within and between members of both families ([@pbio-0020258-Kim1]; [@pbio-0020258-Ulmasov3]). ARF proteins bind to a conserved auxin-responsive element (AuxRE) found upstream of many auxin-regulated genes ([@pbio-0020258-Ulmasov4]).
Previous studies have suggested that auxin and BRs may have a particularly close relationship among plant hormones. In a variety of bioassays representing diverse species, BRs have been shown to synergistically promote cell elongation when supplied with auxin ([@pbio-0020258-Mandava1]). Clouse and colleagues examined the effect of the two hormones on gene transcription more than a decade ago, and found that while BRs could activate the expression of some auxin-responsive genes, others appeared to be auxin specific ([@pbio-0020258-Clouse2]; [@pbio-0020258-Zurek1]). They also noted that detectable BR effects required much longer treatments compared with the extremely rapid effects of auxin, and concluded that BR-mediated cell elongation effects were likely independent from the auxin signal transduction pathway. Microarray experiments, assaying approximately one-third of the *Arabidopsis* genome, rekindled interest in the interaction between auxin and BRs, as it was found that a significant percentage of the BR genomic response comprised genes annotated as auxin responsive ([@pbio-0020258-Goda1]; [@pbio-0020258-Mussig1]; [@pbio-0020258-Yin1]). Recent work from Nakamura and colleagues has shown that three genes---*IAA5, IAA19,* and *SAUR-AC1*---are induced by both auxin and BRs and that induction requires BR biosynthesis ([@pbio-0020258-Nakamura1], [@pbio-0020258-Nakamura2]).
In this work, genetic, physiological, and genomic approaches were used to dissect the relationship between auxin and BRs in seedling growth. Together these techniques demonstrated that the relationship between these hormones is far more deeply intertwined than previously suspected. Auxin and BR effects on cell elongation were found to be interdependent, and this physiological interdependency was mirrored at the transcriptional level. In addition, growth and transcriptional effects of exogenous BR treatment could be largely superceded by overstimulation of the auxin pathway. Several lines of evidence suggested that auxin:BR synergism did not depend upon biosynthetic regulation of hormone levels; rather, the two response pathways are likely to converge at the promoters of shared target genes. New computational approaches detected a number of known transcription factor--binding motifs enriched in promoters induced by both hormones, as well as motifs which are overrepresented in promoters activated specifically by auxin or BRs. This multifaceted approach elucidates the mechanism of action of both auxin and BRs in cell expansion, and serves as a model for interrogating complex signaling networks.
Results {#s2}
=======
Auxin and BRs Interact Synergistically to Promote Hypocotyl Elongation {#s2a}
----------------------------------------------------------------------
Early studies of BR effects in a variety of bioassays suggested that there was a synergistic interaction between auxin and BRs ([@pbio-0020258-Mandava1]). We confirmed and extended these studies to the reference plant *Arabidopsis thaliana,* using hypocotyl (primary stem) length as a quantitative measure of growth. Both hormones are known to induce cell elongation, and exogenous BR treatment has been shown to increase hypocotyl length ([@pbio-0020258-Nemhauser2]). In contrast, addition of auxin to media has only modest effects on seedling hypocotyl elongation, likely as a result of inefficient acropetal transport from root to shoot ([@pbio-0020258-Gray1]). However, increased temperature has been demonstrated previously to be an effective method of altering auxin levels in the shoot and leads to robust increases in hypocotyl length ([@pbio-0020258-Gray1]; [@pbio-0020258-Zhao2]).
In our conditions, hypocotyls of plants grown at 29 °C were approximately 1.8 times longer than those of plants grown at 22 °C, consistent with what has been observed by others ([@pbio-0020258-Gray1]; [@pbio-0020258-Zhao2]). When exogenous brassinolide (BL), the most biologically active BR, was applied, hypocotyls of plants grown at elevated temperature exhibited a "kinked" morphology and agravitropic growth, typical of saturating BL conditions (data not shown). In order to examine the relationship between auxin and BRs, it was necessary to find conditions where auxin levels were increased but at subsaturating levels for the hypocotyl growth-promoting response. Plants grown at 26 °C versus 22 °C showed measurable increases in both hypocotyl elongation and levels of auxin intermediates ([@pbio-0020258-Zhao2]). Using these conditions, it was possible to observe that plants grown at higher temperatures were more sensitive to exogenous BR treatment, both in threshold levels for response as well as in terms of absolute growth ([Figure 1](#pbio-0020258-g001){ref-type="fig"}A).
::: {#pbio-0020258-g001 .fig}
Figure 1
::: {.caption}
###### BR and Auxin Pathways Are Interdependent, as Measured by Hypocotyl Elongation
\(A) Mild temperature elevation causes elongation of the hypocotyl and BR hypersensitivity in WT plants. Columbia ecotype is shown but results are similar for Wassilewskija. Hypocotyls of 3-d-old plants grown at either 26 °C (diamonds, dashed line) or 22 °C (circles, solid line) were measured.
\(B) *det2-1* plants are defective in BR biosynthesis and are also insensitive to the temperature increase. As the *det2* deficiency is rescued by exogenous BL, temperature sensitivity is restored.
\(C) Plants with the weak *bri1-5* mutation are insensitive both to temperature and exogenous BR.
(D--H) BR response depends upon auxin response. WT is shown in circles with a solid thin line and mutants are shown in squares with a thick dashed line. Known auxin response mutants *axr2-1* (D), *axr1-12* (E), *tir1-1* (F), and *axr3-1* (G) have decreased BR response. (F) *tir1* has no hypocotyl elongation phenotype in the absence of exogenous hormone treatment and only very modest effects on BR sensitivity. Response is significantly reduced in *tir1* mutants at 100 nM BL, as measured by Student\'s t-test (*p* = 0.03, using Bonferroni adjustment for multiple tests; [@pbio-0020258-Hochberg1]).
\(H) *yucca* plants, which overproduce auxin, also show reduced BR response.
Error bars represent standard error. Data in (F) and (G) were collected in a separate experiment from other panels, resulting in small differences in the values for WT hypocotyl length.
:::
:::
BR- and auxin-mediated growth promotion required both pathways to be intact. As has been shown previously, hypocotyls of *det2* mutants defective in BR biosynthesis ([@pbio-0020258-Li2]) fail to elongate with increased temperature ([@pbio-0020258-Gray1]; [@pbio-0020258-Zhao2]). Importantly, the hypersensitivity of *det2* plants to exogenous BR was enhanced by increased temperature, suggesting that these two responses are tightly linked ([Figure 1](#pbio-0020258-g001){ref-type="fig"}B). Weak *bri1* mutants were also unresponsive to temperature, suggesting that auxin response was dependent on a functional BR signal transduction pathway ([Figure 1](#pbio-0020258-g001){ref-type="fig"}C). The dramatic growth enhancement caused by overproduction of auxin in the *yucca* mutant ([@pbio-0020258-Zhao1]) requires functional *BRI1,* as *yucca bri1* mutants are dwarfs ([Figure 2](#pbio-0020258-g002){ref-type="fig"}). Conversely, BR response was dependent on a functional auxin signal transduction pathway as *axr1* ([@pbio-0020258-Lincoln1]) and *axr2* ([@pbio-0020258-Timpte1]) mutants with reduced auxin response showed significantly reduced sensitivity to BR treatment (see [Figure 1](#pbio-0020258-g001){ref-type="fig"}D and [1](#pbio-0020258-g001){ref-type="fig"}E). The degree of BR insensitivity is correlated with the level of reduced auxin responsiveness, as *tir1* mutants, which show only subtle phenotypes in the absence of exogenous auxin ([@pbio-0020258-Ruegger1]), exhibited only a modest reduction in BR response (see [Figure 1](#pbio-0020258-g001){ref-type="fig"}F). *axr3* mutants, which in many assays display a constitutive auxin response ([@pbio-0020258-Leyser1]), were insensitive to BRs (see [Figure 1](#pbio-0020258-g001){ref-type="fig"}G). This suggests that the BR insensitivity observed in *axr1* and *axr2* mutants is not simply a block in cell elongation, and that regulated turnover of Aux/IAA proteins, such as those encoded by *AXR2* and *AXR3,* is required for normal BR response. *yucca* mutants were also largely insensitive to exogenous BR and appeared saturated for the BR response (see [Figure 1](#pbio-0020258-g001){ref-type="fig"}H).
::: {#pbio-0020258-g002 .fig}
Figure 2
::: {.caption}
###### Enhanced Hypocotyl Elongation of *yucca* Mutants Requires Functional BRI1
\(A) Average hypocotyl lengths of 3-d-old plants. Error bars represent standard error.
\(B) Ten-day-old WT, *yucca, yucca bri1-116,* and *bri-116* seedlings.
:::
:::
Auxin and BR Transcriptional Responses Substantially Overlap {#s2b}
------------------------------------------------------------
Previous studies have identified several auxin-responsive genes that are also regulated by BRs ([@pbio-0020258-Goda1]; [@pbio-0020258-Mussig1]; [@pbio-0020258-Yin1]; [@pbio-0020258-Nakamura1], [@pbio-0020258-Nakamura2]). To comprehensively compare the genomic effects of treatment with each hormone, Affymetrix oligonucleotide microarrays, representing approximately 22,000 genes, were hybridized with probes from two biological replicates following mock or BR treatment. Linear models were used to identify 342 transcripts whose levels were increased following BR treatment ([Figure 3](#pbio-0020258-g003){ref-type="fig"}A; Tables [S1](#st001){ref-type="supplementary-material"} and [S3](#st003){ref-type="supplementary-material"}). The levels of 296 transcripts were decreased in the same treatment ([Figure 3](#pbio-0020258-g003){ref-type="fig"}A; Tables [S2](#st002){ref-type="supplementary-material"} and [S4](#st004){ref-type="supplementary-material"}). Comparison with newly analyzed data from a similar experiment using auxin-treated seedlings ([@pbio-0020258-Zhao3]) showed that nearly a quarter of genes upregulated by either auxin or BR treatment were regulated by both hormones ([Figure 3](#pbio-0020258-g003){ref-type="fig"}A and [3](#pbio-0020258-g003){ref-type="fig"}C; [Tables S1--S6](#st001){ref-type="supplementary-material"}). This is a much larger overlap than that reported in the recent study by [@pbio-0020258-Goda2], likely reflecting substantial differences in experimental design and analysis methods, including the use of different microarrays. In addition, at least 75% of the genes identified as BR inducible were late responders (only observed after 12 or 24 h of BR treatment) and therefore were not included in the analysis described here.
::: {#pbio-0020258-g003 .fig}
Figure 3
::: {.caption}
###### BR and Auxin Have Shared Genomic Effects
\(A) Venn diagram showing relative proportion of BR- and auxin-responsive genes and the degree of overlap.
\(B) Functional categories of BR--auxin shared genes reveal a potential growth signature.
(C and D) Effects of auxin on BR-regulated gene expression. Transcripts which show elevated levels are shown in orange, those with decreased levels are shown in blue, and those transcripts whose levels are not changed are shown in yellow. (C) Relative ratios were derived from the following comparisons (from left to right): BR versus mock treatment (WT plants; B), auxin versus mock treatment (WT plants; A), and *yucca* versus WT (Y). The three columns to the left are BR-upregulated genes and the three columns to the right are BR-downregulated genes. Among the BR-upregulated genes, there are a large number that are also induced by auxin treatment or in a *yucca* background. Few BR-repressed genes are repressed by auxin. nc, no change. (D) Effect of BR treatment in *yucca* background. Relative ratios represent BR versus mock treatment in WT plants (WT) or in *yucca* mutants (YB). Approximately two-thirds of BR-regulated genes were not affected by BR treatment of *yucca* plants.
\(E) Quantitative PCR shows that shared target genes are synergistically induced when treated with both auxin and BRs. At5g64770 encodes a protein with unknown function. At1g18400 encodes BEE1, a bHLH-containing protein known to be required for the BR response ([@pbio-0020258-Friedrichsen1]). At1g10550 and At4g30290 are putative endoxyloglucan transferases. Asterisks indicate response under an additive model.
:::
:::
As much of the auxin response is transient, *yucca* plants which continuously experience high levels of auxin have a different profile of altered transcript levels than plants exposed to exogenous auxin for a short time period ([@pbio-0020258-Zhao2]). To produce a more complete list of auxin-responsive genes, RNA from *yucca* seedlings was isolated and used to probe additional microarrays. More than 20% of all BR-upregulated genes were also differentially regulated in a *yucca* background ([Figure 3](#pbio-0020258-g003){ref-type="fig"}C; Tables [S1](#st001){ref-type="supplementary-material"} and [S2](#st002){ref-type="supplementary-material"}). In combination, 40% of the BR-upregulated genes were altered either by auxin treatment or in *yucca* mutants (see [Table S1](#st001){ref-type="supplementary-material"}). Members of all known auxin-responsive gene families were identified, as has been seen in previous microarray experiments representing a smaller fraction of the genome ([@pbio-0020258-Goda1], [@pbio-0020258-Goda2]; [@pbio-0020258-Mussig1]; [@pbio-0020258-Yin1]).
While auxin treatment had no effect on ARF gene expression, transcripts of *ARF4* (At4g30080) and *ARF8* (At5g37020) were negatively regulated by BR treatment (see Tables [S2](#st002){ref-type="supplementary-material"} and [S4](#st004){ref-type="supplementary-material"}). This is the first evidence of transcriptional regulation of ARF genes. In addition, BRs repressed the expression of several auxin transport--related transcripts, including *PIN3* (At1g70940), *PIN4* (At2g01420), *PIN7* (At1g23080), and an *AUX1*-like gene (At1g77690). Auxin induced the expression of *BRI1* and a close paralog, *BRL3* (At3g13380), and repressed the expression of another *BRI1*-like gene, *VH1/ BRL2* (At2g01950) ([@pbio-0020258-Clay1]; [@pbio-0020258-Yin2]). It is possible that the genes identified here as auxin and BR responsive may represent a common growth signature regulated by many factors during seedling development. The majority of these genes do not have known functions; however, many of the rest are known or predicted to be involved in cell expansion, metabolism, and signal transduction ([Figure 3](#pbio-0020258-g003){ref-type="fig"}B).
Integration between Auxin and BR Signals Occurs in the Nucleus {#s2c}
--------------------------------------------------------------
Many plant hormones directly regulate the levels of other hormones ([@pbio-0020258-Alonso1]). This complicates analysis of cross-talk, which is defined by shared signal transduction components. The interdependency between auxin and BRs does not function primarily through regulation of hormone levels. Auxin does not induce BR biosynthesis. *det2* plants, which are hypersensitive to exogenous BR treatment, were insensitive to growth at elevated temperature (see [Figure 1](#pbio-0020258-g001){ref-type="fig"}B). Auxin treatment does not affect the subcellular localization of BES1 ([@pbio-0020258-Yin1]), and growth at elevated temperature does not alter BES1 levels or phosphorylation state (unpublished data). Conversely, BRs do not regulate auxin biosynthesis. [@pbio-0020258-Nakamura1] reported that *det2* mutants make at least normal amounts of auxin and that BR treatments do not alter auxin levels. It was recently reported that the stability of an IAA1:luciferase fusion protein was unchanged following BR treatment, though the data were not shown ([@pbio-0020258-Zenser1]). Here, we used a heat shock--inducible β-glucuronidase (GUS) reporter fused to the N-terminal portion of AXR3 described by [@pbio-0020258-Gray2]. This construct was rapidly turned over in the presence of auxin but showed no change in stability following BR treatment ([Figure 4](#pbio-0020258-g004){ref-type="fig"}D).
::: {#pbio-0020258-g004 .fig}
Figure 4
::: {.caption}
###### Endogenous BR Levels Affect Expression of an Auxin-Responsive Reporter but Do Not Induce Aux/IAA Protein Turnover
\(A) WT, (B) *det2*, and (C) *DW4FOX* plants carrying the DR5::GUS transgene.
\(A) GUS staining is particularly strong in young leaves (yellow arrow).
\(B) *det2* plants show no GUS staining in aerial tissues.
\(C) *DWF4OX* plants show increased intensity of staining, particularly at the tips of emerging leaves (yellow arrow) and in the hypocotyl (orange arrows). Inset shows hypocotyl-root junction.
\(D) Aux/IAA stability does not appear to be affected by treatment with BRs. Plants carrying a heat shock--inducible fusion of the N-terminal portion of AXR3 and GUS reporter were subjected to 2 h at 37 °C and then treated with mock or hormone treatments for the time periods listed.
:::
:::
Together, these results suggested that the interaction between the auxin and BR pathways was likely at the promoters of shared target genes. To test whether the auxin:BR synergism was detectable at the level of gene transcription, transcript levels from four genes identified in the microarray studies were quantified in plants exposed to exogenous treatment of either hormone or both in combination (see [Figure 3](#pbio-0020258-g003){ref-type="fig"}E). In all cases, levels of these transcripts were regulated nonadditively in the presence of both hormones. If, as suggested by these results, BR and auxin response pathways converge at the level of gene activation, we reasoned that *yucca* plants, which are largely insensitive to BR for growth promotion, might also show a reduced BR genomic response. RNA was isolated from *yucca* plants treated with BR and used to probe additional microarrays. Approximately two-thirds of genes showing BR responsiveness in wild-type (WT) plants were no longer affected by BR treatment in a *yucca* background (see [Figure 3](#pbio-0020258-g003){ref-type="fig"}D; Tables [S1](#st001){ref-type="supplementary-material"} and [S2](#st002){ref-type="supplementary-material"}). This result strongly suggests that auxin and BR treatment affect transcription of these target genes by a common mechanism.
Promoters of Coordinately Regulated Genes Share Regulatory Motifs {#s2d}
-----------------------------------------------------------------
Computational analysis of coordinately regulated genes is an emerging tool for dissecting regulatory networks (e.g., [@pbio-0020258-DeRisi1]; [@pbio-0020258-Harmer1]; [@pbio-0020258-Tullai1]). To identify potential regulatory elements acting in these pathways, a list of all genes regulated by either auxin or BR was generated, and 500 bp upstream of each gene were identified. These promoters were split into three groups: those with increased transcript levels following treatment with BR only (B group; *n* = 258), those with increased transcript levels following auxin treatment only (A group; *n* = 254), and those genes whose transcripts were induced following treatment with either hormone (AB group; *n* = 82). Known plant promoter elements and their annotations were downloaded from PLACE ([@pbio-0020258-Higo1]) and used to screen each promoter list. The expected number of occurrences of each PLACE motif was estimated using 1,000 sets of *n* promoters randomly sampled from the genome, where *n* is equal to the number of promoters in each group (A, B, or AB). This approach offers a significant advantage over other background models used to assess enrichment. Permuted distributions reflect real expected frequencies and do not rely on assumptions about genome architecture. In addition, the normal distribution of site frequencies observed with large numbers of permutations allows for the use of powerful parametric statistical methods. Moreover, the ease of filtering based on relative probabilities makes this approach ideally suited to comparisons of promoters regulated in different conditions. In this study, matches were considered significant if a motif was overrepresented in a given set (*p* \< 0.1) and present in at least 10% of group promoters. This analysis identified several motifs specifically enriched in a given group ([Table 1](#pbio-0020258-t001){ref-type="table"}), as well as several motifs found to be enriched in multiple groups ([Table 2](#pbio-0020258-t002){ref-type="table"}).
::: {#pbio-0020258-t001 .table-wrap}
Table 1
::: {.caption}
###### PLACE Motifs Enriched Specifically in AB, A, or B Promoters
:::
Motifs with related patterns are grouped together by color
^a^Total number of sites identified
^b^Expected number of sites based on 1,000 randomly sampled groups of promoters
^c^Percentage of promoters containing at least one site
^d^Transcription factor family known to bind this element
\<, value is less than 0.001; SA, salicylic acid
DOI:10.1371/journal.pbio.0020258.t001
:::
::: {#pbio-0020258-t002 .table-wrap}
Table 2
::: {.caption}
###### PLACE Motifs Enriched in Promoters of Multiple Groups
:::
See text and [Table 1](#pbio-0020258-t001){ref-type="table"} caption for abbreviations
DOI:10.1371/journal.pbio.0020258.t002
:::
One of the sequences enriched in the B group was TGTCTC, previously identified as an auxin-responsive element ([@pbio-0020258-Ulmasov1]) termed ARFAT in the PLACE database. Surprisingly, this sequence was not significantly enriched in the A set (*p* = 0.78). However, the A, B, and AB groups showed significant enrichment of the core ARF-binding element TGTC in their promoters, perhaps reflecting some sequence divergence between *Arabidopsis* and soybean, where the element was first identified. A well-characterized synthetic element containing the ARFAT called DR5 ([@pbio-0020258-Ulmasov2]) could be used to test the BR responsiveness of this element and was therefore introduced into plants with altered BR levels. In *det2* plants with lower endogenous levels of BRs ([@pbio-0020258-Li2]), DR5 expression was greatly reduced, particularly in the shoot ([Figure 4](#pbio-0020258-g004){ref-type="fig"}A versus [4](#pbio-0020258-g004){ref-type="fig"}C). Conversely, in plants with increased levels of BRs caused by overexpressing a BR biosynthetic gene, *DWF4* ([@pbio-0020258-Wang1]), DR5 expression was increased ([Figure 4](#pbio-0020258-g004){ref-type="fig"}A versus [4](#pbio-0020258-g004){ref-type="fig"}B). DR5 expression was also increased following transient BR treatment of WT plants carrying the DR5 reporter (unpublished data). [@pbio-0020258-Nakamura1] also recently demonstrated the BR inducibility of DR5::GUS and found no change in endogenous IAA levels following BR treatment, providing further evidence that BR transcriptional effects are direct. These data strongly suggest that the ARF-binding element requires both hormones for proper expression and should be considered a Brassinosteroid-Auxin Response Element. This finding raises questions about the utility of the DR5 element as a reporter of auxin response, as it likely reflects regions of regulatory overlap between the two pathways.
Consensus binding sites for several families of transcription factors were identified as enriched in the AB set (see [Table 1](#pbio-0020258-t001){ref-type="table"}). The presence of a MYC consensus site in more than 80% of AB promoters was quite striking, especially in light of the BR and auxin inducibility of the bHLH-containing *Brassinosteroid Enhanced Expression 1* (*BEE1;* At1g18400) gene, which is known to function in BR response ([@pbio-0020258-Friedrichsen1]). Many of the other AB consensus motif matches were implicated in regulation by light or abscisic acid (ABA), both of which have been linked previously to BR-mediated growth response by physiology and genetics ([@pbio-0020258-Nemhauser1]). For the B set, there was widespread occurrence of a GT-1 consensus binding motif, as well as evidence for a MYB-binding site distinct from that found in the A set. Identification of several elements specific for the A set, including those known to bind WRKY-family members, suggests attractive targets for designing new reporters which may not be BR dependent. Several instances of light-regulated motifs are intriguing given the strong evidence for a close relationship between auxin and light responses ([@pbio-0020258-Tian1]).
Several of the promoter elements identified in the A, B, and AB promoters were found as multiple copies within promoters, including the core ARF-binding element TGTC. Recent studies have suggested that ARF dimerization is not required for activation of ARFAT-mediated transcription ([@pbio-0020258-Tiwari1]). Interestingly, a scan of AB promoters revealed that nearly half of all AB promoters contain at least one instance of multiple copies of the core TGTC element within a 50-bp window. Clustering of TGTC sites was also seen in the A set (42% of promoters contain at least one pair of sites within 50 bp) and somewhat less frequently in the B set (33%). This finding suggests that interactions between ARFs may be important for hormone responsiveness of natural promoters, in addition to enhancing auxin inducibility of synthetic multimerized ARFATs. As specific binding factors are not known for most of the other elements identified, exact nucleotides required for factor binding are not known. Therefore, this analysis is likely a conservative estimate for the number of true transcription factor--binding sites present in each promoter.
Discussion {#s3}
==========
With the notable exception of auxin, most plant hormones are produced and perceived throughout the plant body. Modulation of hormone response stems from regulation of hormone levels and/or signal transduction components, as well as from interactions with other signaling pathways. There are many examples of cross-talk between hormones in plant biology. In addition to auxin and BRs, gibberellins (GAs), ethylene, ABA, and cytokinin have all been shown to affect hypocotyl elongation (reviewed in [@pbio-0020258-Nemhauser1]). As mentioned previously, some of these hormones interact through biosynthetic regulation. For example, auxin, ABA, and cytokinin stimulate ethylene biosynthesis, particularly when supplied at high levels ([@pbio-0020258-Yang1]; [@pbio-0020258-Vogel1]; [@pbio-0020258-Ghassemian1]). Physiological and genetic evidence suggests that auxin, GAs, and ethylene promote hypocotyl growth by largely independent means ([@pbio-0020258-Gray1]; [@pbio-0020258-Collett1]). Similarly, BRs and GAs interact additively in most cell elongation bioassays ([@pbio-0020258-Mandava2]), and analysis of *bri1* mutants suggests that the two hormones independently and antagonistically regulate transcription of some target genes ([@pbio-0020258-Bouquin1]). In contrast, auxin and BRs interact synergistically and interdependently to promote hypocotyl cell elongation, making their relationship unique among plant growth regulators.
The nature of hormone interactions may be tissue specific. A recent study demonstrated that auxin acts primarily through GAs to promote root elongation, and proposed that the DELLA family of negative regulators was a point of convergence between the two pathways ([@pbio-0020258-Fu1]). One possible complication for this interpretation is that auxin is required for normal GA biosynthesis in pea ([@pbio-0020258-Ross1]) and thus, the effects of auxin on DELLA protein stability may be indirect. We have preliminary evidence that interactions between auxin and BRs may be different in aerial tissues than in roots. While auxin and BRs promote hypocotyl elongation, the hormones have opposite effects on root hair growth (J. L. Nemhauser and J. Chory, unpublished data). In addition, reduced BR levels or response may actually increase auxin effects on root pericycle proliferation (J. L. Nemhauser, N. Geldner, and J. Chory, unpublished data).
While auxin and BRs stimulate elongation of the hypocotyl, light antagonizes this effect. The AB genes induced by auxin and BRs may be targets for repression by the light response. Plants with reduced BR levels or response show a light-grown phenotype even when grown in the dark, including a short hypocotyl, expansion of cotyledons, and production of leaves. Many mutants with stabilized Aux/IAA proteins also show this deetiolated phenotype ([@pbio-0020258-Tian1]). Levels of BRs may be light regulated ([@pbio-0020258-Kang1]), and response to BRs is affected by light quality and intensity ([@pbio-0020258-Nemhauser2]). Interestingly, two photoreceptors, PHOT1 (At3g45780) and Phytochrome E (At4g18130), are both downregulated by BRs. Two potential negative regulators of the light response, PKS1-like (At5g04190) and DRT100 (At3g12610), are upregulated by both auxin and BRs. Differential regulation of target genes by auxin, BRs, and light may allow fine-tuning of the photomorphogenetic response.
Bioinformatic Analysis of Signaling Networks {#s3a}
--------------------------------------------
The regulation of gene expression in eukaryotes is complex and is largely mediated by multiple transcription factors that bind within regulatory regions upstream of the coding sequence. In the simplest model, coexpressed genes exhibit similar expression characteristics because they are regulated by the same transcription factors. A number of algorithms have been developed to identify potential regulatory motifs overrepresented in the promoter sequences of coregulated genes (reviewed in [@pbio-0020258-Rombauts1]). Each algorithm requires a background model to calculate the expected frequency for each motif. The simplest background model estimates the expected frequency for a given motif based on the single nucleotide composition of the analyzed sequences ([@pbio-0020258-Bailey1]; [@pbio-0020258-Roth1]). Improvements on these methods use so-called higher-order models based on Markov chain statistics ([@pbio-0020258-Thijs1], [@pbio-0020258-Thijs2]; [@pbio-0020258-Marchal1]), building the background model by estimating the probability at each nucleotide position based on the previous bases in the sequence. Other approaches include enumerative methods that generate background models based on whole-genome motif counts from noncoding intergenic ([@pbio-0020258-van1]) or randomly sampled ([@pbio-0020258-Marino-Ramirez1]) genomic sequences.
Because biological sequences are inherently nonrandom, we chose another approach to build our background model. For each motif under consideration, we modeled the expected frequency distribution by randomly sampling sets of promoter sequences from among all the genes represented on the microarrays used in our study. Therefore, we could directly estimate the statistical significance for each motif from its Z score, which is the number of standard deviations by which the observed frequency exceeds the expected frequency based on the distribution observed in the permutation sampling. In contrast to other methods, our approach uses a background model based on a real distribution of motif counts derived from annotated promoter sequences, rather than estimating expected word frequencies from simulated or randomly selected genomic sequences or from models based on distribution functions. Thus, given any set of *Arabidopsis* genes clustered on the basis of similar expression, we could easily identify overrepresented known transcription factor--binding motifs or overrepresented novel presumptive promoter elements. For example, new experiments assaying genomic effects of different hormone treatments or environmental conditions could be used to define finer groupings of coregulated genes and could be readily integrated into our current analysis.
A Model for Auxin:BR Synergy {#s3b}
----------------------------
Auxin:BR synergism results from convergence of the two response pathways on a common mechanism for promoting cell elongation. The integration of these hormone signals occurs very late in signal transduction, likely at the promoters of more than 80 genes whose expression is induced by short treatments with either hormone. Several known regulatory elements have been identified in these common target genes. The well-characterized auxin-response element ARFAT is one crucial node of intersection between the BR and auxin pathways, as it is BR responsive and requires BR synthesis for normal expression. More than 20 ARFs have been identified in the *Arabidopsis* genome ([@pbio-0020258-Liscum1]). Many have been shown to bind the ARFAT motif and promote auxin-inducible gene expression ([@pbio-0020258-Ulmasov3]; [@pbio-0020258-Tiwari1]). Stabilization of Aux/IAA proteins, such as AXR2 and AXR3, completely blocks BR growth responses. We propose a model where auxin and BR pathways converge on regulation of ARF transcription factors ([Figure 5](#pbio-0020258-g005){ref-type="fig"}).
::: {#pbio-0020258-g005 .fig}
Figure 5
::: {.caption}
###### A Model of BR--Auxin Interaction
Auxin and BR signals are likely integrated on promoters of shared target genes. The node(s) of intersection between auxin and BR pathways must be downstream of BES1 and Aux/IAAs and upstream of gene expression. One likely mechanism is via regulation of transcriptional complexes, such as those containing the ARFs.
:::
:::
Cross-talk is a common feature of animal growth regulator pathways. For example, glucocorticoids synergistically enhance the effects of retinoic acid in mouse cells ([@pbio-0020258-Subramaniam1]). Upon ligand binding, the glucocorticoid receptor directly interacts with the homeodomain protein Pbx1 and activates transcription of Hoxb-1. In *Xenopus,* transcriptional activation of several genes, including *twin, siamois,* and *nodal-related-3,* requires stimulation of both TGFβ and WNT pathways. Similarly, two transcription factors, SOX10 and KROX20, have been recently reported to interdependently regulate expression of a neural crest--specific enhancer conserved among mouse, human, and chicken ([@pbio-0020258-Ghislain1]). This type of coregulation is also seen in plants. One example is the synergistic interaction between osmotic stress and ABA response, which is likely mediated by interaction between DREB and AREB transcription factors ([@pbio-0020258-Narusaka1]). In all of these cases, signal integration is achieved by formation of a complex containing transcription factors independently regulated by each pathway, often binding to composite regulatory elements. By integrating the inputs of multiple pathways, these mechanisms provide cellular or regional specificity for a given response.
ARFAT was originally identified as part of a composite element ([@pbio-0020258-Ulmasov1]). However, DR5 has been characterized as a multimerized simple response element ([@pbio-0020258-Ulmasov3]) and can be activated by either auxin or BRs. So, unlike in the systems described above, auxin and BR signals likely converge on the same family of transcription factors. Such a relationship has recently been described for ethylene and jasmonate in plant defense responses ([@pbio-0020258-Lorenzo1]). Both ethylene and jasmonate pathways are required to induce expression of the transcription factor ERF1, which in turn regulates the expression of a number of defense-related genes. Neither auxin nor BRs have large effects on ARF transcription, and several AB targets are early-response genes not requiring de novo protein synthesis for activation ([@pbio-0020258-Friedrichsen1]; [@pbio-0020258-Liscum1]). Auxin and BRs likely regulate ARF complex activity posttranslationally rather than through transcriptional regulation. Auxin is already known to modulate ARF activity by regulating the stability of the interacting Aux/IAA repressor proteins ([@pbio-0020258-Gray2]; [@pbio-0020258-Tiwari1]). BR perception could increase ARF activity by leading to modification of the ARFs themselves or through interactions with a BR-regulated transcriptional coactivator. The additional transcriptional regulation of some ARFs by BRs, together with auxin and BR effects on a number of Aux/IAA genes, could favor formation of particular transcriptional complexes promoting growth. Five genes encoding proteins with DNA-binding motifs were induced by both hormones, including members of the MYC, EREBP, and leucine zipper families. Higher-order interactions among several transcription factor complexes, perhaps directly involving members of the BES1/BZR1 family, could provide additional control of the shared auxin:BR response pathway.
A longstanding question in plant biology has been how a small number of hormones with overlapping functions can provoke a wide range of responses. Combinatorial control has long been suggested as one possible explanation (e.g., [@pbio-0020258-Singh1]). The detailed analysis of BR and auxin pathways in this work suggests that hormone response is determined by the cellular milieu. Additional factors, including other hormones and environmental stimuli, can be incorporated into this model, leading ultimately to a detailed map of plant growth processes.
Materials and Methods {#s4}
=====================
{#s4a}
### Hypocotyl measurements {#s4a1}
Seeds were sterilized for 15 min in 70% ethanol, 0.01% Triton X-100, followed by 10 min of 95% ethanol. After sterilization, seeds were suspended in 0.1% low-melting-point agarose and spotted on plates containing 0.5× Murashige Minimal Organics Medium (Gibco-BRL, San Diego, California, United States), 0.8% phytagar (Gibco-BRL), and one of five concentrations of BL (0, 1, 10, 100, or 1,000 nM). Seeds on plates were then stratified in the dark at 4 °C for 2 d. Plants were grown in approximately 35 μmol m^−2^s^−1^ white light with a red:far-red light ratio near 1. Plate position was changed every 24 h to minimize position effect. Hypocotyl lengths were measured from 10 to 14 3-d-old seedlings. Seedlings were removed from one plate at a time and scanned between two transparencies on a flatbed scanner. NIH Image 1.62 was used to perform length measurements. All dose-response experiments were performed in duplicate. *bri1-5* is a weak allele in a Wassilewskija background. All other mutants used in this work are in a Columbia background.
### GUS staining {#s4a2}
GUS staining protocol was as described in [@pbio-0020258-Sessions1]. Induction of AXR3-NT-GUS lines was as described in [@pbio-0020258-Gray2].
### Microarray studies {#s4a3}
Nine-day-old, light-grown *Arabidopsis* seedlings were immersed in 1 μM BL in 0.5× Murashige Minimal Organics Medium (Invitrogen, Carlsbad, California, United States) or medium alone for 2.5 h before they were harvested for total RNA preparation. Total RNA from the treated seedlings was used for preparing probes for the microarray experiments, which were carried out according to the protocols provided by the gene chip manufacturer Affymetrix (Santa Clara, California, United States). All experiments used two independent biological replicates. Details of the auxin experiment have been described previously ([@pbio-0020258-Zhao3]). Data analysis was performed in R ([@pbio-0020258-Ihaka1]). Genes were normalized using rma in the Bioconductor affy package (<http://www.bioconductor.org>; [@pbio-0020258-Irizarry1]) and subsequently analyzed using linear models and Empirical Bayes analysis (limma package; [@pbio-0020258-Smyth1]). To be considered differentially expressed, genes were required to have a false discovery rate adjusted *p* value of less than 10% and an empirical Bayes log odds of differential expression (B) greater than 0. Data are available at Gene Expression Omnibus; see [Supporting Information](#s5){ref-type="sec"} for accession numbers.
### Quantitative PCR {#s4a4}
Plants were treated with hormones as above using treatments of either 1 μM BL, 1 μM indole-3-acetic-acid (auxin), both hormones, or a mock treatment. Total RNA was extracted using a Qiagen (Valencia, California, United States) RNAeasy kit and first-strand cDNA was synthesized using an Invitrogen Superscript First-Strand cDNA Synthesis kit. cDNAs were diluted 20-fold and combined with SYBR master mix (PE Biosystems, Wellesley, California, United States) for PCR. Primers were as follows: At5g64770 (5′-CTTCTCATACTCTTCATTTCCTCTCCTACT-3′, 5′-TTCTCGTAAGCTTCGTGCTTGA-3′), At1g18400 (5′-CTAGCGGCGTCTCCGATAAT-3′, 5′-AAGAACCTGTTTCAGTGGCAATAAC-3′), At1g10550 (5′-AAGCTTCCCGCTGGATTTG-3′, 5′-TTGATAAATAGAAAGCAACCACAACAC-3′), and At4g30290 (5′-TCCCTGGTAACTCTGCTGGAA-3′, 5′-CCGGAGATTTAAGATAGAATGTTGTGA-3′). At5g15400 (ubiquitin) was used to normalize all values (5′-TGCGCTGCCAGATAATACACTATT-3′, 5′-TGCTGCCCAACATCAGGTT-3′). PCR reactions were performed in triplicate and analyzed using an ABI PRISMA 7700. A standard curve was constructed for each primer using an equal mixture of all cDNAs.
### Sequences for promoter analysis {#s4a5}
We used a Perl script to extract the 500 bp of sequence preceding the 5′ end of each annotated transcription unit in the AGI pseudomolecules annotation (14-May-2003) downloaded from NCBI. These putative promoter sequences begin immediately upstream of the 5′ UTR for transcription units with an annotated 5′ UTR, and upstream of the annotated translational start for the remainder.
### Promoter analysis and significance calculations {#s4a6}
We analyzed putative promoter regions upstream of auxin- and BR-regulated genes to identify overrepresented promoter elements. One thousand surrogates of each promoter set were created by randomly shuffling the list of genes represented on the Affymetrix ATH1 arrays and then sampling *n* genes and extracting 500-bp promoter sequences for the sampled set of genes. Known plant promoter elements and their annotation were downloaded from PLACE ([@pbio-0020258-Higo1]). For each set of *n* promoters, the null distribution for each PLACE motif was modeled by counting the number of occurrences for each word within each of the 1,000 surrogate sets of *n* promoters. Using this approach we could then ask how well the observed frequency of a certain motif in a set of *n* promoters matched the frequency that would be expected for a random set of *n* promoters. We estimated the one-tailed *p* value for each motif based on the Z score of the difference of the actual word count of the promoter set (C~true~) minus the mean count from the 1,000 surrogates (C~surr~) relative to the SD from the 1,000 surrogates (SD~surr~) \[i.e., Z = (C~true~ − C~surr~)/SD~surr~\]. Thus for each motif the *p* value we calculated was the probability to the right of the observed count calculated on the null distribution derived from sampling promoters randomly from the genome. We considered a motif to be significantly overrepresented if this probability was less than 0.1. These calculations were implemented using Perl scripts and a relational database (MySQL).
Supporting Information {#s5}
======================
Table S1
::: {.caption}
###### Fold Change of BL-Upregulated Genes following Exposure to BL or IAA (Auxin) Treatment
Effects of increased auxin levels in the *yucca* mutant are shown as compared to WT and following BL treatments. The comparisons from left to right are WT BL- versus mock-treated, WT IAA- versus mock-treated, *yucca* mock-treated versus WT mock-treated, and *yucca* BL- versus mock-treated. nc, no change.
(160 KB XLS).
:::
::: {.caption}
######
Click here for additional data file.
:::
Table S2
::: {.caption}
###### Fold Change of BL-Downregulated Genes following Exposure to BL or IAA (Auxin) Treatment
Effects of increased auxin levels in the *yucca* mutant are shown as compared to WT and following BL treatments. The comparisons from left to right are WT BL- versus mock-treated, WT IAA- versus mock-treated, *yucca* mock-treated versus WT mock-treated, and *yucca* BL- versus mock-treated. nc, no change.
(169 KB XLS).
:::
::: {.caption}
######
Click here for additional data file.
:::
Table S3
::: {.caption}
###### Normalized Values of BL-Upregulated Genes
ave, average; se, standard error.
(64 KB XLS).
:::
::: {.caption}
######
Click here for additional data file.
:::
Table S4
::: {.caption}
###### Normalized Values of BL-Downregulated Genes
ave, average; se, standard error.
(64 KB XLS).
:::
::: {.caption}
######
Click here for additional data file.
:::
Table S5
::: {.caption}
###### Values of IAA-Upregulated Genes
ave, average; se, standard error.
(63 KB XLS).
:::
::: {.caption}
######
Click here for additional data file.
:::
Table S6
::: {.caption}
###### Normalized Values of IAA-Downregulated Genes
ave, average; se, standard error.
(26 KB XLS).
:::
::: {.caption}
######
Click here for additional data file.
:::
Accession Numbers {#s5a7}
-----------------
The Gene Expression Omnibus (<http://www.ncbi.nlm.nih.gov/geo/>) accession numbers for the genes and gene products discussed in this paper are WTBR1 (GSM13423), WTBR2 (GSM13424), WTmock1 (GSM13420), WTmock2 (GSM13421), wtzm1 (GSM13430), wtzm2 (GSM13432), wtzmIAA1 (GSM13433), wtzmIAA2 (GSM13434) and, *yucca*BR1 (GSM13428), *yucca*BR2 (GSM13429), *yucca*mock1 (GSM13426), and *yucca*mock2 (GSM13427).The associated experimental descriptions are available at accession numbers GSE862 (BR effects on WT and *yucca* seedlings) and GSE863 (auxin effects on seedlings).
We thank J. Ecker, J. Long, J. Umen, and M. Offenbacher for careful reading of the manuscript, Y. Huang and J. Borevitz for invaluable assistance with and insights into microarray analysis, Y. Zhao and B. Burger for stimulating discussions, and M. Estelle and O. Leyser for providing seeds. The BR microarray experiments were done in collaboration with S. Mora-Garcia and Y. Yin. This work was supported by grants from the National Institutes of Health (NIH) (GM52413), United States Department of Agriculture, and National Science Foundation (MCB-0315845) to JC. JLN and TCM are NIH postdoctoral fellows (F32 GM20742 and F32 GM69090); TCM was also supported by the Bechtel Foundation. JC is an investigator of the Howard Hughes Medical Institute.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** JLN and JC conceived and designed the experiments. JLN performed the experiments. JLN and TCM analyzed the data. TCM wrote scripts and performed bioinformatic analysis. JLN, TCM, and JC wrote the paper.
Academic Editor: Jeffrey Dangl, University of North Carolina
Citation: Nemhauser JL, Mockler TC, Chory J (2004) Interdependency of brassinosteroid and auxin signaling in *Arabidopsis.* PLoS Biol 2(9): e258.
ABA
: abscisic acid
ARF
: Auxin Response Factor
BL
: brassinolide
GA
: gibberellin
GUS
: β-glucuronidase
WT
: wild-type
| PubMed Central | 2024-06-05T03:55:46.801315 | 2004-8-24 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509407/",
"journal": "PLoS Biol. 2004 Sep 24; 2(9):e258",
"authors": [
{
"first": "Jennifer L",
"last": "Nemhauser"
},
{
"first": "Todd C",
"last": "Mockler"
},
{
"first": "Joanne",
"last": "Chory"
}
]
} |
PMC509408 | Introduction {#s1}
============
In the primary visual cortex (V1), different trials of presentation of an identical stimulus yield highly variable firing rates ([@pbio-0020264-Heggelund1]). This trial-to-trial variability is not inherited from subcortical inputs, as these respond in a much more consistent fashion ([@pbio-0020264-Kara1]). Instead, variability has been related to spontaneous variations in cortical state ([@pbio-0020264-Arieli1]; [@pbio-0020264-Buracas1]; [@pbio-0020264-Tsodyks1]; [@pbio-0020264-Kenet1]). These variations may reflect the perceptual effects associated with a stimulus, rather than the presence of the stimulus itself ([@pbio-0020264-Ress1]).
A key property of trial-to-trial variability is that it depends on the strength of the stimulus: Response variance across trials is approximately proportional to response mean ([@pbio-0020264-Tolhurst1]). An example of this effect can be seen in the responses of a cell in cat V1 to drifting gratings ([Figure 1](#pbio-0020264-g001){ref-type="fig"}A--[1](#pbio-0020264-g001){ref-type="fig"}C). Different trials of an identical stimulus elicit firing rates that vary greatly ([Figure 1](#pbio-0020264-g001){ref-type="fig"}A). As a result, the standard deviation of the firing rates is roughly comparable to their mean amplitude ([Figure 1](#pbio-0020264-g001){ref-type="fig"}B and [1](#pbio-0020264-g001){ref-type="fig"}C). The ratio of variance to mean is close to the value predicted for a Poisson process ([Figure 2](#pbio-0020264-g002){ref-type="fig"}A, dashed line). For a Poisson process, the variance of the spike counts is equal to the mean. Once spike counts are converted to firing rate by binning in 10-ms windows (i.e., at 100 Hz), the ratio of variance to mean becomes 100. The Poisson-like behavior of firing rates is well known, although reports differ on the exact value of the ratio of variance to mean ([@pbio-0020264-Tolhurst1]; [@pbio-0020264-Bradley1]; [@pbio-0020264-Vogels1]; [@pbio-0020264-Geisler1]; [@pbio-0020264-Gur1]; [@pbio-0020264-Reich1]; [@pbio-0020264-Buracas1]; [@pbio-0020264-Kara1]).
::: {#pbio-0020264-g001 .fig}
Figure 1
::: {.caption}
###### Variability in the Responses of a Simple Cell
\(A) Firing rate in response to a cycle of an optimal drifting grating. Three trials are shown.
\(B) Firing rate averaged over seven trials. Shaded area indicates 2 s.d.
\(C) Same, for three other stimuli: a grating drifting in the orthogonal direction (top), a grating drifting in the opposite direction (middle), and a blank stimulus (bottom).
\(D) Membrane potential trace measured for the first cycle. Dashed line is resting potential *V~rest~.* Dotted line is firing threshold *V~thresh~* (from \[H\]).
(E--G) As in (A--C), for coarse potential.
\(H) Relation between firing rate and coarse potential. Curve is fit of rectification equation.
(I--K) As in (A--C), for predictions of rectification model.
:::
:::
::: {#pbio-0020264-g002 .fig}
Figure 2
::: {.caption}
###### Relation between Response Variance and Mean for Three Cells
\(A) Variance versus mean for firing rate of the simple cell in [Figure 1](#pbio-0020264-g001){ref-type="fig"} measured with 13 stimuli (the four in [Figure 1](#pbio-0020264-g001){ref-type="fig"} plus nine additional orientations). Line is linear regression. Diagonal line is prediction for a Poisson process.
\(B) Variance versus mean for coarse potential. Error bars are 2 s.d. Curve is linear fit to standard deviation versus mean. Dashed line is resting potential *V~rest~.* Dotted line is firing threshold *V~thresh~.*
\(C) Variance versus mean for firing rate predicted by the rectification model. Details as in (A).
(D--F) As in (A--C) for a complex cell.
(G--I) As in (A--C) for a third neuron, whose behavior is intermediate between those of simple cells and complex cells.
:::
:::
Because the production of firing rates within a neuron introduces remarkably little noise ([@pbio-0020264-Calvin1]; [@pbio-0020264-Mainen1]; [@pbio-0020264-Carandini2]), trial-to-trial variability is thought to arise from the membrane potential fluctuations that result from summated synaptic input ([@pbio-0020264-Calvin1]; [@pbio-0020264-Stevens1]). I have tested this hypothesis by considering membrane potential responses recorded intracellularly in vivo.
Results {#s2}
=======
From traces of membrane potential obtained at high temporal resolution ([Figure 1](#pbio-0020264-g001){ref-type="fig"}D), I obtained an estimate of overall synaptic drive by removing the action potentials and low-pass filtering the resulting traces ([@pbio-0020264-Carandini1]; [@pbio-0020264-Volgushev1]). The outcome of this procedure ([Figure 1](#pbio-0020264-g001){ref-type="fig"}E) is a coarse potential (or "generator potential"; [@pbio-0020264-Lankheet1]) that approximates the synaptic current ([@pbio-0020264-Anderson1]). This technique allows one to estimate synaptic currents while concurrently recording firing rates.
Variability of Coarse Potential during Visual Stimulation {#s2a}
---------------------------------------------------------
We can now consider the mean and variance across trials for coarse potential. The mean, *V~mean~,* is the "signal" reflecting the stimulus-driven synaptic input to the neuron ([Figure 1](#pbio-0020264-g001){ref-type="fig"}F and [1](#pbio-0020264-g001){ref-type="fig"}G, traces). The variance, instead, is the "noise" reflecting the synaptic input\'s trial-to-trial variability ([Figure 1](#pbio-0020264-g001){ref-type="fig"}F and [1](#pbio-0020264-g001){ref-type="fig"}G, shaded areas).
The variability of potential depended only slightly on stimulus strength. Variance was slightly higher when the stimuli depolarized the cell than when they hyperpolarized it ([Figure 1](#pbio-0020264-g001){ref-type="fig"}F and [1](#pbio-0020264-g001){ref-type="fig"}G). For the example simple cell in [Figure 1](#pbio-0020264-g001){ref-type="fig"}, standard deviation of potential was 2.8 ± 1.2 mV (s.d.) for *V~mean~* between --70 and --65 mV, and 4.0 ± 1.7 mV for *V~mean~* between --55 and --50 mV. The relation between standard deviation of potential and *V~mean~* can be described by a regression line (*r* = 0.27 ± 0.04, s.d., bootstrap) whose slope is 0.08 ± 0.01 and whose intercept at *V~rest~* = −60.4 mV is 3.3 ± 0.1 mV. Similar values were obtained in the rest of the population (e.g., [Figure 2](#pbio-0020264-g002){ref-type="fig"}E and [2](#pbio-0020264-g002){ref-type="fig"}H): correlation coefficient was *r* = 0.40 ± 0.19 (s.d., *N* = 22), intercept at *V~rest~* was 3.3 ± 1.4 mV, and mean slope was a shallow 0.14 ± 0.09. In occasional cells (such as that of [Figure 2](#pbio-0020264-g002){ref-type="fig"}H), the standard deviation of potential did not grow monotonically with *V~mean~.*
The ratios of variance to mean seen in membrane potentials were negligible when compared to those seen in firing rate. For the example simple cell, over the entire range of mean potentials the variance of potential grew by less than a factor of four ([Figure 2](#pbio-0020264-g002){ref-type="fig"}B). By contrast, over the entire range of firing rates the variance of firing rate grew by a factor of almost 100 ([Figure 2](#pbio-0020264-g002){ref-type="fig"}A). Similar results were obtained in the remaining cells, such as the complex cell of [Figure 2](#pbio-0020264-g002){ref-type="fig"}D and [2](#pbio-0020264-g002){ref-type="fig"}E and the intermediate cell of [Figure 2](#pbio-0020264-g002){ref-type="fig"}G and [2](#pbio-0020264-g002){ref-type="fig"}H. In the last cell, the difference between potential and firing rate was particularly striking, as the former shows a downward slope that is clearly absent in the latter. These differences in variability are meaningful because potential and firing rate were recorded from the same responses to the same set of stimuli. They are not simply due to differences in time scale ([@pbio-0020264-Buracas1]; [@pbio-0020264-Kara1]) because firing rate and potential were sampled at the same resolution (100 Hz).
Accounting for the Variability of Firing Rate {#s2b}
---------------------------------------------
The origin of the large variability in firing rate lies not in an unforeseen source of noise, but rather in a deterministic mechanism, the nonlinear transformation of potentials into firing rates. This transformation ([Figure 1](#pbio-0020264-g001){ref-type="fig"}H) can be fitted by a simple rectification model ([@pbio-0020264-Granit1]) describing how firing rate *R* grows with potential *V* once this potential is above a threshold *V~thresh~.* As expected ([@pbio-0020264-Anderson2]; [@pbio-0020264-Carandini1]), this rectification model captures the relation between potential and firing rate ([Figure 1](#pbio-0020264-g001){ref-type="fig"}H, curve) and can be used to predict the rough features of firing rate both in individual trials (compare [Figure 1](#pbio-0020264-g001){ref-type="fig"}A and [1](#pbio-0020264-g001){ref-type="fig"}I) and in averages across trials (compare curves in [Figure 1](#pbio-0020264-g001){ref-type="fig"}B and [1](#pbio-0020264-g001){ref-type="fig"}C with those in [Figure 1](#pbio-0020264-g001){ref-type="fig"}J and [1](#pbio-0020264-g001){ref-type="fig"}K).
Of course, rectification is not a full account of the transformation between synaptic inputs and firing rates. Indeed, the relationship between firing rate and potential exhibits substantial error bars ([Figure 1](#pbio-0020264-g001){ref-type="fig"}H). These error bars do not denote noise involved in generating spikes, which is negligible ([@pbio-0020264-Calvin1]; [@pbio-0020264-Mainen1]; [@pbio-0020264-Carandini2]). They simply indicate that (as evident in the Hodgkin--Huxley equations) instantaneous potential is only one of the determinants of firing rate; additional determinants include the membrane potential\'s recent history ([@pbio-0020264-Azouz1]) and frequency content ([@pbio-0020264-Carandini2]; [@pbio-0020264-Volgushev2]).
Despite its simplicity, the rectification model is sufficient to predict the large variability of firing rate, and the increase of firing-rate variance with firing-rate mean. The predicted standard deviation resembles the measured one both in amplitude and in time course (compare shaded areas in [Figure 1](#pbio-0020264-g001){ref-type="fig"}B and [1](#pbio-0020264-g001){ref-type="fig"}C with those in [Figure 1](#pbio-0020264-g001){ref-type="fig"}J and [1](#pbio-0020264-g001){ref-type="fig"}K). Indeed, a plot of variance versus mean for the predicted firing rate ([Figure 2](#pbio-0020264-g002){ref-type="fig"}C) indicates almost as much variability as that seen for the actual firing rate ([Figure 2](#pbio-0020264-g002){ref-type="fig"}A). Similar results were obtained in the other example cells (compare [Figure 2](#pbio-0020264-g002){ref-type="fig"}D to [2](#pbio-0020264-g002){ref-type="fig"}F, and [2](#pbio-0020264-g002){ref-type="fig"}G to [2](#pbio-0020264-g002){ref-type="fig"}I) and in the rest of the population ([Figure 3](#pbio-0020264-g003){ref-type="fig"}A and [3](#pbio-0020264-g003){ref-type="fig"}B). While the rectification model often underestimated the vertical intercept of the line relating mean and variance ([Figure 3](#pbio-0020264-g003){ref-type="fig"}A), it generally captured the line\'s slope ([Figure 3](#pbio-0020264-g003){ref-type="fig"}B). The model, therefore, accounts for the growth of firing-rate variance with the mean.
::: {#pbio-0020264-g003 .fig}
Figure 3
::: {.caption}
###### Performance of the Rectification and Gaussian--Rectification Models in Predicting Firing-Rate Variability
Distributions of firing-rate variance versus firing-rate mean were fitted with a line in logarithmic scale, corresponding to the equation variance = *a* mean*^b^,* where *a* is the intercept of the line and *b* is the slope of the line. Fitting was performed on the measured distributions (e.g., [Figure 2](#pbio-0020264-g002){ref-type="fig"}A), on the distributions predicted by the rectification model (e.g., [Figure 2](#pbio-0020264-g002){ref-type="fig"}C), and on those predicted by the Gaussian--rectification model (e.g., [Figure 6](#pbio-0020264-g006){ref-type="fig"}B). Dashed lines indicate predictions for a Poisson process.
\(A) Comparison of measured intercept versus predicted intercept. Diagonal line indicates equality between measured and predicted values.
\(B) Same, for the slope.
(C and D) Same as in (A) and (B), for the predictions of the Gaussian--rectification model.
:::
:::
The reason why the rectification model explains the large variability of firing rate is rather intuitive. Trial-to-trial fluctuations in potential are critical to obtain spikes, because many visual stimuli (such as the 210° grating in [Figure 1](#pbio-0020264-g001){ref-type="fig"}) elicit a mean potential that barely reaches the firing threshold ([@pbio-0020264-Anderson2]). Therefore, small fluctuations in membrane potential make the difference between a trial with few or no spikes and one with plenty of spikes. In other words, the firing threshold amplifies small fluctuations in potential into large fluctuations in firing rate.
Perhaps less intuitive is the reason why the rectification model explains the growth of firing-rate variance with firing-rate mean. One may think that a necessary condition for this effect is the growth in potential variance observed with increasing mean potential ([Figure 2](#pbio-0020264-g002){ref-type="fig"}B). This is not the case: The variance of potential could stay constant or even decrease (as it does for the cell in [Figure 2](#pbio-0020264-g002){ref-type="fig"}H), and the variance of firing rate would still grow with the mean ([Figure 2](#pbio-0020264-g002){ref-type="fig"}G).
Predicting the Variability of Firing Rate {#s2c}
-----------------------------------------
An intuition and a quantitative account for these properties can be obtained by applying the rectification model to an idealized random distribution of potentials, which we take to be Gaussian. Such a Gaussian--rectification model has been used to explain the dependence of mean firing rate on mean synaptic input ([@pbio-0020264-Anderson2]; [@pbio-0020264-Hansel1]; [@pbio-0020264-Miller1]). It resembles a model proposed by [@pbio-0020264-Abeles1], [@pbio-0020264-Abeles2]) to study neuronal integration time.
In the Gaussian--rectification model, the stimulus determines the mean of the Gaussian ([Figure 4](#pbio-0020264-g004){ref-type="fig"}B), and the portion of Gaussian that crosses threshold determines the distribution of firing rates ([Figure 4](#pbio-0020264-g004){ref-type="fig"}A). The mean of the Gaussian is the average potential *V~mean~* evoked by the stimulus at that instant ([Figure 4](#pbio-0020264-g004){ref-type="fig"}B). The rectification function ([Figure 1](#pbio-0020264-g001){ref-type="fig"}H) operates on this distribution and determines the distribution of firing rates ([Figure 4](#pbio-0020264-g004){ref-type="fig"}A): Each potential contributes a firing rate given by the rectification function, with a probability given by the value of the Gaussian at that potential. When mean potential *V~mean~* is low, the Gaussian lies mostly below the threshold *V~thresh~,* so the predicted firing rate is mostly zero ([Figure 4](#pbio-0020264-g004){ref-type="fig"}A, *a*). When *V~mean~* is higher, however, the tail of the Gaussian that lies above threshold becomes substantially larger, and the distribution of firing rates reaches higher rates ([Figure 4](#pbio-0020264-g004){ref-type="fig"}A, *e*). The large peak at 0 spikes/s corresponds to the area of the Gaussian that lies below *V~thresh~.*
::: {#pbio-0020264-g004 .fig}
Figure 4
::: {.caption}
###### The Gaussian--Rectification Model
(A and B) Distributions across trials of model potential *V* (B) and of model firing rate *R* (A) for five values of the mean potential *V~mean~.* Firing rate is obtained from potential by applying the rectification model ([Figure 1](#pbio-0020264-g001){ref-type="fig"}H). The value for *R* = 0 is shown at 1/3 of veridical height.
(C and D) Mean (data points) and standard deviation (error bars) for the distributions in (A) and (B) as a function of mean potential *V~mean~.* Curve and shaded area indicate model predictions for the full range of mean potentials. Arrows indicate the five mean potentials (± 2 mV) used in (A) and (B). Throughout, dashed lines indicate resting potential *V~rest~* and dotted lines indicate firing threshold *V~thresh~.*
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Such a simple model is sufficient to predict that the variance of firing rate should increase with mean firing rate. As mean potential *V~mean~* increases, the distribution of firing rate becomes broader ([Figure 4](#pbio-0020264-g004){ref-type="fig"}A), increasing not only in mean but also in standard deviation ([Figure 4](#pbio-0020264-g004){ref-type="fig"}C). This phenomenon occurs even though in the model the standard deviation of potential is the same at all mean potentials ([Figure 4](#pbio-0020264-g004){ref-type="fig"}D).
The main assumption of the model, that of a Gaussian distribution of potentials, is generally borne out by the data. In most cells, the distribution of potential is close to a Gaussian, especially at the lowest values of mean potential, where spiking seldom occurs ([Figure 5](#pbio-0020264-g005){ref-type="fig"}B). For the example simple cell, the distribution of *z*-scores (the difference between potential and mean potential, normalized by the standard deviation at that potential) appears remarkably Gaussian ([Figure 6](#pbio-0020264-g006){ref-type="fig"}A). Similar results were obtained in the other cells (e.g., [Figure 6](#pbio-0020264-g006){ref-type="fig"}C and [6](#pbio-0020264-g006){ref-type="fig"}E), although in some cells the tails of the distributions exceeded those of a Gaussian, and a large skewness clearly favored the more depolarized tails (not shown). A Gaussian distribution of potentials is commonly predicted in the theoretical literature (e.g., [@pbio-0020264-Svirskis1]; [@pbio-0020264-Amemori1]; [@pbio-0020264-Rudolph1]). It would be expected in a passive membrane summating many independent, high-rate presynaptic spike trains ([@pbio-0020264-Rice1]; [@pbio-0020264-Tuckwell1]).
::: {#pbio-0020264-g005 .fig}
Figure 5
::: {.caption}
###### Application of the Gaussian--Rectification Model to the Data from the Example Simple Cell
(A and B) Distributions across trials of potential *V* (B) and of firing rate *R* (A) for five values of the mean potential *V~mean~.* Curves are best-fitting Gaussians (B) and predicted distributions of firing rate (A). Bin for *R* = 0 is shown at 1/3 of veridical height (and is three times wider than the others so that area is veridical).
(C and D) Mean (data points) and standard deviation (error bars) for the distributions in (A) and (B), as a function of mean potential *V~mean~.* Curve and shaded area indicate model predictions for the full range of mean potentials. Arrows indicate the five mean potentials (± 2 mV) used in (A) and (B). Even a reduced model with constant standard deviation of potential (D, shaded area) predicts a growing standard deviation (A, shaded area).
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::: {#pbio-0020264-g006 .fig}
Figure 6
::: {.caption}
###### Variability of Potential and Predictions of the Gaussian--Rectification Model for Three Cells
\(A) Distribution of normalized deviations from the mean (*z*-scores) for the potential of the simple cell in [Figure 1](#pbio-0020264-g001){ref-type="fig"} and [Figure 2](#pbio-0020264-g002){ref-type="fig"}A--[2](#pbio-0020264-g002){ref-type="fig"}C. These were computed by subtracting from each potential the corresponding mean potential *V~mean~* (the abscissa in [Figure 2](#pbio-0020264-g002){ref-type="fig"}B) and dividing by the standard deviation (the square root of the ordinate in [Figure 2](#pbio-0020264-g002){ref-type="fig"}B). The results were cumulated. The curve is a normal Gaussian.
\(B) Variance versus mean for firing rate for the same cell and its prediction by the Gaussian--rectification model. Data points are same as [Figure 2](#pbio-0020264-g002){ref-type="fig"}A. Red curve: prediction of Gaussian--rectification model Shaded area: region where the Gaussian--rectification model predicts the occurrence of 75% of the points. Line is linear regression.
(C and D) Same as (A) and (B) for the complex cell in [Figure 2](#pbio-0020264-g002){ref-type="fig"}D--[2](#pbio-0020264-g002){ref-type="fig"}F.
(E and F) Same, for the intermediate cell in [Figure 2](#pbio-0020264-g002){ref-type="fig"}G--[2](#pbio-0020264-g002){ref-type="fig"}I.
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The Gaussian--rectification model has four parameters. Three of these parameters describe the rectification stage and are thus fully constrained by the measured relationship between potential and firing rate ([Figure 1](#pbio-0020264-g001){ref-type="fig"}H). The remaining parameter, the standard deviation of the Gaussian, σ, was obtained from maximum likelihood estimation, i.e., by searching for the standard deviation that maximized the probability of observing the distributions of firing rate ([Figure 5](#pbio-0020264-g005){ref-type="fig"}A). The result, σ = 4.6 mV, slightly overestimates the standard deviation observed for low mean potentials, but correctly estimates it at higher mean potentials ([Figure 5](#pbio-0020264-g005){ref-type="fig"}D, compare shaded area to error bars).
The model predicts the main features of the distributions of firing rate ([Figure 5](#pbio-0020264-g005){ref-type="fig"}A). It predicts that when mean potential is low (e.g., *V~mean~* = −64 mV; [Figure 5](#pbio-0020264-g005){ref-type="fig"}A, *a*), the firing rate is always zero, whereas larger mean potentials yield a distribution of firing rates that spans values from 0 to 300 spikes/s (e.g., *V~mean~* = −54 mV; [Figure 5](#pbio-0020264-g005){ref-type="fig"}A, *d*). Deviations from the predictions are largest where they are least significant, i.e., at high firing rates for the high values of *V~mean~* (e.g., *V~mean~* = −50 mV; [Figure 5](#pbio-0020264-g005){ref-type="fig"}A, *e*). These high values were achieved seldom; for example, only 21 data points were obtained at *V~mean~* = −50 mV ([Figure 5](#pbio-0020264-g005){ref-type="fig"}A, *e*), compared to 273 at *V~mean~* = −54 mV ([Figure 5](#pbio-0020264-g005){ref-type="fig"}A, *d*) and 1,575 at *V~mean~* = −64 mV ([Figure 5](#pbio-0020264-g005){ref-type="fig"}A, *a*).
In fact, the model closely predicts both the firing rate\'s mean and standard deviation ([Figure 5](#pbio-0020264-g005){ref-type="fig"}C). It predicts the two key effects of increasing mean potential: (1) an increase in the firing rate\'s mean (as a power law; [@pbio-0020264-Anderson2]; [@pbio-0020264-Hansel1]; [@pbio-0020264-Miller1]), and (2) an increase in the firing rate\'s standard deviation.
Crucially, the model closely predicts how firing-rate variance depends on firing-rate mean ([Figure 6](#pbio-0020264-g006){ref-type="fig"}B, red curve). Because of noise in the estimation of variance from a limited number of measurements (in this experiment, seven trials), the data are not expected to fall exactly on the model\'s prediction; Monte Carlo simulations with a matched number of trials determined the area in which 75% of the observations are predicted to fall ([Figure 6](#pbio-0020264-g006){ref-type="fig"}B, gray area). Similar results were obtained in the remaining cells of the population, except that the model has a mild tendency to underestimate the intercept and overestimate the slope of the relation between variance and mean ([Figure 3](#pbio-0020264-g003){ref-type="fig"}C and [3](#pbio-0020264-g003){ref-type="fig"}D).
Overall, the Gaussian--rectification model applied to the trace of mean potential performed as well as the rectification model applied to the individual traces of potential. Both models underestimated the intercept of the lines fitted to the relationship between firing-rate variance and mean: the rectification model by 25 ± 42% ([Figure 3](#pbio-0020264-g003){ref-type="fig"}A), and the Gaussian--rectification model by 44 ± 26% ([Figure 3](#pbio-0020264-g003){ref-type="fig"}B). Both models correctly estimated the slope of the line (the growth in variance with increasing mean), with insignificant errors of 0.10 ± 0.25 for the rectification model ([Figure 3](#pbio-0020264-g003){ref-type="fig"}C), and −0.01 ± 0.22 for the Gaussian--rectification model ([Figure 3](#pbio-0020264-g003){ref-type="fig"}D). This performance is remarkable, given that the Gaussian--rectification model replaces detailed knowledge of potential in individual trials with just one free parameter, the standard deviation σ of potential.
These results illustrate how the key element in producing the steep growth in firing-rate variance observed with growing stimulus strength is the nonlinear transformation between potential and firing rate ([Figure 1](#pbio-0020264-g001){ref-type="fig"}H). Indeed, the model was intentionally implemented with the constraint that the standard deviation of potential, σ, be constant. This constraint serves to demonstrate that a mild growth in variance of potential ([Figure 5](#pbio-0020264-g005){ref-type="fig"}D, error bars) is not necessary to produce the steep growth in firing-rate variance ([Figure 5](#pbio-0020264-g005){ref-type="fig"}C, error bars).
Variability of Responses to Current Injection {#s2d}
---------------------------------------------
The predictions of the Gaussian--rectification model apply to any neuron that meets minimal criteria: a relationship between synaptic input and firing rate that is monotonic and includes a threshold, and noise in the input that has a Gaussian distribution.
As an example, let us consider a neuron that is closer to biological reality than the Gaussian--rectification model, one that receives currents (not potentials) in its input and produces individual spikes (not continuous firing rates). In particular, consider an enhanced integrate-and-fire neuron, where each spike is accompanied by a temporary increase in spike threshold and by the entry of calcium, which in turn determines an after-hyperpolarization potassium current (see [Materials and Methods](#s4){ref-type="sec"}).
To ensure realism, I fitted the model parameters to responses to injected currents of a regular spiking neuron. This neuron was recorded in vitro in the visual cortex of the guinea pig, in the near absence of synaptic inputs ([@pbio-0020264-Carandini2]). The injected currents include sinusoids ([Figure 7](#pbio-0020264-g007){ref-type="fig"}A, top four panels) and approximately Gaussian-distributed noise ([Figure 7](#pbio-0020264-g007){ref-type="fig"}A, bottom panels). Once its parameters are appropriately tailored, the enhanced integrate-and-fire model accurately predicts the cell\'s responses, both in the subthreshold membrane potential waveforms and in the timing of individual spikes ([Figure 7](#pbio-0020264-g007){ref-type="fig"}B and [7](#pbio-0020264-g007){ref-type="fig"}C).
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Figure 7
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###### Responses of a Regular-Spiking Neuron in the Visual Cortex to Current Injection, and Predictions by an Enhanced Integrate-and-Fire Model Neuron
\(A) Injected currents were sinusoids or noise waveforms. Noise was obtained by summing eight sinusoids with incommensurate frequencies.
\(B) Membrane potential responses of a regular-spiking neuron (cell 19s2, experiment 4) recorded with sharp electrodes in a study of guinea pig visual cortex in vitro ([@pbio-0020264-Carandini2]).
\(C) Predictions of an enhanced integrate-and-fire neuron model fine-tuned to resemble the responses of the cell.
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Just as predicted, this spiking neuron responds to noisy injected currents with a firing rate whose variance grows with the mean ([Figure 8](#pbio-0020264-g008){ref-type="fig"}). To simulate the synaptic drive to a simple cell recorded in vivo ([Figure 1](#pbio-0020264-g001){ref-type="fig"}A--[1](#pbio-0020264-g001){ref-type="fig"}D) I injected sinusoidal currents, to which I added Gaussian noise. The model responses ([Figure 8](#pbio-0020264-g008){ref-type="fig"}A--[8](#pbio-0020264-g008){ref-type="fig"}D) resemble those seen in vivo ([Figure 1](#pbio-0020264-g001){ref-type="fig"}A--[1](#pbio-0020264-g001){ref-type="fig"}D). The firing rate is highly variable ([Figure 8](#pbio-0020264-g008){ref-type="fig"}B), with a standard deviation that is roughly comparable to the mean ([Figure 8](#pbio-0020264-g008){ref-type="fig"}C and [8](#pbio-0020264-g008){ref-type="fig"}D), even though the standard deviation of the injected current is constant ([Figure 8](#pbio-0020264-g008){ref-type="fig"}H and [8](#pbio-0020264-g008){ref-type="fig"}I). In fact, for firing rate the variance grows proportionally to the mean ([Figure 8](#pbio-0020264-g008){ref-type="fig"}E), even though for injected current the variance is constant ([Figure 8](#pbio-0020264-g008){ref-type="fig"}J).
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Figure 8
::: {.caption}
###### Variability in the Responses of the Spiking Model Neuron
\(A) Response of the model neuron to a 0.6-nA sinusoidal current in the presence of Gaussian noise (s.d. 0.25 nA).
\(B) Corresponding firing rate. Three trials are shown.
\(C) Firing rate averaged over 16 trials. Shaded area indicates 2 s.d.
\(D) Same, for three other stimuli: a 0.4-nA sinusoid (top), a 0.2-nA sinusoid (middle), and noise alone (bottom).
\(E) Variance versus mean for firing rate. Diagonal line is prediction for a Poisson process. Red curve: prediction of Gaussian--rectification model, with no parameters allowed to vary to fit the data. Shaded area: region where the Gaussian--rectification model predicts the occurrence of 75% of the points.
\(F) Relation between firing rate and injected current. Curve is fit of rectification equation.
(G--I) As in (B--D), for injected current.
\(J) Variance versus mean for injected current.
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The Gaussian--rectification model captures the essence of this behavior. Once it is given the standard deviation of the noise and the relationship between injected current and firing rate ([Figure 8](#pbio-0020264-g008){ref-type="fig"}F), the Gaussian--rectification model makes a parameter-free prediction of the relationship between variance and mean ([Figure 8](#pbio-0020264-g008){ref-type="fig"}E, curve). This prediction is not perfect (it consistently underestimates firing-rate variance), but it does capture the most important behavior: that variance grows with the mean for firing rate ([Figure 8](#pbio-0020264-g008){ref-type="fig"}E, curve) but not for injected current ([Figure 8](#pbio-0020264-g008){ref-type="fig"}J, horizontal line).
Similar results were obtained when the stimulus parameters were changed to simulate synaptic inputs to a complex cell, or when the parameters of the spiking neuron were changed to simulate other cells measured in vitro, or even chosen randomly within reasonable bounds. As predicted, as long as the relationship between synaptic input and firing rate involved a threshold and the input noise was Gaussian, the variance grew with the mean for firing rate but not for injected current.
Role of Firing-Rate Encoder {#s2e}
---------------------------
Having validated the Gaussian--rectification model, we can now investigate the role of its parameters in determining the curves relating firing-rate variance and mean ([Figure 9](#pbio-0020264-g009){ref-type="fig"}). The model has four parameters (see [Materials and Methods](#s4){ref-type="sec"}): (1) the standard deviation σ of potential, (2) the firing threshold, *V~thresh~,* (3) the gain *k* of the relationship between firing rate and potential above threshold, and (4) the exponent *n* of this relationship. For the purpose of studying the model, we can assume, without loss of generality, that potential is unitless and has standard deviation σ = 1. Then, because *V~thresh~* can only determine the range of firing rates that is achieved, only *k* and *n* control the shape of the variance versus mean curves ([Figure 9](#pbio-0020264-g009){ref-type="fig"}).
::: {#pbio-0020264-g009 .fig}
Figure 9
::: {.caption}
###### Role of Parameters of Gaussian--Rectification Model
The standard deviation of potential was set to σ = 1, so that the shape of the curves relating firing-rate variance to firing-rate mean depends entirely on the gain *k* and the exponent *n* of the curves relating firing rate to membrane potential. The effects of these two parameters are explored: varying *n* (columns) and varying *k* (rows). Red curves: predictions of the Gaussian--rectification model; shaded areas: regions where the model predicts the occurrence of 75% of the points. Insets illustrate the corresponding curves relating firing rate to membrane potential.
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The gain *k* controls curve position, and the exponent *n* controls curve shape ([Figure 9](#pbio-0020264-g009){ref-type="fig"}). Increasing the gain *k* lifts the curves upward by twice as much as it shifts them rightward ([Figure 9](#pbio-0020264-g009){ref-type="fig"}, rows). These shifts occur because variance grows with *k* ^2^ and mean grows with *k.* Decreasing the exponent *n* causes the curves to saturate ([Figure 9](#pbio-0020264-g009){ref-type="fig"}, columns): The variance saturates to a plateau if *n* = 1 ([Figure 9](#pbio-0020264-g009){ref-type="fig"}, middle), and it reaches a maximal value and then decreases if *n* \< 1 (as in [Figure 9](#pbio-0020264-g009){ref-type="fig"}, left). Saturation occurs because when potential goes well above threshold, increases in mean potential cease to reveal ever larger portions of the Gaussian. If the curves relating firing rate to potential saturate (*n* \< 1), variations in potential are compressed into proportionally ever smaller variations in firing rate; the opposite occurs if the curves expand (*n* \> 1).
This analysis predicts that it should be fairly common for the firing-rate variance to saturate at high firing rates, possibly showing a plateau or even a decrease. Indeed, in the sample of V1 neurons recorded intracellularly, exponents are typically close to unity (*n* = 1.1 ± 0.6). Accordingly, a mild form of saturation is common in the plots of firing-rate variance and mean ([Figure 6](#pbio-0020264-g006){ref-type="fig"}B). To quantify the saturation, however, one needs reliable estimates of firing-rate variance. These estimates are not very reliable in the intracellular sample, which typically involves only a few hundred spikes per cell, leading to large clouds of points in the scatters of variance versus mean ([Figure 6](#pbio-0020264-g006){ref-type="fig"}B).
Variability of Extracellularly Recorded Firing Rates {#s2f}
----------------------------------------------------
To test the model\'s prediction rigorously, I considered a set of V1 responses obtained with extracellular recordings. Thanks to the large number of spikes (commonly \>4,000 per cell), measurements in this dataset yield more precise estimates of firing-rate variance over a wider range of firing rates than are available in the intracellular sample.
An analysis of firing-rate variance versus mean for these extracellularly recorded cells supports the predictions of the model ([Figure 10](#pbio-0020264-g010){ref-type="fig"}). Extracellular data do not afford independent estimates of gain *k* and exponent *n* of the transformation of potential into firing rate. I thus first computed the model predictions for a variety of combinations of *k* and *n* (such as those shown in [Figure 9](#pbio-0020264-g009){ref-type="fig"}). I then made Bayesian estimations of the values of *k* and *n* that maximize the likelihood of the data, while imposing a broad prior for *n* = 1.1, the median value measured intracellularly. The quality of these two-parameter fits was excellent ([Figure 10](#pbio-0020264-g010){ref-type="fig"}), of higher quality than could be obtained by fitting a line, the two-parameter "model" commonly used to describe data of this kind ([Figure 2](#pbio-0020264-g002){ref-type="fig"}A). Moreover, a number of cells exhibited the saturation in variance predicted by the model. The eight representative cells shown in [Figure 10](#pbio-0020264-g010){ref-type="fig"} are arranged in order of increasing exponent *n.* The first three (*n* = 0.9 to 1.0) show evident saturation in firing-rate variance as mean firing rate increases. The remaining five show a milder saturation, as expected from their higher exponents (*n* = 1.1 to 1.2). Saturation was common, as the median *n* was 1.06, with *n* \< 1 in 13/37 cells. Yet to my knowledge, except for an anecdotal account ([@pbio-0020264-Mechler1]), this common property had not been previously reported. It constitutes further support for the usefulness of the Gaussian--rectification model.
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Figure 10
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###### Relationship between Variance and Mean for Eight Cells Recorded Extracellularly in Cat V1, and Fits by the Gaussian--Rectification Model
For each mean firing rate, data point and error bars indicate mean ± 1 s.d. of the observed variance. Red curves and shaded areas are predictions of the model. Values of exponent *n* and gain *k* are reported next to each graph. Cells are arranged in order of increasing exponent *n.*
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Discussion {#s3}
==========
We have seen that a large amplification takes place between the trial-to-trial variability of synaptic input and that of firing rate: The variance of synaptic input is small compared to the dynamic range, and it is roughly constant. The amplification of variability arises from the threshold in the transformation of synaptic input into firing rate.
A Gaussian--rectification model attributes this amplification to very simple causes: approximately constant Gaussian noise in the input, and rectification due to threshold in the output. It indicates that firing-rate variance would grow with the mean even if the variance of synaptic input were constant. Both of the assumptions of the model, constant Gaussian noise and rectification, are borne out by the data. These assumptions are rather minimal, so they are naturally satisfied by more realistic models. For example, a realistic integrate-and-fire model behaves as predicted: Once it is given constant Gaussian noise in the input, it produces a firing-rate variance that grows with the firing-rate mean. Further support for the Gaussian--rectification model comes from its novel, and correct, prediction that firing-rate variance should saturate at high firing rates. In confirming this prediction I showed that the model can be used to account for variability in firing rate without knowledge of cellular properties. The extension to extracellular data is important because extracellular methods constitute the norm in visual neurophysiology, especially in awake animals, and are the ones used in previous studies of firing-rate variability.
These results further lengthen a list of properties of V1 neurons that are simply explained by the firing threshold. In addition to the amplification of trial-to-trial variability demonstrated here, these include the sharpening of tuning for stimulus direction and orientation ([@pbio-0020264-Jagadeesh1]; [@pbio-0020264-Carandini1]; [@pbio-0020264-Volgushev1]), the power-law behavior of firing rate at low contrast ([@pbio-0020264-Heeger1]; [@pbio-0020264-Anderson2]; [@pbio-0020264-Hansel1]; [@pbio-0020264-Miller1]), and even the establishment of the dichotomy between simple and complex cells ([@pbio-0020264-Carandini1]; [@pbio-0020264-Mechler2]; [@pbio-0020264-Priebe1]). It is remarkable that a mechanism as simple as the firing threshold can determine phenomena that might prima facie require more complex explanations at the level of the network.
Limitations of the Approach {#s3a}
---------------------------
One limitation of this study lies in the use of coarse potential. Coarse potential is not completely independent of firing rate: Even when spikes are removed and the traces smoothed, there is still a likely contribution of active conductances that has not been removed. Fortunately, this limitation strengthens my observation that coarse potential is not nearly as variable as firing rate: Any unwanted remaining echo of the spikes would make coarse potential more similar to firing rate and, thus, more variable. Therefore, in reality the variance of the actual synaptic input might be even less dependent on the mean than appears, for example, in [Figure 2](#pbio-0020264-g002){ref-type="fig"}B, [2](#pbio-0020264-g002){ref-type="fig"}E, and [2](#pbio-0020264-g002){ref-type="fig"}H. A partial control for these effects would be to perform some of the measurements while blocking spikes. However, blocking spikes would prevent the key measurements of this study, which require concurrent measurement of firing rate and estimation of synaptic input.
Another limitation of the approach is that I have mostly considered firing rates, not individual spikes. Unlike firing rates, individual spikes can occur only in integer numbers and are separated by refractory periods. These properties can become relevant to response variability, for example, if firing rates become so high that refractory period becomes a limiting factor ([@pbio-0020264-Kara1]). Such concerns are assuaged by the realistic integrate-and-fire model ([Figure 7](#pbio-0020264-g007){ref-type="fig"}), which shows an increase of firing-rate variance with the mean similar to that predicted by the Gaussian--rectification model. As to the saturation in firing-rate variance that was observed in some neurons, it invariably occurred at firing rates much lower than predicted from the refractory period.
A more serious limitation of coarse potentials and firing rates is that they make sense only in a limited range of time windows. The windows should be long enough to be able to contain more than one spike, and short enough that mean potential is approximately constant within the window. An informal analysis of the effect of time window indicates that a range of 5--20 ms is satisfactory. This range, however, might be appropriate only for V1 neurons; further investigations are required before applying these methods elsewhere.
Finally, a broader limitation of this work is that it concentrates on variability across trials, with little bearing on another form of variability, the one observed within trials in the irregularity of spike trains ([@pbio-0020264-Softky1]; [@pbio-0020264-de1]; [@pbio-0020264-Reich1]; [@pbio-0020264-Troyer1]; [@pbio-0020264-Buracas1]; [@pbio-0020264-Shadlen1]; [@pbio-0020264-Stevens1]). Thanks to recent advances, however, the cellular origins of this form of variability have been largely explained ([@pbio-0020264-Reich1]; [@pbio-0020264-Stevens1]). In particular, it is now clear that high variability within trials is to be expected if neurons receive synaptic inputs with slow temporal correlation ([@pbio-0020264-Svirskis1]). In fact, variability within trials is most evident with visual stimuli that provide a roughly stationary response, being greatly diminished with richer stimuli, which elicit highly precise responses ([@pbio-0020264-Bair1]; [@pbio-0020264-Reich1]; [@pbio-0020264-Buracas1]). Conversely, trial-to-trial variability is endemic, being present regardless of type of visual stimulus ([@pbio-0020264-Reich1]; [@pbio-0020264-Buracas1]).
Implications for Cortical Processing {#s3b}
------------------------------------
What computational advantage might cortical neurons derive by amplifying the variability that they receive in their input? Why reduce the signal/noise ratio? To answer these questions, it might help to clarify the sources of "signal" and "noise." The main source of variability in synaptic inputs to a V1 neuron is likely to be intracortical because thalamic responses are half as variable ([@pbio-0020264-Kara1]). Variability thus results largely from ongoing cortical activity ([@pbio-0020264-Arieli1]; [@pbio-0020264-Buracas1]; [@pbio-0020264-Tsodyks1]; [@pbio-0020264-Kenet1]). It appears to us as noise simply because it is not synchronized with stimulus onset. By contrast, the mean across trials of potential or firing rate constitutes a signal that is driven entirely by the stimulus.
The results of this study suggest that threshold affects the interaction between stimulus-driven activity and ongoing activity, turning it from additive to multiplicative. At the level of firing rates, this interaction is largely multiplicative because the variance of firing rate grows proportionally to the stimulus-driven mean firing rate. At the level of synaptic inputs, instead, this interaction is nearly additive because the variance of potential barely depends on the stimulus-driven mean potential. Indeed, additivity has been seen between local field potentials and ongoing voltage-sensitive dye signals ([@pbio-0020264-Arieli1]). We have seen that the rectification due to firing threshold is single-handedly responsible for the variability of firing rate and is, thus, responsible for turning a largely additive interaction into a multiplicative interaction.
It is thus conceivable that the computational role of firing threshold is to multiply stimulus-driven responses by ongoing cortical activity, i.e., to multiply what we call "signal" by what we call "noise." What may appear as lowering the signal/noise ratio can in fact be seen as a useful process, one that progressively amplifies the ongoing activity that ultimately guides our actions.
Materials and Methods {#s4}
=====================
{#s4a}
### Data acquisition in vivo {#s4a1}
Measurements in vivo were obtained in paralyzed, anesthetized cats. Methods for animal preparation and maintenance have appeared elsewhere ([@pbio-0020264-Carandini1]) and were approved by the Animal Care and Use Committees at Northwestern University and at the Smith-Kettlewell Eye Research Institute.
The 22 cells recorded intracellularly belong to a sample that has been analyzed in two previous studies by [@pbio-0020264-Carandini1] and by [@pbio-0020264-Anderson1]. These studies describe in detail the recording methods, which involved the whole-cell patch technique. The electrical noise associated with this technique is commonly \<0.1 mV (as judged from records obtained after losing the patch). From the sample I excluded a few cells that produced less than ten spikes per block of stimuli, or that failed to satisfy other minimal requirements (firing rate \>2 spikes/s, spike height \>10 mV). Stimuli were optimal gratings drifting in 12 directions in 30° intervals, and a blank screen of uniform gray. The resting potential *V~rest~* was taken as the mean potential measured with the blank screen. Coarse potential traces were obtained from traces of membrane potential sampled at 4 kHz by removing spikes ([@pbio-0020264-Lankheet1]) and by applying a low-pass filter with a cutoff of 50 Hz ([@pbio-0020264-Carandini1]; [@pbio-0020264-Volgushev1]). The same low-pass filter was applied to spike trains sampled at 4 kHz to yield firing rate. Both coarse potential and firing rate were subsampled at 100 Hz.
The 37 neurons recorded extracellularly are part of a study of the organization of receptive fields and suppressive surrounds in area V1 ([@pbio-0020264-Bonin1]). This dataset was chosen because it involved lengthy experiments that yielded many thousands of spikes per cell at a variety of firing rates. Recordings were made with quartz-coated platinum/tungsten microelectrodes; methods for data acquisition and animal maintenance have been described by [@pbio-0020264-Freeman1]. Stimuli were drifting gratings presented at the optimal orientation, spatial frequency, and temporal frequency, and enclosed in one of 66 possible windows. The windows were stationary square gratings with variable period and orientation. Stimuli typically lasted 2 s, and each block of stimuli was typically repeated three to six times. Firing rates were extracted from the spike train by low-pass filtering at 50 Hz and were subsampled at 100 Hz.
### Data acquisition in vitro {#s4a2}
Measurements in vitro were made with sharp intracellular electrodes from slices of guinea pig visual cortex. Methods for this preparation were approved by the Animal Care and Use Committee at New York University. The cells are part of the dataset presented by [@pbio-0020264-Carandini2]; the cell in [Figure 7](#pbio-0020264-g007){ref-type="fig"} is the one whose responses are extensively illustrated in that study (cell 19s2).
### Rectification model {#s4a3}
The relation between potential *V* and firing rate *R* (e.g., [Figure 1](#pbio-0020264-g001){ref-type="fig"}H) was fitted with an extension of the rectification model ([@pbio-0020264-Mechler2]), where *R*(*V*) = *k*\[*V*−*V~thresh~\]^*n*^~+~* , with \[.\]~+~ indicating rectification, *k* a proportionality factor, and *n* an exponent. Fitted parameters were *V~thresh~* = −55.3 mV, *k* = 16.7, and *n* = 1.2 for the simple cell in [Figure 1](#pbio-0020264-g001){ref-type="fig"}, and *V~thresh~* = −46.6 ± 10.5 mV, *k* = 12.4 ± 7.9, and *n* = 1.1 ± 0.6 for the whole intracellular population (*N* = 22). The distance between *V~thresh~* and *V~rest~* was 5.1 mV for the simple cell in [Figure 1](#pbio-0020264-g001){ref-type="fig"}, and 8.0 ± 4.2 mV for the population.
### Gaussian--rectification model {#s4a4}
The mean potential *V* ~mean~ in response to a stimulus was defined as the mean across trials of coarse potential.
In the Gaussian--rectification model, the probability of observing a firing rate *r* ([Figure 4](#pbio-0020264-g004){ref-type="fig"}A) given a mean potential *V~mean~* is
where *R*(*V*) is the relation between firing rate and potential *V* ([Figure 1](#pbio-0020264-g001){ref-type="fig"}H), and *N*\[*V~mean~, σ*\] is the probability distribution of potential ([Figure 5](#pbio-0020264-g005){ref-type="fig"}B), a Gaussian with mean *V~mean~* and standard deviation *σ.* The value of *p*(*r*) depends on whether *r* is zero or positive:
for *r* \> 0, and
for *r* = 0. The first expression is simply the value of the Gaussian for *V* = *R^−1^*(*r*). The second expression is the area of the portion of Gaussian that is below threshold (*erf* is the error function).
These expressions allow maximum likelihood estimation of model parameters from measured firing rates. When parameters of the relation between firing rate and potential *R*(*V*) are obtained independently (in intracellular recordings; [Figure 1](#pbio-0020264-g001){ref-type="fig"}H), the only free parameter was the standard deviation σ of potential. Across the intracellular population, the average value of σ obtained by the fits was 5.4 ± 2.0 mV (s.d., *N* = 22). The required σ was always larger (by 2.1 ± 1.5 mV) than the standard deviations observed when *V~mean~* = *V~rest~,* but it was comparable (larger by only 0.9 ± 1.6 mV) to the standard deviations observed when *V~mean~* = *V~thresh~.*
### Statistics {#s4a5}
Let *V* ~mean~(*t*) be the mean potential at time *t* from stimulus onset. Because the sample rate is 100 Hz, each time sample corresponds to a 10-ms interval. Of course, *V~mean~*(*t*) depends on the stimulus. To simplify the notation, however, consider the case of a single stimulus.
Distributions for potential at a given mean potential ([Figure 5](#pbio-0020264-g005){ref-type="fig"}B) were computed as follows: (1) Select a value of interest, *v* (e.g., *v* = −55 mV; [Figure 5](#pbio-0020264-g005){ref-type="fig"}B, *a*); (2) find the times (*t~k~*) when the mean potential *V~mean~*(*t~k~*) is within 2 mV of *v;* (3) pooling across trials *j,* look at the distribution of potential \[*V~j~*(*t~k~*)\] (e.g., [Figure 5](#pbio-0020264-g005){ref-type="fig"}B, *a*).
Distributions for *z*-scores (normalized deviations from the mean) of potential ([Figure 6](#pbio-0020264-g006){ref-type="fig"}A, [6](#pbio-0020264-g006){ref-type="fig"}C, and [6](#pbio-0020264-g006){ref-type="fig"}E) were computed as follows: (1) Divide the range of values of *V~mean~* in 1-mV intervals, with centers (*v~i~*); (2) for each interval *i,* find the set of times (*t~ik~*) when the mean potential is in the *i*-th bin; (3) pooling across trials *j,* compute *σ~i~,* the standard deviation of *V~j~*(*t~ik~*); (4) transform each *V~j~*(*t~ik~*) into a *z*-score: z*~ijk~*= \[*V~j~*(*t~ik~*) − *v~i~*\]/*σ~i~;* (5) look at the distribution of (z*~ijk~*) (e.g., [Figure 6](#pbio-0020264-g006){ref-type="fig"}A).
### Enhanced integrate-and-fire model {#s4a6}
The enhanced integrate-and-fire model was derived in collaboration with Davide [@pbio-0020264-Boino1] by simplifying a model by [@pbio-0020264-Wang1]. The model neuron has a single compartment with membrane equation
where the currents are:
with *t~spike~* the time of the last spike. *Ca*(*t*) is the (unitless) calcium concentration:
where the sum extends over all spikes with *t~spike~ \< t.*
Spikes result from stereotyped conductances *g~Na~*(*t*) and *g~K~*(*t*) derived from Hodgkin--Huxley equations and are scaled to approximate the spikes from the recorded neuron. They occur when *V~m~* exceeds a threshold, which depends on the time since the last spike:
The reversal potentials for sodium and potassium were set to *V~k~* = −80 mV and *V~Na~* = 55 mV. Passive parameters of the membrane (*C~m~* = 120 pF, *g~leak~* = 12.4 nS, *V~leak~* = −60.3 mV) were obtained by fitting the membrane potential responses to sinusoids. The remaining parameters (*g~AHP~* = 23.0 nS, *Ca~50~* = 10, τ*~Ca~* = 200 ms, V~thresh~ = −43.5 mV, τ*~thresh~* = 36 ms) were obtained by a search algorithm aimed at maximizing the quality of the predictions for firing rate.
Thanks to Jeffrey Anderson and David Ferster for participating in intracellular experiments (supported by National Eye Institute grant NEI-EY04726 to David Ferster), to Vincent Bonin and Valerio Mante for participating in extracellular experiments, and to Davide Boino for help with the integrate-and-fire model (supported by the Swiss National Science Foundation). This work has benefited from comments by Michael Shadlen, Massimo Scanziani, Moshe Gur, Nicholas Priebe, and David Ferster, by the members of my laboratory, and by attendees of a meeting of the Sloan-Swartz Centers for Theoretical Neurobiology (La Jolla, California, July 2003), whom I thank collectively. Supported by the James S. McDonnell Foundation 21st Century Research Award in Bridging Brain, Mind and Behavior.
**Conflicts of interest.** The author has declared that no conflicts of interest exist.
**Author contributions.** MC participated in the experiments, analyzed the data, and wrote the paper.
Academic Editor: Charles Stevens, Salk Institute for Biological Studies
Citation: Carandini M (2004) Amplification of trial-to-trial response variability by neurons in visual cortex. PLoS Biol 2(9): e264.
V1
: primary visual cortex
| PubMed Central | 2024-06-05T03:55:46.804888 | 2004-8-24 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509408/",
"journal": "PLoS Biol. 2004 Sep 24; 2(9):e264",
"authors": [
{
"first": "Matteo",
"last": "Carandini"
}
]
} |
PMC509409 | Introduction {#s1}
============
The androgen receptor (AR) is the cellular mediator of the actions of the hormone 5-α dihydrotestosterone (DHT). Androgen binding to AR leads to activation of genes involved in the development and maintenance of the male reproductive system and other tissues such as bone and muscle. However, it is the pivotal role of AR in the development and progression of prostate cancer that has led to increasing interest in this nuclear receptor. Presently, hormone-dependent prostate cancer is treated with a combination of strategies that reduce circulating levels of androgens, such as the administration of antiandrogens that compete for the androgen-binding pocket in the core of the C-terminal ligand-binding domain (LBD). The benefits of these treatments are typically transient, with later tumor growth associated with increases in expression levels of AR or its cofactors, or mutations that render AR resistant to antiandrogens ([@pbio-0020274-Gregory1]; [@pbio-0020274-Culig1]; [@pbio-0020274-Lee1]). Alternative approaches to inhibiting AR transcriptional activity may therefore lie in disrupting critical protein associations the receptor needs for full function.
The precise details of how AR binds the dozens of coregulator proteins reported to associate with different regions of AR in vivo remain poorly understood ([@pbio-0020274-Lee1]). Many nuclear receptors activate transcription by binding short leucine-rich sequences conforming to the sequence LxxLL (where "x" is any amino acid), termed nuclear receptor (NR) boxes, which are found within a variety of NR coactivators including the p160 family. Hormone binding to the LBD stabilizes the C-terminal helix of the receptor, helix 12, in a conformation that completes a binding surface for these LxxLL motifs ([@pbio-0020274-Darimont1]; [@pbio-0020274-Nolte1]; [@pbio-0020274-Shiau1]; [@pbio-0020274-Bledsoe1]). The structural elements composing this binding interface, consisting of helices 3, 4, 5, and 12 of the receptor, are synonymous with a previously defined hormone-dependent activation function that lies within the LBD termed activation function (AF)--2. Association of p160 coactivators allows the recruitment and assembly of a number of other cofactors that together modulate the state of chromatin and interactions with components of the basal transcription machinery to initiate transcription ([@pbio-0020274-Glass1]).
AR, however, utilizes multiple mechanisms to activate gene transcription. Generally, AR activity is dependent on contributions from multiple transactivation functions that lie within the N-terminal domain (NTD) collectively called AF-1. Although the AR AF-2 can bind to a restricted set of LxxLL motifs ([@pbio-0020274-Ding1]; [@pbio-0020274-He2]; [@pbio-0020274-Needham1]) and is relatively potent ([@pbio-0020274-Wang1]), it usually displays weak independent activity at typical androgen-regulated genes, with significant activity observed only in the presence of high levels of p160 coactivators, as detected in some prostate cancers ([@pbio-0020274-He2]; [@pbio-0020274-Gregory1]). Instead, the AR AF-2 exhibits a distinct preference among NRs for phenylalanine-rich motifs conforming to the sequence FxxLF ([@pbio-0020274-He3]; [@pbio-0020274-He1]). Such motifs have been identified in the AR NTD and in an AR cognate family of coactivators that includes AR-associated protein (ARA) 54, ARA55, and ARA70 ([@pbio-0020274-He3], [@pbio-0020274-He5]; [@pbio-0020274-Lee1]). The NTD FxxLF motif (residues 23--27) mediates a direct, interdomain, ligand-dependent interaction between the NTD and LBD (N/C interaction) that is thought to facilitate dimerization, stabilize androgen binding, and possibly regulate AF-1 and AF-2 activity ([@pbio-0020274-Langley1]; [@pbio-0020274-He3]). In addition, the NTD also contains a related hydrophobic motif, WxxLF (residues 433--437), that nucleates formation of an alternative N/C interaction that may serve to inhibit AR activity ([@pbio-0020274-He3], [@pbio-0020274-He4]; [@pbio-0020274-Hsu1]).
Presently, how the AR AF-2 surface can accommodate residues with bulky aromatic side chains and distinguish FxxLF motifs from LxxLL motifs is not known. To understand the structural basis of this unusual coactivator recognition preference, we characterized the full repertoire of interacting sequences using phage display to define amino acids preferred at the AR coactivator binding interface. Crystal structures of the AR LBD in complex with several phage display--derived peptides reveal the structural basis of FxxLF motif specificity and an induced fit of the receptor that allows accommodation of other related hydrophobic motifs. Comparisons of the structures suggest strategies for the design of AR coactivator antagonists.
Results {#s2}
=======
AR Preference for Aromatic Groups in Coregulator Recognition {#s2a}
------------------------------------------------------------
Phage display has been used to study coactivator recognition specificity and to identify coactivator motif sequence variants preferred by the estrogen receptor (ER), thyroid hormone receptor (TR) β, and most recently AR ([@pbio-0020274-Chang1]; [@pbio-0020274-Norris1]; [@pbio-0020274-Paige1]; [@pbio-0020274-Northrop1]; [@pbio-0020274-Hsu1]). Using phage display, we screened more than 2 × 10^10^ randomized peptides against DHT-bound AR LBD. Selections identified sequences containing hydrophobic motifs that were primarily aromatic in character, consistent with another recent study ([@pbio-0020274-Hsu1]) ([Figure 1](#pbio-0020274-g001){ref-type="fig"}). Of these aromatic motifs, FxxLF and related motifs with substitutions of phenylalanine or tryptophan for leucine at positions +1, +5, or both, dominated the selections. (Peptide residues are numbered in reference to the first hydrophobic residue of the core motif, which is numbered +1. Residues preceding the first hydrophobic residue are numbered negatively in descending order starting with −1.) Substitutions of tyrosine at the +5 position were also observed, but to a much lesser extent (unpublished data). At the +4 position, valines, methionines, and even the aromatic residues phenylalanine and tyrosine were observed ([Figure 1](#pbio-0020274-g001){ref-type="fig"}; unpublished data). In general, LxxLL motifs were not selected. The LxxLL motif shown in [Figure 1](#pbio-0020274-g001){ref-type="fig"} was derived from prior phage selections with ER and subsequently demonstrated to bind AR in FRET-based screens in vitro (unpublished data).
::: {#pbio-0020274-g001 .fig}
Figure 1
::: {.caption}
###### AR LBD--Interacting Peptides Selected by Phage Display
Hydrophobic residues of the core motif are highlighted in yellow. Residues in bold were ordered in electron density maps.
:::
:::
Preliminary characterization of the subset of AR-interacting peptides shown in [Figure 1](#pbio-0020274-g001){ref-type="fig"} confirmed that each competed for binding of in vitro translated AR cofactors to bacterially expressed AR LBD in pulldown assays, and generally did so with modestly improved efficiency relative to the native FxxLF motif from the AR NTD and significantly greater efficiency than a native LxxLL motif from glucocorticoid receptor-interacting protein 1 (GRIP1) NR box 3 (P. Webb, personal communication). The equilibrium dissociation constants *(K~d~)* were directly determined for the interaction between the AR LBD and FxxLF and LxxLL peptides and one variant tryptophan-containing peptide, FxxLW, using surface plasmon resonance ([Table 1](#pbio-0020274-t001){ref-type="table"}). The *K~d~* for FxxLF was 1.1 μM, similar to the affinities of physiologically derived FxxLF motifs determined previously by isothermal titration calorimetry ([@pbio-0020274-He1]). The affinity of LxxLL was less than 2-fold weaker, with a *K~d~* of 1.8 μM, but more than three times stronger than the tightest binding p160-derived LxxLL motif, NR box 3 of transcriptional intermediary factor 2 (TIF2) ([@pbio-0020274-He1]). Surprisingly, the affinity of FxxLW, with a *K~d~* of 920 nM, was slightly better than FxxLF, in spite of the presence of the tryptophan residue at the +5 position. Together, our results are consistent with the notion that the phage display peptides interact with the same AR surface that binds FxxLF and LxxLL motifs in native cofactors, and that they do so with similar or improved affinities relative to their natural counterparts.
::: {#pbio-0020274-t001 .table-wrap}
Table 1
::: {.caption}
###### Rate and Dissociation Constants for the Interaction between the AR LBD and Selected Peptides
:::
Surface plasmon resonance data were best fit using the two-state conformational change model ([@pbio-0020274-Warnmark1], [@pbio-0020274-Warnmark2]). Dissociation constants were calculated from rate constants as described previously ([@pbio-0020274-Warnmark1])
:::
One Site Fits All {#s2b}
-----------------
To understand the binding mode of different AR coactivators, we determined the crystal structures of DHT-bound AR LBD without peptide and in complex with each of the seven peptides listed in [Figure 1](#pbio-0020274-g001){ref-type="fig"}. All complexes crystallized in the space group *P*2~1~2~1~2~1~ with one molecule per asymmetric unit and unit cell dimensions similar to those observed in previous AR LBD crystal structures ([@pbio-0020274-Matias1]; [@pbio-0020274-Sack1]). Overall structural features of the complexes are shown in [Figure 2](#pbio-0020274-g002){ref-type="fig"}. Peptides assumed short α helical conformations centered on the core hydrophobic motif and bound in a solvent channel relatively free of crystal contacts on a groove formed by helices 3, 4, 5, and 12 of the receptor ([Figure 2](#pbio-0020274-g002){ref-type="fig"}A). Detailed data collection and refinement statistics, as well as buried surface areas for each complex, are listed in [Table 2](#pbio-0020274-t002){ref-type="table"}. The structures confirm previous suggestions that AR utilizes a single binding interface for LxxLL and noncanonical aromatic-rich motifs ([@pbio-0020274-He3], [@pbio-0020274-He4]). Only side chains move to accommodate the array of peptides, sometimes considerably, with the unbranched side chains of Lys720, Met734, and Met894 making the largest conformational changes upon binding of peptide ([Figure 2](#pbio-0020274-g002){ref-type="fig"}B).
::: {#pbio-0020274-g002 .fig}
Figure 2
::: {.caption}
###### A Structural Profile of the AR Coactivator Binding Interface
AR--peptide complexes are colored as follows: FxxLF, yellow; FxxLW, orange; WxxLF, wheat; WxxVW, purple; FxxYF, green; FxxFF, blue; LxxLL, pink; unbound, grey.
\(A) Cα trace of the peptides superimposed onto the AF-2. For clarity only the LBD of AR--FxxLF is shown.
\(B) Superposition of the LBD of the AR--peptide complexes in the region of the coactivator interface. Backbone atoms are shown as a Cα trace. Side chains of residues composing the interface are shown as sticks.
\(C) Hydrophobic side chains of the core motif superimposed as in (B).
:::
:::
::: {#pbio-0020274-t002 .table-wrap}
Table 2
::: {.caption}
###### Summary of Structures and Crystallographic Statistics
:::
^a^Numbers in parenthesis denote values for the highest resolution shell
^b^ *R* ~sym~ = Σ\|*I* − \<*I*\>\| / Σ (*I*)
^c^ *R* ~cryst~ = Σ \|*F* ~o~ − *F* ~c~\| / Σ \|*F* ~o~\|, where *F* ~o~ and *F* ~c~ are observed and calculated structure factors, respectively; *R* ~free~ was calculated similarly with a randomly selected set of reflections consisting of 5% of total reflections that were excluded from refinement
^d^Values for side chain atoms only
:::
FxxLF {#s2c}
-----
The mechanisms that permit AR to accommodate motifs with bulky phenylalanine residues were assessed in a crystal structure of the AR LBD in complex with the FxxLF peptide. The FxxLF peptide recapitulates the binding mode of p160-derived LxxLL motifs to other nuclear receptors ([@pbio-0020274-Darimont1]; [@pbio-0020274-Nolte1]; [@pbio-0020274-Shiau1]; [@pbio-0020274-Bledsoe1]). The peptide forms a short α helix whose hydrophobic face, composed of Phe+1, Leu+4, and Phe+5, binds an L-shaped groove formed by helices 3, 4, 5, and 12 of the LBD that is composed of three subsites that accommodate each hydrophobic residue ([Figures 2](#pbio-0020274-g002){ref-type="fig"}A and [3](#pbio-0020274-g003){ref-type="fig"}A). The conserved charged residues at either end of the cleft, Lys720 and Glu897, the so-called charge clamp residues, make electrostatic interactions with the main chain atoms at the ends of the peptide helix: Lys720 with the carbonyl group of Phe+5, and Glu897 with the amide nitrogens of Phe+1 and Arg−1 ([Figure 3](#pbio-0020274-g003){ref-type="fig"}C). Glu897 also interacts with the side chain of Arg−1. The two interior residues of the motif, Glu+2 and Ser+3, are solvent exposed and do not interact with the receptor.
::: {#pbio-0020274-g003 .fig}
Figure 3
::: {.caption}
###### Interactions of FxxLF and LxxLL with the AR LBD
(A and B) FxxLF (A) and LxxLL (B) bound to the AR AF-2 interface. FxxLF and LxxLL are shown as yellow and pink Cα coils, respectively. Helices 3, 4, and 5 of the LBD are shown as blue ribbons; Helix 12 is shown in green. LBD residues interacting with peptides are depicted as white sticks. For clarity only peptide side chains making significant interactions with the LBD are shown.
(C and D) Hydrogen-bonding interactions between backbone atoms of FxxLF (C) and LxxLL (D) with Glu897 of the LBD. Peptide alpha carbons are labeled.
:::
:::
Comparison of AR alone and AR in complex with FxxLF (and other aromatic-rich peptides described below) reveals that the AF-2 cleft reorganizes to accommodate the bulky peptide side chains (see [Figures 2](#pbio-0020274-g002){ref-type="fig"}B and [4](#pbio-0020274-g004){ref-type="fig"}). The unbranched side chains of Lys720 and Met734 move from an extended conformation over the +5 pocket to one almost perpendicular to the surface of the protein. The pockets for Phe+1 and Phe+5 are arranged in a line, forming a deep, extended cleft on the LBD spanning the length of the two side chains on the face of the peptide helix (see [Figures 3](#pbio-0020274-g003){ref-type="fig"}A and [4](#pbio-0020274-g004){ref-type="fig"}B). Phe+1, almost entirely solvent inaccessible, binds face down at the base of this groove, making hydrophobic contacts with Leu712, Val716, Met734, Gln738, Met894, and Ile898, which define the +1 pocket. The top of the groove, composed of Val716, Lys720, Phe725, Ile737, Val730, Gln733, and Met734, narrows to form the +5 pocket. Met734 and the aliphatic portion of Lys720 constrict this subsite, forming van der Waals interactions with opposite faces of the Phe+5 benzyl ring. Together, the +1 and +5 residues are almost entirely solvent inaccessible. In contrast, Leu+4 binds in a shallow hydrophobic patch consisting of Leu712 and Val716 lined at the ridges by Val713 and Met894 and is largely solvent exposed.
::: {#pbio-0020274-g004 .fig}
Figure 4
::: {.caption}
###### Induced Fit of the AR AF-2 Interface
Surface representations of the AR AF-2 interface. The unbound structure is shown in (A), the FxxLF bound in (B), and the LxxLL bound in (C). Side chains of the hydrophobic residues of the core motifs of FxxLF and LxxLL are shown as spheres.
:::
:::
LxxLL {#s2d}
-----
The preference of AR for motifs with aromatic groups over leucine-rich motifs was assessed with a crystal structure of the AR LBD in complex with the LxxLL peptide. The structure reveals similarities between the binding modes of the LxxLL and FxxLF motifs to AR, and other LxxLL motifs to other nuclear receptors. The LxxLL motif adopts a helical conformation, and interactions of the motif with the AF-2 cleft are predominantly hydrophobic, with the three leucine residues of the motif contributing most of the interactions. However, significant differences can be seen between the binding mode of the LxxLL motif to AR and that of p160-derived LxxLL motifs to other nuclear receptors. First, flanking residues were largely disordered, with only two N-terminal flanking residues and one C-terminal residue visible in electron density maps (see [Figures 1](#pbio-0020274-g001){ref-type="fig"} and [3](#pbio-0020274-g003){ref-type="fig"}B). This contrasts with extended structures seen in the p160-derived LxxLL motifs in complex with their cognate receptors ([@pbio-0020274-Darimont1]; [@pbio-0020274-Nolte1]; [@pbio-0020274-Shiau1]; [@pbio-0020274-Bledsoe1]). Second, the LxxLL peptide backbone forms hydrogen bonds with only one of the two conserved charge clamp residues, Lys720. A shift in the position of the LxxLL peptide helix precludes direct interactions with Glu897 (see [Figures 2](#pbio-0020274-g002){ref-type="fig"}A and [3](#pbio-0020274-g003){ref-type="fig"}D). This shift results from changes in the geometry of the +1 and +5 subsites mediated by Met734, which moves 2.5Å toward the +1 pocket (see [Figures 2](#pbio-0020274-g002){ref-type="fig"}B and [4](#pbio-0020274-g004){ref-type="fig"}C) and enables binding of a leucine at the +5 subsite by a simultaneous widening and shallowing of the pocket. This movement of Met734 causes displacement of the +1 residue, resulting in a rotation of the peptide helix away from helix 12, toward helix 3. A slight translation of the peptide helix also occurs away from helix 12 because of the shorter side chain length of leucine (see [Figure 2](#pbio-0020274-g002){ref-type="fig"}A).
Side chains of residues flanking the first leucine of the motif make additional hydrophobic interactions with the AR surface (see [Figure 3](#pbio-0020274-g003){ref-type="fig"}B). Trp+2 reaches over Met734, clamping the methionine in between itself and Leu+1. Leu−1 extends over Met894, abutted against Glu893. These interactions likely explain the moderate affinity of AR for this particular LxxLL motif despite suboptimal complimentarity with the residues of the core motif (as discussed below) and the loss of main chain interactions with Glu897.
WxxLF, FxxLW, and WxxVW {#s2e}
-----------------------
To understand how the AR AF-2 accommodates tryptophan residues, structures of AR in complex with peptides containing tryptophan substitutions at the +1 or +5 position, or both, were determined ([Figure 5](#pbio-0020274-g005){ref-type="fig"}). Surprisingly, WxxLF, analogous to the only tryptophan-containing motif known in vivo, WHTLF in the AR NTD, was relatively disordered, with the peptide displaying the highest B-factor and least well defined density, suggesting that it binds with the lowest affinity ([Table 2](#pbio-0020274-t002){ref-type="table"}). Nonetheless, each of the tryptophan peptides adopted similar helical conformations. As described above for the LxxLL motif, substitutions at the +1 and +5 positions for non-phenylalanine residues result in shifts of the peptide helix (see [Figure 2](#pbio-0020274-g002){ref-type="fig"}A). Consequently, backbone interactions with Lys720 are maintained, but interactions with the other charge clamp residue, Glu897, are lost. Once again, however, flanking residues within the peptide make additional contacts with the AR surface, and, unlike the LxxLL peptide, these contacts include Glu897. In FxxLW and WxxVW, the −2 serine ([Figure 6](#pbio-0020274-g006){ref-type="fig"}) forms a bidentate hydrogen-bonding interaction, making hydrogen bonds to both Glu897 and the backbone amide group of the +2 residue. Ser−2 of WxxLF similarly interacts with Glu897, but is too distant for helical-capping interactions with the +2 amide group. Instead, Glu893, in a more typical interaction with the +1 amide nitrogen, caps the WxxLF helix ([Figure 6](#pbio-0020274-g006){ref-type="fig"}B). Thus, tryptophan substitutions are tolerated, but they induce a shift in the peptide backbone that precludes interactions with one of the charge clamp residues. This suboptimal interaction is compensated partially by interactions of flanking residues with the AR surface.
::: {#pbio-0020274-g005 .fig}
Figure 5
::: {.caption}
###### Interactions of the Tryptophan Motifs with the AR LBD
FxxLW (A), WxxLF (B), and WxxVW (C) bound to the AR AF-2 interface. FxxLW, WxxLF, and WxxVW are shown as orange, beige, and purple Cα coils, respectively. The LBD is depicted as in [Figure 3](#pbio-0020274-g003){ref-type="fig"}.
:::
:::
::: {#pbio-0020274-g006 .fig}
Figure 6
::: {.caption}
###### Interactions of Ser−2 with Glu897
Interactions between Ser−2 of the peptides (A) FxxLW, (B) WxxLF, (C) WxxVW, and (D) FxxFF and Glu897 of the LBD. Peptide alpha carbons are labeled.
:::
:::
FxxFF and FxxYF {#s2f}
---------------
Finally, effects of substitutions at the +4 position were assessed in structures of AR in complex with peptides containing FxxFF and FxxYF motifs ([Figure 7](#pbio-0020274-g007){ref-type="fig"}). Surprisingly, the binding mode of FxxFF to AR resembled that of the tryptophan peptides more closely than the binding mode of FxxLF (see [Figures 2](#pbio-0020274-g002){ref-type="fig"}A and [7](#pbio-0020274-g007){ref-type="fig"}B). Like the tryptophan peptides, interactions with Glu897 are mediated by Ser−2 instead of the peptide backbone (see [Figure 6](#pbio-0020274-g006){ref-type="fig"}D). Deviations from ideal helical geometry allow Phe+4 to bind facedown in the +4 pocket with the benzyl ring stacked against Val713.
::: {#pbio-0020274-g007 .fig}
Figure 7
::: {.caption}
###### Interactions of FxxYF and FxxFF with the AR LBD
FxxYF (A) and FxxFF (B) bound to the AR AF-2 interface. FxxYF and FxxFF are shown as yellow and orange Cα coils, respectively. The LBD is depicted as in [Figure 3](#pbio-0020274-g003){ref-type="fig"}.
:::
:::
By contrast, the conformation of FxxYF was the closest to FxxLF (see [Figure 2](#pbio-0020274-g002){ref-type="fig"}A). Other than FxxLF, only FxxYF makes direct backbone interactions with Glu897. Unlike the facedown orientation of Phe+4 observed in the FxxFF peptide, Tyr+4 is bound edgewise into the shallow +4 pocket, making interactions with Val713, Val716, and the aliphatic portion of Lys717. FxxYF was the most ordered of all the peptides, with 12 out of 15 residues observed in the electron density (see [Figures 1](#pbio-0020274-g001){ref-type="fig"} and [7](#pbio-0020274-g007){ref-type="fig"}A). Significant interactions were observed involving residues other than hydrophobic residues of the motif. Lys+2 and Met+6 are predominantly solvent exposed, extending out over the protein surface. Met+6 is bound on top of Phe+5, while Lys+2 makes a water-mediated hydrogen bond with Asp731. Thr−3 of the peptide defines a new subsite, with the hydroxyl group forming a hydrogen bond to Gln738 and the methyl group making hydrophobic contacts in a pocket formed by Glu897, Ile898, and Val901. Similar interactions were observed in the glucocorticoid receptor (GR)--TIF2 complex involving the −3 glutamine of the TIF2 NR box 3 motif ([@pbio-0020274-Bledsoe1]). However a valine to asparagine substitution at the residue corresponding to 901 in AR creates a pocket with a more polar character in GR ([Figure 8](#pbio-0020274-g008){ref-type="fig"}).
::: {#pbio-0020274-g008 .fig}
Figure 8
::: {.caption}
###### Sequence Alignment of the AF-2 Region of NRs
Residues composing the coactivator interface of AR are highlighted in yellow. The absolutely conserved glutamate and lysine composing the charge clamp are highlighted in pink and blue, respectively. Residue numbering is that of AR.
:::
:::
Restrictions of the Three Subsites {#s2g}
----------------------------------
Together, the structures described above permit an assessment of the way that individual subsites of the AR AF-2 cleft accommodate hydrophobic groups. The indole rings of tryptophan and the phenyl rings of phenylalanine fit into their pockets analogously with the +1 and +5 residues bound facedown and edgewise, respectively, into the AF-2 cleft. On the other hand, the position of the +4 residue is variable, with binding in this shallow pocket largely dictated by the position of the peptide backbone caused by the bound conformations of the +1 and +5 residues (see [Figure 2](#pbio-0020274-g002){ref-type="fig"}C). Small shifts in the position of the N-terminal of helix 12 can be seen, which reposition Met894 for more optimal contacts with +4 residues bound at that subsite (see [Figure 2](#pbio-0020274-g002){ref-type="fig"}B).
The binding mode detected in the +1 pocket is the most conserved of the three hydrophobic subsites (see [Figure 2](#pbio-0020274-g002){ref-type="fig"}C). The benzyl moiety of the indole side chains superimpose with the corresponding benzyl side chains of the phenylalanine-rich motifs, effectively mimicking interactions of a phenylalanine residue. However, the presence of a hydrogen-bonding partner on the indole side chain enables an additional polar interaction not seen in the phenylalanine-rich motifs between the indole nitrogen and Gln738 (see [Figure 5](#pbio-0020274-g005){ref-type="fig"}B). Unexpectedly, this additional interaction in the +1 pocket does not occur with Trp+1 of WxxVW (see [Figure 5](#pbio-0020274-g005){ref-type="fig"}C). While similarly distanced to make the same interaction, the plane of the indole ring is rotated about 20° relative to that of WxxLF, causing it to be at a poor angle for strong hydrogen bonding to Gln738.
Binding of tryptophans in the +5 pocket is slightly more variable (see [Figure 2](#pbio-0020274-g002){ref-type="fig"}C). Trp+5 of WxxVW is bound similarly to phenylalanine residues at the same position. Only the six-membered ring of the indole group is fully buried in the pocket. The five-membered ring of the indole side chain sticks out, solvent exposed. In contrast, the +5 indole group of FxxLW is rotated almost 90°, resulting in burial of both rings of the indole group, as well as the formation of a strong hydrogen bond between the indole nitrogen and Gln730 (see [Figure 5](#pbio-0020274-g005){ref-type="fig"}A). Binding in this orientation appears to be highly favorable, as the FxxLW peptide deviates from helical geometry at the +5 position to do so.
Discussion {#s3}
==========
The crystal structures reported here reveal how AR binds coactivator motifs with bulky aromatic hydrophobic groups and permit construction of a profile of the AR coregulator interface (see [Figure 2](#pbio-0020274-g002){ref-type="fig"}). In some ways, this interface resembles those of other nuclear receptors: it is an L-shaped hydrophobic cleft comprised of three distinct subsites that bind hydrophobic groups at the +1, +4, and +5 positions in cognate peptides. Moreover, the so-called charge clamp residues (Lys720 and Glu897) bracket the cleft. Nonetheless, the AR coregulator recognition site is unique in that it rearranges upon motif binding to form a long, deep, and narrow groove that accommodates aromatic residues at the +1 and +5 positions ([Figure 9](#pbio-0020274-g009){ref-type="fig"}). Sequence alignments of AR with other NRs suggest that a unique combination of substitutions at Val730, Met734, and Ile737 combine to permit the formation of a smoother, flatter interaction surface that displays a higher complimentarily to aromatic substituents than to branched aliphatic (see [Figure 8](#pbio-0020274-g008){ref-type="fig"}). Of these, methionine, the only unbranched hydrophobic amino acid and the most accommodating, at a key position between the +1 and +5 sites, allows the AR AF-2 interface to vary the size and shape of its pockets to associate with a more diverse set of coregulators. GR also contains a methionine residue at this position, raising the possibility that it may also employ induced fit to broaden motif recognition. While naturally occurring mutations in AR have yet to be observed at Met734, it is interesting to note that mutations at Val730 and Ile737 have been reported in patients with prostate cancer and androgen insensitivity, respectively ([@pbio-0020274-Newmark1]; [@pbio-0020274-Quigley1]; [@pbio-0020274-Gottlieb1]).
::: {#pbio-0020274-g009 .fig}
Figure 9
::: {.caption}
###### Surface Complimentarity of Hydrophobic Motifs in the AR, ERα, and GR AF-2 Clefts
\(A) AR--FxxLF, (B)AR--LxxLL, (C) ERα--GRIP1 (LxxLL) ([@pbio-0020274-Shiau1]), and (D) GR-TIF2 (LxxLL) ([@pbio-0020274-Bledsoe1]). The inside surfaces of the AF-2 cleft in AR, ERα, and GR are depicted. The LBD is additionally shown as a Cα trace with key side chains shown as white sticks. Phenylalanines and leucines of the FxxLF and LxxLL motifs are shown as spheres.
:::
:::
The same characteristics that make the AR AF-2 ideal for binding of longer, aromatic side chains also make it less well suited for binding of shorter, branched side chains. Although changes in the position of Met734 widen the groove towards the +5 subsite to permit binding of leucine residues, the gross features of the groove remain largely the same (see [Figure 9](#pbio-0020274-g009){ref-type="fig"}B). As a result, the +1 and +5 leucines bind in a smooth, elongated groove and interactions between the +1 and +5 residues on the face of the peptide helix, or with a hydrophobic "bump" present in other receptors caused by a isoleucine to leucine substitution between the +1 and +5 subsites, are absent. Thus, a smaller proportion of the available surface area is available for van der Waals interactions.
Unlike the conserved interaction modes of aromatic residues with the +1 and +5 sites, binding interactions at the +4 site are variable and characterized by nonspecific interactions. This finding agrees with the relatively high conservation of residues at the +1 and +5 positions of AR-interacting motifs and suggests that these residues drive peptide interaction with the LBD, whereas the +4 site is less critical. Indeed, the +4 pocket is shallow, surface exposed, and relatively featureless, explaining the assortment of residues selected at the +4 position. It is likely that any hydrophobic residue that does not clash with surrounding residues would be suitable at this subsite.
While peptide motif recognition is governed by hydrophobic interactions, polar interactions from backbone atoms and residues outside the core motif also contribute. With the exception of FxxFF, motifs containing phenylalanines at the +1 and +5 positions present canonical main chain interactions with both charge clamp residues, Lys720 and Glu897. This finding stands in contrast to predictions of previous studies ([@pbio-0020274-Alen1]; [@pbio-0020274-He2]; [@pbio-0020274-Slagsvold1]; [@pbio-0020274-He1]), which concluded that Lys720 was dispensable for FxxLF binding and that Glu897 was required for binding to FxxLF and LxxLL motifs. Lys720 comprises a significant portion of the +5 subsite, making important van der Waals interactions with the Phe+5 benzyl group in addition to hydrogen bonds to the motif backbone. These results suggest that Lys720 is required for binding of FxxLF motifs. However, it may be that enough binding energy is provided by the other residues of the +5 subsite (i.e., Met734), as well as by the other subsites themselves, such that removal of Lys720 would have little effect on binding. Observations that Lys720 plays a greater role in LxxLL motif binding are likely due to the fact that there is less surface area contributing to van der Waals contacts in LxxLL motifs. Disrupting binding contributions from Lys720 would thus have a more detrimental effect on binding.
On the other hand, Glu897 interacts with the FxxLF peptide backbone, but is disengaged from the LxxLL peptide backbone. One possible explanation for the apparent requirement for Glu897 in LxxLL binding is that it might interact with residues outside of the core motif. The corresponding glutamate of GR, Glu 755, forms hydrogen bonds with the −3 asparagine of TIF2 NR box 3 ([@pbio-0020274-Bledsoe1]), and Glu897 of AR participates in noncanonical interactions with the hydroxyl group of a Ser−2 residue that was selected in all of our tryptophan-containing peptides. This is especially intriguing given that the only WxxLF motif known in vivo, located in the AR NTD, also possesses a Ser−2 residue. WxxLF also makes backbone interactions with an alternate charge clamp residue, Glu893, pointing towards adaptability in AR AF-2 charge clamp formation.
Sequence alignment of NR coactivator sequences shows that positively charged residues are favored N-terminal to the core hydrophobic motif while negatively charged residues are favored C-terminal to the motif ([@pbio-0020274-He1]). Our phage-selected peptides are consistent with this trend. Arginines and lysines were observed at the N-terminal −1 position in all peptides, except for LxxLL, in which Arg was present at the −3 position. Moreover, four out of seven peptides contained negatively charged aspartate or glutamate residues C-terminal to the core motif. While previous studies have shown that complementary interactions between charged residues flanking coactivator signature motifs of coactivators and charged residues surrounding the AF-2 cleft modulated binding to the receptor ([@pbio-0020274-He1]), we find that the flanking charged residues are typically disordered in the electron density, with only Arg−1 of FxxLF interacting with Glu897, and Lys+2 of FxxYF forming a water-mediated hydrogen bond to Asp731. Thus, if charge--charge interactions between flanking peptide residues and the AR surface occur, they are too weak to be detected crystallographically.
Finally, the AR AF-2 surface is an attractive target for pharmaceutical design. Selective peptide inhibitors that bind the AF-2 surface of liganded ERα, ERβ, and TRβ have been developed ([@pbio-0020274-Geistlinger1]), and similar α-helix--mediated protein--protein interfaces have successfully been targeted with tight binding small molecule inhibitors ([@pbio-0020274-Asada1]; [@pbio-0020274-Vassilev1]). Drugs that directly interfere with coactivator binding or formation of the AR N/C interaction would likely inhibit AR activity, perhaps even in androgen-resistant prostate cancers in which conventional therapies have failed. Strategies for designing AR coactivator antagonists are revealed in spite of the changes to the structure at the interface. Together the +1, +4, and +5 subsites contribute the majority of buried surface area of the peptide--LBD interaction ([Table 2](#pbio-0020274-t002){ref-type="table"}). Inhibitors may be designed by varying hydrophobic constituents at these hotspots. The +1 and +5 subsites of AR have a unique preference for aromatic side chains and provide the most viable starting points for designing AR-specific inhibitors. Aromatic groups, possibly with polar constituents to exploit hydrogen bonding interactions with Gln733 and Gln738 in the +1 and +5 subsites, respectively, may provide promising leads. Indeed, initial screens have yielded compounds that bind to the +1 subsite in such a manner (E. Estébanez-Perpiñá, personal communication). Poorly conserved binding and a lack of strong structural features at the +4 subsite suggest that this site may be incorporated for achieving other characteristics important for inhibitors besides fit. Synthetic strategies that link together groups that bind with moderate affinity to the +1, +5, and possibly +4 subsites may yield tight binding inhibitors of AR coactivator association.
Materials and Methods {#s4}
=====================
{#s4a}
### Protein purification {#s4a1}
Expression and purification of the AR LBD for crystallization were performed essentially as described ([@pbio-0020274-Matias1]). The cDNA encoding the chimp AR LBD (residues 663--919---human numbering), which displays 100% identity to the human form in protein sequence, was cloned into a modified pGEX-2T vector (Amersham Biosciences, Piscataway, New Jersey, United States) and expressed as glutathione S-transferase (GST) fusion protein in the E. coli strain BL21 (DE3) STAR in the presence of 10 μM DHT. Induction was carried out with 30 μM IPTG at 17 °C for 16--18 h. E. coli cells were lysed in buffer (10 mM Tris, \[pH 8.0\], 150 mM NaCl, 10% glycerol, 1 mM TCEP, 0.2 mM PMSF) supplemented with 0.5 μg/ml lysozyme, 5 U/ml benzonase, 0.5% CHAPS, and 10 μM DHT. All buffers for further purification steps contained 1 μM DHT. Soluble cell lysate was adsorbed to Glutathione Sepharose 4 Fast Flow resin (Amersham Biosciences), washed with buffer containing 0.1% n-octyl β-glucoside, and eluted with 15 mM glutathione. After cleavage of the GST moiety with thrombin, final purification of the AR LBD was carried out using a HiTrap SP cation exchange column (Amersham Biosciences). Eluted AR LBD was dialyzed overnight at 4 °C against buffer containing 50 mM HEPES (pH 7.2), 10% glycerol, 0.2 mM TCEP, 20 μM DHT, 150 mM Li~2~SO~4~, and 0.1% n-octyl β-glucoside, then concentrated to greater than 4 mg/ml for crystallization.
Purification of AR LBD for use in phage affinity selection was carried out as above without the final dialysis and concentration steps. The expression construct contained the AR LBD as an inframe fusion with GST in a modified pGEX-2T vector containing both a flexible region and an AviTag sequence (Avidity, Denver, Colorado, United States) allowing in vivo biotinylation. The GST--AR LBD fusion expression plasmid was cotransformed with a plasmid-encoding E. coli biotin ligase (Avidity) into BL21 (DE3) STAR cells. Protein expression was carried out as above but with induction supplemented with 50 μM biotin to ensure quantitative biotinylation of AR LBD.
### Phage affinity selections and peptide identification {#s4a2}
Phage affinity selections were performed essentially as described ([@pbio-0020274-Paige1]). Biotinylated AR LBD (10 pmol/well) was incubated in streptavidin-coated Immulon 4 96-well plates (Dynatech International, Edgewood, New Jersey, United States) in TBST (10 mM Tris-HCl \[pH 8.0\], 150 mM NaCl, 0.05% Tween 20) with 1 μM DHT for 1 h at 4 °C. Affinity selections were performed in TBST containing 1 μM DHT. M13 phage distributed among 24 libraries displaying a total of greater than 2 × 10^10^ different random or biased amino acid sequences were added to the wells containing immobilized AR LBD and incubated for 3 h at 4 °C. After washing, bound phage were eluted using pH 2 glycine. Enrichment of phage displaying target-specific peptides was monitored after each round of affinity selection using an anti-M13 antibody conjugated to horseradish peroxidase in an ELISA--type assay.
Synthetic peptides corresponding to the deduced amino acid sequences from receptor-specific phage were tested for their ability to interact with purified AR LBD using a FRET--based assay format. Peptides were synthesized according to the deduced amino acid sequence displayed on phage with an additional C-terminal amino acid sequence consisting of SGSGK to allow the attachment of a biotin tag (Anaspec, San Jose, California, United States). Flourophor conjugates were prepared by incubating either biotinylated peptides with streptavidin-cryptate (Cis Bio International, Bagnols Sur Ceze Cedex, France), or biotinylated AR LBD with streptavidin-XL665 (Cis Bio). Interaction between peptide and AR LBD was monitored by the ratio of energy transfer by excitation at 320 nm and emission at 625 nm and 665 nm.
### Surface plasmon resonance {#s4a3}
Affinities of peptides to the AR LBD were determined with a Biacore (Piscataway, New Jersey, United States) 2000 instrument. A peptide derived from silencing mediator for RXR and TR 2 (SMRT2) served as a negative control. 1 mM peptide stock solutions in DMSO were diluted into HBS-P buffer (10 mM HEPES \[pH 7.4\], 150 mM NaCl, 0.005% Surfactant P20) to generate 10 μM working solutions. HBS-P buffer was flowed through the cells to achieve a stable baseline prior to immobilization of the biotinylated peptides. To achieve the binding of approximately 250 RU of peptides to individual cells, working solutions of peptides were diluted to 100 nM in HBS-P buffer. Unbound streptavidin sites were blocked by injection of a 1 mM biotin solution at a rate of 10 μl/min.
Purified AR LBD was diluted into HBS-P buffer to a concentration of 10 μM and injected into all four Flowcells using the Kinject protocol at a flow rate of 10 μl/min (contact time 360 s, dissociation time 360 s). Following the dissociation phase, the surface of the chip was regenerated to remove residual AR LBD by QuickInject of buffer containing 10 mM HEPES and 50% ethylene glycol (pH 11). Following the establishment of a stable baseline, the same procedure was repeated using a series of AR LBD dilutions (5 μM, 1 μM, and 300 nM) in an iterative manner. Analysis of the data was performed using BIAevaluation 3.0 software (Biacore). The SMRT2 signals were subtracted as background from the three remaining peptide signals. Data were best fit using the two-state conformational change model ([@pbio-0020274-Warnmark1], [@pbio-0020274-Warnmark2]).
### Crystallization, data collection, and refinement {#s4a4}
Purified, concentrated AR LBD was combined with 3x to 6x molar excess of peptide and incubated 1 h at room temperature before crystallization trials. Complexes were crystallized using the hanging drop vapor diffusion method. Protein--peptide solution was combined in a 1:1 ratio with a well solution consisting of 0.6--0.8 M sodium citrate and 100 mM Tris or HEPES buffer (pH 7--8). Crystals typically appeared after 1--2 d, with maximal size attained within 2 wk. For data collection, crystals were swiped into a cryo-protectant solution consisting of well solution plus 10% glycerol before flash freezing in liquid nitrogen. The addition of ethylene glycol to a well concentration of 10%--20% was later found to both improve crystal quality and enable the freezing of crystals directly out of the drop.
Datasets were collected at 100K at the Advanced Light Source (Lawrence Berkeley Laboratory, Berkeley, California, United States), beamline 8.3.1, with either a ADSC Quantum 315 or Quantum 210 CCD detector. Data were processed using Denzo and Scalepack ([@pbio-0020274-Otwinowski1]). Molecular replacement searches were performed with rotation and translation functions from CNS ([@pbio-0020274-Brunger1]). Initial searches for AR--FxxLF were performed using the structure of AR--R1881 (PDB: 1E3G) with R1881 omitted from the search model. Subsequent searches for all other complexes were performed using the refined LBD structure from the AR--FxxLF complex. To minimize the possibility of model bias, FxxLF peptide and DHT were omitted from all molecular replacement searches. Protein models were built by iterative rounds of simulated annealing, conjugate gradient minimization, and individual B-factor refinement in CNS followed by manual rebuilding in Quanta 2000 (Accelrys, San Diego, California, United States) using σ~A~-weighted 2*F* ~o~ − *F* ~c~, *F* ~o~ − *F* ~c~, and simulated annealing composite omit maps. Superposition of structures was performed with LSQMAN ([@pbio-0020274-Kleywegt1]). Buried surface area calculations were performed with CNS. All figures were generated with PyMOL ([@pbio-0020274-DeLano1]). Coordinates and structure factors for all complexes have been deposited in the Protein Data Bank. Accession numbers are listed in [Table 2](#pbio-0020274-t002){ref-type="table"}.
Supporting Information {#s5}
======================
Accession Numbers {#s5a1}
-----------------
The Swiss-Prot (<http://www.ebi.ac.uk/swissprot>) accession numbers for the gene products discussed in this paper are AR (P10275), ARA54 (Q9UBS8), ARA55 (Q9Y2V5), ARA70 (Q13772), ER (P03372, Q92731), glucocorticoid receptor-interacting protein 1 NR box 3 (Q61026 ), GR (P04150), NR box 3 of TIF2 (Q15596), and TR β (P10828).
The Protein Data Bank (<http://www.rcsb.org/pdb>) accession numbers for the structures used in this paper are FxxFF (1T73), FxxLF (1T7R), FxxLW (1T79), FxxYF (1T7M), LxxLL (1T7F), unbound (1T7T), WxxLF (1T74), and WxxVW (1T76).
We would like to thank Erin Anderson-Chisenhall for assistance in protein purification, James Holton and the staff at ALS beamline 8.3.1 for assistance in data collection, and Paul Webb for critical review of the manuscript. This work was supported by funds from the Prostate Cancer Foundation and National Institutes of Health grant R21 CA95324 to RJF.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** EH, BB, and RF conceived and designed the experiments. EH, SP, ESP, HG, and BB performed the experiments. EH, SP, ESP, HG, BB, and RF analyzed the data. BB and RF contributed reagents/materials/analysis tools. EH, BB, and RF wrote the paper.
Academic Editor: Ueli Schibler, University of Geneva
Citation: Hur E, Pfaff SJ, Payne ES, Gron H, Buehrer BM, et al. (2004) Recognition and accommodation at the androgen receptor coactivator binding interface. PLoS Biol 2(9): e274.
AF
: activation function
AR
: androgen receptor
ARA
: androgen receptor-associated protein
DHT
: 5-α dihydrotestosterone
ER
: estrogen receptor
FxxFF
: phenylalanine-x-x-phenylalanine-phenylalanine
FxxLF
: phenylalanine-x-x-leucine-phenylalanine
FxxLW
: phenylalanine-x-x-leucine-tryptophan
FxxYF
: phenylalanine-x-x-tyrosine-phenylalanine
GR
: glucocorticoid receptor
GRIP1
: glucocorticoid receptor-interacting protein 1
GST
: glutathione S-transferase
*K~d~*
: equilibrium dissociation constant
LBD
: ligand-binding domain
LxxLL
: leucine-x-x-leucine-leucine
N/C interaction
: interaction between the N-terminal domain and the ligand-binding domain
NR
: nuclear receptor
NTD
: N-terminal domain
SMRT2
: silencing mediator for RXR and TR 2
TIF2
: transcriptional intermediary factor 2
TR
: thyroid hormone receptor
WxxLF
: tryptophan-x-x-leucine-phenylalanine
WxxVW
: tryptophan-x-x-valine-tryptophan
| PubMed Central | 2024-06-05T03:55:46.809249 | 2004-8-24 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509409/",
"journal": "PLoS Biol. 2004 Sep 24; 2(9):e274",
"authors": [
{
"first": "Eugene",
"last": "Hur"
},
{
"first": "Samuel J",
"last": "Pfaff"
},
{
"first": "E. Sturgis",
"last": "Payne"
},
{
"first": "Hanne",
"last": "Grøn"
},
{
"first": "Benjamin M",
"last": "Buehrer"
},
{
"first": "Robert J",
"last": "Fletterick"
}
]
} |
PMC509410 | Introduction {#s1}
============
Skeletal muscle fibers are generally classified as type I (oxidative/slow) or type II (glycolytic/fast) fibers. They display marked differences in respect to contraction, metabolism, and susceptibility to fatigue. Type I fibers are mitochondria-rich and mainly use oxidative metabolism for energy production, which provides a stable and long-lasting supply of ATP, and thus are fatigue-resistant. Type II fibers comprise three subtypes, IIa, IIx, and IIb. Type IIb fibers have the lowest levels of mitochondrial content and oxidative enzymes, rely on glycolytic metabolism as a major energy source, and are susceptible to fatigue, while the oxidative and contraction functions of type IIa and IIx lie between type I and IIb ([@pbio-0020294-Booth1]; [@pbio-0020294-Berchtold1]; [@pbio-0020294-Olson1]). Adult skeletal muscle shows plasticity and can undergo conversion between different fiber types in response to exercise training or modulation of motoneuron activity ([@pbio-0020294-Booth1], [@pbio-0020294-Jarvis1]; [@pbio-0020294-Pette1]; [@pbio-0020294-Olson1]; [@pbio-0020294-Hood1]). This conversion of muscle fiber from type IIb to type IIa and type I is likely to be mediated by a calcium signaling pathway that involves calcineurin, calmodulin-dependent kinase, and the transcriptional cofactor Peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) ([@pbio-0020294-Naya1]; [@pbio-0020294-Olson1]; [@pbio-0020294-Lin1]; [@pbio-0020294-Wu1]). However, the targeted transcriptional factors directly responsible for reprogramming the fiber-specific contractile and metabolic genes remain to be identified.
Muscle fiber specification appears to be associated with obesity and diabetes. For instance, rodents that gain the most weight on high-fat diets possess fewer type I fibers ([@pbio-0020294-Abou1]). In obese patients, skeletal muscle has been observed to have reduced oxidative capacity, increased glycolytic capacity, and a decreased percentage of type I fibers ([@pbio-0020294-Hickey1]; [@pbio-0020294-Tanner1]). Similar observations have been made in type 2 diabetic patients ([@pbio-0020294-Lillioja1]; [@pbio-0020294-Hickey1]). Recently, it has been shown that increasing oxidative fibers can lead to improved insulin action and reduced adipocyte size ([@pbio-0020294-Luquet1]; [@pbio-0020294-Ryder1]).
We have previously established that peroxisome proliferator-activated receptor (PPAR) δ is a major transcriptional regulator of fat burning in adipose tissue through activation of enzymes associated with long-chain fatty-acid β-oxidation ([@pbio-0020294-Wang1]). Although PPARδ is the predominant PPAR isoform present in skeletal muscle, its in vivo function has not been determined. Our current study uncovers PPARδ as the first transcription factor able to drive the formation of functional type I muscle fibers, whose activation entrains complex pathways both enhancing physical performance and creating a state of obesity resistance.
Results {#s2}
=======
Activation of PPARδ Leads to Muscle Fiber Transformation {#s2a}
--------------------------------------------------------
A role of PPARδ in muscle fiber was suggested by its enhanced expression---at levels 10-fold and 50-fold greater than PPARα and γ isoforms, respectively (unpublished data). An examination of PPARδ in different muscle fibers reveals a significantly higher level in type I muscle (soleus) relative to type II--rich muscle (extensor digitorum longus) or type I and type II mixed muscle (gastrocnemius) ([Figure 1](#pbio-0020294-g001){ref-type="fig"}A); this expression pattern closely resembles that of PGC-1α ([@pbio-0020294-Lin1]). A similar pattern but with more pronounced differences was found at the protein level ([Figure 1](#pbio-0020294-g001){ref-type="fig"}B).
::: {#pbio-0020294-g001 .fig}
Figure 1
::: {.caption}
###### Expression of Endogenous PPARδ and VP16-PPARδ Transgene in Muscle
\(A) Pooled RNA isolated from various muscles of five wild-type male C57B6 mice was hybridized with indicated probes. EDL, extensor digitorum longus; Gastro, gastrocnemius.
\(B) Pooled nuclear proteins (15 μg/lane) isolated from muscles of five wild-type male C57B6 were probed with anti-PPARδ antibody. RNA polymerase II (Pol II) is shown as a loading control.
\(C) Expression of the VP16-PPARδ transgene in various tissues. 10 μg of total RNA from each tissue was hybridized with a VP16 cDNA probe. Gastrocnemius muscle was used here.
\(D) Nuclear proteins (15 μg/lane) isolated from gastrocnemius muscle of the transgenic mice (TG) and the wild-type littermates (WT) were probed with indicated antibodies. The upper, nonspecific band that cross-reacted with the anti-PPARδ antibody serves a loading control.
:::
:::
To directly assess the role of activation of PPARδ in control of muscle fiber plasticity and mitochondrial biogenesis, we generated mice expressing a transgene in which the 78-amino-acid VP16 activation domain was fused to the N-terminus of full-length PPARδ, under control of the 2.2-kb human α-skeletal actin promoter. In agreement with the previous characterization of this promoter ([@pbio-0020294-Brennan1]; [@pbio-0020294-Clapham1]), the VP16-PPARδ transgene was selectively expressed in skeletal muscle, with 10-fold less in the heart ([Figure 1](#pbio-0020294-g001){ref-type="fig"}C). Among different types of muscle fibers, the levels of VP16-PPARδ expression appeared to be similar (unpublished data). As shown in [Figure 1](#pbio-0020294-g001){ref-type="fig"}D for gastrocnemius muscle, VP16-PPARδ fusion protein was produced at a level similar to that of endogenous PPARδ in wild-type littermates. Interestingly, the level of endogenous muscle PPARδ protein in the transgenic mice was much higher than in the control littermates. The substantial increase of endogenous PPARδ may have been caused by a switch to type I fiber (see below), which intrinsically expresses higher levels of PPARδ ([Figure 1](#pbio-0020294-g001){ref-type="fig"}A and [1](#pbio-0020294-g001){ref-type="fig"}B).
Type I muscle can be readily distinguished from type II or mixed muscle by its red color, because of its high concentration of myoglobin, a protein typically expressed in oxidative muscle fibers. We found that muscles in the transgenic mice appeared redder ([Figure 2](#pbio-0020294-g002){ref-type="fig"}A), which is particularly evident in the mixed type I/II fibers of the hindlimb ([Figure 2](#pbio-0020294-g002){ref-type="fig"}B). Indeed, metachromatic staining revealed a substantial muscle fiber transformation ([Figure 2](#pbio-0020294-g002){ref-type="fig"}C). In gastrocnemius muscle, we estimated that there was a 2-fold increase of type I fibers. A diagnostic component of oxidative fibers is their high myoglobin and mitochondrial content, which is supported by the mRNA analysis shown in [Figure 3](#pbio-0020294-g003){ref-type="fig"}A. In addition to myoglobin, mitochondrial components for electron transfer (cytochrome c and cytochrome c oxidase \[COX\] II and IV) and fatty-acid β-oxidation enzymes were elevated ([Figure 3](#pbio-0020294-g003){ref-type="fig"}A; unpublished data). These effects appear to be direct consequences of PPARδ activation, as levels of PGC-1α, a coactivator involved in muscle fiber switch and mitochondrial biogenesis ([@pbio-0020294-Wu2]; [@pbio-0020294-Lehman1]; [@pbio-0020294-Lin1]), remained unchanged. Southern blot analysis detected a substantially higher copy number of the mitochondrial genome--encoded COXII DNA in the transgenic mice ([Figure 3](#pbio-0020294-g003){ref-type="fig"}B). Mitochondrial DNA was increased 2.3-fold in gastrocnemius muscle of the transgenic mice ([Figure 3](#pbio-0020294-g003){ref-type="fig"}C). These results reveal a marked stimulation of mitochondrial biogenesis and further support the idea that there is a muscle fiber switch. This conclusion was also confirmed by Western blot analysis. As shown in [Figure 3](#pbio-0020294-g003){ref-type="fig"}D, the characteristic type I fiber proteins, such as myoglobin and cytochrome c and b, were significantly increased. More importantly, the specialized contraction protein troponin I (slow) of type I fiber was robustly induced; this was accompanied by a marked reduction of the specialized contraction protein troponin I (fast) of type II fiber, indicating a high degree of fiber transformation. We next examined whether acute activation of endogenous PPARδ would induce similar target genes. In agreement with the chronic effects in the transgenic mice, we found that, after treatment of wild-type C57B6J mice with the PPARδ-specific agonist GW501516 for only 10 d, genes for slow fiber contractile proteins, mitochondrial biogenesis, and β-oxidation were all upregulated ([Figure 3](#pbio-0020294-g003){ref-type="fig"}E). This indicates that rapid, systematic, and coordinated changes of muscle fiber properties toward type I can be achieved by activation of the endogenous PPARδ pathway.
::: {#pbio-0020294-g002 .fig}
Figure 2
::: {.caption}
###### Increased Oxidative Type I Fibers in the Transgenic Mice
(A and B) Muscles in transgenic mice (TG) are redder than those in wild-type mice (WT).
\(C) Metachromatic staining of the type II plantaris muscle. Type I fibers are stained dark blue.
:::
:::
::: {#pbio-0020294-g003 .fig}
Figure 3
::: {.caption}
###### Activation of PPARδ Induces Genes Typical for Type I Fibers and Promotes Mitochondrial Biogenesis
\(A) Total RNA (10 μg/lane) prepared from gastrocnemius muscle of transgenic (TG) and wild-type (WT) littermates was probed with indicated probes. The fold increase of induction of each gene is shown.
\(B) Total genomic DNA (10 μg/lane) prepared from gastrocnemius muscle was digested with Nco1 and subjected to Southern analysis with COXII (mitochondrial genome--encoded) and MCIP1 (nuclear genome--encoded) DNA probes.
\(C) Equal amounts of gastrocnemius muscle were collected from both transgenic mice and control littermates. Total mitochondrial DNA was isolated and separated on 1% agarose gel. The relative abundance of mitochondrial DNA in transgenic and wild-type mice is presented.
\(D) Western blot analysis of muscle fiber markers and mitochondrial components. Each lane was loaded with 80 μg of total gastrocnemius muscle extracts.
\(E) Wild-type C57B6 mice were treated with vehicle or PPARδ agonist GW501516 for 10 d. Total RNA (10 μg/lane) prepared from the gastrocnemius muscle was probed with indicated probes.
:::
:::
Muscle Fiber Switch by PPARδ Protects Against Obesity {#s2b}
-----------------------------------------------------
A number of previous studies have shown that obese individuals have fewer oxidative fibers, implying that the presence of oxidative fibers alone may play a part in obesity resistance. To test this possibility, we fed the transgenic mice and their wild-type littermates with a high-fat diet for 97 d. Although the initial body weights of the two groups were very similar, the transgenic mice had gained less than 50% at day 47, and only one-third at day 97, of the weight gained by the wild-type animals ([Figure 4](#pbio-0020294-g004){ref-type="fig"}A). The transgenic mice displayed significantly higher oxygen consumption on the high-fat diet than the control littermates (unpublished data). By the end of this experiment, the control littermates became obese, whereas the transgenic mice still maintained a normal body weight and fat mass composition ([Figure 4](#pbio-0020294-g004){ref-type="fig"}A). A histological analysis of inguinal fat pad revealed a much smaller cell size in the transgenic mice ([Figure 4](#pbio-0020294-g004){ref-type="fig"}B), due to the increased muscle oxidative capacity. While there was no significant difference in intramuscular glycogen content, the triglyceride content was much less in the transgenic mice ([Figure 4](#pbio-0020294-g004){ref-type="fig"}C and [4](#pbio-0020294-g004){ref-type="fig"}D), which may explain their improved glucose tolerance ([Figure 4](#pbio-0020294-g004){ref-type="fig"}E). We also placed wild-type C57BJ6 mice on the high-fat diet and treated them with either vehicle or the PPARδ agonist GW501516 for 2 mo. GW501516 produced a sustained induction of genes for type I muscle fibers; this, at least in part, resulted in an only 30% gain in body weight, a dramatically reduced fat mass accumulation, and improved glucose tolerance, compared to the vehicle-treated group ([Figure 5](#pbio-0020294-g005){ref-type="fig"}). Thus, muscle fiber conversion by stimulation with the PPARδ agonist or the activated transgene has a protective role against obesity.
::: {#pbio-0020294-g004 .fig}
Figure 4
::: {.caption}
###### Resistance to High-Fat-Induced Obesity in the Transgenic Mice
\(A) Seven-week-old transgenic (TG) and wild-type (WT) littermates (*n* = 5--6 for each group) were fed with a high-fat diet for 97 d. Left panel shows net body weight gain, which was calculated for individual mice and then averaged. Right panel shows the body weights before (Day 0) and after (Day 97) high-fat feeding.
\(B) Histology of inguinal fat pad in the transgenic and wild-type littermates under a high-fat diet for 2 mo.
(C and D) Intramuscular glycogen content (C) and triglyceride content (D) of mice in (A) after high-fat feeding (*n* = 6).
\(E) Glucose tolerance test. Mice in (A) after high-fat feeding were fasted for 6 h and then injected with glucose at a concentration of 1g/kg body weight. Then blood glucose levels were measured periodically over 2 h (*n* = 6).
:::
:::
::: {#pbio-0020294-g005 .fig}
Figure 5
::: {.caption}
###### PPARδ Agonists Counteract Obesity Induced by High-Fat Diet
\(A) Eleven-week-old wild-type C57B6 mice were fed a high-fat diet in combination with vehicle or GW501516 for 57 d. Total RNA (10 μg/lane) prepared from the gastrocnemius muscle was probed with indicated probes.
\(B) Net body weight gain for mice in (A) after treatment was calculated for individual mice and averaged. Initial body weights were 28.54 ± 1.04 g for vehicle group (*n* = 5) and 28.86 ± 0.80 g for GW501516 group (*n* = 5).
\(C) Various tissue weights for mice in (A) after treatment. ifat, inguinal fat; rdfat, reproductive fat; retrofat, retroperitoneal fat.
\(D) Glucose tolerance test. Mice in (A) after treatment were fasted for 6 h and then injected with glucose at a concentration of 1g/kg body weight. Blood glucose levels were then measured periodically over 2 h.
:::
:::
Activation of PPARδ Enhances Physical Performance {#s2c}
-------------------------------------------------
Muscle oxidative capacity is a crucial factor for determining endurance and fatigue. Indeed, type I fibers adaptively generated through exercise training are considered to be fatigue resistant. However, whether the type I fibers generated molecularly via PPARδ expression can contribute to enhanced performance in the absence of previous training is unclear. In fact, the consequence of genetically induced fiber switch on running capacity has to our knowledge never been evaluated. We thus compared exercise performance between untrained, body-weight-matched transgenic and wild-type littermates. Mice were run on oxygen-infused, enclosed treadmills until exhaustion. Strikingly, the running time and distance the transgenic mice were able to sustain were increased by 67% and 92%, respectively ([Figure 6](#pbio-0020294-g006){ref-type="fig"}A; also see Videos [S1](#sv001){ref-type="supplementary-material"} and [S2](#sv002){ref-type="supplementary-material"}). The transgenic mice ran about 1 h longer than the controls, which translates to nearly a kilometer further. No significant differences in muscle mass (unpublished data) and daily activity (total counts of activity per hour: 1618 ± 209 for transgenic versus 1987 ± 301 for wild-type, *p* \> 0.35, *n* = 4) were observed between the transgenic and control mice. Thus, the remarkable increase in endurance is the physiologic manifestation of muscle fiber transformation. This suggests that genetically directed muscle fiber switch is physiologically and functionally relevant. In addition, we looked at what effect the absence of PPARδ function has on exercise endurance. In the treadmill test, the PPARδ-null mice could sustain only 38% of the running time and 34% of the distance of their age- and weight-matched wild-type counterparts ([Figure 6](#pbio-0020294-g006){ref-type="fig"}B). These results further support a role for PPARδ in enhancement of physical performance.
::: {#pbio-0020294-g006 .fig}
Figure 6
::: {.caption}
###### PPARδ Regulates Exercise Endurance
\(A) Enhanced exercise performance in the transgenic mice. Fourteen-week-old male transgenic and wild-type littermates with similar body weights (*n* = 4 for each group) were subjected to a forced treadmill exercise test.
\(B) Compromised exercise performance in PPARδ-null mice. Two-month-old male PPARδ-null mice and wild-type controls with similar body weights (*n* = 6 for each group) were subjected to a forced treadmill exercise test.
\(C) Functions of PPARδ in skeletal muscle.
:::
:::
Discussion {#s3}
==========
Our data reveal that a PPARδ-mediated transcriptional pathway can regulate muscle fiber specification, enabling the generation of a strain of mice with a "long-distance running" phenotype. We show that targeted expression of an activated form of PPARδ produces profound and coordinated increases in oxidation enzymes, mitochondrial biogenesis, and production of specialized type I fiber contractile proteins---the three hallmarks for muscle fiber type switching ([Figure 6](#pbio-0020294-g006){ref-type="fig"}C). While induction of muscle oxidation enzymes by PPARδ has been seen both in vivo and in vitro ([@pbio-0020294-Muoio1]; [@pbio-0020294-Dressel1]; [@pbio-0020294-Luquet1]; [@pbio-0020294-Tanaka1]; [@pbio-0020294-Wang1]), its effects shown here on muscle fiber switching are unexpected. These progressive changes in oxidative capacity in conjunction with eventual changes in type I muscle fiber lead to a dramatically improved exercise profile and protection against obesity. This does not solely depend on achieving a directed muscle fiber type switch but also requires all the associated changes in neural innervation, motor neuron function, and peripheral metabolic adaptation to enable a new integrated physiological response. Accordingly, activation of muscle PPARδ essentially recapitulates the effects of exercise training even in the absence of training itself. To our knowledge, this has not been directly described for any other transcriptional factor.
The muscle phenotypes described here are remarkably similar to those of transgenic mice expressing either calcineurin, calmodulin-dependent kinase, or PGC-1α ([@pbio-0020294-Naya1]; [@pbio-0020294-Lin1]; [@pbio-0020294-Wu1]), indicating that PPARδ could be one of the hypothetical downstream transcription factors of these pathways. It is important to note that, from our ligand and gain-of-function transgenic studies, PPARδ needs to be activated in order to direct the muscle fiber switch. Indeed, in a recent report by [@pbio-0020294-Luquet1], simple overexpression of wild-type PPARδ in muscle was found not to be sufficient to promote a fiber switch or obesity resistance, although certain oxidation enzymes were increased. This supports the model in [Figure 6](#pbio-0020294-g006){ref-type="fig"}C that the activating signal or ligand, but not the receptor, is limiting. Thus, PPARδ activation, rather than merely an increase of PPARδ levels, is an essential element for fiber switching and its associated functional manifestations. How might endogenous PPARδ become activated naturally by exercise training? First, it is possible that exercise generates or increases endogenous ligands for PPARδ as tissues are undergoing substantial increases in fatty-acid internalization and burning. Fatty acids and their metabolites can activate PPARδ. A second model is that exercise may induce expression of PGC-1α ([@pbio-0020294-Goto1]) and thereby activate PPARδ. This is consistent with previous work in which we have shown that PGC-1α physically associates with PPARδ in muscle tissue and can powerfully activate it even in the absence of ligands ([@pbio-0020294-Wang1]). Alternatively, PPARδ may be activated by a distal upstream signaling component such as a kinase cascade. Further dissecting the interactions between PPARδ and its regulatory components will be necessary to fully understand the molecular basis of muscle fiber determination pertinent to exercise training.
Skeletal muscle is a major site to regulate whole-body fatty-acid and glucose metabolism. We show that mice with increased oxidative fibers are resistant to high-fat-induced obesity and glucose intolerance. Moreover, ligand studies provide compelling evidence that activation of endogenous PPARδ can produce similar effects. Might PPARδ have any beneficial effects on glucose metabolism in the lean condition? This has not been explored; however, insulin resistance in the elderly is confined mostly to skeletal muscle and may be due to reduction of mitochondrial number and/or function ([@pbio-0020294-Petersen1]). The ability of PPARδ to stimulate mitochondrial biogenesis and oxidative function suggests that PPARδ could be important for control of insulin resistance during normal aging. Together, these data indicate that PPARδ and its ligands comprise a key molecular switch to regulate muscle fiber specification, obesity resistance, insulin sensitivity, and, most surprisingly, physical endurance. This work demonstrates that complex physiologic properties such as fatigue, endurance, and running capacity can be genetically manipulated.
Materials and Methods {#s4}
=====================
{#s4a}
### Animals. {#s4a1}
The transactivation domain (78 amino acid residues, corresponding to residues 413--490) of VP16 was fused in frame with the N-terminus of mouse PPARδ. The VP16-PPARδ fusion cDNA was placed downstream of the human α-skeletal actin promoter ([@pbio-0020294-Brennan1]), and upstream of the SV40 intron/poly(A) sequence. The transgene was purified and injected into C57BL/6J × CBA F1 zygotes. Transgenic mice were backcrossed with C57BL/6J for two generations. Wild-type littermates were used as controls throughout the study. On normal chow diet, the transgenic mice and control littermates used here had similar body weights. PPARδ-null mice were previously generated ([@pbio-0020294-Barak1]). Mice were fed either a standard chow with 4% (w/w) fat content (Harlan Teklad, Harlan, Indianapolis, Indiana, United States) or a high-fat diet containing 35% (w/w) fat content (product F3282, Bioserv, Frenchtown, New Jersey, United States) as indicated. For ligand experiments, we synthesized the GW501516 compound and mice were orally gavaged daily (10 mg/kg or vehicle alone).
### Gene expression analysis and physiological studies {#s4a2}
Mouse EST clones were obtained from ATCC (Manassas, Virginia, United States), verified by sequencing, and used as Northern probes. Antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, California, United States). Total muscle protein extracts ([@pbio-0020294-Lin1]) and nuclear proteins ([@pbio-0020294-Wang1]) were prepared as described.
Prior to the exercise performance test, the mice were accustomed to the treadmill (Columbus Instruments, Columbus, Ohio, United States) with a 5-min run at 7 m/min once per day for 2 d. The exercise test regimen was 10 m/min for the first 60 min, followed by 1 m/min increment increases at 15-min intervals. Exhaustion was defined when mice were unable to avoid repetitive electrical shocks.
### Muscle fiber typing and mitochondrial DNA isolation {#s4a3}
Muscle fiber typing was essentially performed using metachromatic dye--ATPase methods as described ([@pbio-0020294-Ogilvie1]). Muscle mitochondria were isolated ([@pbio-0020294-Scholte1]). Mitochondrial DNA was prepared and analyzed on 1% agarose gel.
### Statistical analysis {#s4a4}
Number of mice for each group used in experiments is indicated in figure legends. Values are presented as mean ± SEM. A two-tailed Student\'s t test was used to calculate *p-*values.
Supporting Information {#s5}
======================
Video S1
::: {.caption}
###### Beginning of Running Test
This video shows the exercise performance of a representative of the transgenic mice (right chamber) and a representative of wild-type control littermates (left chamber) on the treadmill 15 min into the exercise challenge.
(52.4 MB MOV).
:::
::: {.caption}
######
Click here for additional data file.
:::
Video S2
::: {.caption}
###### Running Test 90 Min Later
This video shows the exercise performance of a representative of the transgenic mice (right chamber) and a representative of wild-type control littermates (left chamber) on the treadmill 90 min into the exercise challenge.
(41.7 MB MOV).
:::
::: {.caption}
######
Click here for additional data file.
:::
We thank Dr. M. Downes for providing the α-skeletal actin promoter; Dr. G. D. Barish for comments on the manuscript; M.A. Lawrence for histology; Dr. S. Pfaff for the use of microscopes and photographic equipment; M. Lieberman and K.L. Schnoeker for photography; J.M. Shelton for advice on fiber staining; and E. Stevens and E. Ong for administrative assistance. YXW was supported by a postdoctoral fellowship from California Tobacco-Related Disease Research Program. JH was supported by the BK21 program, Ministry of Education, Korea. HK was supported by grant M1--0311-00--0145 from the Molecular and Cellular BioDiscovery Research Program, Ministry of Science and Technology, Korea. RME is an Investigator of the Howard Hughes Medical Institute at the Salk Institute for Biological Studies and March of Dimes Chair in Molecular and Developmental Biology. This work was supported by the Howard Hughes Medical Institute and the Hillblom Foundation.
**Conflicts of interest.** The authors have declared that no conflicts of interest exist.
**Author contributions.** YXW and RME conceived and designed the experiments. YXW, CLZ, RTY, MCN, and CRBO performed the experiments. YXW, CLZ, and RME analyzed the data. HKC, JH, and HK contributed reagents/materials/analysis tools. YXW and RME wrote the paper.
Academic Editor: Steve O\'Rahilly, University of Cambridge
Citation: Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, et al. (2004) Regulation of muscle fiber type and running endurance by PPARδ. PLoS Biol 2(10): e294.
COX
: cytochrome c oxidase
mCPT1
: muscle carnitine palmitoyltransferase-1
PGC-1α
: Peroxisome proliferator-activated receptor-gamma coactivator 1α
PPAR
: peroxisome proliferator-activated receptor
UCP
: uncoupling protein
| PubMed Central | 2024-06-05T03:55:46.813024 | 2004-8-24 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509410/",
"journal": "PLoS Biol. 2004 Oct 24; 2(10):e294",
"authors": [
{
"first": "Yong-Xu",
"last": "Wang"
},
{
"first": "Chun-Li",
"last": "Zhang"
},
{
"first": "Ruth T",
"last": "Yu"
},
{
"first": "Helen K",
"last": "Cho"
},
{
"first": "Michael C",
"last": "Nelson"
},
{
"first": "Corinne R",
"last": "Bayuga-Ocampo"
},
{
"first": "Jungyeob",
"last": "Ham"
},
{
"first": "Heonjoong",
"last": "Kang"
},
{
"first": "Ronald M",
"last": "Evans"
}
]
} |
PMC509411 | Anyone who thinks plants are passive inhabitants of their environment has never seen time-lapse footage of a seedling bursting from its protective shell or a climbing vine coiling around a tree. Such films dramatize a fundamental fact of plant life: survival depends on responding to environmental cues. Shoots grow toward light and against gravity. Stems and roots curl around obstacles that block their paths.
In plants, environmental cues trigger hormonal changes that in turn regulate cells\' shapes and proliferation. In this way, subtle changes in the environment affect plant growth. Auxin, the first known plant hormone, spurs growth and shapes growth patterns in nearly every plant tissue throughout a plant\'s lifecycle. Brassinosteroids---a class of hormones chemically similar to animal steroids like testosterone---are linked to many of the same processes as auxin.[](#pbio-0020299-g001){ref-type="fig"}
::: {#pbio-0020299-g001 .fig}
::: {.caption}
###### Emerging leaf tips (yellow arrow) and hypocotyl (orange arrows) of an Arabidopsis mutant
:::
:::
Early physiological and molecular experiments gave conflicting evidence about whether auxin and brassinosteroids had similar effects. For many years, biologists believed that these hormones acted through independent signal transduction pathways---chains of molecules that relay stimuli and elicit cellular responses, such as gene expression. But in the last few years, microarray studies, which can measure the transcription of thousands of genes simultaneously, showed that auxin and brassinosteroids do regulate expression of several genes in common.
In this issue of *PLoS Biology*, Jennifer Nemhauser et al. assay the entire genome of Arabidopsis thaliana, a favorite for plant genetics studies, for effects of auxin and brassinosteroids. The group\'s microarray analyses show that these hormones affect transcription of about 80 genes in common---including many known players in the hormones\' signal transduction pathways. To see how this regulation could occur, the research team looked at the genes turned on by both hormones to find common promoter sequences---regions of the genome that do not code for protein but instead help regulate gene transcription. They used a new computational approach to tease out promoter regions that auxin and brassinosteroid pathways both act upon, showing how these hormones have overlapping effects on gene transcription.
The group also compared the effects of auxin and brassinosteroids on seedlings\' stem growth and gene expression in a variety of mutant Arabidopsis lines. They showed that auxin and brassinosteroids greatly enhance each other\'s effects on stem growth, demonstrating that the interaction of these hormones is important for normal plant development. Mutants with a disabled auxin pathway don\'t respond normally to brassinosteroids, and vice versa. Also, mutants with abnormally high levels of auxin have a reduced number of genes that respond to brassinosteroids. Thus, these hormones act through overlapping, interdependent pathways---but they don\'t regulate each other directly. Instead, the researchers suggest, the pathways likely converge on the promoters of a few key genes.
It\'s still an open question why plants use these hormones with such redundant effects. Nemhauser speculates that---as is known to be the case in animals---by having dual, interdependent pathways, plants can finely tune how these ubiquitous hormones act in different cells and tissues to shape patterns of growth. By showing clearly that auxin and brassinosteroids act together and how they affect many of the same genes, Nemhauser and colleagues have set the stage for more detailed studies of how these hormones act in specific parts of plants to shape growth.
| PubMed Central | 2024-06-05T03:55:46.815410 | 2004-8-24 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509411/",
"journal": "PLoS Biol. 2004 Sep 24; 2(9):e299",
"authors": []
} |
PMC509412 | In June, muscular dystrophy patients lost one of their most passionate advocates to a rare form of this degenerative neuromuscular disorder---thirteen-year-old Mattie Stepanek. In his short life, Stepanek wrote five volumes of inspirational poetry, topping the *New York Times* bestseller list and winning accolades from the likes of Jimmy Carter. A wide range of inherited disorders falls under the rubric of muscular dystrophy, but all involve some form of progressive muscle wasting. Stepanek\'s condition impaired nearly all of his body\'s functions, but other more common forms, including Emery-Dreifuss muscular dystrophy (EDMD), selectively target skeletal muscle and induce cardiac abnormalities.
EDMD is caused by mutations in either of two genes: one encodes lamin A, a structural protein associated with the nucleus, and the other encodes a nuclear membrane protein called emerin. Lamins, a major component of the structural network that supports the nuclear envelope, help the nuclear envelope maintain structural integrity and absorb mechanical stress without rupturing. (Structures that support the nucleus and regulate molecular traffic between the cytoplasm and nucleus are collectively referred to as the nuclear envelope. They include the inner and outer nuclear membranes, the nuclear pore complexes, and a network of lamin filaments, called the nuclear lamina, near the inner membrane.) Emerin binds to proteins that regulate gene transcription. Emerin and lamins are found in most cell types, yet EDMD attacks only skeletal muscles, major tendons, and the cells that regulate cardiac muscle contraction. So where does this tissue specificity come from?
One theory suggests that emerin selectively targets proteins that specifically regulate gene expression in EDMD-affected tissues. Another theory proposes that emerin provides structural support to the nuclear envelope and that emerin mutations are most destructive in tissues subjected to mechanical stress---like skeletal muscle and tendons. Current evidence supports both models. Recent studies suggest that emerin forms complexes with actin---the mother of all structural proteins. Actin proteins can join together (polymerize) to form a variety of filaments. However, given longstanding doubts that actin exists in the nucleus, let alone functions there, researchers were unsure what the findings might indicate. Now James Holaska, Amy Kowalski, and Katherine Wilson propose that emerin not only functions as a structural protein in the nucleus but that it does so by interacting with actin.[](#pbio-0020300-g001){ref-type="fig"}
::: {#pbio-0020300-g001 .fig}
::: {.caption}
###### Interactions of structural proteins at the nuclear membrane
:::
:::
Evidence that emerin and lamin A can form multiprotein complexes comes primarily from experiments in test tubes. To get a sense of the physiological significance of these findings, Wilson and colleagues purified emerin-binding proteins from the nuclei of living cells. They found that emerin binds to polymerized actin and, in fact, appears to stimulate polymerization. By binding and "capping" a specific end of the actin filament, emerin prevents filament de-polymerization (disassembly), effectively increasing the rate of actin polymerization by four- to twelve-fold. The authors propose that emerin "promotes the formation of a nuclear actin cortical network," which could serve to anchor membrane proteins and lamin filaments to the inner nuclear membrane and thus enhance the structural integrity of the nuclear envelope. Whether emerin also interconnects the lamin and actin filament networks at the nuclear envelope---which could significantly reinforce its mechanical strength---will have to await further study.
Muscle contraction places enormous stress on cell membranes. These results suggest that actin-based networks, in addition to lamin networks, support the structural integrity of the nuclear envelope. Defects in proteins involved in either network could compromise nuclear structure, which could in turn disrupt the cell\'s gene expression program, for example, or rupture the cell membrane, killing the cell. Subtle defects in proteins important for muscle cell integrity can cause several forms of muscular dystrophy. Now it appears that emerin defects could cause EDMD in part by compromising the mechanical integrity of nuclei in muscle cells and tendons.
| PubMed Central | 2024-06-05T03:55:46.815883 | 2004-8-24 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509412/",
"journal": "PLoS Biol. 2004 Sep 24; 2(9):e300",
"authors": []
} |
PMC509413 | One of the major players in prostate cancer is a nuclear signaling protein called the androgen receptor. Prostate growth and development is regulated by androgen hormones (like testosterone) that activate the androgen receptor. When an androgen binds the receptor, the receptor binds other proteins, called coactivators, to activate genes controlling cell growth, survival, and differentiation. Unlike other receptors that function in the nucleus, the androgen receptor normally shuns coactivators with a leucine-rich binding domain in favor of those with "aromatic" domains. (Aromatic amino acids are defined by their ring structure.) But during prostate cancer, the receptor interacts with both coactivator types, to promote disease progression.
The secret to a protein\'s binding preference rests in the underlying sequence of its amino acids, which determines the protein\'s structure and ultimate behavior. Robert Fletterick and colleagues set out to identify the "full repertoire" of amino acid sequences that might conceivably consort with the androgen receptor. Their findings help explain the unusual behavior of the androgen receptor during prostate cancer progression---a first step toward developing new anticancer therapies.
Treatment for hormone-dependent prostate cancers focuses primarily on reducing androgen levels by using chemicals that compete for androgen receptor docking rights in the hormone-binding pocket of the ligand-binding domain, or LBD. (A ligand is a molecule, like the androgen hormone, that binds to a receptor.) But cancer cells eventually circumvent these chemical assaults through increased levels of either androgen receptors or their coactivators, or through mutations that make androgen receptors immune to chemotherapy. That\'s why Fletterick and colleagues turned their attentions to the receptor\'s consorts. Since targeting the hormone-binding pocket of the receptor offers limited benefits, a better strategy might involve disrupting associations with the receptor\'s coactivators.
Dozens of proteins interact with different regions of the androgen receptor, but the details of these interactions were not known. When a hormone binds to the LBD of other nuclear receptors, it triggers a conformational change that creates a binding surface called AF-2 for the leucine-rich domains of the coactivator proteins. It was not clear, however, how the AF-2 region of the androgen receptor distinguishes between aromatic and leucine-rich domains. To characterize the receptor\'s binding selectivity, Fletterick and colleagues tested 20 billion peptides, or protein fragments, to see whether they interacted with the LBD region of a hormone-bound androgen receptor.
As expected, most of the peptides that associated with the LBD domain were aromatic. And they interacted with the same region that naturally occurring coactivators bind to. Next, Fletterick and colleagues determined the three-dimensional structure of both the receptor bound to just the androgen hormone and the androgen--receptor pair bound to a subset of seven peptides. The different structures showed that the androgen receptor uses a single surface to bind both leucine-rich and aromatic peptides; when the aromatic peptides have bulky appendages, the receptor\'s AF-2 domain reorganizes to accommodate them.[](#pbio-0020305-g001){ref-type="fig"}
::: {#pbio-0020305-g001 .fig}
::: {.caption}
###### Surface complimentarity of hydrophobic motifs
:::
:::
The various structures and binding affinities for the different receptor--peptide complexes described here show how the receptor can interact with a diverse array of proteins. The androgen receptor, unlike other nuclear receptors, has specific amino acid sequences that better support aromatic peptide binding. Interestingly, mutations in one of these amino acid sequences have been found in prostate cancer. Altogether, the authors conclude, the unique properties of the receptor\'s AF-2 surface make it "an attractive target for pharmaceutical design." Drugs that directly interfere with coactivator binding, they explain, are likely to inhibit androgen receptor activity. Here, the authors recommend novel sites on the receptor as promising targets for androgen-receptor-specific inhibitors.
| PubMed Central | 2024-06-05T03:55:46.816649 | 2004-8-24 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509413/",
"journal": "PLoS Biol. 2004 Sep 24; 2(9):e305",
"authors": []
} |
PMC509414 | People are unpredictable. One night you may crave Italian food, but another only Thai will do. One day you might finish a crossword puzzle in record time, and the next not a single clue prompts an answer. Such behavioral variation has been found in laboratory studies, too: a person\'s ability to find a faint image on a screen varies widely from one viewing to the next. Similarly, when an animal repeatedly receives the same stimulus---for example, a faint image---a neuron in a region of the animal\'s visual brain might be very active upon one presentation and relatively quiet the next.
Across the cerebral cortex---the brain region that integrates the senses and controls voluntary movement---neurons are notorious for their unpredictable behavior. The neurons themselves don\'t create this noise; when directly stimulated with an electrode multiple times, neurons will give the same response every time. Most neurons, however, receive signals from a host of other neurons. These various signals combine to form a seemingly noisy electrical input, which shows up as fluctuations in the recipient neuron\'s membrane potential---a difference in electrical charge between the inside and outside of the cell\'s membrane. Neuron function is intimately tied to the membrane potential, which is usually maintained within a narrow range, called the resting potential. But incoming signals can push the resting potential higher or lower. If the membrane potential rises above a certain threshold, the neuron fires, sending an electrical signal down its length. In this way, the brain relays and processes information.
Since the 1960s, neuroscientists trying to account for the cortex\'s variable responses have pointed to noisy inputs from other parts of the brain as the prime suspect. In this issue of *PLoS Biology*, Matteo Carandini addresses this longstanding mystery of neuron variability and comes up with a different answer. Carandini simultaneously measured the membrane potentials and firing patterns of individual neurons in the cat visual cortex. He found, surprisingly, that the membrane potentials varied much less than the firing patterns, ruling out noisy inputs as the cause of neurons\' noisy outputs. Instead, the neurons amplified noise in the signals they received.[](#pbio-0020314-g001){ref-type="fig"}
::: {#pbio-0020314-g001 .fig}
::: {.caption}
###### Noise and threshold make neurons unpredictable
:::
:::
Carandini then used a simple model of neuron behavior to explain why this would occur. He started with a tried-and-true approximation of neuron behavior, called the rectification model: a neuron doesn\'t fire until its membrane potential rises above a threshold, but once it crosses this threshold, its firing rate is correlated with the strength of incoming signals. Then he added the assumption that the neurons receive signals with some randomness. Given these minimal assumptions, Carandini showed that neurons fed a noisy signal will tend to amplify the noise in the signal. Importantly, his model reproduced a well-known phenomenon: as cortical neurons\' average firing rate goes up, their firing rate also becomes more variable---that is, they get noisier.
Carandini\'s model also predicted something new: as the firing rate continues to increase, the firing rate should become more consistent and less noisy---which he calls saturation of variability. Carandini\'s measurements in cats showed neurons actually behave this way, a key validation of his model.
It\'s not clear whether this amplification of variability is something that helps or hampers the brain. Despite being a nuisance to neuroscientists, such fluctuations could be crucial to how the brain functions, Carandini speculates. Without some variability in their cortex, animals would act like cameras or other simple machines that respond the same way each time to a stimulus. It\'s advantageous for behavior, and hence brains, to be adaptable. But amplifying noise in a signal seems to run counter to relaying and processing the information in the signal. Carandini suggests that what appears as noise in the experiments are signals from other parts of the cortex---that is, noise is in the eye of the beholder. Now that the source of the variability is clear, neuroscientists can study whether it serves a function in the brain.
| PubMed Central | 2024-06-05T03:55:46.817178 | 2004-8-24 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509414/",
"journal": "PLoS Biol. 2004 Sep 24; 2(9):e314",
"authors": []
} |
PMC509415 | Have you ever noticed that long-distance runners and sprinters seem specially engineered for their sports? One\'s built for distance, the other speed. The ability to generate quick bursts of power or sustain long periods of exertion depends primarily on your muscle fiber type ratio (muscle cells are called fibers), which depends on your genes. To this extent, elite athletes are born, not made. No matter how hard you train or how many performance-enhancing drugs you take, if you\'re not blessed with the muscle composition of a sprinter, you\'re not going to break the 100-meter world record in your lifetime. (In case you\'d like to try, that\'s 9.78 seconds for a man and 10.49 seconds for a woman.)
Of course that doesn\'t prevent those at the highest levels from using the latest performance enhancer to get that extra 1% edge. But wait until trainers hear about the Marathon Mouse. A new study by Ronald Evans and colleagues provides evidence that endurance and running performance can be dramatically enhanced through genetic manipulation.
Skeletal muscles come in two basic types: type I, or slow twitch, and type II, or fast twitch. Slow-twitch fibers rely on oxidative (aerobic) metabolism and have abundant mitochondria that generate the stable, long-lasting supplies of adenosine triphosphate, or ATP, needed for long distance. (For more on muscle fiber metabolism, see synopsis titled "A Skeletal Muscle Protein That Regulates Endurance") Fast-twitch fibers, which produce ATP through anaerobic glycolysis, generate rapid, powerful contractions but fatigue easily. Top-flight sprinters have up to 80% type II fibers while long-distance runners have up to 90% type I fibers. Coach potatoes have about the same percentage of both.[](#pbio-0020322-g001){ref-type="fig"}
::: {#pbio-0020322-g001 .fig}
:::
Endurance training can enhance the metabolic performance of muscle types, and now it appears that training can also induce conversion between fiber types. Specific changes in gene expression trigger this oxidative fiber transformation, but the transcription factor responsible for engineering this shift was unknown. Evans and colleagues suspected that a nuclear receptor called PPARδ---a major regulator of fat burning in fat tissue that is also prevalent in skeletal muscle---might be involved.
To investigate this possibility, the authors genetically engineered mice to express an activated form of the PPARδ protein in skeletal muscle. Type I fibers normally express higher levels of PPARδ than type II fibers, and the transgenic mice showed much higher levels of the protein than their normal littermates. The transgenic mice also had much redder muscles than the controls---type I fibers have high levels of myoglobin, the red-pigmented protein that facilitates the movement of oxygen within muscle---and elevated levels of proteins associated with mitochondrial biogenesis and operation. A final line of evidence indicating a type I fiber switch was the elevated level of specialized type I contractile proteins and decreased level of specialized type II contractile proteins in the transgenic mice. Interestingly, these same results were seen when naturally occurring (endogenous) PPARδ levels were stimulated in the normal mice (with an orally active compound). This suggests that muscle fibers can be transformed into type I endurance fibers by simply activating the endogenous PPARδ pathway.
In a weight-conscious world, oxidative fibers are thought to offer resistance against obesity since obese individuals have fewer type I fibers than average-weight individuals. Sure enough, transgenic mice fed a high-fat diet gained far less weight than normal mice fed the same diet, even in the absence of exercise. The transgenic mice had much smaller fat cells, which the authors attribute to enhanced oxidative capacity of the muscle tissue, and improved glucose tolerance. (Obese individuals lose the ability to metabolize glucose, which puts them at risk for diabetes.) But what about performance? Remarkably, the marathon mice ran about an hour longer than controls, showing dramatic improvement in both running time and distance---increases of 67% and 92%, respectively.
Altogether, these results show that PPARδ drives the conversion of type I muscle fibers by activating pathways that enhance physical performance and protect against obesity. The finding that endurance and running capacity can be genetically manipulated suggests that muscle tissue is far more adaptable than previously thought. Maybe Olympiads can be made after all---but don\'t give up on training just yet. A full understanding of the molecular basis of muscle fiber determination, including the interactions between PPARδ and its regulatory components, awaits further study.
| PubMed Central | 2024-06-05T03:55:46.817717 | 2004-8-24 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509415/",
"journal": "PLoS Biol. 2004 Oct 24; 2(10):e322",
"authors": []
} |
PMC509416 | Background
==========
Skeletal muscle adapts to neural and use-dependent signals by altering its mass and phenotypic properties, including the proportion of slow (I) and fast (IIa, IIx, and IIb) myosin heavy chain (MHC) isoforms \[[@B1],[@B2]\]. Calcineurin has been implicated as a regulatory molecule involved in the transduction of contractile activity-based signals to molecular signals involved in the regulation of muscle growth and phenotypic gene expression in skeletal muscle \[[@B3]-[@B14]\]. Calcineurin is a phosphatase (protein phosphatase 2B) that is activated by calmodulin upon binding Ca^2+^and has been shown to play an important role in the regulation of cytokine gene expression in lymphocytes \[[@B15],[@B16]\]. Substrates for calcineurin include the phosphorylated isoforms of a transcription factor known as the nuclear factor of activated T cells (NFAT) \[[@B17]\]. Upon dephosphorylation, NFAT isoforms translocate into the nucleus where they can bind to a conserved DNA binding site known as the NFAT response element (NRE) and alter transcription, leading to enhanced expression of specific genes \[[@B15]-[@B17]\].
It has been proposed that in skeletal muscle nuclear NFAT can activate the expression of slow muscle phenotypic genes \[[@B3]-[@B5],[@B8],[@B11],[@B14]\]. In addition, MEF2 and NFAT appear to act synergistically in turning on slow muscle fiber specific genes \[[@B18]\]. Thus, increased muscle contractile activity and the resulting sustained elevations in cytosolic Ca^2+^could potentially result in altered phenotype-specific gene expression via calcineurin \[[@B4]\]. To date, however, no studies have reported the influence of chronically elevated calcineurin activity on the MHC isoform composition and fiber cross-sectional areas (CSAs) of limb and respiratory skeletal muscles. Therefore, in the present study, transgenic mice containing a highly expressed transgene consisting of a constitutively active form of calcineurin (CN\*) driven by the muscle creatine kinase (MCK) enhancer were used to assess the influence of chronic calcineurin activation on MHC isoform protein and muscle mass and fiber cross-sectional area (CSA). Previous studies using the same line of transgenic mice demonstrated that calcineurin activation elevates the expression of some slow phenotypic genes \[[@B11]\], the percentage of EDL fibers with MHC-IIa \[[@B19]\], and proteins related to insulin-stimulated glucose uptake \[[@B20]\].
Results
=======
The MCK-CN\* transgenic mice expressed the transgene in all muscles tested as shown in Figure [1](#F1){ref-type="fig"}. As expected, due to transgene expression being driven by a fast muscle-specific enhancer (MCK enhancer), the fast MG muscle displayed the highest level of expression. The soleus (a mixed fast and slow muscle in mice), diaphragm and plantaris showed relatively similar levels of transgene expression.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
A\) SYBR^®^Green I stained agarose gel of PCR products following RT-PCR for the MCK-CN\* mRNA product. No specific product (\~900 bp, arrow) is observed in the DNase-treated RNA samples (first 4 lanes) of either wild-type (WT) or MCK-CN\* transgenic (CN\*) mouse gastrocnemius. Therefore, there is no contaminating genomic DNA in the RNA samples used for RT-PCR. As expected, RT-PCR of the cDNA (second four lanes) reveals that expression of the transgene is only observed in the MCK-CN\* mice. A positive (+) control of tail DNA from an MCK-CN\* mouse is shown on the right. B) Western blot of CN\* and WT mouse soleus (SOL), diaphragm (DIA), plantaris (PLANT) and gastrocnemius (GAST) muscles for calcineurin. The polyclonal MAb recognized both the endogenous (CN, \~60 kDa) and constitutively active (CN\*, \~45 kDa) forms. As expected, muscle from WT muscles did not contain the CN\* protein. C) Relative semi-quantitative analysis of CN\* mRNA (via RT-PCR) and protein (western blotting) in muscles of MCK-CN\* mice. All values were normalized to the same soleus muscle (n = 3 -- 6 per group). The asterisk (\*) denotes significantly different (p ≤ 0.05) from other muscles.
:::
:::
As shown in Figure [2](#F2){ref-type="fig"}, the muscles from MCK-CN\* mice showed significant shifts towards slower MHC isoforms compared to WT. However, the specific isoforms that were either up- or down-regulated were muscle specific. For instance, the soleus of MCK-CN\* mice showed an elevation in MHC-I at the expense of MHC-IIa. The MG showed a reduction in MHC-IIb, the fastest of the MHC isoforms expressed in mice, and elevations in MHCs-I, -IIa, and -IIx. The diaphragm showed an elevation in MHC-I and reductions in MHCs-IIa and -IIx. The diaphragm of the MCK-CN\* mice also showed a complete absence of MHC-IIb, which was present in small proportions in WT mice. The plantaris of MCK-CN\* mice showed similar directional adaptations in MHC isoforms as did the MG, i.e., higher proportions of MHCs-I, -IIa, and -IIx, and reduced MHC-IIb relative to WT mice. Thus, in MCK-CN\* mice, MHC-I was consistently elevated and MHC-IIb reduced (if it was present in the WT muscle). In contrast, MHC-IIa was increased in the MG and plantaris, but decreased in the soleus and diaphragm of MCK-CN\* mice. Also, MHC-IIx was increased in the MG and plantaris, decreased in the diaphragm, and unchanged in the soleus of MCK-CN\* mice relative to WT.
::: {#F2 .fig}
Figure 2
::: {.caption}
######
Adult myosin heavy chain (MHC) isoform proportions of the soleus (A), medial gastrocnemius (B), diaphragm (C), and plantaris (D) muscles of wild-type (dark bars) and MCK-CN\* transgenic (gray bars) mice as determined by SDS-PAGE of whole muscle extracts. The \* denotes significantly different from wild-type at p ≤ 0.05. Note the elevated proportions of slower MHC isoforms in the transgenic mice compared to wild-type.
:::
:::
The plantaris muscle of MCK-CN\* mice showed a significant shift in fiber type proportions from fast to slow as measured by the proportions of fast and slow isoforms of MHC, sarco(endo)plasmic reticulum Ca^2+^-ATPase (SERCA), and troponin I (Figure [3](#F3){ref-type="fig"}). Plantaris fibers that stained positively for the slow isoform of one protein stained positively for the slow isoforms of the other proteins and likewise for fast protein isoforms. In contrast, immunohistochemical techniques did not detect significant differences in the proportions of fibers containing specific MHC isoforms in the soleus of MCK-CN\* mice (Figure [4](#F4){ref-type="fig"}). However, there was a non-statistically significant tendency for increased proportions of fibers with slower MHC isoforms.
::: {#F3 .fig}
Figure 3
::: {.caption}
######
Proportions of plantaris fibers staining positively for slow and fast isoforms of MHC, sarco(endo)plasmic reticulum Ca^2+^ATPase (SERCA), and troponin I (Tn I) in wild-type and MCK-CN\* transgenic mice. MHC-I, SERCA2, and TN I slow are the slow muscle isoforms and MHC-II, SERCA1 and Tn I fast are the fast muscle isoforms. The \* denotes significantly different from wild-type at p ≤ 0.05. Note the elevated proportions of fibers staining positively for the slow isoforms in the transgenic mice compared to wild-type. All fibers that stained positively for the slow MHC (MHC-I) were positive for the slow isoforms of SERCA and Tn I and all fibers that stained positively for the fast MHC (MHC-II) were positive for the fast isoforms of SERCA and Tn I.
:::
:::
::: {#F4 .fig}
Figure 4
::: {.caption}
######
Proportions of soleus fibers staining positively for specific fast and slow MHC isoforms in wild-type and MCK\* transgenic mice. No fibers stained positively for MHC-IIb with mAb BF-F3, thus antibody RT-D9 which is specific for MHC-IIx and MHC-IIb indicated the presence of MHC-IIx. No significant differences in fiber proportions of the soleus were found between wild-type and MCK-CN\* mice.
:::
:::
To ascertain if other phenotypic properties of muscle fibers were influenced by calcineurin activation, plantaris fibers of MCK-CN\* and WT mice were analyzed for marker enzymes of oxidative (succinate dehydrogenase, SDH) and glycolytic (α-glycerophosphate dehydrogenase, GPD) capacity. Fast plantaris fibers of MCK-CN\* mice showed a significant increase in SDH staining intensity, indicative of elevated mitochondrial content, and a decrease in glycolytic staining intensity, indicative of reduced glycolytic capacity (Figure [5](#F5){ref-type="fig"}). In contrast, slow plantaris fibers of MCK-CN\* mice were not different from WT (Figure [5](#F5){ref-type="fig"}).
::: {#F5 .fig}
Figure 5
::: {.caption}
######
Oxidative and glycolytic enzymatic profiles of individual plantaris fibers of wild-type and MCK-CN\* transgenic mice. The \* denotes significantly different from wild-type at p ≤ 0.05. A) The specific activity of succinate dehydrogenase (SDH, oxidative marker) was increased in fast (type II) plantaris fibers, but unchanged in slow (type I) fibers. The specific activity of α-glycerophosphate dehydrogenase (GPD, glycolytic marker) was significantly decreased in fast plantaris fibers, but not slow fibers. B) The integrated activity (see methods for explanation of integrated activity) of succinate dehydrogenase (SDH, oxidative marker) was increased in fast (type II) plantaris fibers, but unchanged in slow (type I) fibers. The integrated activity of α-glycerophosphate dehydrogenase (GPD, glycolytic marker) was significantly decreased in fast plantaris fibers, but not slow fibers. C) The GPD/SDH ratio (glycolytic/oxidative ratio) was significantly decreased in fast fibers, but unchanged in slow fibers of the plantaris of MCK-CN\* mice relative to wild-type.
:::
:::
As shown in Table [1](#T1){ref-type="table"}, the mass of a characteristically slow muscle, i.e., the soleus, was greater; whereas, the mass of characteristically fast muscles, i.e., the MG and tibialis anterior were lower in MCK-CN\* vs. WT mice. The masses of muscles that characteristically display a more intermediate phenotype, with relatively high proportions of fast oxidative (i.e., IIa and IIx) fibers, i.e., the plantaris and the extensor digitorum longus, were unchanged.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Body and absolute and relative muscle masses.
:::
Wild-type (n = 6) MCK-CN\* (n = 10)
--------------------------------------- ------------------- -------------------
Body Mass (g) 23.3 ± 0.9 20.7 ± 0.4\*
Absolute Muscle Mass (mg)
Soleus 8.2 ± 0.6 10.2 ± 0.6\*
MG 46.7 ± 1.8 34.5 ± 1.9\*
PLT 16.8 ± 0.8 16.2 ± 0.7
TA 43.0 ± 1.2 36.0 ± 1.3\*
EDL 8.3 ± 1.3 7.8 ± 0.5
Relative Muscle Mass (mg/g body mass)
Soleus 0.35 ± 0.02 0.48 ± 0.02\*
MG 2.01 ± 0.04 1.66 ± 0.09\*
PLT 0.72 ± 0.03 0.78 ± 0.04
TA 1.85 ± 0.04 1.74 ± 0.06
EDL 0.36 ± 0.05 0.38 ± 0.03
Abbreviations: MG, medial gastrocnemius; PLT, plantaris; TA, tibialis anterior; EDL, extensor digitorum longus. Values with asterisks are significantly different from wild-type at p ≤ 0.05.
:::
As expected, based on the overall differences in muscle mass between MCK-CN\* and WT mice, the soleus contained fibers with CSAs greater than WT, whereas the MG contained fibers with CSAs smaller than WT (Figure [6A](#F6){ref-type="fig"}). The plantaris, which showed no change in mass, also showed no change in fiber CSA (Figure [6B](#F6){ref-type="fig"}). In the soleus, both slow and fast fibers were larger (Figure [6C](#F6){ref-type="fig"}). However, slow fibers were increased in CSA by 49% and fast fibers by only 22%. This resulted in a \~20% increase in the proportion of the muscle CSA occupied by slow fibers in MCK-CN\* mice (Figure [6D](#F6){ref-type="fig"}). Thus, the changes in overall phenotype of the soleus can be partially accounted for by changes in the size of specific types of fibers.
::: {#F6 .fig}
Figure 6
::: {.caption}
######
Individual fiber cross sectional areas (CSAs) in muscles of wild-type and MCK-CN\* mice. A) Mean fiber CSAs in soleus and medial gastrocnemius (MG) muscle regardless of fiber type. B) Mean fiber CSAs of slow (type I) and fast (type II) fibers of the plantaris muscle. C) Mean fiber CSAs of slow (type I) and fast (type II) fibers of the soleus muscle. D) The percent of the entire soleus muscle cross-section occupied by slow (type I) and fast (type II) fibers. The \* denotes significantly different from wild-type at p ≤ 0.05.
:::
:::
Discussion
==========
Elevated calcineurin activity altered the phenotypic profile and mass of both fast and slow muscles and muscle fibers. The phenotypic changes were indicated by the general fast to slow adaptation in MHC isoform expression in MCK-CN\* versus WT mice. The changes in phenotype were apparently not restricted to MHC isoforms since the plantaris of MCK-CN\* mice had higher proportions of fibers that stained positive for the slow isoforms of MHC, SERCA and troponin I, suggesting a coordinated increase in the expression of slow phenotypic proteins upon calcineurin activation. Fast plantaris fibers also had elevated oxidative and reduced glycolytic profiles, suggesting that calcineurin activation influenced the metabolic profile of fibers promoting a more oxidative phenotype. These data suggest that fibers that did not show adaptations in MHC, SERCA or troponin I isoforms and remained fast were still influenced by calcineurin activation to acquire an oxidative phenotype. These changes are consistent with the proposed role of calcineurin as a sensor of contractile activity and transducer of contractile signals into molecular signals that induce the expression of a slow oxidative phenotype \[[@B4]\].
The overall changes in MHC isoform composition for some muscles, such as the soleus, were apparently aided by fiber type-specific changes in the CSA of the fibers, such that type I fibers were increased and type II fibers decreased (or increased only slightly in the soleus) in CSA as a result of elevated calcineurin activity. These data are consistent with the hypothesis that elevated calcineurin activity induces increased expression of slow muscle phenotypic genes resulting in elevated mass of slow muscles and muscle fibers. In addition, fibers from fast muscles may attain a smaller CSA in MCK-CN\* mice relative to WT as a result of the change in phenotype from a fast glycolytic type of fiber (typically the largest fibers) towards a slow or fast oxidative type of fiber (typically the smallest fibers), see Rivero et al. \[[@B21],[@B22]\].
Although the general adaptations in phenotype were in the direction of faster to slower isoforms, the actual transitions in MHC isoform content were muscle-specific. This is reminiscent of the muscle-specific influence of thyroid hormone treatment on muscle phenotype \[[@B23]\]. Of the muscles examined, the diaphragm of MCK-CN\* mice showed the greatest absolute elevation in MHC-I content. Also, in the soleus and diaphragm of MCK-CN\* mice, MHC-I was up-regulated at the expense of MHC-IIa. However, in the MG, MHCs-I, -IIa, and -IIx were up-regulated at the expense of MHC-IIb. The mechanisms by which some muscles (MG and plantaris) respond to calcineurin activation by up-regulating MHC-IIa and IIx whereas others (soleus and diaphragm) down-regulate these MHCs is currently unknown. *In vitro*data suggest that MHC-IIa is highly (100 fold) and MHC-IIx and -IIb moderately (\~5 -- 10 fold) stimulated by calcineurin activation in mouse C~2~C~12~cells \[[@B24]\]. Because the adaptations were muscle-specific it is likely that additional mechanisms must be invoked in the *in vivo*state to modulate the ultimate response to calcineurin activation. Since the diaphragm showed the greatest response to calcineurin activation and the diaphragm is a chronically active muscle it is tempting to speculate that the chronic motor activity of this muscle results in an up-regulation of parallel signaling pathways that may support a more pronounced calcineurin activity-induced shift in fiber phenotype.
The observation that MHC-IIa and MHC-IIx were up-regulated in fast muscles and MHC-I was up-regulated in slow muscles of MCK-CN\* mice is in agreement with the idea of a limited adaptive range for muscles of varying phenotype. The limited adaptive range theory essentially states that muscles from a slow developmental lineage have the capacity to adapt in the range of I ↔ IIa ↔ IIx, whereas muscles from a fast developmental lineage can adapt in the range of IIa ↔ IIx ↔ IIb \[[@B25]\]. Thus, MG and plantaris fibers may be restricted to express MHC-IIa as the \'slowest\' MHC isoform. In contrast, soleus and diaphragm fibers have the capacity to express MHC-I as the slowest MHC. However, recent data demonstrate that the suggested limited adaptive range for slow muscle is not strict. For example, combined treatment of rats with thyroid hormone and hindlimb suspension results in substantial expression of MHC-IIb by the soleus \[[@B26]\].
Another potential explanation for the muscle-specific response to chronic calcineurin activation is a possible obligatory step-wise transition from MHC-IIb ↔ IIx ↔ IIa ↔ I \[[@B27]\]. Following this scheme a fast muscle fiber would initially express MHC-IIx and then IIa as it transitioned towards a slower phenotype. However, this is a temporal scheme and the animals in this study were 3 months of age, therefore, sufficient time was available for a complete transition of fast muscles to MHC-I. In addition, previous studies from our \[[@B28]-[@B31]\] and other \[[@B26],[@B32]\] laboratories have demonstrated that the obligatory step-wise transition scheme is not always followed. For instance, some rat soleus fibers can transition from MHC-I to MHC-IIx without apparent expression of MHC-IIa protein following hindlimb suspension, spaceflight, or paralysis induced by either spinal cord transection or spinal cord isolation \[[@B28]-[@B31]\].
The molecular mechanisms involved in directing the muscle-specific response are unknown, but could involve parallel signaling pathways, perhaps involving CAMKinase, MEF2, PGC-1, or altered histone acetylation at specific sites. It is also possible that variability in inherent motor-neuron activity influences the expression of specific sets of parallel signaling pathways which in turn allows for alteration of only specific sets of phenotypic proteins in muscles of differing phenotype. Our data showing that the diaphragm, a chronically active muscle, had the greatest absolute increase in MHC-I protein lends support to this idea.
Ultimately, these data support the idea that calcineurin activity plays a role in the regulation of muscle phenotype. Since the expression level of the transgene in these mice results in basal calcineurin activity levels that are \~10 times the activity in WT mice \[[@B33]\], and since the phenotypic adaptations were somewhat modest, this suggests that parallel pathways likely need to be invoked to induce a more complete fiber type shift. In addition, the activation of calcineurin did not completely supersede the developmental-based phenotypic lineage. For instance, in the MCK-CN\* mice the soleus retained \~40% fast (type II) fibers and the MG was predominantly composed of fast MHC isoforms. However, it must be noted that the transgene is active in the MCK-CN\* mice during all stages of development. Thus, negative feedback mechanisms that may reduce the down-stream effectiveness of calcineurin activation may be enhanced or invoked during the development and maturation of the skeletal musculature. It is also possible that parallel pathways must be invoked with calcineurin to fully activate the slow muscle specific phenotype, as suggested by Dunn et al., \[[@B7]\] and demonstrated by Wu et al., \[[@B18]\]. Presence of the transgene will only directly influence the basal calcineurin activity and it is possible that higher sustained levels of calcineurin activity, such as may occur with sustained contractile activity-induced elevations in myoplasmic calcium, would support a more complete fiber transformation. Since the Ca^2+^-calmodulin stimulation of calcineurin can be as great as 100-fold \[[@B15]\], it is possible that normal *in vivo*calcium-induced activation of endogenous calcineurin would result in a much greater elevation in calcineurin activity and a greater degree of fiber type transformation.
Several studies have suggested that calcineurin activation does not influence muscle mass \[[@B7],[@B34]-[@B36]\] or phenotype \[[@B37]\]. One of these studies, Dunn et al., \[[@B7]\], made use of a similar transgenic mouse model as that used in this study. However, the level of basal calcineurin activity in the mice used by Dunn et al., \[[@B7]\] was reported to be several times lower (\~same as WT) than that reported for the mice used in our study, \~10× greater than WT, i.e., transgenic mouse line \#1 from reference \[[@B33]\]. Thus, the lack of effect in some of the previous studies may be related to the level of transgene expression and the lower basal calcineurin activity. It is interesting to note that the transgenic mice used by Dunn et al. \[[@B7]\], also did not display differences in fiber phenotype compared to WT. In contrast, other studies have demonstrated that calcineurin activation and or the accumulation of nuclear NFAT may be involved in the mediation of growth inducing factors, such as IGF-1 \[[@B10],[@B13],[@B38]\].
Curiously, our data suggest that chronic calcineurin activation results in elevated mass of slow muscle and reduced mass of fast muscle. This suggests that the changes in mass and fiber CSA may be secondary to the changes in phenotype. The differential effect of chronic calcineurin activation on slow vs. fast muscle mass may be explained as follows. Elevated expression of slow phenotypic genes in a slow muscle could theoretically result in an increased accretion of slow phenotypic proteins and elevated size of slow fibers. Fast muscle fibers showed an elevation in oxidative enzyme activity, i.e., transitioned from a fast glycolytic (FG) to a fast oxidative glycolytic (FOG) phenotype. Typically, FG fibers are larger than FOG fibers in mammalian muscle. A strong inverse correlation exists between oxidative capacity and fiber CSA \[[@B39],[@B40]\]. Thus, as fast fibers transitioned from FG towards FOG in response to calcineurin activation, they would achieve a smaller CSA.
Conclusions
===========
Collectively, the data support the hypothesis that calcineurin activation plays an important role in the modulation of skeletal muscle phenotype. The responses to calcineurin activation were muscle specific, suggesting that inherent differences in signaling mechanisms within distinct muscles likely modulate the response to calcineurin activation. The observation that a 10-fold increase in basal calcineurin activity results in alterations in the expression of fast versus slow phenotypic proteins and alterations in the metabolic capacities of fibers that do not show a transition to slow fiber type isoforms suggests that the activation of this pathway can modulate multiple aspects of fiber phenotype. However, parallel pathways likely need to be invoked to completely alter the phenotype of all fibers in a muscle.
Methods
=======
Animals
-------
Breeding pairs of MCK-CN\* transgenic mice were obtained from the primary colony at the University of Texas Southwestern Medical Center \[[@B11]\] and housed at the Animal Care Facility at Virginia Polytechnic Institute and State University. This specific line of transgenic mice has been used previously to study the influence of calcineurin activation on the expression of utrophin \[[@B19]\], and insulin-sensitive glucose uptake \[[@B20]\]. Female B6C3F1 mice expressing a transgene consisting of 4800 bp of the MCK enhancer, the coding sequence of the activated form of calcineurin, which codes for amino acids 1 to 398 and lacks the autoinhibitory and calmodulin binding domains \[[@B41]\], and 620 bp of the human growth hormone polyadenylation signal (MCK-CN\* mice) were crossed with the C57Bl6J strain. Transgenic offspring were identified by PCR amplification of tail DNA using primers specific for sequences within the calcineurin coding region (5\'-GAA CCA GCA GTT CCT GTG TGT ACA CG-3\') and the hGH polyadenylation signal (5\'-CAC TCC AGC TTG GTT CCC GAA TAG AC-3\'). Since the transgenic mice were generated in the B6C3F1 strain and were then crossed with the C57Bl6J line, all comparisons were made to wild-type (WT) littermate controls, which would share any strain dependent variables with the MCK-CN\* transgenic mice. All animal procedures were approved by the Virginia Polytechnic Institute and State University and California State Polytechnic University Institutional Animal Care and Use Committees and conform to the American Physiological Society\'s guiding principles for the care and use of animals.
MCK-CN\* (n = 10) and WT littermate controls (n = 6) were sacrificed by pentobarbital injection at 3 months of age. Animals from two independent litters were used. The diaphragm, soleus, medial gastrocnemius (MG), plantaris, tibialis anterior, and extensor digitorum longus muscles were dissected free, trimmed of excess connective tissue, and weighed (diaphragm weights were not obtained). The muscles (except tibialis anterior and extensor digitorum longus) were then rapidly frozen in melting isopentane cooled with liquid nitrogen and stored at -70°C.
Muscle RNA analyses
-------------------
Total RNA was isolated from frozen muscle (soleus, MG, plantaris, and diaphragm) samples using Trizol reagent according to the manufacturer\'s instructions (Invitrogen Corp., Carlsbad, CA, USA). The final RNA pellet was dissolved in nuclease-free water, treated with DNA-free™ (Ambion Inc, Austin, TX, USA) to remove potential contaminating genomic DNA, and then quantified by UV (A~260~) light absorbance. All RNA samples had A~260~:A~280~ratios of greater than 1.8. Two μg of the isolated RNA were reverse transcribed to cDNA with Superscript II (Invitrogen Corp.) in a final volume of 20 μl. The same PCR primers used for genotyping were used to amplify the specific cDNA fragment from transgenic mice expressing the MCK-CN\* transgene. The PCR reaction included: 1 μl of cDNA, 1X Platinum Taq PCR buffer (Invitrogen Corp.), 1.5 mM MgCl~2~, 0.2 mM dNTPs, 1 μM each primer, and 1 unit Platinum Taq (Invitrogen Corp.) in a final volume of 25 μl. The PCR cycle conditions were 94°C for 4 min and then 40 cycles of 94°C for 30 sec, 61°C for 30 sec, and 72°C for 30 sec, followed by a final step at 72°C for 5 min. The PCR products were separated on 1.5% agarose gels, stained with SYBR^®^green I (Molecular Probes Inc., Eugene, OR, USA) and quantified using a ChemiImager 5500 (Alpha Innotech, San Leandro, CA, USA).
Muscle protein analyses
-----------------------
One MG, plantaris and soleus muscle from each animal were serially sectioned perpendicular to the fiber axis using a Microm cryostat, stained imuunohistochemically for fast or slow myosin heavy chain (MHC) isoforms using either anti-fast or anti-slow MHC monoclonal antibodies (mAb, Novocastra Laboratories, Inc.) according to Talmadge et al., \[[@B30]\]. Soleus tissue sections were also analyzed for the presence of MHC-IIa, MHC-IIx and MHC-IIb using mAbs specific for MHC-IIa (mAb SC-71), MHCs-IIx & -IIb (mAb RT-D9) and MHC-IIb (mAb BF-F3). Serial sections of the plantaris were also immunohistochemically stained for the presence of fast and slow isoforms of troponin I (Research Diagnostics Inc.) and sarco(endo)plasmic reticulum Ca^2+^ATPase (SERCA, Novocastra Laboratories, Inc.) isoforms using similar techniques. The sections treated for immunohistochemistry were used to measure the cross-sectional area (CSA) of fifty individual muscle fibers per muscle using a calibrated microscopy-based image analysis system consisting of a GMS-300 grayscale microscopy system (Scion, Frederick, MD) and a Nikon Eclipse E400 microscope.
The MG, plantaris and soleus from the opposite limb and the whole diaphragm (including costal and crural portions) were subjected to high resolution SDS-PAGE for analysis of myosin heavy chain isoforms as detailed in Talmadge and Roy \[[@B42]\]. These muscles were also analyzed for constitutively active and endogenous calcineurin protein levels by semi-quantitative western blotting as described in detail previously by Spangenburg et al. \[[@B43]\]. The primary antibody used for western blotting was a rabbit anti-calcineurin Pan A polyclonal (AB1695) from Chemicon International (Temecula, CA, USA) which recognized both the constitutively active (CN\*) and endogenous forms of calcineurin. The western blot results obtained using this primary antibody were verified (data not shown) with other antibodies that showed similar patterns of reactivity, including antibodies CN-A1 (Sigma Chemical Co., St. Louis, Mo, USA), SC-9070 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and AB1696 (Chemicon International).
Quantitative histochemistry of individual muscle fibers
-------------------------------------------------------
Quantitative histochemical analyses of oxidative and glycolytic enzyme activities were measured on 50 plantaris fibers per muscle that were immunohistochemically typed for fast or slow MHC isoform content in serial sections. According to Rivero et al., \[[@B21],[@B22]\], succinate dehydrogenase (SDH; EC 1.3.5.1) was used as a marker for oxidative capacity and α-glycerophosphate dehydrogenase (GPD) was used as a marker for glycolytic capacity of individual muscle fibers. Histochemical staining and image analysis were performed as previously described in detail \[[@B21],[@B22]\]. The specific enzymatic activity per unit fiber volume (i.e., enzyme concentration) is expressed as change in OD per minute of reaction. The integrated enzymatic activities (ISDH and IGPD) were calculated as the product of specific enzymatic activity (OD/min for a given fiber) and CSA of that fiber which reflects the enzyme content per fiber and is expressed as OD/min × microns^2^.
Statistical analyses
--------------------
Student\'s t-tests were used for comparing mean data between transgenic and WT mice at each age. Statistical differences were significant at p ≤ 0.05.
Authors\' contributions
=======================
RJT: conception, design, data collection and analysis, manuscript preparation, research funds collection.
JSO: quantitative histochemical analyses, genotyping, statistical analysis, figure preparation.
MRR: electrophoretic analyses, statistical analysis, figure preparation.
NDG: mRNA and electrophoretic analyses, figure preparation.
SRS: western blotting, figure preparation.
SJL: muscle sample collection.
FJN: supplied transgenic mice, provided genotyping expertise.
Acknowledgments
===============
We thank Shannon Weber for excellent technical assistance. The authors thank R.W. Grange for help with transgenic animal identification methods and Jay H. Williams for laboratory facilities. The current address for Simon J. Lees is Department of Veterinary Biomedical Sciences, University of Missouri, Columbia, MO 65211; for Jeffrey S. Otis is Emory University, Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30329; and for Francisco J. Naya is Biology Department, Boston University, Boston, MA. This study was supported in part by NIH Grant S06 GM53933 to RJT, and an NIH Postdoctoral Fellowship to FJN while at the University of Texas Southwestern Medical Center.
| PubMed Central | 2024-06-05T03:55:46.818284 | 2004-7-28 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509416/",
"journal": "BMC Cell Biol. 2004 Jul 28; 5:28",
"authors": [
{
"first": "Robert J",
"last": "Talmadge"
},
{
"first": "Jeffrey S",
"last": "Otis"
},
{
"first": "Matthew R",
"last": "Rittler"
},
{
"first": "Nicole D",
"last": "Garcia"
},
{
"first": "Shelly R",
"last": "Spencer"
},
{
"first": "Simon J",
"last": "Lees"
},
{
"first": "Francisco J",
"last": "Naya"
}
]
} |
PMC509417 | Background
==========
The typical antbirds (Thamnophilidae) is a speciose family within the furnariid radiation (sensu \[[@B1]\]) of the New World suboscine clade. The family includes fully 200 species \[[@B2]\] that all are restricted to neotropical forests. Most species are arboreal or undergrowth inhabitants, while only a few members are clearly terrestrially adapted, which otherwise seems to be the commonest lifestyle for most members in closely related clades (e.g., gnateaters Conopophagidae, antpittas Grallariidae, tapaculos Rhinocryptidae, and antthrushes Formicariidae). The highest diversity of typical antbirds is found in the Amazonian basin, and differences in ecological specializations make it possible to find as many as 40 species in the same area \[[@B3]\]. Morphologically typical antbirds shows considerable variation in size and patterns and colors of the plumage (black and shades of grey, buff and chestnut, with sexual plumage dimorphism in many species), while the variation in shape is more restricted. Many insectivorous niches are occupied, but the specialization of some species to follow army ants (to capture escaping insects) is perhaps the most well known. This habit has also given raise to the vernacular family name.
In traditional classifications, the antpittas (Grallariidae) and antthrushes (Formicariidae) were grouped together with typical antbirds in an even larger family. However, the support for the expanded antbird family was indeed weak, and both morphological \[[@B4]-[@B6]\] and molecular \[[@B1],[@B7]\] evidence suggests that antpittas and antthrushes are distantly related to typical antbirds. DNA sequence data \[[@B1],[@B8]\] suggests that gnateaters (Conopophagidae) forms the sister clade to typical antbirds, while antpittas and antthrushes are more closely related to tapaculos (Rhinocryptidae), woodcreepers and ovenbirds (Furnariidae).
Even though the monophyly of typical antbirds seems to be well supported by both syrinx morphology \[[@B6]\] and molecular data \[[@B1],[@B7]\] the phylogenetic relationships within this assemblage are poorly understood, and the confusion extending to all taxonomic levels. Both the monophyly of several genera of typical antbirds has been questioned \[[@B3],[@B9],[@B10]\], as well as the delimitation of certain species \[[@B2],[@B11]-[@B14]\]. Some species have also been moved from one genus to another (e.g., the black-hooded antwren that has been moved from the genus *Myrmotherula*to *Formicivora*\[[@B15]\]). The current knowledge of the phylogenetic relationships among typical antbirds rests mainly on interpretations drawn from external features, mostly of bill and feet, and has remained essentially the same for 150 years \[[@B2]\].
As typical antbirds are morphologically and ecologically diverse, they form a challenging group for studies of, e.g. adaptive evolution. However, such studies, as well as biogeographic interpretations, are difficult to make as long as there is no phylogenetic hypothesis. The aim of this study is therefore to create a hypothesis of generic relationships of typical antbirds that could be used as a framework for more detailed studies of the evolution of the group. Two nuclear introns, intron 2 in myoglobin and intron 11 in the glyceraldehyde-3-phosphodehydrogenase gene (G3PDH), and the mitochondrial cytochrome *b*gene, have been sequenced for 51 typical antbird taxa representing 38 out of the 45 genera recognized by Ridgely and Tudor \[[@B3]\]. We have used Bayesian inference and Markov chain Monte Carlo (MCMC) to estimate the phylogenetic relationships.
A common assumption made by molecular systematists is that gene trees accurately reflect species trees. Nevertheless, different data partitions may have different phylogenies due to processes as lineage sorting, gene duplication followed by extinction, and lateral transfer by hybridization and introgression (reviewed in \[[@B16]-[@B18]\]).
Primarily, there are two contradictory strategies utilized to handle data sets with significant phylogenetic incongruence between independent data partitions. Advocates for a \"total evidence approach\" (e.g., \[[@B19],[@B20]\]) suggest that available data always should be combined, even though individual data partitions might be partly incongruent. The arguments are that a combination of different data partitions might improve the total resolution as different data partitions might be useful to resolve different areas of the tree, and that additive data sets might enhance phylogenetic informative characters that have been hidden by noise in the individual partitions. Opponents to this view (e.g., \[[@B21],[@B22]\]) advice that data partitions with a significant level of incongruence should not be combined, as reliable characters might be obscured by random or systematic errors and in the worse case result in an erroneous topology (even though individual data partitions might provide consistent estimates).
However, when independent evidence is lacking and incongruence occurs between individual data partitions, it may be difficult to determine whether particular partitions are better estimates of the species tree than others. Researchers might favor the \"total evidence approach\" for this particular reason (even though the argument for not combining data partitions with significant levels of incongruence have strong merits). However, the degree of incongruence between individual gene trees could be used to determine whether the phylogenetic conclusions should be based on the combined data set, or only those parts that are similar among the different partitions. A commonly used approach for analysing combined data with maximum likelihood is to assume a single (the same) substitution model for all of the combined genes (for exceptions, see \[[@B23],[@B24]\]). A significant result of incongruence between the combined result and the individual genes can then be hard to explain, since the incongruence could be due to both true difference in gene phylogeny and a misfit in the assumed model of evolution for the combined data \[[@B21],[@B25]\]. This misfit could, for example, be a result of not allowing a heterogeneous model, that is, not allowing the different genes to have separate substitution models in the combined analysis \[[@B26]\]. We have thus explored our data partitions (the individual genes) by the congruence test described by Nylander et al. \[[@B27]\], which utilizes Bayes factors. The test is not an explicit significance test but compares the strength of evidence between two models of character evolution.
Although nuclear genes (as when situated on different chromosomes) may be considered as members of different linkage groups, the maternally inherited mitochondrial genome is effectively independent from the nuclear genome. Organelle genomes have also been suggested to more susceptible to \"flow\" between taxa during hybridization (although much less common in animals than in plants). In birds Degnan and Moritz \[[@B28]\] and Degnan \[[@B29]\], for example, have demonstrated that the mitochondrial tree in Australian white-eyes misrepresented the tree of nuclear loci and the expected species tree, possibly due to previous hybridization events. We have thus primarily been interested in the potential incongruence between the mitochondrial cytochrome *b*and the two nuclear genes (myoglobin and G3PDH), but all combinations of the three genes were examined. However, limitations in the substitution models might be the most important explanation to observed incongruence between data partitions, rather than an intrinsic phylogenetic incongruence \[[@B27]\]. We also explored how changes in substitution models affected the observed incongruence in our data set.
Results
=======
Molecular variation and sequence distances
------------------------------------------
After alignment, the concatenated sequences become 2173 bp long. A total of between 679 bp (*Sclerurus scansor*) and 723 bp (*Myrmotherula leucophthalma*) was obtained from myoglobin intron 2, between 351 bp (*Rhegmatorina melanosticta*) and 400 bp (*Myrmeciza griseiceps*) from G3PDH intron 11, and 999 bp from cytochrome *b*. The observed, pairwise distances between ingroup taxa range between 0, 7% and 10, 7% in myoglobin, between 0, 3% and 19, 3% in G3PDH and between 6, 5% and 23, 9% in cytochrome *b*. Indels were found both in the myoglobin intron 2 and in the G3PDH intron 11. In most cases these are autapomorphic indels or occur in especially variable and repeatable regions. Given the tree topologies obtained from the Bayesian analyses, some synapomorphic indels were observed. For example, all *Thamnophilus*representatives share with *Sakesphorus bernardi*an insertion in the G3PDH intron, and, together with *Dysithamnus mentalis*and *Herpsilochmus atricapillus*, an insertion in the myoglobin intron.
Phylogenetic inference and molecular models
-------------------------------------------
A priori selection of substitution models showed that fairly parameter rich models were the best fit for all data partitions. Importantly, modeling rate variation seemed to be an important component. For the cytochrome *b*partition the GTR+I+Γ was the best fit, and for myoglobin intron 2, it was the GTR+ Γ. For the G3PDH intron 11 the somewhat simpler HKY+ Γ model was chosen. These models were used in the consecutive MCMC of the individual genes as well in the combined analysis. The parameter estimates from the two separate MCMC runs for each data set were found to be very similar (data not shown), thus allowing an inference from the concatenated output. After discarding the burn-in phase the inference for the cytochrome *b*was based on a total of 36, 000 samples from the posterior, for myoglobin the inference was based on 38, 000 samples, and for G3PDH and the combined data, inference were based on 38, 000, and 55, 600 samples, respectively. For the phylogenetic inference, the mode of the posterior distribution of topologies was presented as a majority-rule consensus tree from each analysis (Figures [1](#F1){ref-type="fig"},[2](#F2){ref-type="fig"},[3](#F3){ref-type="fig"},[4](#F4){ref-type="fig"}).
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**The G3PDH majority rule consensus tree.**The 50% majority rule consensus tree obtained from the Bayesian analyses of the G3PDH (glyceraldehydes-3-phosphodehydrogenase) intron 11 data set. Posterior probability values are indicated to the right of the nodes.
:::
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**The myoglobin majority rule consensus tree.**The 50% majority rule consensus tree obtained from the Bayesian analyses of the myoglobin intron 2 data set. Posterior probability values are indicated to the right of the nodes.
:::
:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**The cytochrome b majority rule consensus tree.**The 50% majority rule consensus tree obtained from the Bayesian analyses of the cytochrome b data set. Posterior probability values are indicated to the right of the nodes.
:::
:::
::: {#F4 .fig}
Figure 4
::: {.caption}
######
**The combined majority rule consensus tree.**The 50% majority rule consensus tree obtained from the analyses of the combined data set (G3PDH intron 11, the myoglobin intron 2 and the cytochrome b data sets). Clades A, B and C are major groups of typical antbirds discussed in the text. Posterior probability values are indicated to the right of the nodes.
:::
:::
The trees obtained from the Bayesian analyses of the individual genes (cytochrome *b*, myoglobin and G3PDH) and the combined data set all differ in topology and degree of resolution. The G3PDH gene produced the poorest resolved tree (Figure [1](#F1){ref-type="fig"}) and also contains the smallest number of nodes with posterior probability values above 0.90. The myoglobin (Figure [2](#F2){ref-type="fig"}) and cytochrome *b*(Figure [3](#F3){ref-type="fig"}) genes produced trees with similar degree of resolution and nodal supports, but there is a weak tendency for cytochrome *b*giving better resolution and support at terminal nodes. The combined data set (cytochrome *b*, myoglobin and G3PDH) produced the most resolved tree (Figure [4](#F4){ref-type="fig"}) with the highest number of strongly supported nodes (exceeding 0.90 posterior probability). Overall, the myoglobin, the cytochrome *b*and the combined trees are topologically rather similar, while the G3PDH tree is the most deviant. A common pattern in all trees is that several nodes are unresolved, or short with low or intermediate posterior probabilities support values (0.50--0.90). The observed topological conflicts between the obtained trees generally occur at these short nodes, and there are only a few nodes with posterior probabilities values above 0.90 that are in conflict between the trees. Of these, one concerns the outgroup relationships (the G3PDH tree supports with 0.96 posterior probability a position of *Pteroptochos tarnii*that differs from all other trees). The other two conflicts concern internal relationships within well supported sub-clades: The cytochrome *b*tree places with 0.98 posterior probability *Myrmotherula menetriesii*basal to a clade consisting of *Myrmotherula axillaris, Myrmotherula behni*and *Formicivora rufa*. In the combined tree *Myrmotherula menetriesii*instead is nested within this clade with 1.00 posterior probability. The myoglobin tree suggests with 0.94 posterior probability that *Taraba major*is basal to *Batara cinerea*and *Hypoedaleus guttatus*, while *Taraba major*is basal also to *Mackenziaena severa*and *Frederickena unduligera*with 0.99 posterior probability in both the combined and the cytochrome *b*trees.
However, most suggested relationships are congruently supported by more than one of the trees obtained from the individual genes and by the combined data set. Several clades are also supported by all three genes trees as well as by the combined data set, including the recognition of a monophyletic origins of 1) the \"large antshrikes\" (*Taraba major*, *Batara cinerea*, *Hypoedaleus guttatus*, *Mackenziaena severa*, and *Frederickena unduligera*), 2) the \"professional\" ant-following antbirds (*Pithys albifrons, Phlegopsis erythroptera*, *Phaenostictus mcleannani*, *Rhegmatorhina melanosticta*and *Gymnopithys leucaspis*), 3) a *Sakesphorus*-*Thamnophilus*antshrike lineage (*Sakesphorus bernardi*and the five representatives of the genus *Thamnophilus*), and 4) a clade consisting of the wing-banded antbirds (*Myrmornis torquata*), the spot-winged antshrike (*Pygiptila stellaris*) and the russet antshrike (*Thamnistes anabatinus*). Sistergroup relationships between antvireos (*Dysithamnus mentalis*) and *Herpsilochmus*antwrens (*Herpsilochmus atricapillus*), as well as between *Myrmotherula obscura*and *Myrmochanes hemileucus*are also recognized by all trees.
Based on the tree obtained from the Bayesian analysis of the combined data set, typical antbirds could also be divided into three major clades (marked as A, B and C in Figure [4](#F4){ref-type="fig"}). The first clade (clade A) includes four genera that are suggested to have a basal position in relation to all other typical antbirds (1.00 posterior probability in the combined tree). This basal group (supported by 0.72 posterior probability in the combined tree) includes the representative of *Terenura*antwrens (*Terenura humeralis*), the wing-banded antbird (*Myrmornis torquata*), the spot-winged antshrike (*Pygiptila stellaris*) and the russet antshrike (*Thamnistes anabatinus*).
The second clade (clade B, Figure [4](#F4){ref-type="fig"}) is supported by 0.95 posterior probability in the combined tree and includes all antshrike genera (except the spot-winged antshrike and the russet antshrike, see clade A), antvireos (*Dysithamnus*), *Herpsilochmus*antwrens and the banded antbird (*Dichrozona cincta*). Within this large clade several lineages occur that receives more than 0.95 posterior probability. Noticeable within this clade is that neither the analyses of the individual genes nor the combined data set conclusively support that the representative of the antshrike genus *Sakesphorus*(*Sakesphorus bernardi*) is phylogenetically separated from the *Thamnophilus*antshrikes.
The last clade (clade C, Figure [4](#F4){ref-type="fig"}), including the *Myrmeciza*antbirds, most antwren genera (e.g., *Myrmotherula*and *Formicivora*), the \"professional\" ant-following antbirds, and some allied species, is supported by a 1.00 posterior probability value. Also within this clade several lineages are supported by posterior probability values above 0.90. However, the most interesting observation is the strong support for a polyphyletic origin of the *Myrmeciza*antbirds and the *Myrmotherula*antwrens.
Tests of incongruence
---------------------
The Bayes factor tests showed extensive incongruence between partitions, at least in the sense that relaxing the assumption of a common topology parameter always gave a better model likelihood (Table [1](#T1){ref-type="table"}). For example, allowing the cytochrome *b*partition to have a separate topology from the two nuclear partitions myoglobin and G3PDH, gave a 2logB~12~of 60.8. This value strongly suggests that an unlinked model is superior to the model assuming a common topology parameter for all partitions. This would also suggest that there is strong conflict between the mitochondrial and the nuclear partitions. However, this conclusion is far from conclusive when we consider the linking of the topology parameter for other combinations of the data. Combining the topology parameter for either one of the nuclear partitions with the mitochondrial, actually gives a better model (higher Bayes factors) than considering the mitochondrial vs. the nuclear partition (Table [1](#T1){ref-type="table"}). For example, compared to the model that assumes a common topology parameter, unlinking the myoglobin partition from the other gave a 2logB of 102.26. Unlinking the G3PDH partition gave an even better model, with a 2logB of 118.12. Furthermore, if we would have to choose the one partitioning scheme that had the highest model likelihood, the model allowing a separate topology parameter for all partitions would be the clear choice (having a 2logB of 241.36 compared to the common model).
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Summary of Bayes factor tests of incongruence. Entries are twice the log of the Bayes factor in the comparison between models M~1~and M~2~(2log*B*~12~). The row models are arbitrarily labeled M~1~; thus, positive values indicate support for the column model over the row model. A dash (-) indicates which partitions that have linked topology parameters.
:::
Model Cyt b-Myo-G3PDH Cyt b, Myo-G3PDH Cyt b-Myo, G3PDH Cyt b-G3PDH, Myo Cyt b, Myo, G3PDH
------------------- ----------------- ------------------ ------------------ ------------------ -------------------
Cyt b-Myo-G3PDH 0 60.84 118.12 102.26 241.36
Cyt b, Myo-G3PDH 0 57.28 41.42 180.52
Cyt b-Myo, G3PDH 0 -15.86 123.24
Cyt b-G3PDH, Myo 0 139.1
Cyt b, Myo, G3PDH 0
:::
The parsimony based ILD-test did not find a significant incongruence between the three gene partitions (p = 0.967).
Discussion
==========
Phylogenetic incongruence between gene partitions
-------------------------------------------------
Allowing the gene partitions to have separate topology parameters clearly increased the model likelihood. That is, the unlinked models clearly had a better fit to the data than the linked models. Judging from the absolute value of the 2logB (Table [1](#T1){ref-type="table"}), we are inclined to conclude that we should treat each partition as having its own posterior distribution of trees. However, the question is if we from these results really can say that the gene partitions evolved on different phylogenies? There are several reasons why different data partitions may have different phylogenies, although being sampled from the same taxa, or even the same individuals (se above). We cannot completely rule out the occurrence of any of these processes in our data. However, we believe that the interpretation based solely on Bayes factors might be hazardous. For instance, is it plausible that all three gene partitions had evolved on three different phylogenies, or that the linking of cytochrome *b*and myoglobin is a more reasonable partition of the data, instead of the mitochondrial versus the nuclear partitions? Nylander et al. \[[@B27]\], speculate that limitations in the substitution models might be more reasonable explanations to the high Bayes factors observed when comparing unlinked and linked models. Changing a component of the nucleotide substitution model, e.g. adding parameters to model rate variation, had much higher impact on the model likelihood than unlinking parameters among data partition. To illustrate the impact of changing the substitution model in our data, we run additional MCMC analyses under a different set of models, and compared them with the previous analyses using Bayes factors. The results were striking (Table [2](#T2){ref-type="table"}). For example, we compared two models without rate variation, one with linked and the other with unlinked topologies (in both models GTR was used for cytochrome *b*and for myoglobin, and HKY for the G3PDH). The 2logB was 295.98 in favor for the unlinked model. However, adding parameters for modeling rate variation to either of the two models increased the model likelihood tremendously. The 2logB in favor of a model having parameters for rate variation (applying the same substitution models as the ones chosen a priori using AIC, see material and methods), varied between 5125.22 and 5662.56, depending on the model being compared (Table [2](#T2){ref-type="table"}). Similar observations of magnitude changes in Bayes factors were made by Nylander et al. \[[@B27]\], when allowing rate variation. Another striking feature was that once parameters for modeling rate variation had been incorporated into the model, unlinking topologies did not seem to have as pronounced effect on the model likelihood (Table [2](#T2){ref-type="table"}), compared to the models without rate variation. This observation is in concordance with previous findings that many functional genes have a strong among-site rate variation and that adding the relevant parameters to the model is likely to have a large effect on the likelihood \[[@B23],[@B27],[@B30],[@B31]\].
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Summary of Bayes factor tests showing the effect of changing substitution model components. Entries are twice the log of the Bayes factor in the comparison between models M~1~and M~2~(2log*B*~12~). The row models are arbitrarily labeled M~1~; thus, positive values indicate support for the column model over the row model. A dash (-) indicates which partitions that have linked topology parameters. Asterisks (\*) indicate models where the rates are assumed to be equal.
:::
Model Cyt b-Myo-G3PDH Cyt b, Myo, G3PDH Cyt b-Myo-G3PDH\* Cyt b, Myo, G3PDH\*
--------------------- ----------------- ------------------- ------------------- ---------------------
Cyt b-Myo-G3PDH 0 241.36 -5421.2 -5125.22
Cyt b, Myo, G3PDH 0 -5662.56 -5366.58
Cyt b-Myo-G3PDH\* 0 295.98
Cyt b, Myo, G3PDH\* 0
:::
It is worth noting that the parsimony based ILD-test did not find a significant incongruence between the three gene partitions. The value of this observation is uncertain, however, as the ILD test is based on another optimality criterion (parsimony). Furthermore, the strength of the test and interpretation of the results have also been questioned (e.g., \[[@B32]\])
In conclusion, allowing partitions to have separate topology parameters put fewer restrictions on the data. Hence, we should expect to find a better fit of the model to the data. Bayes factors seem promising for evaluating the relative contribution of components to an evolutionary model. However, judging from the relative increase in model likelihood when unlinking topologies compared to e.g., adding parameters for rate variation, we would anticipate components in the substitution model (for example, allowing rate variation among lineages) to have more effects on accommodating incongruence in the data. That is, even if we find strong evidence for a model allowing separate topology parameters, this might not mean strong evidence for separate gene phylogenies, as long as vital components of the substitution model are still missing. For further discussions on Bayesian approaches to combined data issues see e.g., \[[@B25],[@B26],[@B33]\].
Phylogeny and morphological variation in typical antbirds
---------------------------------------------------------
Even though we are unable to conclusively tell whether the observed phylogenetic incongruence between the individual gene partitions is due to genuine differences in phylogeny, or to limitations in the models used, we believe that the tree obtained from the combined data set represents the best estimate of the true relationships within the typical antbird assemblage. Obviously, several relationships are strongly supported, by congruent recognition by the individual gene trees and/or by high nodal support values. Nevertheless, other relationships have to be regarded as tentative, and especially those where any of the individual gene trees gives a strong nodal support for an alternative topology.
It is noticeable that, although the individual genes congruently support several terminal groups, basal relationships are generally less well resolved and more often in conflict. Even though this observation might be biased due to the use of improper molecular models when calculating the trees, biased mutation rate in studied genes, or a biased taxon sampling, it could indicate that the diversifications of typical antbirds was characterized by some rapid speciation bursts. There are only a few recent studies of typical antbirds with taxon samplings that includes representatives from several genera, but these studies show similar difficulties in resolving generic relationships. For example, in a study of phylogenetic relationships of *Myrmotherula*antwrens that included representatives from several other typical antbird genera, Hackett and Rosenberg \[[@B10]\] obtained considerably different topologies from plumage characters, allozyme and morphometric data, respectively. In addition, the phylogenetic relationships suggested from mitochondrial DNA sequence data within a partly comparable taxon sampling \[[@B9]\], have little resemblance to those in Hackett and Rosenberg \[[@B10]\]. The nodes between typical antbirds in the DNA-DNA hybridization \"tapestry\" by Sibley and Ahlquist \[\[[@B7]\]: Figure 372\] also contain a high degree of short branches.
It is also apparent that earlier antbird taxonomists, using external morphology, had difficulties in their taxonomic decisions and interpretations of higher-level relationships. Ridgway \[\[[@B34]\]: p. 9\] expressed that \"The classification of this group is very difficult, more so probably than in the case of any American family of birds\". Hackett and Rosenberg \[[@B10]\] concluded that antwren speciation mainly has been followed by plumage differentiation (and to some degree size differentiation) rather than changes in body proportions. Overall, this evolutionary pattern, with great changes in plumage and more limited changes in body proportions, seems to characterize the entire typical antbird assemblage (in contrast to the situation in ovenbirds, where there is a great variation in body proportions but not in plumage characters). However, Hackett and Rosenberg \[[@B10]\] suggested that neither plumage nor morphometric data correctly predicted the genetic relationships among the studied taxa. Our results seem to support their assumption as the traditionally used plumage characters in typical antbirds, as stripes, wingbars, and general coloration; seem to be irregularly distributed in the phylogenetic tree. It is reasonable to assume that plumage characters in typical antbirds are variable to such a degree that they are of limited use in studies of higher-level relationships. High levels of homoplasy (convergences and reversals) in plumage characters have also been reported in other passerine birds e.g., in Australian scrubwrens \[[@B35]\] brush-finches \[[@B36]\], and in New World orioles \[[@B37]\].
However, if excluding members in the \"basal\" group (clade A, Figure [4](#F4){ref-type="fig"}) and a few other aberrant taxa, the division of typical antbirds into the two main lineages in our phylogeny (clade B and C, Figure [4](#F4){ref-type="fig"}) is overall in good agreement with their body proportions (although there is a considerable size variation within both clades). The antshrikes (excluding *Tamnistes*and *Pygiptila*), antvireos and *Herpsilochmus*antwrens in clade B (Figure [4](#F4){ref-type="fig"}) are all more or less robust birds with heavy and prominently hooked bills, and many of them have a barred plumage pattern. The taxa in clade C (Figure [4](#F4){ref-type="fig"}), which includes most antwren genera, the *Myrmeciza*antbirds, the \"professional\" ant-following antbirds and some allied species, are generally slimmer birds with longer, thinner bills that have a less prominent hook. Most suggested relationships within clade B and C are in good agreement with traditional classifications. The recognition of monophyletic origins of most of the \"professional\" ant-following taxa (*Phaenostictus*, *Gymnopithys*, *Rhegmatorhina*, *Pithys*and *Phlegopsis*) and the \"large\" antshrikes (*Taraba*, *Hypoedaleus*, *Batara*, *Frederickena*and *Mackenziaena*) are two examples where our results are congruent with traditional classifications. The suggested relationships between the *Hypocnemis*and *Drymophila*antbirds, and the *Herpsilochmus*antwrens and the antvireos (*Dysithamnus*), respectively, have also been proposed previously based on molecular data \[[@B9],[@B10]\]. Unfortunately, the genera *Biatas*, *Clytoctantes*, *Percnostola*, *Rhopornis*, *Stymphalornis*and *Xenornis*were lacking in our study; while most of these should probably be referred to Clade C, *Biatas*is difficult to place.
Some novel relationships and the phylogenetic positions of some aberrant taxa
-----------------------------------------------------------------------------
For certain taxa the position in our combined phylogeny is unexpected considering the external morphology and traditional classification. Most noticeable are the position of the banded antbird (*Dichrozona cincta*), which is nested within the clade with antshrikes, antvireos and *Herpsilochmus*antwrens (clade B, Figure [4](#F4){ref-type="fig"}), and the position of the wing-banded antbird (*Myrmornis torquata*) as sister to the russet antshrike (*Thamnistes anabatinus*) and the spot-winged antshrike (*Pygiptila stellaris*) (clade A, Figure [4](#F4){ref-type="fig"}). However, the increased number of molecular based phylogenies in recent years have led to discoveries of several examples, at different phylogenetic levels, were birds have been misclassified due to significant morphological differences from the taxa to which they are most closely related \[[@B38]-[@B40]\].
The phylogenetic position of the wing-banded antbird (*Myrmornis torquata*) has long been obscured and it was long placed with the typical army-ant followers (e.g., \[[@B2]\]). The wing-banded antbird has also been suspected to be related to ground antbirds (Formicariidae sensu \[[@B7]\]) based on similarities in morphology and general appearance \[[@B7]\]. Our results confidently place it within typical antbirds, a conclusion further supported by its vocalization \[[@B2]\] and choice of nest site and its white egg \[[@B41]\]. The well supported relationship to the arboreal russet antshrike (*Thamnistes anabatinus*) and spot-winged antshrike (*Pygiptila stellaris*), suggested by our data, has apparently been obscured by structural differences caused by its adaptation to a terrestrial life-style shared with for example the antthrushes. A similar explanation may apply to the peculiar position of the banded antbird (*Dichrozona cincta*) in the combined phylogeny, as this taxon is also a mainly terrestrial bird, unlike the other members in the \"antshrike\" clade (clade B, Figure [4](#F4){ref-type="fig"}). The fact that the banded antbird has a rather long branch in the combined tree and that its phylogenetic position alter between the individual gene trees, leads us to consider the phylogenetic position of the banded antbird (*Dichrozona cincta*) as preliminary. However, it is obvious that it is not closely related to the *Hylophylax*antbirds with which it has traditionally been grouped (based on similarities in plumage patterns and weak sexual dimorphism). It should be noted that, due to the peculiar position of *Dichrozona cincta*, a second individual (ZMUC 128217) have been sequenced for all three genes. There were no variation at all found between the two individuals in G3PDH, in myoglobin 1 ambiguous position were found, and in cytochrome *b*24 base pairs (2.4%) that differed as well as 3 ambiguous positions were found. Overall, this variation is within the variation that could be suspected between individuals within a species. Thus, the strange position of *Dichrozona cincta*in our analyses is unlikely to be due to sample or sequence mix-up.
There are several other, less striking examples where the position of taxa in our phylogeny conflicts with relationships suggested in classifications based on external morphology. The *Herpsilochmus*antwrens for example (traditionally placed among *Myrmotherula*, *Microrhopias*and *Formicivora*antwrens), are quite different in appearance from their sister group *Dysithamnus*in being rather slim, lacking a particularly hook-bill, and in having a distinctly patterned plumage (however, as discussed above a close relationship between *Herpsilochmus*and *Dysithamnus*is also supported by an independent molecular study). Other examples are the positions of *Myrmorchilus*and *Neoctantes*, respectively (see discussion below). In these cases their true affinities may have been obscured by morphological adaptations to habitats or food sources that differ from those preferred by their closest relatives.
The strong support in the combined tree for basal positions of *Myrmornis*, *Pygiptila, Thamnistes*and *Terenura*relative to all other typical antbirds is maybe the most unexpected result of our study. In a majority of classifications *Terenura*is placed close to other antwrens, but with no strong data support. Although the precise position of the *Terenura*antwrens is partly ambiguous in our analysis, they obviously belong to an ancient radiation that is only distantly related to the other \"antwrens\". The *Terenura*antwrens differ from other \"antwrens\" in plumage pattern and in being more slender and warbler-like with a thinner bill and longer tail. In a study based on mitochondrial DNA the position of *Terenura*was ambiguous depending on how the data set was analyzed \[[@B9]\] but clearly it was not closely related to the other taxa included in that study (e.g., *Myrmotherula*, *Formicivora, Herpsilochmus*, *Hypocnemis*, *Drymophila*).
The well-supported phylogenetic position of *Pygiptila*and *Thamnistes*as the sistergroup to *Myrmornis*(instead of being close to other antshrikes as suggested in many linear classifications), is novel. However, *Pygiptila*and *Thamnistes*resemble each other in their ways of feeding in the sub-canopy, *Thamnistes*also resembling the *Pygiptila*female in appearance, and differing from most antshrikes in feeding behavior. DNA-DNA hybridization data \[[@B7]\] and protein electrophoresis \[[@B10]\] have previously shown *Pygiptila*to be genetically distant from the *Thamnophilus*antshrikes. The general external resemblance of *Pygiptila*and *Thamnistes*to other antshrikes is therefore best explained, as being plesiomorphic, and this may also be the case with their suspended nest-type.
*The polyphyly of*Myrmotherula *antwrens and*Myrmeciza *antbirds*
-----------------------------------------------------------------
Our results confirm both previous molecular studies that suggest the *Myrmotherula*antwrens are polyphyletic \[[@B9],[@B10]\], and the suspicion based on morphology that also the rather diverse genus *Myrmeciza*constitutes an unnatural taxon \[[@B3]\]. Nevetheless, most *Myrmeciza*antbirds studied herein belong to the same clade, although they are not monophyletic as several other genera (*Myrmoborus*, *Gymnocichla*, *Pyriglena*, *Sclateria*, *Schistocichla*, *Hypocnemoides*, and *Hylophylax*) are nested among them. However, the chestnut-tailed antbird (*Myrmeciza hemimelaena*), which represents a group of small and slim *Myrmeciza*antbirds with prominent wing spots in both sexes, groups with the *Drymophila*, *Hypocnemis*and *Cercomacra*antbirds. The small and slim *Myrmeciza*antbirds resembles morphologically the *Hypocnemis*antbirds in having similar wing spots as well as a rather short and rufous-brown tail.
The clade that includes the remaining *Myrmeciza*antbirds consists of three unresolved lineage. The first includes a group of large and heavily built *Myrmeciza*antbirds (represented by *Myrmeciza fortis*). Next outside this group is the fire-eye (*Pyriglena leuconota*), followed by the bare-crowned antbird (*Gymnocichla nudiceps*) and the *Myrmoborus*antbird representative (*Myrmoborus myotherinus*). These taxa have rather stout bodies and in most cases red eyes. Both the fire-eyes and the bare-crowned antbird were previously assumed to be related to the large, heavy-billed *Myrmeciza*antbirds (e.g., \[[@B3]\]). The second lineage consists of the silvered antbird (*Sclateria naevia*) and the *Schistocichla*antbird representative (*Schistocichla leucostigma*). These relationships are in good agreement with the overall plumage characters in these taxa \[[@B3]\], with the males being rather uniform gray while the females are rufous. Such a plumage is also found in the genus *Percnostola*, with which the *Schistocichla*antbirds are considered to be most closely related (*Schistocichla*and *Percnostola*have even been regarded as congeneric, but it has also been suggested that *Percnostola*could be polyphyletic).
In the third lineage, *Myrmeciza griseiceps*and *Myrmeciza berlepschi*form the sister clade to *Myrmeciza loricata, Hypocnemoides maculicauda*and *Hylophylax naevia*(the latter two are sister taxa). This group consists of rather typical shaped and sized \"*Myrmeciza*\" antbirds. Although it has a shorter tail, *Hylophylax naevia*shares plumage pattern with *Myrmeciza loricata*(*Hypocnemoides maculicauda*is more discretely patterned), while *Myrmeciza griseiceps*and *Myrmeciza berleschi*, on the other hand, are more uniformly colored birds.
A non-monophyletic origin of *Myrmotherula*antwrens, suggested by our data, agrees with the results of previous molecular studies \[[@B9],[@B10]\]. The results also support Hackett and Rosenberg\'s \[[@B10]\] protein electrophoresis data suggesting that the \"gray\" and \"streaked\" forms of *Myrmotherula*antwrens are more closely related to each other than either is to the \"checker-throated\" forms. The combined tree (Figure [4](#F4){ref-type="fig"}, clade C) suggests that the *Myrmotherula*antwrens evolved along two separate phylogenetic lineages. In the first, the \"checker-throated\" forms (*Myrmotherula fulviventris*and *Myrmotherula leucophthalma*) group with the black bushbird (*Neoctantes niger*) and constitute the sister to the dot-winged antwren (*Microrhopias quixensis*) and the stripe-backed antbird (*Myrmorchilus strigilatus*). Based on the external morphology these taxa indeed constitute a rather heterogeneous group. For example, the stripe-backed antbird has previously been suggested to be related to *Formicivora*and *Drymophila*antwrens \[[@B42]\], which are distantly related according to our results. However, *Neoctantes*, *Microrhopias*and *Myrmorchilus*are monotypic genera that lack obvious close relatives. *Myrmorchilu*s is essentially a terrestrial bird, living in chaco scrub, thus differing in habits and habitat from the \"typical\" antwren lifestyle. *Neoctantes*lives in humid forest like most *Myrmotherula*antwrens, but its bill is modified to hammers on stems, vines etc., and to be used as a wedge to pry off strips of bark \[[@B2]\]. The morphological differences between *Neoctantes*and *Myrmorchilus*on one hand, and the \"checker-throated\" *Myrmotherula*antwrens on the other, could thus be the result of adaptive specializations in the former taxa.
In the second lineage of *Myrmotherula*antwrens, the \"streaked\" forms represented by the short-billed antwren (*Myrmotherula obscura*) and the black-and-white antbird (*Myrmochanes hemileucus*) form the sister group to the \"gray\" forms (represented by *Myrmotherula menetriesii*, *axillaris*and *behni*) and *Formicivora rufa*. Although the support for nesting *Formicivora rufa*among the \"gray\" forms of *Myrmotherula*is rather weak, it suggests that the generic boundary between *Formicivora*and \"gray\" *Myrmotherula*antwren is far from unambiguously settled. This is also indicated by the recent transfer of the black-hooded antwren from the genus *Myrmotherula*to *Formicivora*\[[@B15]\]. Bates et al. \[[@B9]\] also found a close relationship between *Myrmotherula longipennis*(belonging to the \"gray\" form of *Myrmotherula*antwrens) and the genus *Formicivora*(*Formicivora grisea*and *Formicivora rufa*).
Conclusions
===========
The phylogenetic results support that most antbirds could be divided into two major clades that are in broad agreement with traditional classifications. The first clade includes most antshrike genera, antvireos and the *Herpsilochmus*antwrens, while the second clade consists of the *Myrmeciza*antbirds, the \"professional\" ant-following antbirds, and allied. However, some relationships within these clades, as well as the support for that *Terenura*antwrens, the wing-banded antbird (*Myrmornis torquata*), the spot-winged antshrike (*Pygiptila stellaris*) and the russet antshrike (*Thamnistes anabatinus*) are basal to all other typical antbirds, are unexpected based on external morphology. Possibly the true affinities of these taxa have been obscured by morphological convergence due to adaptations to new habitats or food sources. Our results also strongly support that both the *Myrmeciza*antbirds and the *Myrmotherula*antwrens are unnatural groupings in need for taxonomic revisions. Also certain other taxa may be unnatural units, but definitive conclusions must await future analyses involving more taxa.
Bayes factors seem promising for evaluating the relative contribution of components to an evolutionary model. However, changing a component of the nucleotide substitution model, e.g. adding parameters to model rate variation, had much higher impact on the model likelihood than unlinking parameters among data partition. Thus, even though strong evidence for a model allowing separate topology parameters is found, this might not mean strong evidence for separate gene phylogenies, as long as vital components of the substitution model are still missing.
Methods
=======
Taxon sampling, amplification and sequencing
--------------------------------------------
Totally 51 typical antbird species were selected for the molecular analyses, including representatives from 38 genera out of 45 genera recognized by Ridgely and Tudor \[[@B3]\]. From some antbird genera (*Myrmeciza*, *Myrmotherula*and *Thamnophilus*) several species were included, as the monophyly for these genera had been questioned \[[@B3],[@B9],[@B10]\]. The phylogenetic trees were rooted using representatives from major furnariid lineages suggested by Irestedt et al. \[[@B1]\]. Sample identifications and GenBank accession numbers are given in Table 3 (see [additional file 1](#S1){ref-type="supplementary-material"} ).
Nucleotide sequence data were obtained from two nuclear introns, myoglobin intron 2 and the glyceraldehydes-3-phosphodehydrogenase (G3PDH) intron 11, and from the mitochondrial cytochrome *b*gene. The complete myoglobin intron 2 (along with 13 bp and 10 bp of the flanking regions of exons 2 and 3, respectively) corresponding to the region between positions 303 (exon 2) and 400 (exon 3) in humans (GenBank accession number XM009949) and the complete G3PDH intron 11 (including 36 bp and 18 bp of exons 11 and 12, respectively) corresponding to the region 3915 to 4327 in *Gallus gallus*(GenBank accession number M11213) were sequenced. From the cytochrome *b*gene 999 bp were obtained corresponding to positions 15037 to 16035 in the chicken mitochondrial genome sequence \[[@B43]\]. Some indels were observed in the alignments of myoglobin intron 2 and the G3PDH intron 11, respectively (see results), but all gaps in the sequences were treated as missing data in the analyses. No insertions, deletions, stop or nonsense codons were observed in any of the cytochrome *b*sequences.
Extraction, amplification and sequencing procedures for cytochrome *b*and myoglobin intron 2 follow the descriptions in Ericson et al. \[[@B44]\] and Irestedt et al. \[[@B1]\]. A protocol described by Fjeldså et al. \[[@B45]\] was followed for the amplification and sequencing of the G3PDH intron.
For each gene and taxon, multiple sequence fragments were obtained by sequencing with different primers. These sequences were assembled to complete sequences with SeqMan II™ (DNASTAR inc.). Positions where the nucleotide could not be determined with certainty were coded with the appropriate IUPAC code. Due to a rather low number of insertions in myoglobin intron 2 and G3PDH intron 11 the combined sequences could easily be aligned by eye.
Phylogenetic inference and model selection
------------------------------------------
We used Bayesian inference and Markov chain Monte Carlo (MCMC) for estimating phylogenetic hypothesis from DNA data (see recent reviews by Holder and Lewis, \[[@B46]\]; Huelsenbeck et al., \[[@B47]\]). Bayesian inference of phylogeny aims at estimating the posterior probabilities of trees and other parameters of an evolutionary model. Importantly, two components need to be specified (apart from the data): the model of nucleotide substitution and the prior distributions for the parameters in that model. The models for nucleotide substitutions were selected for each gene individually, prior to the MCMC, and using the Akaike Information Criterion (AIC \[[@B48]\]). This was done using the program MrModeltest \[[@B49]\] in conjunction with PAUP\* \[[@B50]\]. Specifically, MrModeltest compares 24 standard substitution models, including models allowing rate variation, utilizing the likelihood scores calculated by PAUP\* on an initial, approximate phylogeny (see e.g., \[[@B51]\]).
After models had been selected for the individual gene partitions, prior distributions for the model parameters were specified. For stationary state frequencies, we used a flat Dirichlet prior, Dir(1, 1, 1, 1). A Dirichlet prior, Dir(1, 1, 1, 1, 1, 1) were also used for the nucleotide substitution rate ratios of the general time-reversible model (GTR \[[@B52]-[@B54]\]). A Beta distribution, Beta(1, 1), were used for the transition/transversion rate ratio of the Hasegawa-Kishino-Yano model (HKY \[[@B55]\]). A uniform prior, Uni(0.1, 50), was used on the shape parameter of the gamma distribution of rate variation (Γ \[[@B56]\]), and a Uni(0, 1) prior was used for the proportion of invariable sites (I \[[@B57]\]). An exponential prior, Exp(10), were used for branch lengths, and all trees were assumed to be equally likely (flat prior on topology).
The posterior probabilities of trees and parameters in the substitution models were approximated with MCMC and Metropolis coupling using the program MrBayes \[[@B58]\]. The gene partitions were analyzed both separately and combined. In the combined analysis, each gene partition was allowed to have separate parameters by using a rate multiplier \[[@B27],[@B58],[@B59]\]. One cold and three incrementally heated chains were run for 3 million generations, with a random starting tree and a temperature parameter value of 0.2. Trees were sampled every 100th generations, and the trees sampled during the burn-in phase (i.e., before the chain had reached its apparent target distribution) were discarded. Two runs, starting from different, randomly chosen trees, were made to ensure that the individual runs had converged on the same target distribution \[[@B60]\]. Convergence of parameters was checked by examining parameter means and variances between runs. After checking for convergence, final inference was made from the concatenated output from the two runs.
A Bayesian test of incongruence
-------------------------------
Bayesian methods provide us ways not only to estimate posterior probabilities for trees and parameters in a model, but also to evaluate the model itself. Bayes factors \[[@B61]\], allow us to make sophisticated comparisons between models used in phylogenetic analyses \[[@B27],[@B62],[@B63]\]. Bayes factors measure the strength of evidence in favor of one model M~1~compared to another M~2~, given the data X, and is calculated as the ratio of the model likelihoods, B~12~= *f*(X\|M~1~)/ *f*(X\|M~2~). The model likelihoods *f*(X\|M~*i*~) are difficult to calculate analytically but can be estimated by using the output from an MCMC \[[@B27],[@B62]\].
Here we explore the congruence test described by Nylander et al. \[[@B27]\], which utilizes Bayes factors. The test is not a significance test but merely compares the strength of evidence between two models of character evolution. In the first model, data partitions are allowed to have their own unique set of substitution parameters, but we assume the data as having evolved on the same topology, but with partition-specific branch lengths. Strictly speaking, we are restricting the data partitions to have the same posterior distribution for topologies, but (potentially) different distributions in all other parameters. In the second model we relax the assumption of a single distribution of topologies for all data partitions. That is, if the data partitions (genes) truly evolved on different phylogenies, they are allowed to do so in the model. The comparison or \'test\' is to see if the second model provides compelling evidence as to be accepted as superior. Here we use the log of the Bayes factor and a value of \>10 for 2 logB~12~have been suggested as strong evidence against the alternative model, M~2~\[[@B61]\].
To accomplish the incongruence test we utilized the *unlink*command in MrBayes, which allows the user to let parameters as well as topologies to be unlinked between partitions. We calculated Bayes factors and compared the effects on the model likelihood when linking or unlinking topologies between all the gene partitions. We were primarily interested in the potential incongruence between the mitochondrial cytochrome *b*partition and the two nuclear partitions myoglobin and G3PDH, but all combinations of the three genes in our data set were examined.
For comparison, we also tested whether the different gene partitions were in significant conflict with each other by using the parsimony based incongruence-length differences test (ILD) \[[@B64]\], implemented in PAUP\* \[[@B50]\]. The results are based on 10,000 replicates, with ten iterations (random additions of taxa) per replicate.
Authors\' contribution
======================
MI designed the study, carried out the labwork, participated in the phylogenetic analyses, and drafted the manuscript. JF assisted with the design of the study and with the draft of the manuscript. JN performed the phylogenetic analyses, drafted parts of the results, and material and methods section of the manuscript. PE conceived the study. All authors read and approved the manuscript.
Supplementary Material
======================
::: {.caption}
###### Additional File 1
**Table 3. Samples used in the study**. The classification follows Ridgely and Tudor \[3\] for typical antbirds, and Irestedt et al. \[1\] for families. Abbreviations: AHMN = American Museum of Natural History, New York; FMNH = Field Museum of Natural History, Chicago; LSUMZ = Louisiana State University, Museum of Natural Science; NRM = Swedish Museum of Natural History; ZMCU = Zoological Museum of the University of Copenhagen. References: (1) Irestedt et al. \[1\]; (2) Fjeldså et al. \[45\]; (3) Johansson et al. \[65\]; Fjeldså et al. \[66\].
:::
::: {.caption}
######
Click here for file
:::
Acknowledgements
================
We are grateful to many people and institutions that have been involved in this work. Most tissue and blood samples were obtained from the Zoological Museum, University of Copenhagen (ZMUC) and the Swedish Museum of Natural History. Support to expeditions where tissue samples were acquired and stored at the ZMUC, was provided over many years by the Danish Natural Research Council (mainly grants 11-0380 and 9502155). Many samples at the Swedish Museum of Natural History were collected in Paraguay in collaboration with the Museo Nacional de Historia Natural del Paraguay, San Lorenzo. Important samples have also been obtained from the Field Museum, Chicago (Shannon Hackett, David E. Willard), Museum of Natural Science, Louisiana State University (Donna Dittman), and the American Museum of Natural History, New York (Paul Sweet). Jan Ohlson and Niels Krabbe are thanked for commenting on the manuscript. Mari Källersjö provided logistic support and advice for the work at the Molecular Systematics Laboratory at the Swedish Museum of Natural History, and Dario Zuccon is thanked for practical support at the laboratory. The Swedish Research Council (grant no. 621-2001-2773 to P.E.) funded the laboratory work. Computational facilities were provided by Mikael Thollesson and the Linnaean Center of Bioinformatics, Uppsala.
| PubMed Central | 2024-06-05T03:55:46.820834 | 2004-7-30 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509417/",
"journal": "BMC Evol Biol. 2004 Jul 30; 4:23",
"authors": [
{
"first": "Martin",
"last": "Irestedt"
},
{
"first": "Jon",
"last": "Fjeldså"
},
{
"first": "Johan AA",
"last": "Nylander"
},
{
"first": "Per GP",
"last": "Ericson"
}
]
} |
PMC509418 | Background
==========
Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. Expressed sequence tags (EST) are particularly useful genomic resources because they enable multiple lines of research and can be generated for any organism: ESTs allow the identification of molecular probes for developmental studies, provide clones for DNA microchip construction, reveal candidate genes for mutant phenotypes, and facilitate studies of genome structure and evolution. Furthermore, ESTs provide raw material from which strain-specific polymorphisms can be identified for use in population and quantitative genetic analyses. The utility of such resources can be tailored to target novel characteristics of organisms when ESTs are isolated from cell types and tissues that are actively being used by a particular research community, so as to bias the collection of sequences towards genes of special interest. Finally, EST resources produced for model organisms can greatly facilitate comparative and evolutionary studies when their uses are extended to other, closely related taxa.
Salamanders (urodele amphibians) are traditional model organisms whose popularity was unsurpassed early in the 20^th^century. At their pinnacle, salamanders were the primary model for early vertebrate development. Embryological studies in particular revealed many basic mechanisms of development, including organizer and inducer regions of developing embryos \[[@B1]\]. Salamanders continue to be important vertebrate model organisms for regeneration because they have by far the greatest capacity to regenerate complex body parts in the adult phase. In contrast to mammals, which are not able to regenerate entire structures or organ systems upon injury or amputation, adult salamanders regenerate their limbs, tail, lens, retina, spinal cord, heart musculature, and jaw \[[@B2]-[@B7]\]. In addition, salamanders are the model of choice in a diversity of areas, including vision, embryogenesis, heart development, olfaction, chromosome structure, evolution, ecology, science education, and conservation biology \[[@B8]-[@B15]\]. All of these disciplines are in need of genomic resources as fewer than 4100 salamander nucleotide sequences had been deposited in GenBank as of 3/10/04.
Here we describe results from an EST project for two ambystomatid salamanders: the Mexican axolotl, *Ambystoma mexicanum*and the eastern tiger salamander, *A. tigrinum tigrinum*. These two species are members of the Tiger Salamander Complex \[[@B16]\], a group of closely related species and subspecies that are widely distributed in North America. Phylogenetic reconstruction suggests that these species probably arose from a common ancestor about 10--15 million years ago \[[@B16]\]. *Ambystoma mexicanum*has a long research history of over 100 years and is now principally supplied to the research community by the Axolotl Colony \[[@B17]\], while *A. t. tigrinum*is obtained from natural populations in the eastern United States. Although closely related with equally large genomes (32 × 10^9^bp)\[[@B18]\], these two species and others of the Complex differ dramatically in life history: *A. mexicanum*is a paedomorphic species that retains many larval features and lives in water throughout it\'s life cycle while *A. t. tigrinum*undergoes a metamorphosis that is typical of many amphibians. Like many other traditional model organisms of the last century, interest in these two species declined during the rise of genetic models like the fly, zebrafish, and mouse \[[@B19]\]. However, \"early\" model organisms such as salamanders are beginning to re-attract attention as genome resources can rapidly be developed to exploit the unique features that originally identified their utility for research. We make this point below by showing how the development of ESTs for these two species is enabling research in several areas. Furthermore, we emphasize the value of developing resources in model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research programs.
Results and Discussion
======================
Selection of libraries for EST sequencing
-----------------------------------------
Eleven cDNA libraries were constructed using a variety of tissues (Table [1](#T1){ref-type="table"}). Pilot sequencing of randomly selected clones revealed that the majority of the non-normalized libraries were moderate to highly redundant for relatively few transcripts. For example, hemoglobin-like transcripts represented 15--25% of the sampled clones from cDNA libraries V1, V2, and V6. Accordingly, we chose to focus our sequencing efforts on the non-normalized MATH library as well as the normalized AG library, which had lower levels of redundancy (5.5 and 0.25% globins, respectively). By concentrating our sequencing efforts on these two libraries we obtained transcripts deriving primarily from regenerating larval tissues in *A. mexicanum*and several non-regenerating larval tissues in *A. t. tigrinum*.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Tissues selected to make cDNA libraries.
:::
**ID** **Tissue** **cDNAs sequenced**
-------- ----------------------------------------- ---------------------
GARD limb blastema 1029
MATH limb blastema 16244
V1 tail blastema 1422
V2 brain 3196
V3 liver 792
V4 spleen 337
V5 heart 38
V6 gill 3039
V7 stage 22 embryo 96
AG liver, gonad, lung, kidney, heart, gill 19871
Further information is found in Methods and Materials.
:::
EST sequencing and clustering
-----------------------------
A total of 46,064 cDNA clones were sequenced, yielding 39,982 high quality sequences for *A. mexicanum*and *A. t. tigrinum*(Table [2](#T2){ref-type="table"}). Of these, 3,745 corresponded to mtDNA and were removed from the dataset; complete mtDNA genome data for these and other ambystomatid species will be reported elsewhere. The remaining nuclear ESTs for each species were clustered and assembled separately. We included in our *A. mexicanum*assembly an additional 16,030 high quality ESTs that were generated recently for regenerating tail and neurula stage embryos \[[@B20]\]. Thus, a total of 32,891 and 19,376 ESTs were clustered for *A. mexicanum*and *A. t. tigrinum*, respectively. Using PaCE clustering and CAP3 assembly, a similar number of EST clusters and contigs were identified for each species (Table [2](#T2){ref-type="table"}). Overall contig totals were 11,190 and 9,901 for *A. mexicanum*and *A. t. tigrinum*respectively. Thus, although 13,515 more *A. mexicanum*ESTs were assembled, a roughly equivalent number of contigs were obtained for both species. This indicates that EST development was more efficient for *A. t. tigrinum,*presumably because ESTs were obtained primarily from the normalized AG library; indeed, there were approximately twice as many ESTs on average per *A. mexicanum*contig (Table [2](#T2){ref-type="table"}). Thus, our EST project yielded an approximately equivalent number of contigs for *A. mexicanum*and *A. t. tigrinum*, and overall we identified \> 21,000 different contigs. Assuming that 20% of the contigs correspond to redundant loci, which has been found generally in large EST projects \[[@B21]\], we identified transcripts for approximately 17,000 different ambystomatid loci. If ambystomatid salamanders have approximately the same number of loci as other vertebrates (e.g. \[[@B22]\]), we have isolated roughly half the expected number of genes in the genome.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
EST summary and assembly results.
:::
***A. mex*** ***A. t. tig***
------------------------ -------------- -----------------
cDNA clones sequenced 21830 24234
high-quality sequences 19383 20599
mt DNA sequence 2522 1223
seqs submitted to NCBI 16861 19376
sequences assembled 32891^a^ 19376
PaCE clusters 11381 10226
ESTs in contigs 25457 12676
contigs 3756 3201
singlets 7434 6700
putative transcripts 11190 9901
^a^Includes 16,030 ESTs from \[20\].
:::
Identification of vertebrate sequences similar to *Ambystoma*contigs
--------------------------------------------------------------------
We searched all contigs against several vertebrate databases to identify sequences that exhibited significant sequence similarity. As our objective was to reliably annotate as many contigs as possible, we first searched against 19,804 sequences in the NCBI human RefSeq database (Figure [1](#F1){ref-type="fig"}), which is actively reviewed and curated by biologists. This search revealed 5619 and 4973 \"best hit\" matches for the *A. mexicanum*and *A. t. tigrinum*EST datasets at a BLASTX threshold of *E*= 10^-7^. The majority of contigs were supported at more stringent E-value thresholds (Table [3](#T3){ref-type="table"}). Non-matching contigs were subsequently searched against the Non-Redundant (nr) Protein database and *Xenopus tropicalus*and *X. laevis*UNIGENE ESTs (Figure [1](#F1){ref-type="fig"}). These later two searches yielded a few hundred more \'best hit\' matches, however a relatively large number of ESTs from both ambystomatid species were not similar to any sequences from the databases above. Presumably, these non-matching sequences were obtained from the non-coding regions of transcripts or they contain protein-coding sequences that are novel to salamander. Although the majority are probably of the former type, we did identify 3,273 sequences from the non-matching set that had open reading frames (ORFs) of at least 200 bp, and 911 of these were greater than 300 bp.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Results of BLASTX and TBLASTX searches to identify best BLAST hits for *Ambystoma*contigs searched against NCBI human RefSeq, nr, and *Xenopus*Unigene databases**.
:::
:::
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
*Ambystoma*contig search of NCBI human RefSeq, nr, and *Xenopus*Unigene databases.
:::
***A. mex*** ***A. t. tig***
------------------------ -------------------- --------------------
\# BLASTX Best Matches [6283]{.underline} [5545]{.underline}
\< E^-100^ 630 870
\< E^-50^\> E^-100^ 2015 1990
\< E^-20^\> E^-50^ 2153 1595
\< E^-10^\> E^-20^ 967 745
\< E^-7^\> E^-10^ 518 345
:::
The distribution of ESTs among contigs can provide perspective on gene expression when clones are randomly sequenced from non-normalized cDNA libraries. In general, frequently sampled transcripts may be expressed at higher levels. We identified the 20 contigs from *A. mexicanum*and *A. t. tigrinum*that contained the most assembled ESTs (Table [4](#T4){ref-type="table"}). The largest *A. t. tigrinum*contigs contained fewer ESTs than the largest *A. mexicanum*contigs, probably because fewer overall *A. t. tigrinum*clones were sequenced, with the majority selected from a normalized library. However, we note that the contig with the most ESTs was identified for *A. t. tigrinum:*delta globin. In both species, transcripts corresponding to globin genes were sampled more frequently than all other loci. This may reflect the fact that amphibians, unlike mammals, have nucleated red blood cells that are transcriptionally active. In addition to globin transcripts, a few other house-keeping genes were identified in common from both species, however the majority of the contigs were unique to each list. Overall, the strategy of sequencing cDNAs from a diverse collection of tissues (from normalized and non-normalized libraries) yielded different sets of highly redundant contigs. Only 25% and 28% of the *A. mexicanum*and *A. t. tigrinum*contigs, respectively, were identified in common (Figure [2](#F2){ref-type="fig"}). We also note that several hundred contigs were identified in common between Xenopus and *Ambystoma*; this will help facilitate comparative studies among these amphibian models.
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
Top 20 contigs with the most assembled ESTs.
:::
**Contig ID** **\# ESTs** **Best Human Match** **E-value**
---------------------------- ------------- -------------------------------------------------------- -------------
MexCluster\_4615\_Contig1 415 (NM\_000519) delta globin E^-39^
MexCluster\_600\_Contig1 354 (NM\_182985) ring finger protein 36 isoform a E^-110^
MexCluster\_6279\_Contig1 337 (NM\_000559) A-gamma globin E^-32^
MexCluster\_10867\_Contig1 320 (NM\_000558) alpha 1 globin E^-38^
MexCluster\_5357\_Contig1 307 (NM\_000558) alpha 1 globin E^-37^
MexCluster\_9285\_Contig3 285 (NM\_001614) actin, gamma 1 propeptide 0
MexCluster\_7987\_Contig3 252 (NM\_001402) eukaryotic translation elongation f1 0
MexCluster\_9285\_Contig1 240 (NM\_001101) beta actin; beta cytoskeletal actin 0
MexCluster\_9279\_Contig3 218 (NM\_000223) keratin 12 E^-113^
MexCluster\_11203\_Contig1 181 (NM\_002032) ferritin, heavy polypeptide 1 E^-70^
MexCluster\_8737\_Contig2 152 (NM\_058242) keratin 6C E^-131^
MexCluster\_3193\_Contig1 145 (NM\_004499) heterogeneous nuclear ribonucleoprotein E^-90^
MexCluster\_8737\_Contig7 134 (NM\_058242) keratin 6C E^-131^
MexCluster\_5005\_Contig3 132 (NM\_031263) heterogeneous nuclear ribonucleoprotein E^-124^
MexCluster\_6225\_Contig1 125 (NM\_001152) solute carrier family 25, member 5 E^-151^
MexCluster\_1066\_Contig1 122 \[31015660\] IMAGE:6953586 E^-16^
MexCluster\_8737\_Contig4 114 (NM\_058242) keratin 6C; keratin, epidermal type II E^-132^
MexCluster\_8187\_Contig2 113 (NM\_005507) cofilin 1 (non-muscle) E^-65^
MexCluster\_2761\_Contig1 109 (NM\_001961) eukaryotic translation elongation factor2 0
MexCluster\_9187\_Contig1 105 (NM\_007355) heat shock 90 kDa protein 1, beta 0
***A. t. tigrinum***
TigCluster\_6298\_Contig1 654 (NM\_000519) delta globin E^-38^
TigCluster\_10099\_Contig2 193 (NM\_001614) actin, gamma 1 propeptide 0
TigCluster\_6470\_Contig1 167 (NM\_000558) alpha 1 globin E^-39^
TigCluster\_9728\_Contig2 142 (NM\_000477) albumin precursor E^-140^
TigCluster\_6594\_Contig1 117 (NM\_001402) eukaryotic translation elongation f1 0
TigCluster\_5960\_Contig1 91 (NM\_001101) beta actin; beta cytoskeletal actin 0
TigCluster\_7383\_Contig1 77 (NM\_001614) actin, gamma 1 propeptide 0
TigCluster\_6645\_Contig1 76 (NM\_001063) transferrin 0
TigCluster\_7226\_Contig4 74 (NM\_006009) tubulin, alpha 3 E^-160^
TigCluster\_7191\_Contig1 67 (NM\_019016) keratin 24 E^-89^
TigCluster\_10121\_Contig1 64 (NM\_005141) fibrinogen, beta chain preproprotein 0
TigCluster\_6705\_Contig1 63 (NM\_000558) alpha 1 globin E^-39^
TigCluster\_7854\_Contig1 62 (NM\_021870) fibrinogen, gamma chain isoform E^-121^
TigCluster\_6139\_Contig1 52 (NM\_001404) eukaryotic translation elongation f1 0
TigCluster\_7226\_Contig2 51 (NM\_006009) tubulin, alpha 3 0
TigCluster\_10231\_Contig1 44 (NM\_003018) surfactant, pulmonary-associated prot. E^-08^
TigCluster\_6619\_Contig1 36 (NM\_000041) apolipoprotein E E^-38^
TigCluster\_7232\_Contig2 35 (NM\_003651) cold shock domain protein A E^-46^
TigCluster\_5768\_Contig1 34 (NM\_003380) vimentin E^-177^
TigCluster\_9784\_Contig3 32 \|XP\_218445.1\| similar to RIKEN cDNA 1810065E05 E^-15^
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**Venn diagram of BLAST comparisons among amphibian EST projects.**Values provided are numbers of reciprocal best BLAST hits (*E\<*10^-20^) among quality masked *A. mexicanum*and *A. t. tigrinum*assemblies and a publicly available *X. tropicalis*EST assembly <http://www.sanger.ac.uk/Projects/X_tropicalis>
:::
:::
Functional annotation
---------------------
For the 10,592 contigs that showed significant similarity to sequences from the human RefSeq database, we obtained Gene Ontology (23) information to describe ESTs in functional terms. Although there are hundreds of possible annotations, we chose a list of descriptors for molecular and biological processes that we believe are of interest for research programs currently utilizing salamanders as model organisms (Table [5](#T5){ref-type="table"}). In all searches, we counted each match between a contig and a RefSeq sequence as identifying a different ambystomatid gene, even when different contigs matched the same RefSeq reference. In almost all cases, approximately the same number of matches was found per functional descriptor for both species. This was not simply because the same loci were being identified for both species, as only 20% of the total number of searched contigs shared sufficient identity (BLASTN; *E\<*10^-80^or *E\<*10^-20^) to be potential homologues. In this sense, the sequencing effort between these two species was complementary in yielding a more diverse collection of ESTs that were highly similar to human gene sequences.
::: {#T5 .table-wrap}
Table 5
::: {.caption}
######
Functional annotation of contigs
:::
***A. mex*** ***A. t. tig***
--------------------------------------- -------------- -----------------
**Molecular Function (0016209)**
antioxidant (0016209) 25 29
binding (0005488) 3117 2578
chaparone (0003754) 100 85
enzyme regulation (003023) 193 223
motor (0003774) 73 75
signal transduction (0004871) 344 375
structural protein (0005198) 501 411
transcriptional reg. (0030528) 296 221
translational reg. (0045182) 94 59
bone remodeling (0046849) 8 8
circulation (0008015) 23 78
immune response (000695) 182 263
respiratory ex. (0009605) 254 288
respiratory in. (0009719) 72 58
stress (0006950) 263 320
**Biological Process (0008150)**
[Cellular (0009987)]{.underline}
activation (0001775) 4 6
aging and death (0008219) 158 148
communication (0007154) 701 696
differentiation (0030154) 31 20
extracellular mat. (0043062) 4 4
growth and main. (0008151) 1731 1445
migration (0016477) 8 14
motility (0006928) 163 154
[Developmental (0007275)]{.underline}
aging (0007568) 32 21
embryonic (0009790) 6 1
growth (0040007) 2 2
morphogenesis (0009653) 350 272
pigment (0048066) 13 26
post embryonic (0009791) 8 13
reproduction (0000003) 42 27
[Physiological (0007582)]{.underline}
coagulation (0050817) 22 73
death and aging (0016265) 159 148
homeostasis (0042592) 22 27
metabolism (0008152) 3059 2513
secretion (0046903) 9 16
sex differentiation (0007548) 3 2
Numbers in parentheses reference GO numbers \[23\].
:::
Informatic searches for regeneration probes
-------------------------------------------
The value of a salamander model to regeneration research will ultimately rest on the ease in which data and results can be cross-referenced to other vertebrate models. For example, differences in the ability of mammals and salamanders to regenerate spinal cord may reflect differences in the way cells of the ependymal layer respond to injury. As is observed in salamanders, ependymal cells in adult mammals also proliferate and differentiate after spinal cord injury (SCI) \[[@B24],[@B25]\]; immediately after contusion injury in adult rat, ependymal cell numbers increase and proliferation continues for at least 4 days \[\[[@B26]\]; but see \[[@B27]\]\]. Rat ependymal cells share some of the same gene expression and protein properties of embryonic stem cells \[[@B28]\], however no new neurons have been observed to derive from these cells *in vivo*after SCI \[[@B29]\]. Thus, although endogenous neural progenitors of the ependymal layer may have latent regenerative potential in adult mammals, this potential is not realized. Several recently completed microarray analyses of spinal cord injury in rat now make it possible to cross-reference information between amphibians and mammals. For example, we searched the complete list of significantly up and down regulated genes from Carmel et al. \[[@B30]\] and Song et al. \[[@B31]\] against all *Ambystoma*ESTs. Based upon amino acid sequence similarity of translated ESTs (TBLASTX; *E*\<10^-7^), we identified DNA sequences corresponding to 69 of these 164 SCI rat genes (Table [6](#T6){ref-type="table"}). It is likely that we have sequence corresponding to other presumptive orthologues from this list as many of our ESTs only contain a portion of the coding sequence or the untranslated regions (UTR), and in many cases our searches identified closely related gene family members. Thus, many of the genes that show interesting expression patterns after SCI in rat can now be examined in salamander.
::: {#T6 .table-wrap}
Table 6
::: {.caption}
######
*Ambystoma*contigs that show sequence similarity to rat spinal cord injury genes.
:::
**Ambystoma Contig ID** **RAT cDNA clone** **E-value**
-------------------------------------------------------------------------------------- ------------------------------------------------------- -------------
MexCluster\_7440\_Contig1 gi\|1150557\|c-myc, exon 2 E^-29^
MexCluster\_4624\_Contig1 gi\|1468968\| brain acyl-CoA synthtase II E^-09^
TigCluster\_4083\_Contig1 E^-09^
TigSingletonClusters\_Salamander\_4\_G20\_ab1 gi\|1552375\| SKR6 gene, a CB1 cannabinoid recept. E^-08^
MexSingletonClusters\_NT009B\_B04 gi\|17352488\| cyclin ania-6a E^-46^
TigCluster\_3719\_Contig1 E^-114^
TigCluster\_8423\_Contig1 gi\|1778068\| binding zyginI E^-102^
TigCluster\_7064\_Contig1 gi\|1836160\| Ca2+/calmodulin-dependent E^-20^
MexCluster\_3225\_Contig1 gi\|1906612\| Rattus norvegicus CXC chemokine E^-68^
TigSingletonClusters\_Salamander\_13\_F03\_ab1 E^-38^
MexSingletonClusters\_BL285B\_A06 gi\|203042\| (Na+, K+)-ATPase-beta-2 subunit E^-63^
TigCluster\_6994\_Contig1 E^-65^
MexSingletonClusters\_BL014B\_F12 gi\|203048\| plasma membrane Ca2+ ATPase-isoform 2 E^-112^
TigSingletonClusters\_Salamander\_5\_F07\_ab1 E^-92^
MexCluster\_1251\_Contig1 gi\|203167\| GTP-binding protein (G-alpha-i1) E^-110^
TigSingletonClusters\_Salamander\_3\_P14\_ab1 E^-152^
TigSingletonClusters\_Salamander\_22\_B01\_ab1 gi\|203336\| catechol-O-methyltransferase E^-47^
TigSingletonClusters\_Salamander\_17\_N04\_ab1 gi\|203467\| voltage-gated K+ channel protein (RK5) E^-08^
MexSingletonClusters\_v1\_p8\_c16\_triplex5ld\_ gi\|203583\| cytosolic retinol-binding protein (CRBP) E^-77^
TigCluster\_6321\_Contig1 E^-18^
MexCluster\_5399\_Contig1 gi\|204647\| heme oxygenase gene E^-67^
TigCluster\_2577\_Contig1 E^-67^
MexCluster\_4647\_Contig1 gi\|204664\| heat shock protein 27 (Hsp27) E^-83^
TigSingletonClusters\_Salamander\_12\_M05\_ab1 E^-51^
MexSingletonClusters\_BL285C\_F02 gi\|205404\| metabotropic glutamate receptor 3 E^-41^
TigSingletonClusters\_Salamander\_2\_B24\_ab1 gi\|205508\| myelin/oligodendrocyte glycoprotein E^-26^
TigCluster\_5740\_V2\_p10\_M20\_TriplEx5ld\_ gi\|205531\| metallothionein-2 and metallothionein 1 E^-08^
TigSingletonClusters\_V2\_p5\_A2\_TriplEx5ld\_ gi\|205537\| microtubule-associated protein 1A E^-59^
MexCluster\_1645\_Contig1 gi\|205633\| Na, K-ATPase alpha-2 subunit E^-149^
TigSingletonClusters\_Contig328 0
TigSingletonClusters\_Contig45 gi\|205683\| smallest neurofilament protein (NF-L) E^-63^
MexSingletonClusters\_NT016A\_A09 gi\|205693\| nerve growth factor-induced (NGFI-A) E^-95^
TigSingletonClusters\_I09\_Ag2\_p9\_K24\_M13R E^-24^
MexSingletonClusters\_NT007A\_E07 gi\|205754\| neuronal protein (NP25) E^-64^
TigCluster\_7148\_Contig1 E^-57^
MexCluster\_9504\_Contig1 gi\|206161\| peripheral-type benzodiazepine receptor E^-73^
MexSingletonClusters\_BL016B\_B02 gi\|206166\| protein kinase C type III E^-36^
TigCluster\_981\_Contig1 E^-27^
MexSingletonClusters\_nm\_19\_k3\_t3\_ gi\|206170\| brain type II Ca2+/calmodulin-dependent E^-117^
MexSingletonClusters\_v11\_p42\_j20\_t3\_049\_ab1 gi\|207138\| norvegicus syntaxin B 1e^-079^
MexSingletonClusters\_nm\_14\_h19\_t3\_ gi\|207473\| neural receptor protein-tyrosine kinase E^-40^
TigSingletonClusters\_Contig336 E^-34^
TigSingletonClusters\_E10\_Ag2\_p18\_O19\_M13 gi\|2116627\| SNAP-25A E^-123^
MexCluster\_211\_Contig1 gi\|220713\| calcineurin A alpha E^-63^
TigSingletonClusters\_Salamander\_7\_K14\_ab1 E^-87^
MexSingletonClusters\_NT014A\_G03 gi\|220839\| platelet-derived growth factor A chain E^-21^
TigSingletonClusters\_Salamander\_9\_M15\_ab1 E^-56^
TigSingletonClusters\_Salamander\_19\_M06\_ab1 gi\|2501807\| brain digoxin carrier protein E^-55^
MexSingletonClusters\_Contig100 gi\|2746069\| MAP-kinase phosphatase (cpg21) E^-108^
TigSingletonClusters\_Salamander\_11\_A16\_ab1 E^-70^
MexCluster\_8345\_Contig1 gi\|2832312\| survival motor neuron (smn) E^-40^
TigCluster\_8032\_Contig1 E^-49^
MexCluster\_3580\_Contig1 gi\|294567\| heat shock protein 70 (HSP70) 0
TigCluster\_8592\_Contig2 E^-161^
TigSingletonClusters\_Salamander\_17\_N08\_ab1 gi\|2961528\| carboxyl-terminal PDZ E^-10^
MexSingletonClusters\_BL286C\_D09 gi\|298325\| sodium-dependent neurotransmitter tran. E^-12^
TigSingletonClusters\_Contig95 E^-22^
MexSingletonClusters\_Contig461 gi\|2996031\| brain finger protein (BFP) E^-08^
TigSingletonClusters\_Salamander\_11\_O19\_ab1 E^-23^
TigSingletonClusters\_E16\_Ag2\_p8\_O20\_M13R gi\|3135196\| Ca2+/calmodulin-dependent E^-33^
MexSingletonClusters\_Contig188 gi\|3252500\| CC chemokine receptor protein E^-15^
MexCluster\_6961\_Contig1 gi\|3319323\| suppressor of cytokine signaling-3 E^-08^
MexSingletonClusters\_nm\_14\_p15\_t3\_ gi\|349552\| P-selectin E^-16^
TigCluster\_218\_Contig2 E^-99^
MexSingletonClusters\_Contig506 gi\|3707306\| Normalized rat embryo, cDNA clone E^-14^
TigSingletonClusters\_I16\_Ag2\_p5\_N7\_M13R gi\|3711670\| Normalized rat muscle, cDNA clone E^-35^
MexSingletonClusters\_V1\_p1\_a10\_Triplex5Ld gi\|3727094\| Normalized rat ovary, cDNA clone E^-15^
TigSingletonClusters\_v2\_p1\_D20\_triplex5ld E^-16^
MexSingletonClusters\_NT005B\_F02 gi\|3811504\| Normalized rat brain, cDNA clone E^-35^
TigSingletonClusters\_Salamander\_22\_I04\_ab1 E^-34^
TigSingletonClusters\_Ag2\_p34\_N23\_M13R gi\|405556\| adenylyl cyclase-activated serotonin E^-17^
TigSingletonClusters\_Salamander\_1\_H02\_ab1 gi\|4103371\| putative potassium channel TWIK E^-22^
MexCluster\_4589\_Contig1 gi\|4135567\| Normalized rat embryo, cDNA clone E^-32^
TigSingletonClusters\_Contig220 E^-09^
TigCluster\_4093\_Contig1 gi\|4228395\| cDNA clone UI-R-A0-bc-h-02-0-UI E^-104^
MexSingletonClusters\_nm\_21\_2\_m7\_t3\_ gi\|425471\| nuclear factor kappa B p105 subunit E^-22^
TigCluster\_8535\_Contig1 E^-11^
MexSingletonClusters\_v6\_p1\_j6\_triplex5\_1ld\_ gi\|430718\| Sprague Dawley inducible nitric oxide E^-13^
TigSingletonClusters\_Salamander\_15\_D22\_ab1 E^-41^
MexCluster\_3498\_Contig1 gi\|436934\| Sprague Dawley protein kinase C rec. 0
TigCluster\_6648\_Contig1 0
MexSingletonClusters\_BL279A\_B12 gi\|464196\| phosphodiesterase I E^-49^
TigSingletonClusters\_Salamander\_25\_P03\_ab1 E^-75^
MexCluster\_8708\_Contig1 gi\|466438\| 40kDa ribosomal protein E^-168^
TigCluster\_5877\_Contig1 E^-168^
MexSingletonClusters\_nm\_14\_a9\_t3\_ gi\|493208\| stress activated protein kinase alpha II E^-51^
TigSingletonClusters\_Salamander\_11\_A13\_ab1 gi\|517393\| tau microtubule-associated protein E^-44^
TigSingletonClusters\_Salamander\_12\_J14\_ab1 gi\|55933\| c-fos E^-26^
MexSingletonClusters\_nm\_21\_2\_l13\_t3\_ gi\|56822\| major synaptic vesicel protein p38 E^-39^
TigCluster\_2065\_Contig1 E^-50^
MexCluster\_10965\_Contig1 gi\|56828\| nuclear oncoprotein p53 E^-75^
TigCluster\_5315\_Contig1 E^-66^
MexCluster\_4245\_Contig1 gi\|56909\| pJunB gene E^-50^
TigSingletonClusters\_G05\_Ag2\_p9\_G8\_M13R E^-09^
MexSingletonClusters\_NT013D\_C12 gi\|56919\| region fragment for protein kinase C E^-33^
TigSingletonClusters\_Salamander\_21\_H19\_ab1 E^-24^
MexCluster\_9585\_Contig1 gi\|57007\| ras-related mRNA rab3 E^-61^
TigCluster\_4885\_Contig1 E^-63^
TigSingletonClusters\_Salamander\_1\_M03\_ab1 gi\|57238\| silencer factor B E^-13^
MexSingletonClusters\_NT008B\_D05 gi\|57341\| transforming growth factor-beta 1 E^-13^
TigSingletonClusters\_Salamander\_24\_I16\_ab1 E^-20^
MexCluster\_9533\_Contig1 gi\|57479\| vimentin 0
TigCluster\_5768\_Contig1 0
MexSingletonClusters\_BL283B\_A11 gi\|596053\| immediate early gene transcription E^-12^
TigSingletonClusters\_Salamander\_13\_J19\_ab1 E^-16^
MexSingletonClusters\_v6\_p4\_j2\_triplex5\_1ld\_ gi\|790632\| macrophage inflammatory protein-1alpha E^-22^
TigCluster\_2146\_Contig1 gi\|951175\| limbic system-associated membrane prot. E^-11^
MexSingletonClusters\_v11\_p54\_o4\_t3\_ gi\|971274\| neurodegeneration associated protein 1 E^-09^
TigSingletonClusters\_Salamander\_2\_J12\_ab1 E^-11^
:::
Similar gene expression programs may underlie regeneration of vertebrate appendages such as fish fins and tetrapod limbs. Regeneration could depend on reiterative expression of genes that function in patterning, morphogenesis, and metabolism during normal development and homeostasis. Or, regeneration could depend in part on novel genes that function exclusively in this process. We investigated these alternatives by searching *A. mexicanum*limb regeneration ESTs against UNIGENE zebrafish fin regeneration ESTs (Figure [3](#F3){ref-type="fig"}). This search identified 1357 significant BLAST hits (TBLASTX; *E*\<10^-7^) that corresponded to 1058 unique zebrafish ESTs. We then asked whether any of these potential regeneration homologues were represented uniquely in limb and fin regeneration databases (and not in databases derived from other zebrafish tissues). A search of the 1058 zebrafish ESTs against \> 400,000 zebrafish ESTs that were sampled from non-regenerating tissues revealed 43 that were unique to the zebrafish regeneration database (Table [7](#T7){ref-type="table"}). Conceivably, these 43 ESTs may represent transcripts important to appendage regeneration. For example, our search identified several genes (e.g. *hspc128*, pre-*B-cell colony enhancing factor 1*, *galectin 4*, *galectin 8*) that may be expressed in progenitor cells that proliferate and differentiate during appendage regeneration. Overall, our results suggest that regeneration is achieved largely through the reiterative expression of genes having additional functions in other developmental contexts, however a small number of genes may be expressed uniquely during appendage regeneration.
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**Results of BLASTN and TBLASTX searches to identify best BLAST hits for *A. mexicanum*regeneration ESTs searched against zebrafish EST databases.**A total of 14,961 *A. mexicanum*limb regeneration ESTs were assembled into 4485 contigs for this search.
:::
:::
::: {#T7 .table-wrap}
Table 7
::: {.caption}
######
*Ambystoma*limb regeneration contigs that show sequence similarity to zebrafish fin regeneration ESTs
:::
**Mex. Contigs** **Human ID** **E-value** **Zfish ID** **E-value**
------------------------------ ----------------- ------------- ------------------------ -------------
Contig94 gi\|10835079\| 1e^-63^ gnl\|UG\|Dr\#S12319632 1e^-58^
nm\_30\_a11\_t3\_ gi\|32306539\| 1e^-58^ gnl\|UG\|Dr\#S12312602 1e^-35^
Contig615 gi\|4502693\| 1e^-70^ gnl\|UG\|Dr\#S12313407 1e^-34^
nm\_23\_l13\_t3\_ No Human Hit gnl\|UG\|Dr\#S12320916 1e^-31^
nm\_9\_e22\_t3\_ gi\|4758788\| 1e^-98^ gnl\|UG\|Dr\#S12309914 1e^-29^
nm\_8\_l17\_t3\_ gi\|21361310\| 1e^-16^ gnl\|UG\|Dr\#S12313396 1e^-27^
Contig531 gi\|13775198\| 1e^-27^ gnl\|UG\|Dr\#S12309680 1e^-26^
Contig152 gi\|5453712\| 1e^-32^ gnl\|UG\|Dr\#S12239884 1e^-26^
nm\_32h\_j20\_t3\_ gi\|39777601\| 1e^-79^ gnl\|UG\|Dr\#S12136499 1e^-25^
Contig1011 gi\|39752675\| 1e^-65^ gnl\|UG\|Dr\#S12136499 1e^-24^
v11\_p50\_b24\_t3\_ gi\|41208832\| 1e^-36^ gnl\|UG\|Dr\#S12319219 1e^-23^
Contig589 gi\|4506505\| 1e^-56^ gnl\|UG\|Dr\#S12312662 1e^-22^
Contig785 gi\|33695095\| 1e^-61^ gnl\|UG\|Dr\#S12264765 1e^-22^
Contig157 gi\|21361122\| 1e^-138^ gnl\|UG\|Dr\#S12313094 1e^-21^
v11\_p42\_j20\_t3\_049\_ab1 gi\|47591841\| 1e^-100^ gnl\|UG\|Dr\#S12137806 1e^-21^
Contig610 gi\|10801345\| 1e^-114^ gnl\|UG\|Dr\#S12310326 1e^-20^
nm\_27\_o1\_t3\_ gi\|7706429\| 1e^-72^ gnl\|UG\|Dr\#S12310422 1e^-19^
Contig439 gi\|4504799\| 1e^-25^ gnl\|UG\|Dr\#S12309233 1e^-19^
nm\_31\_d5\_t3\_ gi\|8923956\| 1e^-50^ gnl\|UG\|Dr\#S12264745 1e^-17^
v11\_p41\_h12\_t3\_026\_ab1 No Human Hit gnl\|UG\|Dr\#S12320916 1e^-17^
Contig129 gi\|34932414\| 1e^-103^ gnl\|UG\|Dr\#S12313534 1e^-17^
nm\_14\_j21\_t3\_ gi\|4505325\| 1e^-42^ gnl\|UG\|Dr\#S12136571 1e^-17^
Contig1321 gi\|4501857\| 1e^-80^ gnl\|UG\|Dr\#S12309233 1e^-17^
nm\_19\_k3\_t3\_ gi\|26051212\| 1e^-106^ gnl\|UG\|Dr\#S12137637 1e^-17^
Contig488 gi\|4557525\| 1e^-105^ gnl\|UG\|Dr\#S12311975 1e^-15^
nm\_35h\_k19\_t3\_ gi\|16950607\| 1e^-43^ gnl\|UG\|Dr\#S12196214 1e^-15^
Contig195 gi\|4557231\| 1e^-99^ gnl\|UG\|Dr\#S12309233 1e^-14^
nm\_14\_h19\_t3\_ gi\|4503787\| 1e^-86^ gnl\|UG\|Dr\#S12310912 1e^-13^
v11\_p51\_d20\_t3\_ gi\|30520322\| 1e^-19^ gnl\|UG\|Dr\#S12321150 1e^-13^
g3-n14 gi\|13654278\| 1e^-23^ gnl\|UG\|Dr\#S12318856 1e^-13^
nm\_29\_f2\_t3\_ gi\|4506517\| 1e^-65^ gnl\|UG\|Dr\#S12312662 1e^-13^
g4-h23 gi\|24111250\| 1e^-33^ gnl\|UG\|Dr\#S12312651 1e^-13^
Math\_p2\_A2\_T3\_ No human Hit gnl\|UG\|Dr\#S12078998 1e^-13^
nm\_35h\_f4\_t3\_ gi\|41148476\| 1e^-67^ gnl\|UG\|Dr\#S12319663 1e^-13^
Contig952 gi\|21264558\| 1e^-61^ gnl\|UG\|Dr\#S12318843 1e^-12^
g4-g21 gi\|11995474\| 1e^-65^ gnl\|UG\|Dr\#S12192716 1e^-12^
Contig854 gi\|8922789\| 1e^-117^ gnl\|UG\|Dr\#S12313534 1e^-11^
Contig1105 gi\|6912638\|\| 1e^-83^ gnl\|UG\|Dr\#S12079967 1e^-11^
nm\_26\_f7\_t3\_ gi\|30181238\| 1e^-83^ gnl\|UG\|Dr\#S12319880 1e^-11^
Contig949 gi\|21284385\| 1e^-68^ gnl\|UG\|Dr\#S12290856 1e^-11^
g3-n3 gi\|18490991\| 1e^-64^ gnl\|UG\|Dr\#S12320832 1e^-10^
v11\_p41\_m16\_t3\_007\_ab1 gi\|4885661\| 1e^-33^ gnl\|UG\|Dr\#S12310912 1e^-10^
Contig653 gi\|4505047\| 1e^-124^ gnl\|UG\|Dr\#S12239868 1e^-09^
Contig1349 gi\|9665259\| 1e^-46^ gnl\|UG\|Dr\#S12320840 1e^-09^
6h12 gi\|31317231\| 1e^-43^ gnl\|UG\|Dr\#S12321311 1e^-09^
v11\_p43h\_i14\_t3\_070\_ab1 No Human Hit gnl\|UG\|Dr\#S12320916 1e^-09^
nm\_35h\_d11\_t3\_ gi\|7661790\| 1e^-35^ gnl\|UG\|Dr\#S12196146 1e^-09^
nm\_35h\_k22\_t3\_ gi\|5031977\| 1e^-124^ gnl\|UG\|Dr\#S12242267 1e^-09^
v11\_p48\_g2\_t3\_087\_ab1 gi\|11496277\| 1e^-60^ gnl\|UG\|Dr\#S12312396 1e^-09^
nm\_30\_e11\_t3\_ gi\|32483357\| 1e^-56^ gnl\|UG\|Dr\#S12309103 1e^-08^
nm\_28\_f23\_t3\_ gi\|42544191\| 1e^-25^ gnl\|UG\|Dr\#S12239884 1e^-08^
nm\_12\_p16\_t3\_ gi\|21361553\| 1e^-21^ gnl\|UG\|Dr\#S12310912 1e^-08^
nm\_32h\_a8\_t3\_ gi\|11386179\| 1e^-22^ gnl\|UG\|Dr\#S12312152 1e^-08^
Human RefSeq sequence ID\'s are provided to allow cross-referencing.
:::
DNA sequence polymorphisms within and between *A. mexicanum*and *A. t. tigrinum*
--------------------------------------------------------------------------------
The identification of single nucleotide polymorphisms (SNPs) within and between orthologous sequences of *A. mexicanum*and *A. t. tigrinum*is needed to develop DNA markers for genome mapping \[[@B32]\], quantitative genetic analysis \[[@B33]\], and population genetics \[[@B34]\]. We estimated within species polymorphism for both species by calculating the frequency of SNPs among ESTs within the 20 largest contigs (Table [4](#T4){ref-type="table"}). These analyses considered a total of 30,638 base positions for *A. mexicanum*and 18,765 base positions for *A. t. tigrinum*. Two classes of polymorphism were considered in this analysis: those occurring at moderate (identified in 10--30% of the EST sequences) and high frequencies (identified in at least 30% of the EST sequences). Within the *A. mexicanum*contigs, 0.49% and 0.06% of positions were polymorphic at moderate and high frequency, while higher levels of polymorphism were observed for *A. t. tigrinum*(1.41% and 0.20%). Higher levels of polymorphism are expected for *A. t. tigrinum*because they exist in larger, out-bred populations in nature.
To identify SNPs between species, we had to first identify presumptive, interspecific orthologues. We did this by performing BLASTN searches between the *A. mexicanum*and *A. t. tigrinum*assemblies, and the resulting alignments were filtered to retain only those alignments between sequences that were one another\'s reciprocal best BLAST hit. As expected, the number of reciprocal \'best hits\' varied depending upon the *E*value threshold, although increasing the *E*threshold by several orders of magnitude had a disproportionately small effect on the overall total length of BLAST alignments. A threshold of *E\<*10^-80^yielded 2414 alignments encompassing a total of 1.25 Mbp from each species, whereas a threshold of *E\<*10^-20^yielded 2820 alignments encompassing a total of 1.32 Mbp. The percent sequence identity of alignments was very high among presumptive orthologues, ranging from 84--100% at the more stringent *E*threshold of *E\<*10^-80^. On average, *A. mexicanum*and *A. t. tigrinum*transcripts are estimated to be 97% identical at the nucleotide level, including both protein coding and UTR sequence. This estimate for nuclear sequence identity is surprisingly similar to estimates obtained from complete mtDNA reference sequences for these species (96%, unpublished data), and to estimates for partial mtDNA sequence data obtained from multiple natural populations \[[@B16]\]. These results are consistent with the idea that mitochondrial mutation rates are lower in cold versus warm-blooded vertebrates \[[@B35]\]. From a resource perspective, the high level of sequence identity observed between these species suggests that informatics will enable rapidly the development of probes between these and other species of the *A. tigrinum*complex.
Extending EST resources to other ambystomatid species
-----------------------------------------------------
Relatively little DNA sequence has been obtained from species that are closely related to commonly used model organisms, and yet, such extensions would greatly facilitate genetic studies of natural phenotypes, population structures, species boundaries, and conservatism and divergence of developmental mechanisms. Like many amphibian species that are threatened by extinction, many of these ambystomatid salamanders are currently in need of population genetic studies to inform conservation and management strategies \[e.g. \[[@B13]\]\]. We characterized SNPs from orthologous *A. mexicanum*and *A. t. tigrinum*ESTs and extended this information to develop informative molecular markers for a related species, *A. ordinarium*. *Ambystoma ordinarium*is a stream dwelling paedomorph endemic to high elevation habitats in central Mexico \[[@B36]\]. This species is particularly interesting from an ecological and evolutionary standpoint because it harbors a high level of intraspecific mitochondrial variation, and as an independently derived stream paedomorph, is unique among the typically pond-breeding tiger salamanders. As a reference of molecular divergence, *Ambystoma ordinarium*shares approximately 98 and 97% mtDNA sequence identity with *A. mexicanum*and *A. t. tigrinum*respectively \[[@B16]\].
To identify informative markers for *A. ordinarium*, *A. mexicanum and A. t. tigrinum*EST contigs were aligned to identify orthologous genes with species-specific sequence variations (SNPs or Insertion/Deletions = INDELs). Primer pairs corresponding to 123 ESTs (Table [8](#T8){ref-type="table"}) were screened by PCR using a pool of DNA template made from individuals of 10 *A. ordinarium*populations. Seventy-nine percent (N = 97) of the primer pairs yielded amplification products that were approximately the same size as corresponding *A. mexicanum*and *A. t. tigrinum*fragments, using only a single set of PCR conditions. To estimate the frequency of intraspecific DNA sequence polymorphism among this set of DNA marker loci, 43 loci were sequenced using a single individual sampled randomly from each of the 10 populations, which span the geographic range of *A. ordinarium*. At least one polymorphic site was observed for 20 of the sequenced loci, with the frequency of polymorphisms dependent upon the size of the DNA fragment amplified. Our results suggest that the vast majority of primer sets designed for *A. mexicanum*/ *A. t. tigrinum*EST orthologues can be used to amplify the corresponding sequence in a related *A. tigrinum*complex species, and for small DNA fragments in the range of 150--500 bp, approximately half are expected to have informative polymorphisms.
::: {#T8 .table-wrap}
Table 8
::: {.caption}
######
EST loci used in a population-level PCR amplification screen in *A. ordinarium*
:::
**Locus ID** **Forward Primer 5\' to 3\'** **Reverse Primer 5\' to 3\'**
------------------------- ------------------------------- -------------------------------
1F8 AAGAAGGTCGGGATTGTGGGTAA CAGCCTTCCTCTTCATCTTTGTCTTG
1H3 GGCAAATGCTGGTCCCAACACAAA GGACAACACTGCCAAATACCACAT
2C8 GCAAGCACCAGCCACATAAAG GGCCACCATAACCACTCTGCT
3B10 TCAAAACGAATAAGGGAAGAGCGACTG TTGCCCCCATAATAAGCCATCCATC
5E7 ACGCTTCGCTGGGGTTGACAT CGGTAGGATTTCTGGTAGCGAGCAC
5F4 CCGAGATGAGATTTATAGAAGGAC TAGGGGAAGTTAAACATAGATAGAA
6A3 GTTTATGAAGGCGAGAGGGCTATGACCA ATCTTGTTCTCCTCGCCAGTGCTCTTGT
6B1 TGATGCTGGCGAGTACAAACCCCCTTCT TTTACCATTCCTTCCCTTCGGCAGCACA
6B3 ACCACGTGCTGTCTTCCCATCCAT ACGAAGCTCATTGTAGAAGGTGTG
6B4 CCCACGATGAATTGGAATTGGACAT CTGCCTGCCAGACCTACAGACTATCGT
6C4 ATGGCGCCAAAGTGATGAGTA GGGCCAGGCACACGACCACAAT
6D2 ATCAAGGCTGGCATGGTGGTCA GGGGGTCGTTCTTGCTGTCA
6H8 GAAGAAGACAGAAACGCAGGAGAAAAAC CGGGCGGGGGCGGGTCACAGTAAAAC
BL005B\_A01.5.1 GACAGGTCATGAACTTTTGAAAATAA AAAGTATATGTACCAAATGGGAGAGC
BL006A\_G07.5.1 GATGTCCTCTCCACTATACAAGTGTG GTTTGACTTGTCACCACTTTATCAAC
BL012D\_F02.5.1 ACAGCCAGAAATAGAAACTTTGAACT TGAAAGTATGTATTGTTTTCACAGGG
BL013C\_E01.5.1 AGGATGAAATAATATGCTGTGCTTC ACCGTGATAAACTCCATCCCTT
BL014D\_B11.5.1 AGCAAAACTCCTCTATGAATCTCG ATTGCACACTAAATAGGTGAATACGA
BL279A\_G10.5.1 ATGGCAGGATGAAGAAAGACAT ATGCACTTTGGACCCACTGAG
Et.fasta.Contig1023.5.1 TGTGGTTATTGGACTACTTCACTCTC AAACGTCCATTTGACACTGTATTTTA
Et.fasta.Contig1166.5.1 GAATGAAGAGAAAATGTTTTGAAGGT GCACAGTATTGGCTATGAGCAC
Et.fasta.Contig1311.5.1 AGAAAACTGTGTCAAGCTTATTTTCC CAACTTAGTGTTCACATTTCTGAGGT
Et.fasta.Contig1335.5.1 CCACTTATGGTAGTTCCCACTTTTAT GCTAAAGAATACCAAGAACCTTTGAC
Et.fasta.Contig1381.5.1 GTCACAGGTATAACATTGAAAGGATG TAAATGAATCAAACATTGAAGAGAGC
Et.fasta.Contig1459.5.1 ATAACAAGGACATGTTCTGCTGG CTAGCAGAACCCTGTATAGCCTG
Et.fasta.Contig1506.5.1 AGGATATCCGCTCAGAAATATGAAG CTGACCACTTGCAAAACTTACTACCT
Et.fasta.Contig1578.5.1 CCTAGAACATTACCAAAACAGACTCA AATGAAGAAGTATTGCATGTGAGAAC
Et.fasta.Contig1647.5.1 GTACAACGTCAGGCAAAGCTATTCT ATCTCCAACACCGTGGCTAAT
Et.fasta.Contig1717.5.1 GAACTTGTTGGCAGGTTTCTCTT CTAGTGATAGGTTGGACATACCAGAG
Et.fasta.Contig1796.5.1 TGTGGGTATGTATATGGCTAACTTGT AGATTTTATGTGCTACTGCATTTACG
Et.fasta.Contig1908.5.1 CTCATGACTTAATTGCTGTTCTTCG ATAACCATTCTGAGGTTTTGAGTTG
Et.fasta.Contig1941.5.1 ATCTCCTGCTTCATCTCTTGATTTAT TAACAGATTTAATAAACGTCCCCTTC
Et.fasta.Contig1943.5.1 AGTACGATGAATCTGGTCCTTCAAT CCACAATACTGACATACTCTGGTCTT
Et.fasta.Contig325.5.1 GTGAAGTCAGTGAGTAAAGTCCATGT CTAGGATACCAGTGGGAGAGTGTAAT
Et.fasta.Contig330.5.1 GTCATCACCTCCACTACTTCACAAG TTTTGGCACTGTAAGATTCTATGAAC
Et.fasta.Contig536.5.1 CCTTAGGTAGAACAGACTGAAGCAG GAAACATGAAACTGGACTTGTTTTAG
Et.fasta.Contig917.5.1 GGATGCAGATTCTTCCTATTTTACTC CTGGTCACTTTACTTGTTTTCAGTGT
Et.fasta.Contig926.5.1 TTCATCACATTCTACTTCACAAATCA CTAGGCAAGCAAGCTTTCTAATAGTT
Et.fasta.Contig93.5.1 GAATAAAAGCAACAATTGCAGAGTTA CTCGACTCCTTCTACGATCTCTACTC
Et.fasta.Contig990.5.1 GTTTAGGTTAGTATGAAGGATCCCAA TGCCAGTACTCACCAATTAGTAAAAG
G1-C12 CCCAAATCCAGGAGTTCAAA TGGGACCTGGGGCTTCATT
G1-C13 TTGCCCGAGAAAAGGAAGGACATA CAAGGGTGGGTGAGGGACATC
G1-C5 F-CACTGTTGACTTGGGTTATGTTATT CTGCTCCTAGGGTTTGTGAAG
G1-C7 CCCGTGTGGCTGGCTTGTGC TCGGCTACTTTGGTGTTTTTCTCCCTCAT
G1-C9 TGGTCCGGCAACAGCATCAGA GCTTTTCGGTATTCAACGGCAGAGTG
G1-C9 TGGTCCGGCAACAGCATCAGA GCTTTTCGGTATTCAACGGCAGAGTG
G1-D5 AGACCCTTGCTGTGTAACTGCT GACTGGGACTGACTTCTATGACG
G1-D6 CAGCGTGCCCACCCGATAGAA TCCCAAAAAGTAAAATGTGCAAAGAAAA
G1-D7 CAGCGGTGGAAATGACAAACAGG CCAAGACGACGAGGAACGGTATT
G1-E12 CAACCATGAGAGGAGGCCAGAGAAC AAAACAGCACTACCTACAAAACCCTATT
G1-F1 TTAGTTTGGGTGCAGACAGGA GGTGCTCAACAACAAATCAACT
G1-F20 TCCCCAACAACTCCAGCAGAT GGAAACCACCTAGACGAAAAATG
G1-I18 CATGTTTGTGGGTGTGGTGAA AAAAGCGGCATCTGGTAAGG
G1-I19 ACCCAGACCTGTCCACCTCA GAACAGCTCTCCAATCCACAAG
G1-I21 CCAAGCGAAGGAGGCGTGTG CATGTGGCTCTTTGTTTCTGGA
G1-I5 TAATCGTGTTTGGTGGCATCCTTGAGTC AGCAGCAGTTCCATTTTCCCACACCA
G1-I8 ACCTGCAGTGGGCTAAGACC ATGGAAATAATAAAATAAAATGTT
G1-J10 CGTTCGCTTTGCCTGCCACA GGCTCTTCCCCGGTCGTCCAC
G1-J17 AGCGCCTTCTACACGGACAC TATGCCCCAATTACTCTTCTGC
G1-J2 TACAGTAACTATGCCAAGATGAAATG CAATATGGATAATGGCTGTAGACC
G1-J20 ATCCTCCAAGCTCACTACAACA CCAGCCCCTTCCCAAACAG
G1-J9 CTGTCATTGCCTGCATCGGGGAGAAG TGTTGAGGGGAAGCAGTTTTG
G1-K2 GCTTTCGCCTTTGACACCTC GGCCGGACCATTGCTGAAGAAG
G1-L11 AAAGTGACCATCCAGTGCCCAAACCT CCGGCCGAAACTGACGAGATACATTAG
G1-L13 TCAGCTGCACTAGGTTTGTC CATTTTGATTTGCTCCATAA
G1-L19 GACAACCTTGAATCCTTTATG AGATGTTGGTTGGTGACTTAT
G1-L20 TGGGCATAGATGGCAAGGAAAAA CCCCCAGCATCTCGCATACAC
G1-L7 GTGCTACAGGAAGGAATGGATG TAGCACAGGAACAGCCGACAATAA
G1-M14 CCGCTTGGACATGAGGAGAT TGGCAAAGAAACAGAACACAACTA
G1-M19 GAGAAGTAGTGTCCCGGCAGAAAC ATGGGTGAAAACTTAGGTGAAATG
G1-N9 GCGGGGCAATACATGACGTTCCACAG GACCCCCATCTCCGTTTCCCATTCC
G1-O1 GGGGTAGAGCACAGTCCAGTT TTGCAAGGCCGAAAAGGTG
G1-O12 GGAATTCCGGGGCACTACT TCGCGAGGACGGGGAAGAG
G1-O24 CGGCCTTCCTGCAGTACAACCATC TCGGCAACGTGAAGACCATA
G2-A11 GCCCCTGGAAGCTGTTGTGA GGGGTCCATCCGAGTCC
G2-A7 TTACCCCACAGACAAAATCAACACC GGCGGCCCCTCATAGCAC
G2-B1 GGGCCTAGTCCTGCTGGTC CAAAGAGTGCGGAGAAATGG
G2-B8 CAACATGCGACCACTATAGCCACTTCCT CGCCACCGCCACCACCACA
G2-C2 TTTGCAGGAAGAGTCATAACACAG GTCAACAACACCCTTTTCCCTTCCT
G2-D1 GCAGGTCGGCAAGAAGCTAAAGAAGGAA AGGGTTGGTTTGAAAGGATGTGCTGGTAA
G2-E17 GGAGCACCAAATTCAAGTCAG CGTCCCCGGTCAATCTCCAC
G2-E19 CCAGTTTGAGCCCCAGGAG TCGCGGCAGTCAAGAGGTC
G2-F17 TATCCTCTTATTGCTGCATTCTCCTCAC AGTACGGCCGTTCACCATCTCTG
G2-F2 CACACCACAGACGCATTGAC TCCCCAGCCTGTGTAGAAC
G2-G13 GGGAGGGGAGAAGGCTACCA ATACACGGCTTCCATGCTTCTTCTT
G2-G15 CCACGGCCCCACATCCAGC TCCCGCAGAATTTCCGTATCCAT
G2-G21 TCCAAGAGGGTGTGAGGTGAAC AAAGCCATGCGAAGCGGAAGAC
G2-G23 GGTTTGGTACTTCAGCGGATGT CCAAAGCCTGTACTATGCGAAAAG
G2-G5 CGGTCCCTACTGTGGTCTATGGTTTTCA GGCTCTGCATATCCTCGGTCACACTTCC
G2-G6 CCCATGGCTGCAAGGATTACG CAGGGGTTGTTGGGAGGCAGTGT
G2-H18 TTGTCAAATGGGCGAGTTCA TGTTTTGCACCCAGTTTTTG
G2-I18 GATCTCCTCAGGTCTCTTTCA GATTATGGGCCGGTGTCTCT
G2-I23 TGACTTTCCCAATGTGAGCAGAC CAGAGGTGGTGTTACAGCAGCAGTTT
G2-J12 CCTCTTGTCCCAGTGCCAGTG TCCAGGGATCCGAAACAAAG
G2-J21 CCGCCTCAGCCTGTTTCTCTACTTTT CTTTGAATTTCTGCTTTTGGTGCTCTGC
G2-K12 ACATTAGTCCTGGTTACGAGAGC AAAGGGCAGTCCAGCATTGA
G2-K2 CTGCCCAAGAAGACCGAGAGCCACAAG AGCGCCCCCTGCACCAAAATCA
G2-L16 CCAAGGGTAGGAGAACAAGACA ATGGCATGCTGGGAAATCA
G2-L21 GAATCTAGGTCCAAGCAGTCCCATCT GACCATCACACCACTACCCACACTCA
G2-L3 TGAAAGAGGCCAGAAACAAGTAG TTCCCAAGGTCTCCATAACAAT
G2-L4 TGGCCAAGAAGATGAAACAGGAAGAGGAG TGGCAAAGGACACGACGCAGAG
G2-M14 CGGCCTCCTCGACGCATACG CCAGGCCGGCCCATTGTTC
G2-M24 ACGGAGCACGGTCAGATTTCACG CCCGGCTGGCTCTTCTTGCTCTT
G2-M3 CGATCCGCATTGAACGAGT TGTGGCAGGAAGGAGAAGG
G2-N2 CGTGTTTTCCTCCTATGTCGACTTCTTTG ACGTGCTCTGCCTTTCTTGATCTTGTGTT
G3-D7 AGGATTTCTTGGCCGGTGGAGTGG GAAGTTGAGGGCCTGGGTGGGGAAGTA
NT001D\_E08.5.1 AGAAGTTCCTAGATGAGTTGGAGGAG AATTAATTTCCTAAACCAGGTGACAG
NT010B\_E09.5.1 GAAGAGGTCCTAAAATATCAAGATGC ATGATAGACTTCGTCCTTGTCATAGA
NT014D\_E01.5.1 AAAGAAGTCCCGCATCTAACCT ATTAAATATGAGAAGATGTGTGCAGG
V2\_p1\_b8 AGTCACTGTGTTACATTATCACCCAC ATAATTATACACTGCGGTCTGCATCT
V2\_p1\_c5 AGTACCTGTTCGACAAGCACAC TGAGAACATAGACAAGTTAACATACACC
V2\_p1\_d10 GAGATAGAAAGGCTGCATAAAGAAAT TATGTTTCAACAATGTACAGGAAACC
V2\_p1\_d4 CACCAGAACAAGCTGTATTTTTATGT TGGTTTGCATCATATATTAAAGGGTA
V2\_p1\_g7 GACTTCAAGCACATTGGGAAAC ATTGTAAACTTGATAGGCTGGTGAG
V2\_p2\_g6 AGAATTCCCAATAGCACCTGAAAT CACTTGGTAAATACATACACACAGCA
V2\_p2\_h2 CTTTTTGGCCTGGTCTTTTTG AGATTCTTCAGACTCGTCCTTCTTT
V2\_p3\_a5 TTTACACAGAAACCTTGTTTATTTGGC TTTAAGGATGCTTAGAGGCAAAGTATT
V2\_p3\_b1 AGTCACTGTGTTACATTATCACCCAC TATACACTGCGGTCTGCATCTACT
V2\_p5\_b3 AATGGGATGAAGAGCGAGAAT CTGCCCCATTGACATTTACCTA
V2\_p5\_h3 CCTTCAGACGAAAACAGCACTAAG TACAGTGTATGAGAGCCCAATATTTC
V2\_p6\_a4 AGAAATACATCAAATATCGGGTGG AAAAAGGACAATGTTCAGCTCTCT
V3\_p1\_a21 ACCAAGTTCTTGGAAAGTGGTG CTTAGTGTCTCCTGGGTTTGAATAG
V3\_p1\_b13 GTCTTGGTACTCAATGAAGGAGATG TCAATCTGATGAAGAGTTTACATGTCT
:::
Comparative gene mapping
------------------------
Salamanders occupy a pivotal phylogenetic position for reconstructing the ancestral tetrapod genome structure and for providing perspective on the extremely derived anuran *Xenopus*(37) that is currently providing the bulk of amphibian genome information. Here we show the utility of ambystomatid ESTs for identifying chromosomal regions that are conserved between salamanders and other vertebrates. A region of conserved synteny that corresponds to human chromosome (*Hsa*) 17q has been identified in several non-mammalian taxa including reptiles (38) and fishes (39). In a previous study Voss et al. (40) identified a region of conserved synteny between *Ambystoma*and *Hsa*17q that included collagen type 1 alpha 1 (*Col1a1*), thyroid hormone receptor alpha (*Thra*), homeo box b13 (*Hoxb13)*, and distal-less 3 (*Dlx3)*(Figure [4](#F4){ref-type="fig"}). To evaluate both the technical feasibility of mapping ESTs and the likelihood that presumptive orthologues map to the same synteny group, we searched our assemblies for presumptive *Hsa*17 orthologues and then developed a subset of these loci for genetic linkage mapping. Using a joint assembly of *A. mexicanum*and *A. t. tigrinum*contigs, 97 Hsa 17 presumptive orthologues were identified. We chose 15 genes from this list and designed PCR primers to amplify a short DNA fragment containing 1 or more presumptive SNPs that were identified in the joint assembly (Table [9](#T9){ref-type="table"}). All but two of these genes were mapped, indicating a high probability of mapping success using markers developed from the joint assembly of *A. mexicanum*and *A. t. tigrinum*contigs. All 6 ESTs that exhibited \'best hits\' to loci within the previously defined human-*Ambystoma*synteny group did map to this region (*Hspc009*, *Sui1*, *Krt17*, *Krt24*, *Flj13855,*and *Rpl19*). Our results show that BLAST-based definitions of orthology are informative between salamanders and human. All other presumptive *Hsa*17 loci mapped to *Ambystoma*chromosomal regions outside of the previously defined synteny group. It is interesting to note that two of these loci mapped to the same ambystomatid linkage group (*Cgi-125*, *Flj20345*), but in human the presumptive orthologues are 50 Mb apart and distantly flank the syntenic loci in Figure [4](#F4){ref-type="fig"}. Assuming orthology has been assigned correctly for these loci, this suggests a dynamic history for some *Hsa*17 orthologues during vertebrate evolution.
::: {#F4 .fig}
Figure 4
::: {.caption}
######
**Comparison of gene order between *Ambystoma*linkage group 1 and an 11 Mb region of Hsa17 (37.7 Mb to 48.7 Mb)**. Lines connect the positions of putatively orthologous genes.
:::
:::
::: {#T9 .table-wrap}
Table 9
::: {.caption}
######
Presumptive human chromosome 17 loci that were mapped in *Ambystoma*
:::
**Marker ID** **Primers^a^** **Diagnosis^b^** **LG^c^** **Symbol^d^** **RefSeq ID^e^** **E-value^f^**
----------------- ---------------------------------- ------------------ ----------- --------------- ------------------ ----------------
Pl\_6\_E/F\_6 F-GAAAACCTGCTCAGCATTAGTGT ASA ul PFN1 NP\_005013 E^-34^
R-TCTATTACCATAGCATTAATTGGCAG
Pl\_5\_G/H\_5 F-CTATTTCATCTGAGTACCGTTGAATG PE (A) 23 CGI-125 NP\_057144 E^-56^
R-TAATGTAGAACTAAATGGCATCCTTC
E-CCATGGTGCAGGAAGAGAGCCTATAT
Pl\_0.4\_A/B\_1 F-GTCTCATTATCCGCAAACCTGT SP 1 RPL19 NP\_000972 E^-67^
R-ATTCTCATCCTCCTCATCCACGAC
Pl\_4\_B\_7/8 F-CCTAGAACATTACCAAAACAGACTCA RD (Dpn II) 1 KRT10 NP\_061889 E^-17^
R-AATGAAGAAGTATTGCATGTGAGAAC
Pl\_4\_B\_9/10 F-GAACTTGTTGGCAGGTTTCTCTT RD (AciI) 1 KRT17 NP\_000413 E^-146^
R-CTAGTGATAGGTTGGACATACCAGAG
Pl\_10\_C/D\_4 F-CTCCACTATTTAAAGGACATGCTACA PE (A) 1 SUI1 NP\_005792 E^-48^
R-TTAATATAGCACAACATTGCCTCATT
E-TGCTACATTAATGTAATAAACGGCATCATC
Pl\_6\_E/F\_11 F-AAGAGAAGTTCCTAGATGAGTTGGAG PE (A) 1 HSPC009 NP\_054738 E^-26^
R-TGAAGAGAGAACTCAAAGTGTCTGAT
E-TCATGTTTTGCTCTGCTGTGCAGT
Pl\_9\_A/B\_10 F-TGATAGTTTCTGGATTAAGACGAGTG PE (T) 1 FLJ13855 NP\_075567 E^-15^
R-CTTAGAGCCATTGTTACAAGATGTTC
E-GTGATCTAGTGGGATCAAACCCTAAAGACC
Pl\_10\_C/D\_9 F-AAAGTGCCAAGAAGGAGATTAACTT PE (T) 9 NME1 NP\_000260 E^-71^
R-GAGCTCAGAAAACAAGGCAGTAAC
E-AAATGGATCTACGAGTAGACCTTGACCC
Pl\_9\_C/D\_9 F-GAGTCTCCTTTAGGATTGACGTATCT PE (T) 23 FLJ20345 NP\_060247 E^-17^
R-GCTATGTGAGCAGAGATAAAAGTCAG
E-GTTACAGCATCAGTGGGATGTGGTATGT
Pl\_8\_C/D\_9 F-AGGATACCAACCTCTGTGCTATACAT PE (C) 15 H3F3B NP\_005315 E^-66^
R-TAAATGTATTTACAAACCGAAAGCAA
E-CGTGGCGAGCGTGCCTAGT
Pl\_9\_C/D\_4 F-GTGGTTATTTGTAACATTTCGTTGAC PE (A) 8 SFRS2 NP\_003007 E^-40^
R-AATTACATTTGGGCTTCTCAATTTAC
E-TTTTTAAACGCGTAAAAATGTTAACAGA
Pl\_6\_C/D\_5 F-CCGTAAATGTTTCTAAATGACAGTTG PE (G) 2 ACTG1 NP\_001605 0
R-GGAAAGAAAGTACAATCAAGTCCTTC
E-GATTGAAAACTGGAACCGAAAGAAGATAAA
^a^Sequences are 5\' amplification primers, 3\' amplification primers, or primer extension probes, and are preceded by F-, R-, and E- respectively. ^b^Genotyping methods are abbreviated: allele specific amplification (ASA), size polymorphism (SP), restriction digestion (RD), primer extension (PE). Diagnostic restriction enzymes and diagnostic extension bases are provided in parentheses. ^c^Ambystoma linkage group ID. \"ul\" designates markers that are unlinked. ^d^Official gene symbols as defined by the Human Genome Organization Gene Nomenclature Committee <http://www.gene.ucl.ac.uk/nomenclature/>. ^e^Best BLASTX hit (highest e-value) from the human RefSeq database using the contig from which each marker was designed as a query sequence. ^f^Highest E-value statistic obtained by searching contigs, from which EST markers were designed, against the human RefSeq database.
:::
Future directions
-----------------
Ambystomatid salamanders are classic model organisms that continue to inform biological research in a variety of areas. Their future importance in regenerative biology and metamorphosis will almost certainly escalate as genome resources and other molecular and cellular approaches become widely available. Among the genomic resources currently under development (see \[[@B41]\]) are a comparative genome map, which will allow mapping of candidate genes, QTL, and comparative anchors for cross-referencing the salamander genome to fully sequenced vertebrate models. In closing, we reiterate a second benefit to resource development in *Ambystoma*. Genome resources in *Ambystoma*can be extended to multiple, closely related species to explore the molecular basis of natural, phenotypic variation. Such extensions can better inform our understanding of ambystomatid biodiversity in nature and draw attention to the need for conserving such naturalistic systems. Several paedomorphic species, including *A. mexicanum*, are on the brink of extinction. We can think of no better investment than one that simultaneously enhances research in all areas of biology and draws attention to the conservation needs of model organisms in their natural habitats.
Conclusions
===========
Approximately 40,000 cDNA sequences were isolated from a variety of tissues to develop expressed sequence tags for two model salamander species (*A. mexicanum*and *A. t. tigrinum*). An approximately equivalent number of contigs were identified for each species, with 21,091 unique contigs identified overall. The strategy to sequence cDNAs from a diverse collection of tissues from normalized and non-normalized libraries yielded different sets of highly redundant contigs. Only 25% and 28% of the *A. mexicanum*and *A. t. tigrinum*contigs, respectively, were identified in common. To demonstrate the utility of these EST resources, we searched databases to identify new probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human/*Ambystoma*synteny group with marker loci, and extended PCR primer sets designed for *A. mexicanum*/ *A. t. tigrinum*orthologues to a related tiger salamander species. Over 100 new probes were identified for regeneration research using informatic approaches. With respect to comparative mapping, 13 of 15 EST markers were mapped successfully, and 6 EST markers were mapped to a previously defined synteny group in *Ambystoma*. These results indicate a high probability of mapping success using EST markers developed from the joint assembly of *A. mexicanum*and *A. t. tigrinum*contigs. Finally, we found that primer sets designed for *A. mexicanum*/ *A. t. tigrinum*EST orthologues can be used to amplify the corresponding sequence in a related *A. tigrinum*complex species. Overall, the EST resources reported here will enable a diversity of new research areas using ambystomatid salamanders.
Methods
=======
cDNA library construction
-------------------------
Ten cDNA libraries were constructed for the project using various larval tissues of *A. mexicanum*and *A. t. tigrinum*(Table [1](#T1){ref-type="table"}). Larval *A. mexicanum*were obtained from adult animals whose ancestry traces back to the Axolotl Colony \[[@B17]\]. Larval *A. t. tigrinum*were obtained from Charles Sullivan Corp. The GARD and MATH A. mexicanum limb regeneration libraries were constructed using regenerating forelimb mesenchyme. Total RNAs were collected from anterior and posterior limbs amputated at the mid-stylopod level on 15 cm animals, and from the resulting regenerates at 12 h, 2 days, 5 days and early bud stages. One hundred μg fractions of each were pooled together and polyA-selected to yield 5 μg that was utilized for directional library construction (Lambda Zap, Stratagene). The V1 (*A. mex*), V2 (*A. tig*), V4-5 (*A. tig*), and V6-7 (*A. mex*) libraries were made from an assortment of larval tissues (see Table [1](#T1){ref-type="table"}) using the SMART cDNA cloning kits (Clontech). Total RNAs were isolated and reverse transcribed to yield cDNAs that were amplified by long distance PCR and subsequently cloned into pTriplEX. The V3 and AG libraries were constructed by commercial companies (BioS&T and Agencourt, respectively).
cDNA template preparation and sequencing
----------------------------------------
cDNA inserts were mass excised as phagemids, picked into microtitre plates, grown overnight in LB broth, and then diluted (1/20) to spike PCR reactions: (94°C for 2 min; then 30 cycles at 94°C for 45 sec, 58°C for 45°sec, and 72°C for 7 min). All successful amplifications with inserts larger than \~500 bp were sequenced (ABI Big Dye or Amersham Dye terminator chemistry and 5\' universal primer). Sequencing and clean-up reactions was carried out according to manufacturers\' protocols. ESTs were deposited into NCBI database under accession numbers BI817205-BI818091 and CN033008-CN045937 and CN045944-CN069430.
EST sequence processing and assembly
------------------------------------
The PHRED base-calling program \[[@B42]\] was used to generate sequence and quality scores from trace files. PHRED files were then quality clipped and vector/contaminant screened. An in-house program called QUALSCREEN was used to quality clip the ends of sequence traces. Starting at the ends of sequence traces, this program uses a 20 bp sliding window to identify a continuous run of bases that has an average PHRED quality score of 15. Mitochondrial DNA sequences were identified by searching all ESTs against the complete mtDNA genome sequence of *A. mexicanum*(AJ584639). Finally, all sequences less than 100 bp were removed. The average length of the resulting ESTs was 629 bp. The resulting high quality ESTs were clustered initially using PaCE \[[@B43]\] on the U.K. HP Superdome computer. Multi-sequence clusters were used as input sequence sets for assembly using CAP3 \[[@B44]\] with an 85% sequence similarity threshold. Clusters comprising single ESTs were assembled again using CAP3 with an 80% sequence similarity threshold to identify multi-EST contigs that were missed during the initial analysis. This procedure identified 550 additional contigs comprising 1150 ESTs.
Functional annotation
---------------------
All contigs and singletons were searched against the human RefSeq database (Oct. 2003 release) using BLASTX. The subset of sequences that yielded no BLAST hit was searched against the non-redundant protein sequence database (Feb. 2004) using BLASTX. The remaining subset of sequences that yielded no BLAST hit was searched against *Xenopus laevis*and *X. tropicalis*UNIGENE ESTs (Mar. 2004) using TBLASTX. Zebrafish ESTs were downloaded from UNIGENE ESTs (May 2004). BLAST searches were done with an E-value threshold of E \<10-7 unless specified.
Sequence comparison of *A. mexicanum*and *A. t. tigrinum*assemblies
-------------------------------------------------------------------
All low quality base calls within contigs were masked using a PHRED base quality threshold of 16. To identify polymorphisms for linkage mapping, contigs from *A. mexicanum*and *A. t. tigrinum*assemblies were joined into a single assembly using CAP3 and the following criteria: an assembly threshold of 12 bp to identify initial matches, a minimum 100 bp match length, and 85% sequence identity. To identify putatively orthologous genes from *A. mexicanum*and *A. t. tigrinum*assemblies, and generate an estimate of gene sequence divergence, assemblies were compared using BLASTN with a threshold of *E*\<10^-20^. Following BLAST, alignments were filtered to obtain reciprocal best BLAST hits.
Extending *A. mexicanum*/ *A. t. tigrinum*sequence information to *A. ordinarium*
---------------------------------------------------------------------------------
Polymorphic DNA marker loci were identified by locating single nucleotide polymorphisms (SNPs) in the joint *A. mexicanum*and *A. t. tigrinum*assembly. Polymerase chain reaction (PCR) primers were designed using *Primer 3*\[[@B45]\] to amplify 100 -- 500 bp SNP-containing fragments from 123 different protein-coding loci (Table [8](#T8){ref-type="table"}). DNA was isolated from salamander tail clips using SDS, RNAse and proteinase K treatment, followed by phenol-chloroform extraction. Fragments were amplified using 150 ng DNA, 75 ng each primer, 1.5 mM MgCl~2~, 0.25 U Taq, and a 3-step profile (94°C for 4 min; 33 cycles of 94°C for 45 s, 60°C for 45 s, 72°C for 30 s; and 72°C for 7 min). DNA fragments were purified and sequenced using ABI Big Dye or Amersham Dye terminator chemistry. Single nucleotide polymorphisms were identified by eye from sequence alignments.
Linkage mapping of human chromosome 17 orthologous genes
--------------------------------------------------------
Putative salamander orthologues of genes on human chromosome 17 (*Hsa*17) were identified by comparing the joint *A. mexicanum*and *A. t. tigrinum*assembly to sequences from the human RefSeq (NCBI) protein database, using BLASTX at threshold *E\<*10^-7^. Linkage distance and arrangement among markers was estimated using MapManager QTXb19 software \[[@B46]\] and the Kosambi mapping function at a threshold of p = 0.001. All markers were mapped using DNA from a previously described meiotic mapping panel \[[@B40]\]. All PCR primers and primer extension probes were designed using *Primer 3*\[[@B45]\] and *Array Designer2*(Premier Biosoft) software. Species-specific polymorphisms were assayed by allele specific amplification, restriction digestion, or primer extension, using the reagent and PCR conditions described above. Primer extension markers were genotyped using the AcycloPrime-FP SNP detection assay (Perkin Elmer). See Table [9](#T9){ref-type="table"} for amplification and extension primer sequences, and information about genotyping methodology.
Author\'s contributions
=======================
SP and DK: bioinformatics; JW: clone management and sequencing in support of *A. mexicanum*and *A. t. tigrinum*ESTs; JS: comparative mapping and polymorphism estimation; DW: extending ESTs to *A. ordinarium*; JM, KK, AS, NM: PCR and gel electrophoresis; BH and ET: cDNA library construction and sequencing for spinal cord regeneration ESTs; MR, SB, DG: cDNA library construction and clone management for limb regeneration ESTs; DP and SV conceived of the project and participated in its design and coordination. All authors read and approved the final manuscript.
Acknowledgements
================
We thank the Axolotl Colony. We thank Greg Chinchar and Betty Davidson for providing RNA to make cDNA libraries V3 and V4. We acknowledge the support of the National Science Foundation, the National Center for Research Resources at the National Institutes of Health, the Kentucky Spinal Cord and Head Injury Research Trust, and the NSF EPSCOR initiative in Functional Genomics at University of Kentucky.
| PubMed Central | 2024-06-05T03:55:46.825828 | 2004-8-13 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509418/",
"journal": "BMC Genomics. 2004 Aug 13; 5:54",
"authors": [
{
"first": "Srikrishna",
"last": "Putta"
},
{
"first": "Jeramiah J",
"last": "Smith"
},
{
"first": "John A",
"last": "Walker"
},
{
"first": "Mathieu",
"last": "Rondet"
},
{
"first": "David W",
"last": "Weisrock"
},
{
"first": "James",
"last": "Monaghan"
},
{
"first": "Amy K",
"last": "Samuels"
},
{
"first": "Kevin",
"last": "Kump"
},
{
"first": "David C",
"last": "King"
},
{
"first": "Nicholas J",
"last": "Maness"
},
{
"first": "Bianca",
"last": "Habermann"
},
{
"first": "Elly",
"last": "Tanaka"
},
{
"first": "Susan V",
"last": "Bryant"
},
{
"first": "David M",
"last": "Gardiner"
},
{
"first": "David M",
"last": "Parichy"
},
{
"first": "S Randal",
"last": "Voss"
}
]
} |
PMC509419 | Background
==========
Extracellular nucleotides (ATP, ADP, UTP and UDP) exert a wide range of biological effects in blood cells mediated by multiple ionotropic P2X receptors and G protein-coupled P2Y receptors \[[@B1]-[@B3]\]. So far, the P2Y family is composed of eight cloned and functionally distinct subtypes (P2Y~1~, P2Y~2~, P2Y~4~, P2Y~6~, P2Y~11~, P2Y~12~, P2Y~13~, P2Y~14~) \[[@B4],[@B5]\]; the P2X family is composed of seven cloned subtypes (P2X~1~-P2X~7~) \[[@B6],[@B7]\].
We have previously quantified P2 receptor mRNA expression in platelets (representing megakaryocyte expression), and demonstrated a selective expression of the ADP receptors P2Y~12~and P2Y~1~, together with the ATP receptor P2X~1~\[[@B8]\]. This is consistent with the clinical effect of the P2Y~12~antagonist clopidogrel for the prevention of myocardial infarctions in patients with acute coronary syndromes \[[@B9],[@B10]\]. However, virtually every hematopoietic cell is responsive to nucleotides \[[@B2]\]. Because effects as different as proliferation, differentiation, chemotaxis and release of cytokines are regulated by nucleotides, they could play a role in the atherosclerotic inflammatory process. Human lymphocytes, monocytes and macrophages constitute an important line of defence upon infection and exposure to inflammatory stimuli \[[@B11]\]. Circulating blood monocytes become activated, migrate to tissues, and undergo differentiation into macrophages during inflammation \[[@B12]\]. Monocytes have been shown to express several P2Y receptors and up-regulation of P2X~7~receptor mRNA in monocytes has been observed upon cell differentiation to macrophages \[[@B13],[@B14]\].
Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory \[[@B1],[@B2]\]. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors \[[@B8],[@B15]\]. In addition, we describe for the first time the mRNA expression of P2 receptors in CD34^+^stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function.
Results and Discussion
======================
Our previous studies of P2 receptor mRNA expression in man with real-time PCR has shown a good resemblance with pharmacological and physiological experiments in vascular smooth muscle cells, endothelial cells and platelets \[[@B8],[@B15]\]. It is therefore likely that our present mRNA findings in inflammatory, progenitor and stem cells are physiologically relevant. The lack of selective agonists and antagonists for most of the receptor subtypes combined with the absence of studies focused on several of the more recently cloned receptors makes the findings important. Furthermore, no pharmacological studies have been made on CD34^+^stem and progenitor cells.
Expression of P2Y receptors in lymphocytes
------------------------------------------
In lymphocytes, all the target genes P2Y~1~, P2Y~2~, P2Y~4~, P2Y~6~, P2Y~11~, P2Y~12~, and P2Y~13~could be detected (n = 6). To illustrate expression of the P2 receptors relative to each other the P2Y~1~receptor was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y~1~. Among the P2Y receptor subtypes the P2Y~12~and P2Y~2~had highest expression (Figure [1A](#F1){ref-type="fig"}). The lowest expressed P2Y receptor was P2Y~4~.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Relative P2 gene expression in lymphocytes.**A, Bar graph shows relative P2Y~1~, P2Y~2~, P2Y~4~, P2Y~6~, P2Y~11~, P2Y~12~and P2Y~13~receptor gene expression normalized to GAPDH. B, Bar graph shows relative P2X~1~, P2X~4~and P2X~7~receptor gene expression normalized to GAPDH. P2Y~1~was chosen to be calibrator.
:::
:::
Extracellular nucleotides and their P2 receptors are involved in the regulation, proliferation but also apoptosis and cell death in lymphocytes and monocytes \[[@B3],[@B16]\]. Previous studies have shown that ATP, ADP, UTP and UDP stimulate phospholipase C and Ca^2+^release from intracellular stores, that fits well with the highly expressed P2Y~2~receptor, together with the lesser expressed P2Y~1~and P2Y~6~receptors. ATP and ADP, but not UTP, can also increase cAMP \[[@B17]\]. This is in agreement with the P2Y~11~receptor that had the third highest mRNA expression.
The most interesting finding was that P2Y~12~had the highest expression among the P2Y receptors in lymphocytes. It is not likely that this is the result of platelet contamination, because platelets contain very low amounts of RNA. To the best of our knowledge, there are no studies that have examined the effects of P2Y~12~on lymphocytes, even though selective antagonists exist. It is expected to inhibit cAMP generation and may activate lymphocytes. This could explain the antiinflammatory effect of clopidogrel. Clopidogrel is a P2Y~12~antagonist used in the clinic as a platelet aggregation inhibitor that reduces thrombotic cardiovascular events such as myocardial infarctions. However, it has also been shown to reduce CRP, even though aspirin in antiplatelet doses lacks this effect \[[@B18]\]. This effect may be mediated via P2Y~12~receptors in lymphocytes.
Expression of P2X receptors in lymphocytes
------------------------------------------
The most abundant P2X receptor in lymphocytes was the P2X~4~receptor. As showed in Figure [1B](#F1){ref-type="fig"}, the expression of P2X~4~was 3.2 times higher than P2Y~1~. The expression of P2X~4~was significantly higher than the expression of the other P2X receptors; P2X~1~(P \< 0.001) and P2X~7~(P \< 0.01).
Selective pharmacological tools to discriminate between P2X receptors are scarce. Nevertheless, several studies have suggested the importance of P2X~7~in lymphocyte regulation. However, B lymphocytes stimulated with ATP do not undergo the typical increase in permeability up to 900 Da that is typical for the P2X~7~receptor. On the other hand, P2X~7~mediated effects on Ba^2+^and ethidium influx, phospholipase D activity and shedding of L-selectin have been blocked by the P2X~7~selective antagonist KN-62 in human lymphocytes \[[@B19]\]. Thus it is a surprising finding that the P2X~4~receptor was the highest expressed subtype in lymphocytes at the mRNA level. Even though we have demonstrated that more than 90% of the preparation consists of lymphocytes (see methods), it is possible that a small contamination of monocytes may have influenced the results, at least regarding P2X~4~receptor mRNA expression, because of its high expression levels in monocytes. P2X~4~receptors have indeed been demonstrated at the protein level in human B lymphocytes by confocal immunohistochemistry, in which P2X~1~, P2X~4~and P2X~7~were detected at the protein level \[[@B20]\]. However, the P2X~4~receptor staining was the most variable of the P2X receptors with weak to moderate levels of staining in a large proportion of cells in three patients and weak levels in only a minority of the cells from the other three patients examined \[[@B20]\].
Expression of P2Y receptors in monocytes
----------------------------------------
Again, the P2Y~1~expression was used as calibrator for the others, i. e. the other receptors were expressed as a ratio of the P2Y~1~. Among the P2Y receptors, the P2Y~2~, P2Y~13~and P2Y~11~had highest expression (Figure [2A](#F2){ref-type="fig"}, n = 6). The presence of P2Y receptor mRNA in monocytes and lymphocytes is in agreement with previous studies using regular RT-PCR \[[@B21]\].
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**Relative P2 gene expression in monocytes.**A, Bar graph shows relative P2Y~1~, P2Y~2~, P2Y~4~, P2Y~6~, P2Y~11~, P2Y~12~and P2Y~13~receptor gene expression normalized to GAPDH. B, Bar graph shows relative P2X~1~, P2X~4~and P2X~7~receptor gene expression normalized to GAPDH. P2Y~1~was chosen to be calibrator.
:::
:::
Extracellular nucleotides stimulate interleukin secretion, iNOS-generation in monocytes, are involved in differentiation, cytotoxicity and killing of pathogens. All monocyte/macrophage cell lines express P2Y receptors coupled to IP~3~generation and intracellular Ca^2+^release, but the individual subtypes have not been investigated in detail in monocytes \[[@B2],[@B3]\]. However, both ATP and UTP are active agonists, which is in agreement with the highest mRNA expression of the ATP/UTP receptor P2Y~2~(Fig [2](#F2){ref-type="fig"}). ATP mediated increase in cAMP has suggested the presence of P2Y~11~, with a suggested role in maturation of human monocyte-dendritic cells \[[@B22]\]. A relatively high expression of P2Y~11~was confirmed in our experiments. Interestingly, the P2Y~13~had even higher mRNA levels. To our knowledge, no experiments have addressed the presence of this cAMP inhibitory ADP receptor in monocytes. In fact, it has been an unresolved issue in what tissue this receptor is expressed. High levels in the spleen could be in agreement with monocyte expression \[[@B23]\]. Thus, the presence of P2Y~2~and P2Y~11~receptors are confirmed as expected, with the interesting addition of P2Y~13~receptors. Future experiments addressing the physiological role of P2Y~13~receptors in monocytes are needed.
Expression of P2X receptors in monocytes
----------------------------------------
Early studies demonstrated that ATP activates a receptor on macrophages that increase cell permeability eventually leading to cell death \[[@B2],[@B3]\]. P2X~7~receptor transfection confers susceptibility to ATP-dependent permeabilization and ATP-resistant clones lack the P2X~7~receptor, demonstrating that it is present on macrophages and necessary for permabilization. However, it is not known whether P2X~7~is the only constitutive subunit or if it assembles with other subunits.
As showed in Figure [2B](#F2){ref-type="fig"}, P2X~4~was by far the highest expressed P2 receptor in monocytes and the P2X~1~(P \< 0.01) and P2X~7~(P \< 0.01) had lower levels. Thus, unexpectedly the P2X~7~receptor was not the highest expressed P2X receptor in monocytes. This is in agreement with patch-clamp experiments suggesting that other P2X receptors are involved \[[@B24]\]. Interrelation of these experiments has suggested the contribution of P2X~4~receptors, which is supported by our findings \[[@B25]\]. It should be noted that all the three P2X receptors were expressed at very high levels compared to other cell types (30-fold more than the calibrator gene for P2X~4~and 6--7-fold more for P2X~1~and P2X~7~). A physiological role for all three subtypes can therefore be expected.
Expression of P2 receptors in CD34^+^stem and progenitor cells
--------------------------------------------------------------
CD34^+^stem and progenitor cells are receiving an increasing attention because of their extensive self-renewal and multilineage differentiation ability making them attractive for cellular therapy \[[@B26]\]. Knowledge of their P2 receptor expression could be used for directing differentiation or for further subtype selection of early progenitors types. There are no previous pharmacological or expression studies of P2 receptors on human CD34^+^stem and progenitor cells. We found expression of several P2Y receptors, especially P2Y~1~and P2Y~2~(Figure [3A](#F3){ref-type="fig"}, n = 3). This indicates that both ATP and UTP are agonists for CD34^+^stem and progenitor cells and may stimulate IP~3~and intracellular Ca^2+^release.
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**Relative P2 gene expression in CD34^+^stem and progenitor cells.**A, Bar graph shows relative P2Y~1~, P2Y~2~, P2Y~4~, P2Y~6~, P2Y~11~, P2Y~12~and P2Y~13~receptor gene expression normalized to GAPDH. B, Bar graph shows relative P2X~1~, P2X~4~and P2X~7~receptor gene expression normalized to GAPDH. P2Y~1~was chosen to be calibrator.
:::
:::
Among the P2X receptors the P2X~1~receptor had the highest expression followed by P2X~4~(P2X~1~had significantly higher expression than P2X~7~, P \< 0.05) (Figure [3B](#F3){ref-type="fig"}, n = 3), suggesting a potential role of these receptors in regulation of stem and progenitor cells. P2Y~1~, P2Y~2~and P2X~1~receptors have all been shown to stimulate proliferation, but also to be able to mediate apoptosis \[[@B26]\]. Such roles could be of major importance in the highly proliferative CD34^+^stem and progenitor cells. Antagonists or agonists of these receptors could be used to control their differentiation or proliferation.
Conclusions
===========
The P2X~4~receptor had the highest mRNA expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y~12~and P2Y~2~had highest expression in lymphocytes, while the P2Y~2~and P2Y~13~had highest expression in monocytes. The most interesting findings were the high mRNA expression of P2Y~12~receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y~13~receptors in monocytes and a previously unrecognised expression of P2X~4~in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns have been studied in CD34^+^stem and progenitor cells. Several P2 receptors were expressed (P2Y~2~, P2Y~1~, P2Y~12~, P2Y~13~, P2Y~11~, P2X~1~, P2X~4~), indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.
Methods
=======
The studies were approved by the local Ethics Committee of the Lund University and were conducted according to the principles of the Declaration of Helsinki.
Preparation of monocytes and lymphocytes
----------------------------------------
Peripheral blood was drawn from each of 6 healthy volunteers (after informed consent) into heparin vials. The mononuclear cells were isolated by density gradient centrifugation on Lymphoprep™ (Axis Shield Poc AS, Oslo, Norway) at 605 g for 30 minutes. The lymphocytes and monocytes thus obtained were washed three times in RPMI 1640 medium with L-glutamine (Gibco/BRL, Life Technologies Ltd, Paisleys, Scotland) and 0.1% human serum albumin (Sigma, St Louise, MO, USA), (medium), and centrifuged each time at 605 g for 5 minutes.
The fraction of lymphocytes and monocytes obtained according to this procedure was resuspended in medium with 15% normal human serum (NHS) added to a concentration of 4 × 10^6^cells/ml. Flow cytometry (Epics XL-MCL Beckman-Coulter, Florida, USA) analysis on these cells by detection of cell surface CD14 and CD45 showed that approximately 10% of the cells were monocytes. 800 μl of this cell-suspension was plated on a chamber slide 4 well glass slide (Nalge Nunc International, IL, USA) at 37°C in an atmosphere containing 5% CO~2~and 96% humidity for 1 h in order for the monocytes to adhere. Nonadherent cells were removed by washing three times with medium. Flow cytometry analysis of these nonadherent cells showed that at least 90% were lymphocytes, and were therefore used as source of lymphocytes. The cells attached to the glass slides (\<90% monocytes as assessed by flow cytometry) were detached by adding first PBS and then 0.5 mM EDTA-PBS for 3 min in room temperature.
Preparation of CD34^+^stem and progenitor cells
-----------------------------------------------
Bone marrow samples were obtained from healthy volunteers (n = 3), after informed consent, using guidelines approved by the Ethical Committee, Lund University. Mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque; Pharmacia, Uppsala, Sweden). CD34^+^cells were isolated by 2passages through magnetic columns (MidiMacs;Miltenyi Biotec, Bergish Gladbach, Germany) by using a hapten-conjugated CD34 antibody (Qbend/10) and an antihapten antibody conjugated to magnetic beads (CD34^+^isolation kit; Miltenyi Biotec). CD34 expression was analyzed by immunostaining with a FACSCalibur flow cytometer (Becton Dickinson) by using the CellQuest program (Becton Dickinson) and the purity of isolated populations was reproducibly \> 95% \[[@B27]\].
RNA extraction
--------------
Total cellular RNAs were extracted using TRIzol reagent (Gibco BRL, Life Technology) according to the supplier\'s instructions, dissolved in diethyl-pyrocarbonate (DEPC) treated water and stored at -70°C until used.
Quantitative analysis of P2 receptors by real-time reverse transcription polymerase chain reaction
--------------------------------------------------------------------------------------------------
TaqMan Reverse Transcription Reagents Kit was used to transcribe mRNA into cDNA. Real-time PCR were performed by means of a PRISM 7700Sequence Detector as described previously \[[@B8],[@B15],[@B28],[@B29]\]. Oligonucleotide primers and TaqMan probes were designed using the Primer Express software, based on sequences from the GenBank database \[[@B8],[@B15]\]. Constitutively expressed GAPDH were selected as endogenous control to correct for potential variation in RNA loading or efficiency of the amplification reaction.
Previous analysis showed that amplification efficiencies were almost identical for GADPH and the following receptor mRNAs: P2Y~1~, P2Y~2~, P2Y~4~, P2Y~6~, P2Y~11~, P2Y~12~, P2Y~13~, P2X~1~, P2X~4~, and P2X~7~normalized to GAPDH \[[@B8],[@B15]\]. To confirm equal amplification efficiencies, we used the criterion of a regression slope of less than 0.1 for each gene normalized to GAPDH. This confirms that we could use the comparative C~T~method for the relative quantification of target without running standard curves on the same plate (Perkin-Elmer Applied Biosystems Inc; User Bulletin No. 2, December 1997). The amount of target and endogenous reference was determined from the comparative C~T~method. The target gene normalized to GAPDH was expressed as ΔC~T~(C~T~of target gene minus C~T~of GAPDH). P2Y~1~was arbitrarily chosen to be the calibrator in the comparative analysis and is expressed as ΔC~TP2Y1~(C~T~of target minus C~T~of GAPDH for P2Y~1~). The normalized calibrated value is given by the equation 2^-ΔΔCt^, where ΔΔC~T~is ΔC~T~-ΔC~TP2Y1~. To further verify the specificity of PCR assays, the PCR was performed with non-reverse-transcribed total cellular RNA and samples lacking the DNA template. No significant amplifications were obtained in any of these samples (data not shown).
Drugs
-----
Unless otherwise stated, all reagents and drugs were purchased from Sigma Chemical Corp, St. Louis, MI, USA. PCR consumables were obtained from Perkin-Elmer Applied Biosystems Inc, Foster City, CA, USA.
Statistical methods
-------------------
Data are expressed as mean and standard error of the mean (SEM) unless otherwise stated. n indicates the number of subjects that were tested. Statistical analysis of the normalized C~T~values (ΔC~T~) was performed with a one-way ANOVA, followed by a multiple comparison post test (Tukey\'s test) using GraphPad InStat version 3.00 (GraphPad Software Inc., USA). Significant differences were considered at P \< 0.05 (two-tailed test).
Authors\' contributions
=======================
LW designed the study, carried out the RNA isolation and real-time PCR, and wrote the manuscript.
SEWJ supervised the isolation of CD34^+^stem and progenitor cells, and participated in writing the manuscript.
AB supervised the isolation of monocytes and lymphocytes, and participated in writing the manuscript.
DE conceived the study, guided throughout the study, and wrote the manuscript.
All authors read and approved the final manuscript.
Acknowledgments
===============
The study has been supported by the Swedish Heart and Lung Foundation, Franke and Margareta Bergqvist Foundation, the Wiberg Foundation, the Bergwall Foundation, the Zoegas Foundation, the Tore Nilsson Foundation, and Swedish Medical Research Council Grant 13130.
| PubMed Central | 2024-06-05T03:55:46.837983 | 2004-8-3 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509419/",
"journal": "BMC Immunol. 2004 Aug 3; 5:16",
"authors": [
{
"first": "Lingwei",
"last": "Wang"
},
{
"first": "Sten Eirik W",
"last": "Jacobsen"
},
{
"first": "Anders",
"last": "Bengtsson"
},
{
"first": "David",
"last": "Erlinge"
}
]
} |
PMC509420 | Background
==========
Changes in corneal curvature and axial length have been reported following scleral buckling procedure using keratometer \[[@B1]-[@B7]\]. Videokeratography has also been used to assess the corneal surface changes after buckling procedure\[[@B8]\].
All these studies have reported a change in the corneal curvature in its anterior surface. However, no study has been performed to evaluate the posterior corneal topographic changes with scanning slit topography system following retinal surgeries. Hence, we made an attempt to study the effect of various retinal surgeries on posterior corneal surface using Orbscan II topography system.
Methods
=======
A prospective study was performed by enrolling patients admitted in the Retina Services of Rajendra Prasad Centre for Ophthalmic Sciences, New Delhi for retinal/ vitreoretinal surgeries. Thirty one eyes of 31 patients who underwent retinal/ vitreoretinal surgery and came for regular follow-up as per schedule during the period between December 2001 and October 2002 were included in the study. Only those patients were included who had not undergone any previous ocular surgery & did not have any corneal pathology. An informed consent was obtained from all the patients.
The patients were evaluated preoperatively on parameters of best-corrected visual acuity (BCVA), axial length (AL) measured by Ultrasound A-scan instrument (Sonomed, Inc., NY) and detailed corneal examination on slit-lamp biomicroscope and on Orbscan topography system II (Bausch and Lomb, Salt Lake City, Utah). The parameters that were evaluated by Orbscan topography system were anterior elevation, posterior elevation and simulated keratometry.
All surgeries were performed by a single surgeon (LV) under local anesthesia by peribulbar injection of 6 ml of 2% xylocaine and 2 ml of 0.5% bupivacaine. Eyes with fresh retinal detachment with clear media and absence of advanced proliferative vitreoretinopathy underwent scleral buckling procedure (Group I, n = 11). In all these eyes, the break/ s were localized, cryotherapy was performed and subretinal fluid was drained. Only circumferential buckle of silicone of style 276 (Labtician Ophthalmics, Inc., Oakville, Canada) was used and radial buckle or sponge was not used in any eye. The size of the buckle was 90° to 360° depending upon the requirement in individual cases. Encircling element of style 240 (Labtician Ophthalmics, Inc., Oakville, Canada) was used in all the eyes undergoing scleral buckling.
In eyes with associated vitreous hemorrhage or advanced proliferative vitreoretinopathy changes along with retinal detachment, vitreoretinal surgery was performed along with scleral buckling (Group II, n = 8). Pars plana vitrectomy was performed and vitreoretinal membranes were removed either by peeling or by segmentation or delamination. Air fluid exchange was performed followed by Air-Silicone oil exchange. In eyes with only vitreous hemorrhage without the presence of retinal detachment, only pars plana vitrectomy was performed (Group III, n = 12). An encircling element of style 240 (Labtician Ophthalmics, Inc., Oakville, Canada) was used in all these eyes to counter the anterior traction that could not be fully released by vitrectomy in order to avoid lens damage.
The intraoperative details including the nature of surgery, size of the buckle, encircling element, drainage of subretinal fluid, vitrectomy & use of silicone oil or gas, were noted. Postoperative treatment included topical ciprofloxacin 0.3% QID, topical dexamethasone 0.1% QID and topical Homatropine 2% QID.
The patients were evaluated at 12 weeks following surgery on similar (preoperative) parameters.
Statistical analysis
--------------------
The data of all the patients were managed on an excel spreadsheet. All the entries were checked for any possible keyboard error. Preoperative and postoperative measurements in the three retinal surgery groups were summarized by mean and standard deviation. Changes following surgery within each group were assessed using paired \'t\' test. Preoperative and postoperative values in the three groups were compared using one way analysis of variance (ANOVA), followed by bon ferroni correction for multiple comparison. For the three retinal surgery groups, median was computed for increase in various parameters due to surgery. Kruskal Wallis one way analysis of variance was used to compare median increase in the three groups. STATA 7.0 statistical software was used for data analysis. In this study, p-value smaller than 0.05 was considered as statistically significant.
Results
=======
The mean age of the patients was 45.96 ± 15.17 (range: 18--78) years and majority (83.87%) of the patients (N = 31) were males. Of these, right eye was operated in 19 (61.29%) patients.
The mean preoperative best-corrected visual acuity (BCVA) was hand motion close to face in 26 eyes; counting finger near to face in 4 eyes and 1/60 on snellen\'s acuity chart in 1 eye. The mean decimal postoperative BCVA was 0.20 ± 0.12 at 12 weeks follow-up after surgery.
The mean preoperative anterior corneal elevation as recorded by Orbscan II topography system in group I was 0.006 ± 0.007 mm, which increased to 0.024 ± 0.013 mm at 12 weeks after surgery (p= 0.003). In group II, it increased from 0.009 ± 0.006 mm preoperatively to 0.021 ± 0.010 mm at 12 weeks (p= 0.008) and in group III, it increased from 0.003 ± 0.004 mm preoperatively to 0.012 ± 0.007 mm at 12 weeks follow-up (p = 0.003). On comparative evaluation between the groups, the change in anterior corneal elevation was significant between group I and III (p = 0.04).
The mean posterior elevation in group I increased from a preoperative value of 0.016 ± 0.010 mm to 0.043 ± 0.007 mm at 12 weeks after surgery (p= 0.0000). In group II, it increased from 0.014 ± 0.006 mm preoperatively to 0.043 ± 0.007 mm at 12 weeks (p= 0.0001) and in group III it increased from a preoperative value of 0.012 ± 0.005 mm to 0.029 ± 0.006 mm at 12 weeks after surgery (p = 0.0001). A comparative analysis between the groups indicated that the increase in posterior corneal elevation between groups I & III and groups II & III were found to be highly significant (I vs III: p= 0.001; II vs III: p= 0.001).
Again, the increase in the posterior corneal elevation was greater than the increase in the anterior elevation in all the 3 groups and on comparative evaluation, the difference in the increase in posterior and anterior elevation was significant statistically in each group (group I: p = 0.02; group II: p = 0.01; group III: p = 0.008).
The mean corneal astigmatism in group I increased from 0.89 ± 0.54D preoperatively to 2.50 ± 1.39D at 12 weeks follow-up (p= 0.004). In group II, the average corneal astigmatism increased from 0.87 ± 0.30D to 3.38 ± 2.15D at 12 weeks (p= 0.01) and in group III, the mean preoperative and postoperative corneal astigmatism was 0.85 ± 0.55D and 1.37 ± 0.87D respectively (p= 0.02). A comparative analysis of the change in corneal astigmatism following surgery between groups II & III was significant statistically (p= 0.02).
The mean preoperative axial length in group I was 23.27 ± 0.79 mm which increased to 23.98 ± 0.76 mm at 12 weeks after surgery (p= 0.009). The mean preoperative and postoperative (12 weeks follow-up) axial length in group II were 23.92 ± 1.32 mm and 25.94 ± 2.96 mm respectively (p= 0.03). The mean preoperative and postoperative axial length in group III were 22.69 ± 0.87 mm and 22.71 ± 0.83 mm respectively (p= 0.79). Comparative analysis of increase in axial length following surgery between groups I & III and groups II & III were found to be significant statistically (I & III: p = 0.003; II & III: p = 0.003).
Discussion
==========
Retinal surgery with or without the use of encircling and buckling elements for external tamponade can alter the shape of the globe. This may cause changes in the refractive status of the eye. Scleral buckling is known to cause a change in the shape of the sclera and can cause induced refractive changes, including astigmatic and nonastigmatic changes \[[@B5]-[@B10]\].
We have used Orbscan slit scanning system II to evaluate the corneal topographic changes following retinal/ vitreoretinal surgeries. The data accumulated by Orbscan may be limited by factors such as the accuracy of the system which is ± 20 μm, the measurement noise which leads to both positive and negative difference in the height of the posterior corneal surface and the necessity of aligning the posterior surface before and after surgery which may be a source of error\[[@B11],[@B12]\]. However, this is the best tool available to study the posterior corneal elevation.
In the present study, there was a significant increase in both anterior and posterior corneal elevation as detected by scanning slit topography (Orbscan II topography system) following surgery. This increase in the anterior and posterior corneal elevation is probably due to the use of encircling element and/ or buckle in retinal surgeries resulting in corneal steepening. We noted that the change in the posterior elevation was more significant than anterior elevation. A comparative analysis between the three groups indicated that there was no significant difference in the anterior elevation; however the posterior elevation was significantly more in eyes with buckle. It is possible that the buckle and the encircling element have a greater effect on the posterior corneal surface. The increase in the anterior and posterior corneal elevation might be one of the contributing factors for the non improvement of visual acuity in an eye that has undergone retinal/ vitreoretinal surgery. This change might escape detection by routine videokeratography. The anterior protrusion of the corneal back surface induces an increase in the negative power of the corneal surface. Assessing the corneal surface by keratometry or placido disc videokeratography may provide inadequate information regarding refractive change caused by corneal surface alteration that results in retinal/ vitreoretinal surgery.
There was an increase in mean corneal astigmatism following surgery in all the groups. This is due to the effect of encircling element or buckle on the corneal surface. Studies have reported that induced astigmatism has been associated with radial scleral buckles\[[@B5],[@B13]\], circumferential buckles\[[@B14]\], medial rectus disinsertion\[[@B15]\], anterior location of the scleral buckle\[[@B13]\], and use of sponge material rather than hard silicone\[[@B1]\]. In our study, a comparative analysis between the groups indicated that buckle causes more astigmatic changes than encircling element.
In retinal/ vitreoretinal surgeries, the encircling band creates a circular indentation of the eye, thereby increasing its anteroposterior axial length; the myopic shift may be upto 3 diopters (D)\[[@B9],[@B10],[@B13],[@B16]\]. An increase in axial length by 0.54 mm\[[@B9]\] and 1.7 mm\[[@B14]\] has been reported following scleral buckling in two studies. In the present study, there was an increase in the mean axial length of the eyes in all the three groups (Table 1). This increase in axial length may be attributed to an anteroposterior elongation of the eyeball secondary to the transverse compression by the buckle and/ or an encircling element. The postoperative increase in axial length was found to be more pronounced in eyes with buckle. Buckle being wider and thicker results in greater indentation and thereby a greater increase in axial length than encircling element.
Conclusions
===========
Retinal/ vitreoretinal surgeries result in an increase in the elevation of the corneal surfaces. These changes are more pronounced on posterior corneal surface.
Declaration of competing interest
=================================
None declared.
Individual contribution of authors
==================================
RS designed the study and performed the data collection. NS wrote the manuscript. LKV performed the retinal surgeries. RMP performed the statistical analysis and RBV followed up the patients.
All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2415/4/10/prepub>
| PubMed Central | 2024-06-05T03:55:46.839457 | 2004-8-3 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509420/",
"journal": "BMC Ophthalmol. 2004 Aug 3; 4:10",
"authors": [
{
"first": "Rajesh",
"last": "Sinha"
},
{
"first": "Namrata",
"last": "Sharma"
},
{
"first": "Lalit",
"last": "Verma"
},
{
"first": "RM",
"last": "Pandey"
},
{
"first": "Rasik B",
"last": "Vajpayee"
}
]
} |
PMC509421 | Background
==========
Bipolar disorder is a chronic severe mental illness affecting approximately 1% of the adult population. The most widely used mood stabilizer for this condition is lithium \[[@B1]\], although the exact mechanism by which it is clinically effective remains undetermined. One suggestion is that it acts via effects on choline metabolism. This is based upon findings that lithium can inhibit the membrane transport of choline in both animals \[[@B2]\], and human post-mortem brain tissue \[[@B3]\]. It also increases the accumulation of erythrocyte choline in lithium-treated patients \[[@B4]-[@B7]\]. Also of note is that choline concentrations increase significantly in rats following electroconvulsive shock \[[@B8]\]. Based upon this data choline has been used to treat mania in a some small pilot studies \[[@B9]\], with one open label study reporting that choline augmentation of lithium treatment helped rapid-cyclers \[[@B10]\]. Patients treated with choline also had increased basal ganglia concentrations of choline, suggesting that externally administered choline could alter brain concentrations \[[@B11],[@B12]\].
The most appropriate method to measure brain choline concentrations *in vivo*utilizes proton magnetic resonance spectroscopy (^1^H-MRS). Previous studies of bipolar patients utilizing this methodology have had mixed findings. Overall, while some studies have suggested there may be increased choline concentrations in specific situations \[[@B13]-[@B18]\], more have found no changes \[[@B19]-[@B27]\], and one found a trend towards a decrease in concentrations \[[@B28]\]. In both patients and volunteers lithium also doesn\'t appear to alter choline/creatine peak ratios concentrations \[[@B29],[@B30]\]. Nonetheless, two reviews concluded that the evidence to date suggests that lithium increases brain choline concentrations \[[@B31],[@B32]\], although as noted in these reviews previous studies have varied considerably in terms of patient populations, brain region studied, medications administered, and MRS methodology. Many studies have also examined differing patients (Type I and Type II) in differing mood states (mixed, depressed, manic, and euthymic). This may partially explain the varied results.
Sodium valproate is also widely used as a mood stabilizer, both alone and in combination with lithium \[[@B33]\]. To date there have been few studies which have examined the effects of sodium valproate on choline concentrations or activity. An *in-vitro*study suggested that valproate may inhibit choline acetyltransferase activity \[[@B34]\]. In one study 9 patients taking either lithium or valproate were examined \[[@B35]\], and increased Cho/Cr ratios were seen in the bipolar patients compared to controls. There were no differences between the lithium and valproate treatment groups, although the sample sizes were small. However, another study in epilepsy patients treated with valproate found no changes in choline concentrations \[[@B36]\]. Nonetheless, given the lack of studies to date, the possibility that valproate and lithium may both increase choline concentrations warrants further investigation.
Most of the previous studies have examined Cho/Cr ratios. However, it should be noted that the \"choline\" resonance peak seen in ^1^H-MRS spectra is composed primarily of phosphocholine and glycerophosphocholine, along with free choline, acetylcholine, and cytidine diphosphate choline. Also, we have shown in animal studies that both lithium and valproate can both decrease creatine concentrations \[[@B37]\]. Therefore, when using Cho/Cr ratios it is not possible to be certain that any changes in this peak represent changes in brain choline concentrations. We were therefore interested to determine if there were any differences in results when using different methodologies, and more specifically to determine if studies using a ratio methodology may have different results from studies utilizing metabolite concentrations.
Methods
=======
In the first part of the study patients taking either lithium or valproate were examined using the Cho/Cr ratio method, and both Bipolar Type I and Bipolar Type II patients were included who could also be taking other medications. In the second part of this study only Bipolar Type I patients on valproate monotherapy were included, and quantification of choline concentrations was made. Some of the data from the first part of this study has been reported previously \[[@B38]\].
Subjects and Study Design
-------------------------
All subjects gave full informed consent, and both studies were approved by the ethics committee at the University of Alberta. Healthy controls were examined using a detailed, but non-standardized, psychiatric interview. They were excluded if there was any personal history, or immediate family history, of psychiatric disorder. For patients, diagnoses were made using DSM-IV criteria for Bipolar Disorder Type I or Type II following detailed psychiatric interview, with additional information being available in almost all cases from long-term psychiatric clinic records. They also had to be taking a dose of either lithium or valproate which maintained their blood levels within the ranges of 0.4--1.2 mmol/l for lithium and 200--700 μmol/l for sodium valproate. Serum lithium and valproate levels were also measured on the day of MRS scanning. Other medications taken by the patient were noted. In the second part of the study the same criteria were used, except that only patients meeting diagnostic criteria for Bipolar Disorder Type I were included, and they had to be on sodium valproate monotherapy. This was done to examine Bipolar Type I patients in more detail, and to remove a possible confounding variable. All patients had to be euthymic for the previous 3 months, as determined by interviews with the patient, and additional interviews with their relatives and bipolar clinic records when available. MRS scans were carried out within 24 hours of this interview.
Magnetic Resonance Spectroscopy Methodology
-------------------------------------------
For both studies magnetic resonance experiments were performed using a Magnex 3 T scanner with 80 cm bore equipped with actively shielded gradient, and spectrometer control was provided by an Surrey Medical Imaging System (SMIS) console. The subjects head was immobilized with a restraint system. Signal transmission and reception were achieved using a quadrature birdcage resonator for ^1^H measurements.
Part 1 - Magnetic Resonance Spectroscopy
----------------------------------------
Initially, MRI data were acquired using gradient echo imaging sequences to produce multiple slice images along both coronal and transverse planes. This allowed registration of a 2 × 2 × 3 cm volume-of-interest (VOI) to be selected in the temporal lobe. ^1^H MR spectra were acquired using the PRESS localization method \[[@B39],[@B40]\], with TE = 32 ms, TR = 3 s, and with 128 averages. Baseline correction and deconvolution of the spectra was accomplished using the Peak Research (PERCH) spectrum analysis software package. The metabolite peaks of interest \[choline (Cho) and creatine (Cr)\] in each spectrum were fitted to a Gaussian line-shape for peak area estimation. To determine changes in choline concentrations we examined the Cho/Cr ratio. Figure [1](#F1){ref-type="fig"} shows an individual ^1^H MRS spectra in which all the major metabolite peaks can be seen.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
A typical ^1^H-MRS spectrum of the human brain at 3.0 T. A number of metabolites can be seen. 1: creatine (methylene) + phosphocreatine, 2: glutamate + glutamine, 3: *myo*-inositol + glycine, 4: taurine, **5: total choline compounds**, 6: creatine (methyl) + phosphocreatine, 7: N-acetylaspartate.
:::
:::
Study 2 - Magnetic Resonance Spectroscopy
-----------------------------------------
To accurately quantify the brain concentration of creatine we used a 125 ml glass sphere containing a solution of 4 mmol creatine as an external standard. The PRESS sequence was used to acquire proton MRS data with TE1 = 25 msec, TE2 = 25 msec, TR = 3000 msec, and 128 scan averages. The MRS data were acquired from three 2 × 2 × 2 cm^3^voxels placed in the cortex of the left frontal lobe, the cortex of the left temporal lobe, and in the external standard solution. The average coordinates \[[@B41],[@B42]\] of the centers of the two brain voxels were determined: x = 0.5 mm (SD = 1.6), y = 63.5 mm (SD = 12.1), z = -25.5 mm (SD = 4.2) in the frontal lobe, and x= 32.2 mm (SD = 6.3), y = 20.5 mm (SD = 3.9), z = 10.7 mm (SD = 2.6) in the temporal lobe. In order to measure T~1~and T~2~values of the metabolites in the brain and external standard solution, MRS data were collected with different TE values at a constant TR and different TR values at a constant TE both for the healthy volunteers and the patients and also from external standard solution \[[@B42]\]. However, due to these constraints, the fact that the two studies used different populations at different times, and the size of the external 125 ml container (which limited voxel size to 2 × 2 × 2 cm^3^), it was not possible to exactly match the voxel size or location between the two studies.
MRS Data Analysis
-----------------
For quantitative measurement of brain metabolite concentrations we used previously described methodology \[[@B42],[@B43]\]. In this, \[Met\]~b~, in millimoles per kg of wet brain, the CSF volume fraction, f~csf~, in the spectroscopic voxels must be corrected. Thus, brain metabolite concentrations were calculated as described in the following equation:
where V~voxel~is the volume of a 8 cm^3^spectroscopic voxel \[[@B43]\], and N~b~represents the number of metabolite molecules per unit voxel in brain.
Statistical Analysis for both MRS studies
-----------------------------------------
Means ± SEM were used in the statistical analysis. Sex differences were analyzed using chi-squared, and age differences with ANOVA with *post-hoc*Tukey tests. The MRS data was analyzed using Student\'s unpaired *t*-test using a significance level of p \< 0.05 comparing diagnostic groups (patients vs controls) in each brain region (frontal and temporal).
Results
=======
Study 1
-------
### Subjects
A total of 18 healthy controls, 14 bipolar patients taking lithium, and 11 bipolar patients taking valproate completed this study. Of the 14 bipolar patients taking lithium, 7 were Type I and 7 were Type II. In the valproate group, 7 were Type I and 4 were Type II. These groups were studied both separately and together, but as there were no statistically significant differences between the Type I and Type II patients, the results for both types are presented together. Of the 14 bipolar patients taking lithium 12 patients were taking other psychotropic medications: these were benzodiazepines (7 patients), antidepressants (5 patients), and antipsychotics (2 patients). Of the 11 patients taking sodium valproate 10 patients were taking other psychotropic medications: these were benzodiazepines (5 patients), antidepressants (5 patients), and antipsychotics (4 patients).
The mean age for the lithium group was 40.43 ± 2.96 years, for the valproate group 35.47 ± 2.27 years, and for the control group was 31.35 ± 2.89 years. These differences were statistically significant (F = 3.68, df = 2, p = \<0.05), which was attributable to the lithium group being significantly older than the control group (Tukey *post hoc*, p \< 0.05).
There were no gender differences within the groups: 10 females and 8 males in the control group (χ^2^= 0.167, df 1, p \> 0.05), 5 females and 9 males in the lithium group (χ^2^= 1.143, df 1, p \> 0.05), and 6 females and 5 males in the valproate group (χ^2^= 0.474, df 1, p \> 0.05).
Mean serum lithium levels were 0.79 ± 0.06 mmol/l, and the range was 0.46--1.08 mmol/l. The mean serum valproate levels were 508 ± 42 μmol/l, and the range was 210--912 μmol/l.
MRS Data
--------
### ^1^H MRS
We utilized the ratio of the choline peak to creatine peak (Cho/Cr) as a primary correlate of Choline concentrations. This result has been reported briefly in a previous publication \[[@B38]\]. The mean Cho/Cr ratio with this measure was 1.46 ± 0.04 for controls, 1.18 ± 0.07 for lithium-treated patients, and 1.12 ± 0.08 for valproate-treated patients. These were statistically significant, with a reduction in ratios occurring in both the control vs. lithium comparison (t = 3.628, df = 30, p = 0.001) and the control vs. valproate comparison (t = 4.248, df = 27, p = 0.002).
Study 2
-------
### Subjects
A total of 11 healthy controls and 9 Bipolar Type I patients taking valproate as monotherapy were entered into this study. The mean age for the control group was 37.3 ± 2.2 years, and for the valproate patients 42.4 ± 3.0 years. These differences were not statistically significant (F = 1.49, df = 1, p = 0.27).
There were no gender differences within the groups: 7 females and 2 males in the valproate group and 5 females and 6 males in the control group (χ^2^= 0.474, df 1, p \> 0.05). The mean serum valproate levels were 472 ± 36 μmol/l, and the range was 284--728 μmol/l.
In the frontal lobe the mean choline concentration for the healthy controls was 2.21 ± 0.17 mmol/kg wet brain and for the patients was 2.38 ± 0.12 mmol/kg wet brain. In the temporal lobe the mean choline concentration for the healthy controls was 2.35 ± 0.14 mmol/kg wet brain and for the patients was 2.40 ± 0.19 mmol/kg wet brain. There were no statistically significant differences between the controls and patients in either the frontal (t = 0.78, df = 18, p = 0.44) or temporal (t = 0.203 df = 18, p = 0.84) lobes (Table [1](#T1){ref-type="table"}).
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Concentrations (mmol/kg wet brain) and ratios (Cho/Cre) in frontal and temporal lobes in healthy volunteers and in patients chronically treated with valproate (Study \#2)
:::
Choline (Cho) Creatine (Cre) Cho/Cre
---------------------------- ----------- --- --------------- ---------------- ---------- ---------- ---------- ----------
1 50 M 3.51 2.95 6.67 8.53 0.53 0.35
2 45 M 2.19 3.03 10.1 9.11 0.22 0.33
3 43 F 3.01 2.31 9.97 9.52 0.30 0.24
4 39 M 2.11 2.72 7.94 7.60 0.27 0.24
5 37 F 2.47 2.34 9.98 9.89 0.25 0.24
6 36 F 1.91 1.76 8.28 8.19 0.23 0.22
7 35 M 1.76 2.36 7.93 8.36 0.22 0.28
8 32 F 1.88 1.51 9.56 9.56 0.2 0.16
9 32 M 1.94 2.14 7.04 7.79 0.28 0.28
10 30 F 1.82 2.52 7.8 8.63 0.23 0.29
11 28 M 1.72 2.23 7.16 8.51 0.24 0.26
**Mean** **37.00** **2.21** **2.35** **8.40** **8.70** **0.27** **0.26**
Valproate Treated Patients
1 58 F 2.72 2.1 9.16 10.13 0.30 0.21
2 50 M 2.61 3.42 8.17 10.53 0.32 0.33
3 49 F 2.03 1.79 8.56 7.48 0.24 0.24
4 48 F 2.44 1.88 9.93 8.19 0.25 0.23
5 36 M 2.60 2.53 7.84 7.51 0.33 0.34
6 35 F 2.07 2.77 9.26 10.39 0.22 0.27
7 35 F 2.78 1.89 8.35 9.79 0.33 0.19
8 34 F 1.76 2.93 7.26 8.01 0.24 0.37
9 34 F 2.43 2.27 7.75 7.23 0.31 0.31
**Mean** **42.11** **2.38** **2.40** **8.48** **8.81** **0.28** **0.28**
:::
The Cho/Cr ratios in the frontal lobes were 0.27 ± 0.028 in controls and 0.28 ± 0.015 in patients. In the temporal lobes the Cho/Cr ratios were 0.26 ± 0.021 in controls and 0.28 ± 0.016 in patients. There were no statistically significant differences between the controls and patients in either the frontal (t = 0.367, df = 18, p = 0.72) or temporal (t = 0.539, df = 18, p = 0.59) lobes (Table [1](#T1){ref-type="table"}).
Discussion
==========
The results from the present study vary considerably between the two sections utilizing differing methodologies. This is despite the fact that both studies were carried out by the same group on the same scanner with bipolar patients coming from the same patient pool. This strongly suggests that the methodology used to determine choline concentrations can considerably alter the results. In the first part of the study we found that both the lithium-treated and valproate-treated patients had significantly reduced Cho/Cr peak ratios compared to controls. This is similar to the findings from one previous study which also suggested that there may be a trend towards decreased choline in grey matter \[[@B28]\]. This study was a frontal lobe study that measured metabolite concentrations in a 1.5 T scanner in bipolar type I patients hospitalized for manic (n = 9) or mixed (n = 8) states. In this study most patients were being treated with valproate and an atypical antipsychotic.
These findings, however, differ from those in the second part of the present study in which we found no differences in choline concentrations between valproate-treated patients and controls in either frontal or temporal lobes. This second part of the study was much better controlled in terms of the patients receiving valproate monotherapy, only including bipolar Type I patients, and in using an external choline solution to accurately quantify choline concentrations. This finding of a lack of change is also in keeping with most previous studies. Several studies which have also previously measured metabolite concentrations with 1.5 T scanners also found no changes. These include a study of the hippocampus in 15 euthymic bipolar type 1 patients, of whom 10 were taking either lithium or valproate \[[@B19]\], a study of basal ganglia in 8 rapid cycling patients on lithium \[[@B22]\], a study of the anterior cingulate in 10 bipolar children \[[@B23]\], and a study in frontal lobes of 23 euthymic bipolar patients of whom 13 were on lithium \[[@B25]\]. Several other studies have examined metabolite ratios, mostly in patients on lithium, and those also found no changes in choline concentrations \[[@B20],[@B21],[@B26],[@B27]\]. In a study using metabolite ratios in bipolar children who were off medication for at least one week there was also no change in choline concentrations \[[@B24]\]. In a double-blind placebo-controlled human volunteer study before and after one week of lithium administration we also found no changes in cholinein 10 volunteers \[[@B30]\], which is similar to a patient study which compared 7 patients on lithium to 6 non-lithium treated controls and in which no differences were seen \[[@B29]\].
In contrast, animal studies have suggested that lithium may increase brain choline concentrations, and in lithium-treated patients it also increases the accumulation of choline within erythrocytes \[[@B4]-[@B7]\]. Nonetheless, ^1^H-MRS studies in patients examining this possibility is mixed. To date 6 studies have suggested some support for this \[[@B13]-[@B18]\], but in none of these studies were metabolite concentrations measured, and most of the studies measured choline/creatine ratios \[[@B14]-[@B18]\], the other one measuring metabolite intensity/tissue volume \[[@B13]\]. The first study to examine brain choline in basal ganglia studied only 4 patients, all of whom were on lithium \[[@B18]\]. Another study examined 19 euthymic inpatients and found increased choline/creatine ratios in basal ganglia, but only 10 of these patients were receiving lithium \[[@B17]\]. The third study to report an increase in this ratio (in this case in the left subcortical region) was in a mixed group of patients receiving a wide range of medications \[[@B16]\]. Two other studies have reported increased choline concentrations, but only in limited circumstances. In one study in 11 bipolar children patients were examined before and after lithium administration \[[@B14]\]. There were no significant findings before or after lithium administration, although there was a trend towards increased choline/creatine ratios in the patients before lithium treatment. This latter finding does not suggest that in patients lithium significantly alters the choline/creatine ratio. The final study examined 15 euthymic males who were on either lithium or valproate \[[@B13]\]. This study found that thalamic choline concentrations, determined by measuring metabolite intensity/tissue volume ratios, were significantly increased only if the right and left hemisphere were compared separately, but not if they were compared together.
It is also conceivable that both lithium and valproate may increase Choline concentrations, but that the differences were not large enough for us to detect, or that without lithium or valproate treatment patients would have lower Choline concentrations. The cross-sectional nature of this study does not allow this to be examined. It is also important to recognize other limitations of the present study. Firstly, these MRS studies are not pre- and post-treatments, so may not accurately reflect changes that occur in individual patients. Secondly, part of the study used a ratio-method to assess choline concentrations, the limitations of which are increasingly clear (particularly since creatine concentrations may be altered by medication \[[@B37]\]). Thirdly, the sizes of all groups are small and it therefore possible that a larger study may have been fully powered to identify differences between groups. Fourthly, several patients in the first study (but not the second study) were on other drugs which may have affected the results of this study. Fifthly, we have not determined if age affects the results, and in the first part the groups were not all matched for age. In addition, the voxel locations were not the same in both studies due to the reasons discussed in the methodology section. Nonetheless, despite these limitations we believe the results add significantly to the literature in this under-researched area.
We conclude that, taking all current evidence together including the findings from the present study, it is unlikely that either lithium or valproate significantly alter brain choline concentrations. However, given the large differences in patients populations, medications received, and MRS methodologies it is difficult to directly compare all these studies. In addition, the methodology used to measure choline concentrations can significantly alter the results. Future MRS studies in bipolar patients should, therefore, examine metabolite concentrations rather than a ratio of choline compared to other metabolites.
Competing interests
===================
None declared.
Acknowledgements
================
This work was supported in part by peer-reviewed grants from the Canadian Institutes of Health Research (CIHR) and the Alberta Heritage Foundation for Medical Research (AHFMR).
| PubMed Central | 2024-06-05T03:55:46.840461 | 2004-7-30 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509421/",
"journal": "Ann Gen Hosp Psychiatry. 2004 Jul 30; 3:13",
"authors": [
{
"first": "Ren H",
"last": "Wu"
},
{
"first": "Tina",
"last": "O'Donnell"
},
{
"first": "Michele",
"last": "Ulrich"
},
{
"first": "Sheila J",
"last": "Asghar"
},
{
"first": "Christopher C",
"last": "Hanstock"
},
{
"first": "Peter H",
"last": "Silverstone"
}
]
} |
PMC509422 | Introduction
============
Chromate reductases are a group of enzymes that catalyze the reduction of toxic and carcinogenic Cr(VI) to the less soluble and less toxic Cr(III). These proteins have recently raised enormous interest because of their central role in mediating chromium toxicity and their potential use in bioremediation and biocatalysis. Chromate (Cr(VI)) is generated as by-product of various industrial processes such as leather tanning, chrome-plating, pigment production and thermonuclear weapon manufacture \[[@B1]\]. Its high water solubility facilitates a rapid leaching, provoking a wide dispersion capable to contaminate drinking water supplies. Therefore, the characterization of enzymes that reduce chromate, as well as the study of their induction patterns and gene expression are relevant to complete our understanding of chromium metabolism in order to minimize the toxicity of this compound in the environment.
The chromate-reducing activities have been located in the cell membrane or in the cytoplasm of many bacteria \[[@B2]\]. Their ubiquities in many different organisms suggest that they might share a common role in, for example, physiological redox sensing or detoxification. Recently, two novel dimeric flavoproteins with chromate reductase activity, ChrR (from *Pseudomonas putida*) and YieF (from *E. coli*) have been purified and characterized \[[@B1]\]. These enzymes were able to transform chromate to the less toxic Cr(III). However, while ChrR was not a pure two-electron reducer of chromate, YieF was able to catalyze a three-electron reduction. The role of ChrR and YieF in protection against chromate toxicity was also investigated and the results suggested that both enzymes may have an important role in protection against chromate toxicity \[[@B1]\].
The ability of some microorganism and their enzymes to remove toxic pollutants has been recently reviewed \[[@B3]-[@B6]\]. The identification and characterization of the degradative pathways functioning in microorganism have been the starting point for biotechnological and environmental applications \[[@B3]\].
Discussion
==========
The intensive industrial and agricultural development has been considered as responsible for a widespread contamination of soil, air and groundwater with toxic pollutants, which are harmful for human health and the environment \[[@B6]\]. These contaminants enter the environment through different paths, which may include direct application, combustion processes and natural emissions. Major contaminants are polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbons, phenols, polychlorinated biphenyls, azo dyes, organophosphorus pesticides and heavy metals \[[@B3]\]. In particular, Cr(VI) is a common pollutant due to the use of chromium compounds in tanning and other industries. Chromate shares structural similarities with sulphate ion (SO~4~^-2^) and may be introduced in eukaryotic and bacterial cells by the sulphate transport system \[[@B1]\]. In bacteria, flavoenzymes such as glutathione reductase reduce Cr(VI) by a one electron transfer leading to the formation of the highly unstable radical Cr(V) and the flavin semiquinone form of the enzyme. Both species undergo a further redox cycle in which Cr(VI) is re-generated by one-electron transfer to oxygen, producing and accumulating reactive oxygen species (ROS). The appearance of relatively large quantities of ROS, and the consequent oxidative stress are responsible for the toxic effects and cellular damage attributable to the presence of Cr(VI). On the other hand, trivalent chromium Cr(III) is water insoluble, less bio-available and less toxic \[[@B1]\]. Thus, the strategies employed to eliminate chromate toxicity would involve its reduction to Cr(III) by chemical or biological means.
While chemical methods are expensive at the large scale required to decontaminate waste sites, microorganisms are commonly used for environmental purposes through the exploitation of their natural catalytic activities. Enzymatic treatments have a minimal impact on ecosystems, as they present no risk of biological contamination. Furthermore, enzymes can act over a wide range of pH, temperature and ionic strength and also may be active in the presence of high concentrations of organic solvents in which major pollutant molecules are soluble \[[@B6]\].
Several bacterial enzymes that can be used in bioremediation have been described; they include mainly oxidative enzymes such as mono-and dioxygenases but their use is restricted by the need of cofactors, which can only be efficiently regenerated inside the microorganism \[[@B6]\]. In the last two decades bioremediation has explored the use of the catalytic machinery of white rot fungi to remove toxic pollutants. White rot fungi comprise all those fungi capable to degrade lignin, a polyphenolic polymer highly resistant to bacterial biodegradation. Many strains from the genera *Pleurotus, Bjerkandera, Phanerochaete*, and *Trametes*produce extracellular enzymes with ligninolytic activity. These enzymes are often referred to as lignin-modifying enzymes and include mainly Lignin peroxidase, Manganese dependent peroxidase and laccase \[[@B4]\], though some authors have reported other related enzymes such as a Mn-independent MnP activity \[[@B7]\]. Besides lignin-modifying enzymes, several other enzymes such as the heme-containing peroxidases, chloroperoxidase and horseradish peroxidase, and the non-enzymatic hemeproteins, hemoglobin and cytochrome *c*, are able to oxidize organic compounds in the presence of hydrogen peroxide. An interesting feature of these enzymes is their remarkable low specificity towards substrates that arises from their own catalytic mechanism. *In vivo*, peroxidases use endogenous low-molecular weight compounds, called mediators, to generate free radicals capable to carry out a wide variety of reactions such as oxidations, bond cleavage, hydroxylations, polymerization and demethylation \[[@B4]\]. Several research efforts have been focused on the ability of peroxidases to degrade pollutants such as PAH\'s, azo dyes and organophosphorus pesticides \[[@B8]-[@B10]\].
Strong regulations have been established to push the industrial sector to develop new programs destined to a greater environmental care. Nowadays, industry is strongly dependent on petroleum and its derivatives as a source for raw materials and energy. There are still large reserves of crude oil, which are heavy oils with a high content of sulphur and heavy metals. The use of these fuels generate a great pollution, being one of their most important environmental impacts the formation of the acid rain which takes place by the sulphur oxide production during combustion. Redox enzymes may encounter fields of application not only in the bioremediation of polluted environments, but also in the development of novel clean technologies to avoid or diminish the environmental contamination. Biocatalytic methods for sulphur removal from straight-run diesel fuel have been developed \[[@B11]\]. The removal of heavy metals from the petrophorphyrin-rich fraction of asphaltenes has also been reported \[[@B12],[@B13]\]. Thus, enzymes can play an important role in the development of alternative or complementary biotechnological processes with potential application in polluting industries.
Despite their potential application in bioremediation and clean processes, the activity of oxidative enzymes may be limited, among other factors, by the low bioavailability of the pollutants and by the relatively low operational stability of the enzyme under the environmental conditions required to carry out the bioremediation. Several strategies to increase the catalytic activity of peroxidases have been proposed, including chemical modification of the enzyme \[[@B14],[@B15]\] and genetic tools \[[@B16]\]. Recently, with the cloning and expression in suitable hosts, larger amounts of the desired enzymes may be produced, facilitating their characterization and their direct use in environmental applications. Further, through the use of novel techniques such as directed molecular evolution \[[@B17]\], proteins designed specifically for bioremediation could be made available in a not distant future. In the last few years there has been an extensive research in the application of laboratory evolution for tailoring redox enzymatic systems (laccases, peroxidases, cytochrome P450 monooxygenases) to improve their activities and stabilities against temperature or organic solvents \[[@B18]-[@B20]\]. The application of this powerful approach for bioremediation issues is coming up, but first a big effort in the high-throughput (HTP) screening methodology must be done. So far, little has been reported on the optimisation of suitable HTP for the detection of xenobiotics \[[@B21]\]. Therefore, the success in the enzyme evolution for environmental issues will be highly dependent on the automation of HTP.
Microbial genomics is a new emerging field that enables us to look at parts of the environment that were, until recently, masked to us. Present estimations suggest that more than 99% of the microorganisms in most environments (also those subjected to chronic contamination) are not amenable to grow in pure culture, and thus very little is known about their enzymatic activities. We can now access the genomes of non-culturable microorganisms through creating the \"so-called\" metagenomic-libraries and identify protein-coding genes and biochemical pathways that will shed some light on their properties and function \[[@B22]\]. New enzymatic systems found in contaminated areas can be used as parental types for some rounds of directed evolution with the main aim of improving the catalytic performance for their use towards solving a broad range of environmental problems.
Conclusion
==========
The even more strict regulations on hazard wastes has forced to the development of new environmentally compatible strategies to substitute or complement the conventional ones. Chemical technologies are expensive when applied to large scale and in many cases are technically not feasible. On the other side, by exploitation of the huge diversity of natural activities and metabolic pathways presented by microorganisms, new strategies can be envisaged. The use of oxidative enzymes as biocatalysts for environmental purposes presents a promising potential due to their low specificity and low energetic requirements. However, further characterization of new biocatalysts is needed. The use of novel technologies such as molecular directed evolution may have a large impact in the tailoring and further application of enzymes not only for bioremediation but also for the development of friendly environmental technologies.
| PubMed Central | 2024-06-05T03:55:46.842688 | 2004-8-2 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509422/",
"journal": "Microb Cell Fact. 2004 Aug 2; 3:10",
"authors": [
{
"first": "Humberto",
"last": "Garcia-Arellano"
},
{
"first": "Miguel",
"last": "Alcalde"
},
{
"first": "Antonio",
"last": "Ballesteros"
}
]
} |
PMC509423 | Background
==========
Drugs that may slow or halt the breakdown of cartilage and other joint tissues in osteoarthritis (OA) and possibly improve symptoms and function are now being developed in the pharmaceutical industry. The potential availability of disease modifying OA drugs has focused attention on our relative lack of information on the \'natural disease history\' of OA with regard to changes in symptoms, functional limitations, joint structure and other markers of disease change \[[@B1]\].
Epidemiological studies have identified factors associated with increased risk for incident or progressive OA, such as age, sex, heredity, obesity, and joint injury, pain, alignment, or laxity. There is, however, a paucity of long-term data that document the rate and nature of natural OA disease progression on either group or individual levels. Such information is needed for identification of study cohorts and planning of clinical trials of disease modifying OA drugs. Even more importantly, knowledge of natural disease progression in different patient groups will be needed to select those future groups that may benefit from such drugs.
Only a few of the previously published studies have presented information on longitudinal variation in pain and function in the natural history of knee OA. The \"Bristol 500 OA study\" noted, that although pain changed little on a group level over a 3-year follow-up period, it varied greatly in individuals, with some subjects reporting marked improvements. Similarly, a minority improved functionally \[[@B2]-[@B4]\].
Yet another report suggested that most patients with OA attending rheumatology clinics do not deteriorate radiographically or symptomatically over an 11-year period \[[@B5]\]. A more recent report stated that 42--44% of community-recruited knee OA individuals did not change in physical functioning over a 3-year study \[[@B6]\]. Most investigations of the natural history of OA have been concerned with radiographic rather than clinical changes. For example, it was reported that the radiographic Kellgren and Lawrence classification score of 1 could represent incipient OA and be predictive of later development of more advanced radiographic features of OA \[[@B7]\]. MRI may be more responsive to change in early-stage OA than plain radiography \[[@B8]\].
However, outcome is usually heterogeneous: study subjects may report improvement or deterioration while they do not change radiographically over the time period assessed. It may also be that a few individuals alone generate much of any change detected at group level \[[@B9]-[@B11]\]. A further confounding factor in the longitudinal assessment of OA is the potential influence of the population from which the study group was recruited; a study group recruited from e.g. a specialist outpatient clinic is likely to have, on the average, more severe disease and may be at different risk to progress over time than a study group recruited from the community.
The objective of this investigation was to assess both group and individual variation in knee pain, function and quality of life over two years in a study group enriched in incipient and early-stage radiographic knee OA.
Methods
=======
Patients
--------
Approval was obtained from the Research Ethics Committee of the Medical Faculty of Lund University, Sweden. All patients who underwent meniscectomy between 1983 and 1985 were identified by searching the surgical records at the Department of Orthopedics, Lund University Hospital. In this period 552 meniscectomies were performed. Inclusion and exclusion criteria (Figure [1](#F1){ref-type="fig"}) were used to identify 264 former patients who, in 1998, were sent a self-administered questionnaire evaluating their knee-specific symptoms and knee function.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Flow chart presenting the inclusion and exclusion criteria for patients.**ACL = anterior cruciate ligament, PCL = posterior cruciate ligament, OA = osteoarthritis.
:::
:::
Out of 211 individuals (80%) who returned the questionnaires, 6 were excluded because they matched one of the exclusion criteria. At 2 years after the first assessment 5 subjects had died, but the remaining 200 individuals were asked to provide a second evaluation using an identical questionnaire. Replies were received from 143 (72%). Of these 143 participants, 102 were meniscectomized by open surgery, and 41 by arthroscopy. Nineteen underwent an additional meniscus operation in the index knee.
All re-operations were performed within 3 years after the original meniscectomy. Twenty-three participants were treated with subsequent meniscectomy of the contralateral knee. One of them underwent high tibial osteotomy and 1, because of OA, received a knee prosthesis in the contralateral knee. Data concerning subsequent surgeries were based on the medical records of Lund University Hospital and on self-reported information.
Radiographic assessment
-----------------------
At the time of the participants\' second evaluation with questionnaires, standing anteroposterior (AP) radiographs of both knees were taken in 15 degrees of flexion using a CGR Phasix 60 generator at 70 kV, 16 mA, film-focus distance 1.5 m (CGR, Liège, Belgium). Ten out of the 143 participants (7%) declined the radiographic examination. All AP radiographs of the tibiofemoral joints from the follow-up were assessed for joint space narrowing (JSN) and osteophytes according to the atlas from Osteoarthritis Research Society International (OARSI) \[[@B12]\].
The presence of these features was graded on a 4-point scale (range 0--3, with 0 = no evidence of bony changes or JSN). We considered radiographic knee OA to be present if any of the following criteria was achieved in any of the 2 tibiofemoral compartments: JSN ≥ grade 2 or the sum of the 2 marginal osteophyte grades from the same compartment ≥ 2, or JSN grade 1 in combination with an osteophyte grade 1 in the same compartment \[[@B13],[@B14]\]. This cut-off approximates grade 2 knee OA or worse based on the Kellgren and Lawrence scale \[[@B15]\].
Disease-specific questionnaire
------------------------------
The Knee injury and Osteoarthritis Outcome Score (KOOS, Swedish version LK 1.0) is a 42-item self-administered knee-specific questionnaire based on the WOMAC Osteoarthritis Index \[[@B16],[@B17]\]. KOOS was developed to be used for short- and long-term follow-up studies of knee injuries, and it comprises 5 subscales: Pain, Symptoms, Activities of Daily Living (ADL), Sports and Recreation Function (Sport/Rec) and knee-related Quality of Life (QOL). A separate score ranging from 0 to100, where 100 represents the best result, is calculated for each subscale.
The questionnaire and scoring manual can be downloaded from <http://www.koos.nu>. The KOOS is valid, reliable and responsive in follow-up of meniscectomy \[[@B17]\], anterior cruciate ligament reconstruction \[[@B18]\] and total knee replacement for OA \[[@B19]\]. The participants completed the KOOS questionnaire answering questions on their operated index knee.
Change
------
The minimal perceptible clinical improvement (MPCI) represents the difference on the measurement scale associated with the smallest change in the health status detectable by the individual. Since the KOOS questionnaire contains the full and original version of the WOMAC LK 3.0 index, we used the MPCI as described for WOMAC \[[@B20]\]. Thus, a level of 10 points or more of improvement or decline was operationally used as a cut-off representing a clinically perceptible difference. The sensitivity of the questionnaire has been established \[[@B21]\].
Data collection and statistics
------------------------------
If questions were left unanswered in any part of the questionnaire, we returned the questionnaire to be completed. The questionnaires were then completed fully. The Mann-Whitney U-test was used to determine differences between the groups. *P*-values for categoric data were calculated with Fisher\'s exact test. All tests were 2-tailed and a *P*-value of ≤ 0.05 was considered statistically significant (SigmaStat, version 2.0, for Windows).
Results
=======
Group level
-----------
The study group comprised 143 individuals, of whom 23 (16%) were women. The participants\' mean age at the first follow-up was 51 (range 27--83) years. The assessment was carried out twice: at approximately 14 and 16 years after the surgery, with a median interval of 2.3 (range 2.3 to 3.0) years. Fifty-three (40%) of the 133 individuals who had undergone radiographic examination had radiographic tibiofemoral OA in their index (operated) knee (21% women, age range 29--83, mean 53) and 80 were classified as non having OA (11% women, age range 27--82, mean 50). An additional 25 (19%) (not classified as radiographic OA) had a single osteophyte grade 1 in either one or both tibiofemoral compartments.
Mean scores for the KOOS subscales at the first assessment did not change significantly over the 2-year study period (Table [1](#T1){ref-type="table"}). Moreover, there were no significant changes in group mean subscale scores over 2 years when participants were divided into those with or without radiographic OA in the index knee (Table [1](#T1){ref-type="table"}, Figure [2](#F2){ref-type="fig"}). However, individuals with radiographic OA scored worse at both examinations than did those without radiographic OA. The differences between those with and without OA were statistically significant for KOOS Pain Δ = 11 points (*P*= 0.004), other Symptoms Δ = 9 points (*P*= 0.013), ADL Δ = 10 points (*P*= 0.003), Sport/Rec Δ = 17 points (*P*= 0.005), and QOL Δ = 16 points (*P*= 0.003) assessed in 2000, and in the dimensions Sport/Rec Δ = 14 points (*P*= 0.020) and QOL Δ = 12 points (*P*= 0.041) evaluated in 1998.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
KOOS scores overall and in patients without and with radiological signs of OA
:::
KOOS subscales Patients p-values
---------------- ---------- ---------- --------- --------- --------- --------- --------- -------
pain mean 85 84 88 87 79 76 0.008
median 94 94 94 94 86 83
SD 20 21 16 18 24 25
range 19--100 25--100 39--100 25--100 19--100 25--100
symptoms mean 85 84 87 87 80 78 0.013
median 93 89 93 93 89 82
SD 19 18 17 16 23 21
range 14--100 14--100 25--100 18--100 14--100 14--100
ADL mean 88 88 90 91 83 81 0.004
median 99 97 99 99 94 90
SD 18 18 15 15 23 21
range 18--100 31--100 44--100 34--100 18--100 31--100
sports/rec mean 69 68 74 76 60 57 0.007
median 80 80 80 85 60 60
SD 31 32 28 28 34 34
range 0--100 0--100 0--100 0--100 0--100 0--100
QOL mean 75 73 78 78 67 63 0.005
median 81 81 81 84 69 63
SD 26 27 23 23 30 30
range 0--100 6--100 25--100 6--100 0--100 13--100
Mean, median, standard deviation and range of KOOS scores overall and in patients without and with radiological signs of OA. Note that 10 patients out of 143 did not undergo radiographic examination. *P*-values for comparison between KOOS subscale results in patients with and without OA in year 2000 are presented.
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
**Group mean KOOS scores for patients assessed in 1998 and 2000.**Group mean KOOS scores for patients with (n = 53) and without (n = 80) radiographic osteoarthritis (ROA) assessed in 1998 and 2000. Possible score range 0 to 100, with 100 representing the best result. ADL -- Activities of Daily Living, QOL -- knee-related Quality of Life. Bars present ± 95% confidence intervals. The bars going upwards have wider caps. Note vertical axis break.
:::
:::
We analyzed separately those subjects (N = 57) that did not participate in the second assessment. Their mean KOOS scores at the first examination did not differ significantly from the remainder of the study cohort, indicating little or no inclusion bias for the second follow-up (data not shown). The scores in the 5 patients that underwent additional surgery (e.g. osteotomy, knee arthroplasty) did not differ significantly from the rest of the group.
Individual study subject changes
--------------------------------
In spite of the lack of change on a group level, we found substantial intra-individual variability in the questionnaire subscale scores measured 2 years apart. Out of the total 143 study subjects, 40 had either improved or deteriorated (n = 23 (16%) and n = 17 (12%), respectively) 10 points or more for the KOOS subscale Pain. Of the 23 subjects who had improved in their pain score by these criteria, 14 had also improved in the subscale Symptoms, 17 in ADL, 16 in Sports/Rec, and 17 in QOL.
Only 1 of these subjects deteriorated in Symptoms, and 2 in Sports/Rec, none in the other subscales. Of the 17 subjects who deteriorated in Pain, 13 similarly deteriorated in Symptoms, 12 in ADL, 10 in Sports/Rec, and 10 in QOL. When evaluating those who had undergone radiographic examination, there were no significant differences in variability detected whether the subject had radiographic tibiofemoral OA or not (*P*= 0.24, Table [2](#T2){ref-type="table"}).
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
The percentage of patients improving, not changing, or deteriorating for KOOS subscales over time
:::
non-ROA ROA
------------ ---- ---- --------- ---- ---- ----- ----
pain 10 13 76 11 21 66 13
20 6 88 6 8 87 6
symptoms 10 16 69 15 26 55 19
20 6 86 8 13 77 9
ADL 10 9 79 13 19 64 17
20 5 86 9 15 79 6
sports/rec 10 19 60 21 28 42 30
20 11 76 13 21 64 15
QOL 10 20 56 24 26 57 17
20 5 88 8 15 75 9
The percentage of patients, with and without radiographic osteoarthritis (ROA), improving, not changing, or deteriorating for KOOS subscales over the 2 year study period. For definition of ROA see methods. Two cut-offs for change (≥ 10 and ≥ 20 points) are presented.
:::
We also evaluated a stricter cut-off of 20 points or more as used for the OARSI responder criteria, as opposed to minimal clinically perceptible change \[[@B22]\]. With this cut-off, in total 19 patients fulfilled the criterion for improvement or deterioration (n = 9 (6%), n = 10 (7%), respectively) in KOOS Pain. Among the subjects with radiographic OA, 3 (6%) improved and 4 (7%) deteriorated by 20 points or more. Corresponding numbers for those without radiographic OA were 5 (6%) for both improvement and deterioration.
In order to explore these changes in more detail, the subjects were divided into quartiles, according to KOOS Pain score at the first assessment (Figure [3](#F3){ref-type="fig"}). The most noticeable changes were found in the quartile representing the worst scores: 21 of 36 (58%) subjects showed a change of 10 points or more in either direction. A corresponding change was seen in 11 (31%) individuals from the second worst quartile and in only 9 (25%) from the second best and best quartiles (6 and 3 subjects, respectively). Comparable results were seen for the other subscales of KOOS (data not shown).
::: {#F3 .fig}
Figure 3
::: {.caption}
######
**KOOS Pain subscale.**Patients are divided into 4 subgroups (quartiles) according to the score at entry. Each line represents one patient visualizing the score in 1998 (left endpoint of line) and in 2000 (right endpoint of the same line).
:::
:::
Discussion
==========
We found no significant change over 2 years in the average patient-relevant outcome scores for this study group of individuals who had undergone meniscectomy about 15 years earlier, even though the group was highly enriched in early-stage and incipient radiographic knee OA. However, we found substantial change in the self-report for individual subjects over the same time period.
The generally worse KOOS scores for the individuals with radiographic knee OA, compared to those without, are consistent with earlier reports. Thus, the Baltimore Longitudinal Study of Aging reported that patients with a Kellgren-Lawrence score of 1 were almost twice as likely to report ever having knee joint pain compared with those who had a score of zero. The strength of the association increased with increasing Kellgren and Lawrence score \[[@B23]\].
Similarly, there was in meniscectomized individuals evidence for a graded increase in pain and functional limitations with increasing severity of radiographic signs of OA \[[@B24]\]. However, a discrepancy between knee pain and radiographic features of knee OA has also been noted, both cross-sectionally and longitudinally \[[@B3],[@B24],[@B25]\]. Depression and lack of muscle strength have been shown to better explain pain than radiographic findings \[[@B26]-[@B28]\].
Individual vs. group analysis
-----------------------------
Few reports have explored OA symptom variation on an individual level \[[@B2]-[@B4]\]. A detailed comparison of our results with earlier reports is difficult, since they were conducted before validated and patient-relevant OA disease-specific measurement tools had been widely introduced. The \"Bristol OA 500\" were patients with advanced radiographic knee OA and a mean age of 65 years recruited from a hospital based rheumatology clinic.
In contrast, the mean age of the present study cohort was 50 years, with 2/5 having mild-moderate radiographic OA, and another 1/5 incipient radiographic changes. Further, the cohort reported on here was recruited from a group of individuals that had undergone isolated meniscectomy 15 years earlier, but independent of their subsequent symptom level or disease history. The mean scores of our study group were relatively good and not representative of subjects with advanced OA seeking medical care.
The rationale for investigating this particular cohort at this time after surgery was its enrichment in early-stage knee OA, and that it consequently may represent a study group suitable for future pharmacological disease-modifying intervention. We assessed our patients at an interval of 2 years; this period of time being suggested as a minimum for clinical trials of disease modification in OA to detect both structural and symptom change \[[@B29]\].
It could be that the findings reported here are valid only for post-injury, secondary OA, or for this particular cohort. However, the criteria and delimitations for posttraumatic OA compared to primary OA have recently been shown to be much less clear than thought \[[@B13],[@B14],[@B30]\], and meniscal pathology is common also in primary, garden-variety, knee OA \[[@B31]\]. Tibiofemoral OA was observed in 53 out of 133 patients who were underwent radiographic examination. Isolated patellofemoral OA was rare and, since it did not affect the final results, was not taken into account. A further argument favoring the general applicability of the present results is the concordance of our findings with other longitudinal studies on OA \[[@B2]-[@B5],[@B32]\].
Methodological issues
---------------------
We applied the criteria for minimal perceptible clinical improvement (MPCI) obtained for the WOMAC; since KOOS contains the WOMAC items and is similar in format. The KOOS subscale ADL is equivalent to the WOMAC subscale Function, while new items have been added to the KOOS subscales Pain and Symptoms. The dimensions assessed by the KOOS subscales Sport and Recreation Function and knee-related Quality of Life are not assessed by the WOMAC. The MPCI for the WOMAC is in the range of 8 to 12 points on a 0--100 scale \[[@B20]\].
This threshold coincides with the change observed in KOOS scores between 3 and 6 months postoperatively when assessing rehabilitation following reconstruction of the anterior cruciate ligament and concurs with the OARSI definition of moderate improvement in the knee pain assessment for clinical trials in OA \[[@B18],[@B22]\]. However, the OARSI responder criteria were designed for the evaluation of the patient\'s response to oral NSAID and intra-articular treatment and may differ for other interventions.
It may be argued that the subject-related changes observed in this study represent inherent instrument instability. However, validation studies of KOOS support the reproducibility and stability of the KOOS instrument \[[@B17]-[@B19]\]. Test-retest data on the KOOS subscale pain obtained from 75 patients about to undergo knee arthroscopy \[[@B17]\] was used to determine the number of subjects improving, deteriorating or not changing over an average period of 5 days.
The proportion of subjects changing over 5 days was approximately half of that changing over 2 years in the present study, in further support that the variation in the present study cannot be explained solely by instrument noise (data not shown). A \'frame shift\' in the priorities of the individual patient may occur during long term studies. However, we suggest that a significant frame shift is unlikely to have occurred over this 2 year study period of a cohort with a mean age of 50 years.
Significant change of KOOS scores over time were noted in 1/3 of the cohort studied. About half of those who changed clinically improved. This was true in particular for patients with lower (worse) baseline scores. It is thus possible that the lower proportion of \'changers\' among those with better baseline scores may have been, at least in part, due to a ceiling effect.
Conclusions
===========
We conclude that despite unchanged group mean scores over 2 years, pain, function and quality of life change considerably over time in individuals, in this study cohort enriched in incipient and early-stage knee OA. These findings may be applicable also to other at risk patient groups in different phases of OA development, and motivate further careful examination of the natural history of OA, as well as properties of the OA outcome instrument used. We suggest that longitudinal OA study data should be analyzed both on the individual and group level.
Our findings may have further relevance to clinical trials of OA that seek to document long term benefit in the form of symptom improvement and structural improvement. It is clear that much additional effort will need to be spent on selection of groups at high risk of progression of symptoms and structural joint change, and the identification of predictors for deterioration. Our results also suggest that the use of responder criteria may be an important aspect of analyzing the outcome of such trials \[[@B22],[@B33]\].
Authors\' contributions
=======================
EMR and LSL planned study and collected the data. PTP performed the statistical analysis and drafted the manuscript. ME formed the database of patients and participated in the statistical analysis. ME, EMR, LSL corrected the manuscript. All authors read and approved the manuscript
Acknowledgements
================
This work was supported by grants from the Swedish Institute, the Swedish Rheumatism Association, The Swedish National Center for Research in Sports, The Swedish Research Council, The King Gustaf V 80-year Birthday Fund, Zoega Foundation for Medical Research, Kock Foundations and Lund University Medical Faculty and Region Skane. The authors are indebted to Robert Foltyn for excellent assistance in the preparation of this manuscript.
| PubMed Central | 2024-06-05T03:55:46.844153 | 2004-7-27 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509423/",
"journal": "Health Qual Life Outcomes. 2004 Jul 27; 2:38",
"authors": [
{
"first": "Przemyslaw T",
"last": "Paradowski"
},
{
"first": "Martin",
"last": "Englund"
},
{
"first": "Ewa M",
"last": "Roos"
},
{
"first": "L",
"last": "Stefan Lohmander"
}
]
} |
PMC509424 | Review
======
In multicellular organisms, every cell comprising every tissue, organ, or organ system constantly strives to maintain homeostasis in the face of destabilizing influences. Whether it is external stimuli such as toxic chemical exposure or changes in oxygen tension, or natural alterations in pH or osmolarity due to normal cellular metabolism, each cell in the body is equipped with the molecular machinery required to sense these environmental changes and respond to them. Because these stressful stimuli can impinge on normal cellular functioning, discrete cellular sensing mechanisms have evolved to maintain homeostasis. For example, ATP depletion caused by hypoxia activates AMPK which phosphorylates multiple downstream targets to switch the cell from a mainly anabolic to catabolic state \[[@B1]\]. Similarly, activation of SAPK/JNK by stressors such as ultraviolet (UV) light results in activation of well-defined molecular targets \[reviewed in \[[@B2]\]\].
One interesting question is how do cells translate diverse stressful stimuli into activation of specific molecular pathways? Cellular signaling from membrane to nucleus is typically accomplished through ligand/receptor activation of intracellular second messengers. In many cases these second messengers are kinases which phosphorylate substrates leading to a cascade by which successive macromolecules are triggered. Ultimately, a terminal transcription factor is activated which then translocates to the nucleus to activate specific target genes. This type of cellular signaling has been well-characterized following receptor activation by polypeptide ligands, but some forms of cellular stress such as hypoxia cause activation of specific molecular pathways in the apparent absence of ligand to receptor stimulation. Therefore, there must be alternative routes for direct activation of target genes that circumvents the canonical ligand/receptor/second messenger cascade. One such pathway that may transmit signals to the nucleus by this alternative route is the Janus Activated Kinase/Signal Transducer and Activator of Transcription family of transcription factors (JAK/STAT).
The JAK/STAT pathway includes seven functionally related, latent transcription factors (STAT) and four non-receptor tyrosine kinases (JAK) \[reviewed in \[[@B3]\]\]. In the typical JAK/STAT paradigm, a cytokine binding to its receptor results in activation of receptor-associated JAKs. JAKs then phosphorylate the cytoplasmic receptor chains creating docking sites for recruited STATs. Finally, STATs are phosphorylated on tyrosine by JAKs, dimerize, and then translocate to the nucleus to activate specific target genes. Because the second messenger (STAT) is also the terminal transcription factor, in many ways the JAK/STAT pathway represents a streamlined apparatus for cellular signaling. Thus, the JAK/STAT pathway differs from many signaling cascades in that the usual system of multiple sequential signaling molecules is bypassed.
While signaling through the canonical JAK/STAT pathway has been well-characterized in immune-type cells in response to interleukins and interferons, there is emerging evidence that STATs also mediate cellular responses to various forms of cellular stress. These findings, in addition to mounting evidence suggesting that some STATs are phosphorylated on serine by members of the MAPK family, imply that alternative mechanisms for STAT activation exist. Further, these alternative means of activation may lead to different outcomes with regards to STAT signaling. For example, STAT3 was recently shown to be serine phosphorylated by JNK during UV stress which had a predominately inhibitory role on STAT3 transcriptional activity \[[@B4]\]. Could these alternative routes of STAT activation account for STAT\'s \"yin and yang\" type properties whereby depending on the type of cell stressor, either cell death or cell survival pathways are activated? Although interferons themselves can regulate cellular responses to exogenous stressors such as infection through the canonical JAK/STAT pathway, this literature review will focus mainly on those alternative mechanisms of JAK/STAT signaling during cellular stress.
STATs and cellular stress
=========================
Some of the earliest studies implicating STATs in mediating cell stress responses were performed in cells exposed to UV light. In mouse embryonic fibroblasts (MEFs), UV light treatment resulted in phosphorylation of serine 727 in STAT1 via p38 MAPK \[[@B5],[@B6]\]. Further analysis revealed that STAT1 could be phosphorylated directly by p38 MAPK *in vitro*. Thus, the MAPK and STAT pathways appear to converge during periods of cellular stress. In another study, UV light caused STAT1 tyrosine phosphorylation, nuclear accumulation, and DNA binding in keratinocytes \[[@B7]\]. Together, these studies raise the possibility that STATs can be activated in a ligand-independent manner during cellular stress, resulting in the activation of STAT-dependent target genes.
Cellular stress can also occur in disease states such as diabetes, which is characterized by vascular dysfunction. For example, prolonged elevated glucose can act as a cell stressor through multiple pathways including hyperosmolarity \[[@B8]\], protein kinase C (PKC) activation \[[@B9]\], and oxidative damage \[[@B10]\]. Recent studies have determined that constitutive JAK/STAT phosphorylation was elevated in cultured smooth muscle-like mesangial cells treated with high glucose \[[@B11]\]. Furthermore, in these same cells, angiotensin stimulation of STATs was prolonged by high glucose treatment \[[@B12],[@B13]\]. The authors of these studies related these findings to TGFβ-induced extracellular matrix accumulation because collagen and fibronectin secretion could be inhibited with STAT anti-sense RNAs. This implies that the JAK/STAT pathway may play a role in basement membrane thickening observed in diabetic nephropathy and retinopathy. These studies were some of the first to suggest that perturbed JAK/STAT signaling could play a role in disease states like diabetes and possibly link glucose-mediated cell stress and the JAK/STAT pathway with diabetic sequelae.
Hyperosmotic stress can also activate STATs. For example, in the slime mold *Dictyostelium*, hyperosmotic stress leads to STAT1 phosphorylation without any known involvement of JAK or MAPK \[[@B14]\]. But mammalian cells also utilize STATs during hyperosmotic stress. In one report, sorbitol-induced hyperosmolarity was shown to cause JAK1, JAK2, and TYK2 phosphorylation and subsequent activation of STAT1 and STAT3 in various cell types; this led to the formation of STAT1/STAT3 complexes with the m67SIE oligonucleotide from the c-fos promoter \[[@B15]\]. Interestingly, these authors speculate that the hyperosmotic signal occurred independently of gp130. This suggests an alternative pathway by which JAKs may be activated beyond the canonical JAK/STAT route. In agreement with this study, hyperosmotic shock in COS-7 cells was shown to lead to tyrosine phosphorylation of STAT1 in a MKK6/p38-dependent pathway \[[@B16]\]. In this case, STAT1 but not JAK1 phosphorylation could be inhibited by genistein (a non-specific tyrosine kinase inhibitor) leading the authors to conclude that a tyrosine kinase distinct from JAK1 (possibly novel) represented the link between hypertonicity and STAT activation.
The most well-investigated reports linking cellular stress with the JAK/STAT pathway are studies in cardiomyocytes undergoing hypoxia/reperfusion. Like UV light, hypoxia/reperfusion led to p38 MAPK phosphorylation followed by serine 727 phosphorylation of STAT1 which was associated with activation of pro-apoptotic FAS/FASL and caspase-1 \[[@B17]\]. It was concluded that Fas and caspase-1 expression were directly STAT-1 dependent because their expression could be inhibited by STAT1 anti-sense RNAs. Thus, STAT1-dependent FAS activation plays a leading role in cardiomyocyte death during hypoxia/reperfusion injury \[[@B18]\] and as the authors point out, inhibition of this pathway may prove to be cardioprotective following ischemic insult \[[@B19]\]. Interestingly, while these studies showed that both tyrosine 701 and serine 727 of STAT1 were phosphorylated in response to hypoxia/reperfusion, only phosphorylation on serine was required for FAS expression. Because serine phosphorylation alone is not sufficient for direct DNA binding, these results indicate alternative pathways by which STAT1 may activate target genes during stress. For example, serine phosphorylated STAT1 may associate with other scaffolding proteins as it does in the case of MCM5 \[[@B20]\], BRCA1 \[[@B21]\], or HSF \[[@B22]\] and act instead as a transcriptional co-activator, rather than a direct activator of target genes \[[@B23]\].
*In vivo*models of hypoxia/reperfusion also implicate STAT5 as a player in responses to cellular stress. But unlike STAT1, STAT5 is thought to be mainly protective by activating anti-apoptotic signals. For example, Yamaura *et al*. report that genetic deletion of STAT6 but not the STAT5A causes resistance to myocardial ischemia/reperfusion injury \[[@B24]\]. This resistance was thought to be related to two distinct STAT5A-mediated pathways: one involving a Src/STAT5/PI-3 kinase/Akt pathway, and the other a direct JAK2/STAT5A pathway. Like STAT5, STAT3 was also shown to be protective against cardiac ischemia/reperfusion injury through a JAK2/STAT3-dependent mechanism involving up-regulation of anti-apoptotic Bcl2 and down-regulation of pro-apoptotic Bax \[[@B25],[@B26]\]. Studies in our laboratory indicate that in microvascular endothelial cells, hypoxia caused an increased tyrosine phosphorylation of JAK2, down-regulation of FAS/FASL, and that AG490 (a JAK2 inhibitor) de-repressed FAS transcription (unpublished observations). This may suggest a possible mechanism whereby activated JAK2 could mediate protection during ischemia in endothelial cells by repressing pro-apoptotic FAS transcription through downstream STAT3 or STAT5. Intriguingly, a complex of STAT3 and c-Jun was recently shown to be a FAS repressor \[[@B27],[@B28]\] but it remains to be determined if STAT5 or other STATs can behave like STAT3 and act as transcriptional repressors of pro-apoptotic genes during cellular stress. STAT5 nuclear translocation and DNA binding to the GAS (γ-activated site) implicates STAT5 in hypoxic stress responses, but the biological significance of this observation is not yet clear \[[@B29]\].
Although not as well studied as hypoxia/reperfusion or osmotic stress, reactive oxygen species have also been shown to activate the JAK/STAT pathway. Oxidative stress, such as might occur in diabetes and cardiovascular disease, was shown to activate HSP70 in smooth muscle cells in a JAK-dependent manner \[[@B30]\]. This response is thought to aid in adapting these cells to oxidative damage. Recently it was shown that STAT1 forms a complex with HSF-1 to activate the HSP promoter while STAT3 filled just the opposite role \[[@B22],[@B31]\]. Thus, it appears that STAT1 and STAT3 can perform entirely different functions with regard to cellular stress-type responses. Other studies have determined that peroxide treatment resulted in STAT3 tyrosine phosphorylation and nuclear translocation \[[@B32]\] and JAK2, STAT1, and STAT3 were activated by oxidized LDL \[[@B33]\].
Taken together, the studies reviewed herein support a role for the JAK/STAT pathway in various forms of cellular stress and relate perturbed JAK/STAT signaling to potential disease states. However, consistent with some of the current ideas about STAT biology, it is clear that cellular stress seems to activate STATs in ways that can be both detrimental to and supportive of cell survival. For example, STAT1 activation by hypoxia-reperfusion injury activates cell death pathways, while STAT5 activation by the same type of stressor seems to promote cell survival pathways. Thus, STATs may have evolved to fulfil both sides of a \"yin and yang\" type mechanism where either death or survival pathways can be activated depending on the strength or type of cellular stressor \[[@B34]\]. This may be especially true in endothelial cells, which seem to be able to resist short-term hypoxic-stress compared to other cell types but die by apoptosis following prolonged exposure to hypoxia \[[@B35]\].
The most intriguing aspect of many cellular stress-activated pathways is the apparent absence of ligand-to-receptor stimulation. But the cell must somehow \"sense\" changes in the external milieu and transmit these signals to the nucleus. How does it do this? Two such examples of this type of cellular sensing are activation of a well-described transcription factor known as HIF-1α (hypoxia-inducible factor), and another is the cellular thermostat called HSF (heat shock factor). In the case of HIF, enzymatic modification by an enzyme requiring oxygen as a cofactor is responsible for HIF activation and switching on of target genes when cells are stressed by low oxygen \[[@B36]\]. For HSF multiple stressors such as ATP depletion, ischemia, and intracellular acidosis lead to HSF phosphorylation, unfolding, and its translocation to the nucleus to activate target genes \[[@B37]\]. So these powerful signal transducing pathways are somehow activated directly presumably without ligand stimulation. This is probably the most speedy and efficient way of activating downstream target genes to promote cell survival.
Conclusions
===========
Is it possible that STATs can also act as a cellular rheostat of various stressors? Recent suggestions that STATs can be post-translationally modified in ways other than phosphorylation (e.g. acetylation, methylation, and ubiquitination) make this a possibility \[[@B38]\]. For example, changes in the intracellular redox environment by low oxygen tension may modify STAT conformation leading to enhanced availability of its active centers \[[@B15]\]. This change may facilitate interactions with other STAT-modifying proteins such as p38 MAPK. Alternatively, other previously unknown STAT pathways may be activated during cellular stress, altering its transcriptional capacity. These might include STAT association with other second messengers such as PI-3 kinase and Akt but also STAT upstream activation by molecules like Src \[[@B24]\]. Figure [1](#F1){ref-type="fig"} summarizes the potential role of STATs in cellular stress.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
**Role of STATs in cell stress responses.**(A) Autocrine IFN may activate JAK/STAT through the canonical pathway. This activation would involve tyrosine phosphorylation of STAT by JAK resulting in STAT dimers which are 20% transcriptionally active. This process is thought to \"prime\" STATs for serine phosphorylation by an IFN-inducible serine kinase (possibly PKC) \[42\]. Both tyrosine and serine phosphorylation results in a 100% transcriptionally active STAT1 dimer. (B) Hypoxia-reperfusion injury may directly activate p38 MAPK which phosphorylates STAT1 on SER727. Serine phosphorylated STAT could then participate in protein-protein interactions with other STAT binding proteins and activate the expression of pro-apoptotic genes like FAS. (C) In this case, hypoxia-reperfusion may activate STAT5 resulting in activation of cell survival pathways. STAT5 activation by hypoxia may be mediated by JAK2 and a STAT5/cSrc/PI-3 kinase/Akt pathway. (D) STAT3 may act as a constitutive FAS repressor, but FAS is de-repressed during UV stress which may involve STAT3 inhibition by PI3-kinase/Akt.
:::
:::
Finally, while our understanding of a stress-related p38/STAT1 (pSER) pathway seems to be taking shape, very few studies have investigated the role of serine phosphorylation of other STATs and what the upstream kinase (s) may be during cellular stress. Future studies might focus on identifying whether serine phosphorylation is common to other STATs during cellular stress and how this might relate to activation or inactivation of target genes. Other questions to be answered are what is the function of unphosphorylated STAT dimers found in the nucleus of unstimulated cells and how might other STAT post-translational modifications (other than phosphorylation) mediate STAT signaling beyond the canonical JAK/STAT pathways \[[@B39]-[@B41]\]. Answers to these questions may help to begin to unravel the complex nature of STAT signaling and how some of the alternative routes of STAT activation are related to cellular stress-activated pathways. Ultimately, modulation of the JAK/STAT pathway *in vivo*may prove to be of therapeutic value.
| PubMed Central | 2024-06-05T03:55:46.846418 | 2004-8-6 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509424/",
"journal": "Cell Commun Signal. 2004 Aug 6; 2:8",
"authors": [
{
"first": "Andrew C",
"last": "Dudley"
},
{
"first": "David",
"last": "Thomas"
},
{
"first": "James",
"last": "Best"
},
{
"first": "Alicia",
"last": "Jenkins"
}
]
} |
PMC509425 | Background
==========
Dendritic Cell (DC) vaccination is one of the most promising tools of immunological therapy for cancer. Administration of DC, generated and loaded with tumor antigens *ex vivo*, can be used to circumvent tumor immunotolerance \[[@B1],[@B2]\]. A large number of immature DC (iDC) can be produced by culturing peripheral blood monocytes with GM-CSF and IL-4 *in vitro.*These iDC possess functional characteristics typical of this maturation status, such as phagocytosis, macropinocytosis, receptor-mediated endocytosis and antigen processing \[[@B3],[@B4]\]. After antigen uptake and processing, under inflammatory stimuli, iDC undergo functional changes that result in their maturation (mDC) \[[@B5]\]. Following the up-regulation of HLA class I and II and costimulatory molecules (CD80, CD86) and other specific markers such as CD83, DC-LAMP and CCR7, mDC migrate to the T-cell zone of lymphoid tissue, where they have an optimal stimulatory capacity \[[@B6],[@B7]\]. The migration of DC to regional lymph nodes therefore represents one of the most important requirements for lymphocyte priming. Migration probably occurs through lymphatic pathways, but it is not known whether it is active or passive. Furthermore, factors such as PGE~2~may considerably increase migration, inducing CCR7 expression on the surface of DC. Penetration may be limited to the peripheral zones of lymphoid tissue when the DC are still immature, or may reach the deeper T-cell zones, where a greater number of naïve T-cells are present, when DC are mature and activated.
Surface antigen CCR7, present on the cell membrane of DC, strongly influences migratory capacity through its interaction with transporter molecules, TREM-2, LTC4, LTD4, etc. \[[@B8]-[@B10]\]. The mDC that reach lymph nodes prime naïve T-cells for a limited time and then exhaust their active functions. This can be verified by measuring IL-12 production, which rapidly decreases, and by determining the presence of IL-10, previously absent. Special conditions such as the linkage with lymphocyte ligand CD40 may prolong the active phase of mDC \[[@B11]-[@B13]\].
Recent studies on cancer patients evaluating the efficacy of *in vitro*-generated vaccines have shown that mature, but not immature DC, induce an effective antitumor response \[[@B14]-[@B18]\]. Animal studies have provided direct evidence that subcutaneously injected DC preferentially migrate to draining lymph nodes to induce a measurable antitumor effect \[[@B18],[@B19]\]. Similarly, the use of radiolabelled DC in humans demonstrates the ability of these cells to migrate to draining lymph nodes. It has also been observed that migration efficiency is linked to their maturation status or administration route (intravenous, subcutaneous, or intradermal) \[[@B20]-[@B23]\].
In the course of a vaccination trial using DC pulsed with autologous tumor lysate (ATL) in cancer patients, we evaluated the *in vivo*migration ability of DC by labelling them with ^99m^Tc-HMPAO or ^111^In-Oxine. In particular, migratory activity was assessed in iDC and mDC in terms of time required for migration to lymph nodes, duration of activity, and number of cells that migrated. Migratory capacity was further evaluated by comparing subcutaneous and intradermal administration.
Materials and methods
=====================
Patients
--------
The case series consisted of a subset of the 19 patients enrolled onto a phase I/II vaccination trial for advanced melanoma and renal cell carcinoma in which the first 9 patients were treated with iDC and the remaining 10 received mDC, both pulsed with autologous tumor lysate and keyhole limpet hemocyanin (Biosyn, Fellbach, Germany). In the present study 8 patients were analyzed (7 melanoma, 1 renal carcinoma) for a total of 11 treatments. *In vivo*migration was assessed using a part of the DC obtained for one of the therapy cycles. Three of the 8 patients were evaluated twice.
Two melanoma patients were treated with iDC (one of whom twice), while 4 other patients with melanoma and 1 with renal cell carcinoma (treated twice) received mDC. The remaining melanoma patient was treated with iDC and subsequently with mDC.
The clinical trial was approved by the Italian Ministry of Health and by the Ethical Committee of Forlì Health and Social Services (Azienda USL -- Forlì, Italy). All patients gave written informed consent.
Tumor lysate
------------
Tumor samples surgically removed from the patients were immediately placed in PBS. Adjacent non malignant tissue was removed by scalpel and tumor cells were dispersed to create a single-cell suspension. Cells were lysed by incubation in sterile distilled water. Lysis was monitored by light microscope. Larger particles were removed by centrifugation (10 min at 600 *g*) and the supernatant was passed through a 0.2-μm filter. Protein contents were determined and aliquots were stored at -80°C until use.
Treatment
---------
Patients were generally vaccinated intradermally with DC (4--6 inoculations at the base of the thigh, about 10 cm from the groin, in the absence of visible disease). From days 2--6, IL-2 (Chiron, Milan, Italy) was administered subcutaneously at a dose of 3 million IU/die. This procedure was repeated after two weeks and once a month until progression occurred.
DC generation
-------------
DC were prepared from peripheral blood monocytes (PBMC) obtained by leukapheresis without previous mobilization. 5--9 liters of blood were processed in each collection. PBMC were purified on Ficoll-Paque. An aliquot of PBMC was utilized immediately for DC generation and the rest was frozen in bags for use at a later date (4--5 bags/1 collection).
PBMC were incubated in tissue culture flasks with CellGro DC Medium (Cell Genix, Freiburg, Germany) at 10 × 10^6^cells/ml for 2 h. The non-adherent cells were discarded and the adherent cells were incubated in CellGro DC Medium containing 1000 IU/ml rhIL-4 (Cell Genix) and 1000 IU/ml rhGM-CSF (Shering Plough, Milan, Italy) for 7 days to generate a DC-enriched cell population. On day 6 DC were pulsed with autologous tumor lysate (100 mg/ml) and with KLH (50 mg/ml) and incubated overnight. On day 7, they were defined as iDC. After eliminating the previous culture medium, pulsed iDC were cultured for a further 2 days with a cocktail of cytokines (TNFα, IL-1β, IL-6, Endogen, Pierce Biotechnology, Rockford, USA; PGE~2~, Cayman Chemical, Ann Arbor, MI, USA). On day 9 they were defined as mDC. iDC or mDc were removed, washed and suspended in sterile saline for therapeutic infusion into the patient.
DC labelling and migration evaluation
-------------------------------------
Labelling of DC was performed according to the methods described for leucocyte radiolabelling \[[@B24]-[@B26]\]. A part of both iDC and mDC destined for vaccination (about 9.10^6^) were resuspended in platelet-poor autologous plasma (CFP1) and incubated for 15 min at room temperature with ^99m^Tc-HMPAO (20 mCi) (Nycomed Amersham plc, Little Chalfont, UK) ^111^In-Oxine (1 mCi) (Altana Pharma, Milan, Italy). After two washes to eliminate the unbound isotope, the cells were resuspended in a total volume of 1.5 ml of CFP1. Radiolabelling of the DC and of the culture supernatant was evaluated with a gamma counter, after which DC were inoculated intradermally into the patient near healthy lymph nodes and in the contralateral zone not used for therapeutic vaccination (3 inoculations at 10 cm from inguinal or axillary lymph nodes). The patient then underwent serial acquisitions with gamma-camera positioned at the site of inoculation, with a field of view that included all the lymphatic regions of interest. The first acquisition was performed with a dynamic study of 20 min, followed by 10-min static acquisitions carried out every 30 min for the first 4--6 h and from 18 to 28 h. Other static determinations were made at 36, 48 and 72 h. The maximum duration of observation of DC migratory activity, which depended on the half-life of the radioisotope used, was 72 h for ^111^In-Oxine and 36 h for ^99m^Tc-HMPAO.
The identification of lymph node stations involved in the migratory activity was initially visual, after which we carried out a semiquantitative evaluation of the percentage of DC that migrated to lymph nodes from the inoculation site and an assessment of the speed of DC migration, expressed by activity/time curves obtained through the compartmental mathematical model.
Evaluation of labelling stability
---------------------------------
DC obtained from the culture of frozen PBMC were divided into two parts: one was labelled with ^99m^Tc-HMPAO and the other was labelled with ^111^In-Oxine. The labelled cells were then suspended in CellGro DC Medium, divided into 4--5 culture flasks for each labelling molecule and incubated for 0 h, 4 h, 21 h, 24 h (^99m^Tc-HMPAO) and 0 h, 4 h, 21 h, 24 h, 48 h (^111^In-Oxine). The DC from one flask were removed and centrifuged. The supernatant containing the free molecule, and the pellet containing the labelled cells, were then measured with a gamma counter.
Phenotype analysis
------------------
iDC and mDC phenotypes were determined by single or two-color fluorescence analysis. 3--5·10^5^cells were suspended in 100 μl of buffer (PBS, 2% FCS, 1% sodium azide) and incubated for 30 min at 4°C with 10 μl of appropriate fluorescein isothiocyanate or phycoerythrin-labelled monoclonal antibodies (mAbs). The cells were then washed twice and resuspended in 500 μl of assay buffer. The fluorescence was analyzed by a FACS Vantage flow cytometer (Becton Dickinson, Milan, Italy). mAbs specific for human CD1a, CD14, CD80, CD86, (Becton Dickinson) CD83 (Immunotech, Marseille, France) and CCR7 (BD Pharmingen, Milan, Italy) were used.
Cytokine production
-------------------
At each pre-set time the supernatant was collected and stored at -80°C until analysis was carried out using commercially available ELISA Kit (Biosource, Nivelles, Belgium) to measure the production of IL-12 + p40 (bioactive heterodimer of IL-12) and IL-10 by DC.
Endocytosis evaluation
----------------------
Single cell-based measurement of endocytosis was carried out as described (27). Dendritic cells were incubated for 30 min at 37°C with 0.5 mg/ml FITC-Dextran (40S DX-FITC Sigma, Milan, Italy). DX-FITC (average MW 42,000) was centrifuged before use to remove aggregates. As negative control, cells were incubated with DX-FITC at 4°C. The cells were washed with cold PBS containing 2% FCS and 2 nM sodium azide to exclude dead cells and were then analyzed on a FACS Vantage flow cytometer (Becton Dickinson) \[[@B27]\].
Results
=======
Patient characteristics
-----------------------
All the patients (6 males, 2 females) had advanced disease and all but one had undergone previous treatment. Median age was 49 years (range 46--52 years). Three patients were HLA-A1, 3 were HLA-A3, 1 was HLA-A2 and 1 was HLA-A11 (Table [1](#T1){ref-type="table"}). Two melanoma patients were treated with iDC, while 2 other patients with melanoma and 1 with renal cell carcinoma received mDC. The remaining 3 melanoma patients were treated with iDC and subsequently with mDC (Table [1](#T1){ref-type="table"}). The 8 patients received a total of 73 therapeutic vaccination cycles (20 with DC obtained from fresh PBMC and 53 from frozen PBMC) and 11 labelled DC evaluations were carried out.
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Patient characteristics
:::
**Patients** **Sex/Age** **Pathology** **Site of Metastasis** **Previous Treatment** **i/mDC** **HLA**
-------------- ------------- --------------- --------------------------------------------- ------------------------ ----------- ---------------------------------------
1 M/47 Mel Liver, mediastinal lymph nodes IFN iDC A~1~A~2~B~8~B~35~Bw~6~Cw~4~Cw~7~
2 M/52 Mel Liver BIOCT iDC A~3~A~28~B~35~B~53~Cw~4~
3 M/49 Mel Liver, adrenal glands No treatment iDC + mDC A~11~A~31~B~14~B~60~Bw~6~Cw~3~
4 M/42 Mel Liver, mediastinal and axillary lymph nodes BIOCT iDC+ mDC A~1~A~9~B~17~Bw~4~Bw~6~Cw~3~Cw~4~
5 F/49 Mel Lung, lymph nodes, skin, peritoneum BIOCT iDC+ mDC A~1~A~9~B~7~B~44~Bw~4~Bw~6~Cw~4~Cw~7~
6 M/50 Renal ca. Skin, adrenal glands BIOCT mDC A~2~A~3~B~7~B~51~Bw~4~Bw~6~Cw~1~Cw~7~
7 F/52 Mel Lung, liver HdIFN + CT mDC A~3~A~29~B~44~Bw~4~
8 M/46 Mel Abdominal lymph nodes IFN+BIOCT mDC A~3~A~28~B~21~B~35~Cw~4~
IFN, alpha interferon; BIOCT, biochemotherapy; CT, chemotherapy; HdIFN, high-dose adjuvant alpha interferon (ECOG 1684)
:::
DC characteristics
------------------
The characteristics of iDC and mDC used to evaluate migration activity were similar to those of the DC utilized by us for therapeutic vaccination and to results published in the literature. Data on the purity and vitality of DC, the presence of surface markers and DC functional features (endocytosis and production of IL-12 and IL-10) are reported in Table [2](#T2){ref-type="table"}.
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Biological characteristics of dendritic cells used for vaccination
:::
**iDC median % (range)** **mDC median % (range)**
------------------------------------ -------------------------- --------------------------
**DC surface markers:**
**CD 1a** 20 (4--58) 2 (0--8)
**CD 14** 3 (0--7) 2 (0--11)
**CD 80** 3 (1--23) 37 (27--87)
**CD 86** 30 (10--55) 81 (15--94)
**HLA-DR** 45 (17--82) 78 (56--88)
**CD 83** 2 (0--13) 55 (34--73)
**CCR7** 4 (2--5) 86 (48--92)
**Endocytosis**% of positive cells 70 (39--91) 15 (1--42)
**IL-12 production**pg/ml 49 (17--225) \> 1350
**IL-10 production**pg/ml 0 0
**% purity \*** 74 (66--98) 59 (31--100)
**% vitality \*\*** 75 (68--79) 82 (66--89)
\* viable DC/viable DC + viable lymphocytes \*\* viable DC + viable lymphocytes/total cells
:::
DC labelling efficiency and stability
-------------------------------------
The *in vitro*stability of DC labelled with ^99m^Tc-HMPAO and ^111^In-Oxine was evaluated using DC cultured from frozen PBMC. ^99m^Tc-HMPAO-labelled DC showed a 75% loss of activity 4--24 h after the beginning of *in vitro*culture. ^111^In-Oxine-labelled DC showed a higher labelling stability (50%) that lasted for up to 24 h (Fig. [1](#F1){ref-type="fig"}). This accounts for the differences in lymph node uptake percentages observed in our migration studies. More accurate information on the linkage stability of ^111^In-Oxine-labelled DC over time would permit the opportune correction of the uptake percentage and would enable data to be compared with those obtained using indium.
::: {#F1 .fig}
Figure 1
::: {.caption}
######
A sample of mature dendritic cells cultured *in vitro*for vaccination was divided into two parts, one labelled with ^99m^Tc-HMPAO and the other with ^111^In-Oxine. The DC were then suspended in DC medium and cultured *in vitro*for 24 h (^99m^Tc-HMPAO) and 48 h (^111^In-Oxine). At 0, 4, 21, 24, and 48 h, the activity of the supernatant containing the free molecule and of the pellet containing labelled cells was measured. After 24 h, a 75% and 50% loss of activity was observed for ^99m^Tc-HMPAO-and ^111^In-Oxine-labelled DC, respectively.
:::
:::
Administration routes
---------------------
The migration activity of mDC administered simultaneously by intradermal and subcutaneous injection in the arms of two patients (nos. 7 and 8) was evaluated by comparing radioactive uptake in axillary lymph nodes. The intradermal route showed a threefold higher migration than that observed for the subcutaneous route (Table [3](#T3){ref-type="table"}). Evaluations were made at intervals from 0 to 44 h after inoculation. The sites of inoculation showed an exponential type washout that was virtually identical for both routes of administration (data not shown). The final migration percentage ratio (measured after 44 h) was fairly similar in both patients, but was obviously not statistically significant (Fig. [2A,2B](#F2){ref-type="fig"},[3](#F3){ref-type="fig"}).
::: {#T3 .table-wrap}
Table 3
::: {.caption}
######
Different vaccine administration routes: intradermal vs. subcutaneous lymph node uptake
:::
**Patients** **mDC × 10^6^** **Administration route** **Isotope** **Max uptake (%) \***
-------------- ----------------- -------------------------- ------------------- -----------------------
R.L. (no. 7) 4 Intradermal **^99m^Tc-HMPAO** 0.95
G.D. (no. 8) 4 Intradermal **^99m^Tc-HMPAO** 1.02
R.L. (no. 7) 4 Subcutaneous **^99m^Tc-HMPAO** 0.30
G.D. (no. 8) 4 Subcutaneous **^99m^Tc-HMPAO** 0.37
**UPTAKE RATIO: intradermal/subcutaneous average: 3**\*Maximum uptake = maximum lymph node activity/inoculation site activity at 0 h
:::
::: {#F2 .fig}
Figure 2
::: {.caption}
######
In patients no. 7 (A) and 8 (B), the same number of ^99m^Tc-HMPAO-labelled DC were administered simultaneously: subcutaneously (sc) in the left axilla and intradermally in the right axilla (id). The acquisition times with gamma camera are reported along the X-axis and the Y-axis shows the counts per 10 min. In both patients intradermal administration presents a greater concentration of labelled cells in lymph nodes than the subcutaneous route.
:::
:::
::: {#F3 .fig}
Figure 3
::: {.caption}
######
In patient no. 7 the same number of ^99m^Tc-HMPAO-labelled DC were administered simultaneously: subcutaneously (s.c.) in the left axilla and intradermally (i.d.) in the right axilla. The figure shows acquisition images with gamma camera at 0, 3, 5, and 18 h after inoculation. Greater migration capacity after intradermal administration is clearly visible. (IS, inoculation site: LN, lymph node).
:::
:::
iDC/mDC migration
-----------------
iDC and mDC migration was evaluated in all 8 patients (4 iDC and 7 mDC treatments) (Table [4](#T4){ref-type="table"}). The data presented refer to 4 groups of patients treated with mature or immature cells labelled with ^111^In-Oxine or ^99m^Tc-HMPAO. A simple numerical analysis shows that the maximum uptake ratio between ^99m^Tc-HMPAO-labelled mDC and iDC varies from 2 to 35, with an average of 8.4. The same ratio for ^111^In-Oxine-labelled cells varies from 4 to 7, with an average of 6. ^99m^Tc-HMPAO labelling is influenced by the very low iDC uptake due to its greater binding instability and to the short half-life of the radioisotope, which does not permit the acquisition of reliable counts beyond 24--36 h.
::: {#T4 .table-wrap}
Table 4
::: {.caption}
######
Comparison between iDC and mDC lymph node uptake
:::
**Patients** **DC i/m × 10^6^** **Isotope** **Max uptake (%) \***
---------------- -------------------- ------------- ----------------------- ------
G.C. (no. 1) i 6 **^99m^Tc-HMPAO** 0.22
G.C. (no. 1) i 6.9 **^99m^Tc-HMPAO** 0.05
G.L. (no. 2) i 9 **^99m^Tc-HMPAO** 0.05
P.A.M. (no. 3) i 6 **^111^In-Oxine** 0.42
T.N. (no. 5) m 5.6 **^99m^Tc-HMPAO** 1.75
P.I.M. (no. 4) m 12 **^99m^Tc-HMPAO** 0.53
PA.M. (no. 3) m 7 **^111^In-Oxine** 3.14
S.G. (no. 6) m 6.4 **^99m^Tc-HMPAO** 0.39
S.G. (no. 6) m 6.7 **^111^In-Oxine** 1.88
R.L. (no. 7) m 4 **^99m^Tc-HMPAO** 0.95
G.D. (no. 8) m 4 **^99m^Tc-HMPAO** 1.02
**UPTAKE RATIO: mDC/iDC**^111^**In-Oxine**average: 6 (range 4--7) ^99m^**Tc-HMPAO**average: 8.4 (range 2--35) \*Maximum uptake = maximum lymph node activity/ inoculation site activity at 0 h
:::
A lymph node uptake can be observed in all patients within the first two hours of inoculation, reaching a maximum uptake after 12 h in 7 patients (Fig. [4A,4B](#F4){ref-type="fig"},[5](#F5){ref-type="fig"}). In the last 4 experiments in which a more accurate temporal analysis was performed, the uptake percentage continued to increase for the entire temporal range studied (24--30 h with ^99m^Tc-HMPAO and 48--60 h with ^111^In-Oxine). A curve fitting analysis also seemed to indicate a progressive increase in uptake after the first 60 h, but the number of patients evaluated is too low for any definitive conclusions to be drawn.
::: {#F4 .fig}
Figure 4
::: {.caption}
######
This figure shows the migration activity of mDC (patient no. 5) and iDC (patient no. 2) labelled with ^99m^Tc-HMPAO (A) and of mDC and iDC (patient no. 3) labelled with ^111^In-Oxine (B). The acquisition times with gamma camera are reported along the X-axis and the Y-axis shows the counts per 10 min. mDC migrating to regional lymph node are always higher than iDC. ^99m^Tc-HMPAO-labelled DC were detected in lymph nodes within 1 h of administration and the maximum concentration was reached within 60 min for iDC and between 18 and 20 h after inoculation for mDC.
:::
:::
::: {#F5 .fig}
Figure 5
::: {.caption}
######
The figure shows static acquisition images with gamma camera 2, 24, and 48 h, and 2, 24, 48 and 72 h after inoculation with ^111^In-Oxine-labelled iDC and mDC, respectively, for patient no. 3. Greater migration activity of mDC is clearly visible. (IS, inoculation site: LN, lymph node).
:::
:::
Pure ^99m^Tc-HMPAO injection
----------------------------
After injection of ^99m^Tc-HMPAO alone in the same inoculation sites used for the study, no labelled hot spots were observed. This would seem to suggest that pure tracers move through lymphatic vessels without accumulating inside lymph nodes.
Discussion
==========
DC-based immunotherapy has undergone a remarkable transformation in its development from basic research to clinical application \[[@B10],[@B28]\]. However, many issues remain to be clarified to improve functionality and therapeutic effects and to insure a powerful and wide-ranging antitumor response by T-cells. Two stages of fundamental importance in the therapeutic use of DC are the optimization of the maturation stimulus and the induction of an effective migration to regional lymph nodes to guarantee powerful and long-lasting priming of naïve T-cells \[[@B5],[@B29]\]. Migration activity is therefore one of the functional characteristics of DC that warrants further investigation in an attempt to increase their potential \[[@B30]\].
Recent published data have shown that the choice of maturation stimulus may be crucial for therapeutic success. In particular, it has been seen that PGE~2~is essential for activating DC chemotaxis through the expression of CCR7 on the cell surface \[[@B31],[@B32]\]. This receptor permits migration through a concentration gradient of its ligands CCL19 and CCL21 inside lymphoid organs. The use of PGE~2~may therefore prove to be important for increasing migration activity and DC efficacy. However, it has also been observed that PGE~2~inhibits IL-12 production, resulting in a weaker *in vivo*activation of T-cells \[[@B33],[@B34]\]. These contradictory research data obviously require validation by *in vivo*experimentation.
In the present study we aimed to clarify some issues concerning DC migration activity in a clinical vaccination trial utilizing radiolabelled (^99m^Tc-HMPAO and ^111^In-Oxine) iDC and mDC \[[@B20],[@B35]\]. Technetium has a high labelling intensity and a short half-life, while indium, despite having a lower intensity, has a longer half-life, which enabled us to monitor the migration activity of labelled DC for up to 72 h.
The first step was to evaluate whether radioactive tracer would be scintigraphically visualized in lymph nodes. For this purpose technetium alone was injected intradermally and its progression followed. At the same time labelled DC were administered in the contralateral zone. It was seen that the tracer was not entrapped in lymph node stations and this confirmed that the radioactive molecule detected in contralateral regional lymph nodes was undoubtedly the expression of labelled DC that had migrated to that site.
One of the simplest analyses carried out was the evaluation of mDC migration activity using two different routes of administration: subcutaneous and intradermal. We chose two patients at random and compared migration activity simultaneously in both arms (right intradermal and left subcutaneous); it was observed that intradermal administration had a threefold higher migration to lymph nodes than the subcutaneous route. Although it remains to clarify the extent to which this migratory capacity is active or passive, it is clear that DC must be administered intradermally to obtain a higher migration.
A crucial phase of the study was the comparison between the migratory activity of iDC (used in the first part of the clinical trial) and that of mDC (used in the second half of our ongoing study). The result was once again unequivocal, showing a greater progressive concentration of mDC that was, on average, six-eightfold higher than that of iDC, in accordance with data reported by other authors \[[@B20],[@B23]\] and in contrast to results published by Blocklet \[[@B17]\], who probably used iDC. The phenotype obtained in our study bears witness to the fact that mDC exhibit a much higher CCR7 surface expression than iDC (86% vs. 4%) (Table [2](#T2){ref-type="table"}). Furthermore, mDC have an extremely high IL-12 production, which confirms a marked stimulatory activity.
Notwithstanding the results obtained from the present study, many issues remain to be clarified. It has yet to be determined whether the increased activity detected in lymph nodes corresponds to an effectively greater migratory capacity or whether it is the result of a more effective adhesion capacity between surface molecules. Both hypotheses could even prove to be correct. We also do not know how long DC remain in lymph nodes. The increase in activity in lymph nodes is high in the first few determinations but tends to diminish or stabilize after around 36 h. It remains to be seen whether this presumed stabilization is the result of a sort of saturation or whether it can be attributed to the attainment of a dynamic equilibrium. The former hypothesis would indicate the need for an optimization of the number of DC to administer, as only a limited number would be functionally active. The latter would highlight the importance of the timing of administration and perhaps also the degree of DC maturation. To further investigate this, we plan to administer ^111^In-Oxine-and ^99m^Tc-HMPAO-labelled DC in succession and in the same site, to follow their migratory course.
Finally, we aim to assess the migratory capacity of *in vitro*transiently stimulated DC (semimature DC). The therapeutic use of this type of DC, which have already begun the process of maturation and may be capable of reaching lymph nodes before their functional exhaustion, could increase the duration of their activation and stimulation. If these semimature DC prove to be equipped with a good migratory capacity, further improvement in the therapeutic use of DC may be possible.
Conclusions
===========
The migration activity of DC to regional lymph nodes is one of the many critical factors that influence the therapeutic result of antitumor vaccination. In the present study we used radioisotope-labelled DC and demonstrated that a better migration activity is obtained using intradermal than subcutaneous administration and that mDC show, on average, a six-to eightfold higher migration than iDC. Numerous other issues on DC functionality have yet to be clarified before antitumor therapeutic efficacy can be improved. The next important step will be to closely monitor the quantity and quality of responses observed in T-cells, and it is hoped that a consensus will be reached on standardized criteria for the definition and validation of clinical results obtained.
Abbreviations
=============
DC, dendritic cell; iDC, immature dendritic cell; mDC, mature dendritic cell; ATL, autologous tumor lysate; PBMC, peripheral blood monocytes.
Authors\'s contributions
========================
RR and LR participated in the design of the study and were responsible for the clinical side of the study. AR, MP, LF and MS participated in the design of the study and were responsible for the biological part of the study. GM performed the apheresis collections. RG, AM and GF carried out DC labelling and migration evaluation. GG performed the mathematical and statistical analysis. All authors read and approved the final manuscript.
Competing interests
===================
None declared.
Acknowledgements
================
The authors wish to thank Gráinne Tierney for editing the manuscript. This work was partially supported by Ministry of Health Research Projects 2000, no. 352 and 2002, no. 234.
| PubMed Central | 2024-06-05T03:55:46.847940 | 2004-7-30 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509425/",
"journal": "J Transl Med. 2004 Jul 30; 2:27",
"authors": [
{
"first": "Ruggero",
"last": "Ridolfi"
},
{
"first": "Angela",
"last": "Riccobon"
},
{
"first": "Riccardo",
"last": "Galassi"
},
{
"first": "Gianluigi",
"last": "Giorgetti"
},
{
"first": "Massimiliano",
"last": "Petrini"
},
{
"first": "Laura",
"last": "Fiammenghi"
},
{
"first": "Monica",
"last": "Stefanelli"
},
{
"first": "Laura",
"last": "Ridolfi"
},
{
"first": "Andrea",
"last": "Moretti"
},
{
"first": "Giuseppe",
"last": "Migliori"
},
{
"first": "Giuseppe",
"last": "Fiorentini"
}
]
} |
PMC509426 | Background
==========
It is estimated that nearly 7.2 million people in Asia and the Pacific region are now living with HIV/AIDS, one million of whom acquired the virus in 2002 \[[@B1]\]. Of these, more than 2.4 million are women (ages 15--45) \[[@B1]\]. India\'s national HIV prevalence rate of less than 1% offers little indication of the serious situation facing the country. An estimated 3.97 million people were living with HIV in India at the end of 2001, ranking it second only to South Africa in numbers of people infected. Although recent data suggest that prevention efforts directed at high-risk populations has resulted in greater HIV/AIDS knowledge and condom use \[[@B2],[@B3]\], the prevalence of HIV/AIDS continues to rise. For HIV-positive pregnant women who have the additional risk of transmitting the disease to their unborn child, fewer prevention efforts are in place and are acutely needed. In some states in India such as Andhra Pradesh, Karnataka, Maharashtra, Manipur, Nagaland and Tamil Nadu prevalence rates are greater than 1% \[[@B1]\], underlining the need for well-planned and sustained interventions on a large scale.
In most industrialized countries, voluntary HIV counseling and testing services are a major component of HIV and AIDS control programs targeted toward pregnant women, although such programs are only now being advocated in developing countries \[[@B4],[@B5]\]. Voluntary counseling and testing involves two basic components--an educational component on HIV/AIDS and an informed consent component related to an individual\'s choice to be HIV tested or not. The primary motivations for conducting voluntary HIV counseling and testing in lower risk populations such as pregnant women are that i) immediate knowledge of the woman\'s status can help reduce risk of HIV transmission to the unborn child; ii) if not infected, education at this point can help both the husband and wife to reduce risk behaviors to prevent transmission; and iii) testing of the partner can be encouraged \[[@B6]\]. Because voluntary HIV counseling and testing is the principal entry point for both prevention and care, it is critical that the content of the counseling is well understood and that procedures to ensure true informed consent are in place \[[@B7],[@B8]\].
The basic ethical issues regarding consent may be universal to all countries. One of the potential limitations with the current approach is that consent has been reduced to a technical issue and is often removed from wider ethical considerations. The consent procedures required by international review boards include having to provide detailed scientific and legal information to participants. Although the intent is to increase knowledge, these messages may lose their meaning when administered within certain populations. The challenge then becomes one of how to balance the requirements of providing complete information while also obtaining true informed consent.
To begin establishing voluntary counseling and testing for pregnant women in India, the Indian National AIDS Control Organization (NACO) in 2001 started voluntary HIV counseling and testing for women attending government-run antenatal clinics in 11 sites throughout the country. This study was conducted in one of these sites, Pune, Maharashtra, in order to develop tools to enhance, standardize and improve the communication of messages during the group education and counseling (GEC) sessions, and to enhance the informed consent process.
Methods
=======
This study was conducted in the antenatal clinic of an urban government hospital in Pune. Eligible patients were newly registered pregnant women between 18 and 44 years of age who were less than 36 weeks gestation and had no physical or mental disabilities (as determined by a physician). After each eligible woman registered in the antenatal clinic and met with the attending physician, they were offered the opportunity to attend the HIV/AIDS GEC sessions.
The eight major topics covered in the education component of the GEC included general transmission modes of HIV, sexual transmission, mother-to-infant HIV transmission, precautions to avoid HIV transmission, ways through which HIV does not transmit, identification of HIV in an individual, detection of HIV in the body, and symptoms of AIDS. In addition, nine major topics related to HIV testing and informed consent were covered, including procedural risks of HIV screening, social risks of HIV screening, availability of the report, the right to say no, repercussions of refusing to take the test, confidentiality of the testing procedures, the right to consult others, the benefits of HIV screening, what one\'s signature means, specific procedures of the HIV ELISA test and, if applicable, specific procedures of the rapid HIV test.
Baseline data
-------------
We conducted structured observations of 30 GEC sessions over the period from December 2000 to January 2001 to assess how well the information was being covered. The GEC sessions were chosen so that every clinic day (Monday-Saturday) and session time would be equally represented (there were, on average, four GEC sessions during the clinic hours of 9:30 am to 1:00 pm).
The structured observation tool included a comprehensive checklist of each of the specific points for each main topic covered during the GEC. A topic would be considered adequately covered if the counselor conveyed all the specific points for the topic. Overall session adequacy was assigned if at least 80% of all topics were presented adequately during the session.
To assess women\'s understanding of the key issues covered we interviewed 136 consecutively consenting women immediately after they attended one of the 30 sessions (89% of the total number of women who attended these sessions). Informed consent was obtained from each woman prior to the interview. After the interview, the women met briefly with an individual counselor, where they chose whether to have HIV testing and, if so, signed the appropriate informed consent form.
The women\'s understanding of the GEC sessions was determined through structured interviews containing 17 questions covering each of the main topics discussed in the GEC. Their answers were scored as either adequate or inadequate based on predetermined criteria.
Based in part on these observational and interview data we developed and enhanced the content of the GEC sessions. We also developed a comprehension test to evaluate women\'s understanding of key issues related to informed consent. The key enhancements to the GEC sessions were as follows:
• Areas with greater privacy for both group counseling and individual counseling were established.
• Posters illustrating the main topics were created and placed in the GEC rooms.
• Similar visuals were made into a flipchart and used by the counselor during individual counseling to reinforce the messages.
• All visuals developed included substantial input from the counselors.
• The visuals were simple, using bold colors and conveying only one message each.
• The posters were created to provide informational cues to the counselor to promote and maintain regularity and standardization in presentation.
• The counselors completed further training in the use of the visuals.
Development of the comprehension test of key informed consent issues
--------------------------------------------------------------------
Current standards for ethical treatment of human participants in a study do not require that participants demonstrate comprehension of the study prior to giving informed consent. However, given the volume and complexity of information conveyed during the GEC sessions, we felt it necessary to develop a concise yet complete comprehension test that could be used in HIV screening programs and adapted for future clinical trial enrollments.
The questionnaire was developed by choosing eight of the 17 questions from the previous questionnaire used during the baseline evaluation. The eight questions were identified by the field team of counselors, physicians, and behavioral scientists as key issues related to informed consent. The questionnaires were administered by the hospital counselor. Adequacy of women\'s understanding for any topic was determined if the woman correctly answered the question based on pre-determined criteria. If a woman scored adequately on at least 80%, or six out of eight, questions she became eligible to sign the informed consent form and proceed with the HIV screening. Prior to signing the form, the counselor had the opportunity to clarify any previously misunderstood issues with the woman.
The eight questions used to assess comprehension of informed consent issues were as follows:
1\. What are the modes by which HIV germs are transmitted?
2\. Can you say \"No\" to taking the HIV test?
3\. What happens if you decide not to take the HIV test?
4\. What do we mean by \"the result of the test will be kept confidential\"?
5\. Do you have the right to consult your husband or other family members before taking the test?
6\. What do you think are the benefits of finding out your HIV status?
7\. What problems can a woman face on finding out her HIV-positive status?
8\. What does your signature on the consent form mean?
Post-intervention evaluation
----------------------------
After the enhancements to the GEC were put into place and the comprehension test developed, we conducted structured observations of 40 GEC sessions from May-June 2001. We performed the comprehension test on 224 women who attended one of these 40 sessions (89% of the total women attending). These same women were re-interviewed after the completion of individual counseling. The time between the interview after group counseling and the re-interview after individual counseling was, on average, one-two hours.
Analysis
--------
We compared the adequacy of GEC topic coverage prior to (baseline 30 GEC sessions) and after (40 GEC sessions) the inclusion of the enhancements. Also, improvements in the women\'s comprehension were assessed through a comparison of their responses to the eight key informed consent questions before GEC enhancements (*N = 136*) and after (*N = 224*). In addition, we examined the added improvements in knowledge when the second group of women (*N = 224*) were tested after their individual counseling. Analysis of the improvements in topic coverage and women\'s understanding of topics covered was determined by a chi-square test of proportions or Z-scores (for changes in knowledge in the same population) using SPSS version 10 \[[@B9]\].
This research was approved by the Institutional Review Boards (IRB) at Johns Hopkins University, Baltimore, USA, the local IRBs in Pune, the local medical institutions involved in the research and the National Institutes of Health, USA, which funded this study.
Results
=======
Coverage of topics in GEC sessions
----------------------------------
Topics related to informed consent and HIV testing were not well covered during the GEC sessions. Only three topics--availability of a report, the right to say no, and the benefits of screening--were covered in 90% or more of the sessions. The procedural and social risks of HIV screening, the right to consult others, and confidentiality were discussed in less than 10% of the sessions. The impact of refusing to be screened and what a signature means were discussed in 40% and 70% of the sessions, respectively.
Coverage of informed consent issues with the use of visuals
-----------------------------------------------------------
Focusing only on the eight key issues related to understanding informed consent (see previously listed questions), we compared topic coverage before (Group A) and after (Group B) the improvements were made to the GEC sessions (Table [1](#T1){ref-type="table"}). With the use of visuals, there were improvements in coverage in nearly all topic areas. The coverage of \"the meaning of the signature\" increased significantly from 70% to 90% (*P \< 0.01*). Coverage of \"social risk\" during sessions showed a statistically significant increase from 7% to 23% (*P \< 0.05*), yet the overall coverage was still very low. Similarly, the coverage of \"the right to consult others\" showed a statistically significant increase (*P \< 0.001*), however, it was still only covered in 35% of the sessions. Non-significant improvements occurred in the area of \"consequences of refusal\", increasing from 40% to 55%. Overall, the use of visuals resulted in a statistically significant improvement in adequacy of coverage of the counselling sessions from 30% to 68% of all GEC sessions.
Women\'s understanding of informed consent issues with the use of visuals
-------------------------------------------------------------------------
Table [2](#T2){ref-type="table"} shows both women\'s topic-specific and overall comprehension of informed consent issues. We compare improvements in comprehension in two groups of women, Group A (without use of visual) and Group B (with use of visuals). We then examine improvements in comprehension in the same group of women (Group B), after enhanced group counselling and with the addition of enhanced individual counselling. There was significantly better understanding in three critical areas of appropriate informed consent when visuals were used, namely, \"the right to refuse\", \"consequences of refusal\", and \"the meaning of the signature.\" \"The right to refuse\" rose from 54% at baseline to 79% and 96% (*P \< 0.01*) with enhanced group counseling and enhanced individual counseling, respectively. It should be noted that this topic was well covered in all sessions with or without the addition of visual aids. On the other hand, the discussion of \"social risk if found to be HIV-positive\" or \"the right to consult others\" was rarely covered in any of the counseling sessions. In spite of this, a considerable majority of women understood both these issues. Women\'s understanding of \"consequences of refusal\" showed marked improvements with use of visuals, rising from 19% at baseline to 75% with enhanced group counselling (*P \< 0.01*) and further to 96% (*P \< 0.01*) with enhanced individual counselling.
Overall, based on adequately answering six out of eight questions, women\'s knowledge of informed consent topics in enhanced group counselling improved dramatically, from 38% to 72% (*P \< 0.01*). With the addition of individual counselling with visuals, women\'s overall understanding showed a statistically significant increase in all eight aspects of informed consent, and an overall increase from 72% to 96% (*P*\< 0.01).
Conclusions
===========
There were two primary objectives of this study. The first was to enhance the communication of key concepts within the HIV/AIDS GEC setting and the second was to develop a simple tool to evaluate women\'s comprehension of informed consent issues.
Many international health education and prevention programs have developed and tested creative and culturally appropriate communication strategies to provide information on issues as sensitive and diverse as family planning and changing defecation behaviors. However, innovation in, and evaluation of, the process of delivering the often complex information necessary for informed consent has been limited \[[@B10]\]. The use of simplified visuals and text as a means of communicating messages to populations with little or no literacy has commonly been used to convey information on family planning, health, nutrition and public health activities \[[@B11],[@B12]\]. However, such visuals must be developed with the specific population in mind and by community members in order to ensure their positive impact on understanding \[[@B13]\].
There is very little evidence demonstrating that individuals in resource-poor countries cannot understand the basics of research design or biomedical treatment options just because they have little education or different views about health and illness. It may be difficult to communicate the purposes, conditions and risks of research, but the difficulty of doing so should not detract from the importance of obtaining individual informed consent. In fact, several researchers \[[@B10],[@B14]\] have established that, with some effort, acceptable levels of information can be communicated. This study indicates that the full informed consent process (including both group and individual counselling), when combined with enhanced education and counselling materials, can lead to excellent comprehension of informed consent issues.
The dramatic improvement we found in the women\'s comprehension of informed consent issues, despite their varying socioeconomic and educational backgrounds, is encouraging. Caution should be exercised, however, in interpreting the improvements in comprehension as being a result of the individual counseling and visuals. The second interview was done within one-two hours of the first, and the women may have had time to reconsider their responses to the previous questionnaire.
The major limitation in this study was the experimental design, in that we did not have the opportunity to measure the impact of individual counseling without the use of visuals. This was mainly because during baseline data collection we were interested in comprehension immediately after the GEC session; interaction with the counselor for individual counseling directly following the GEC was very brief and, therefore, was not expected to have improved women\'s knowledge considerably. Moreover, by the time we tested the visuals we had developed, the counselors had gained a lot more experience in HIV counseling and the women might have received more information from various media about HIV.
Despite these limitations, we can assert from our data that simple didactic group education on HIV/AIDS and testing issues is not sufficient to help women in this setting to understand the complexities of informed consent for HIV testing. The use of visuals in the form of posters and flipcharts provided structure and uniformity to the GEC sessions, thereby reinforcing the messages for these women and enhancing the overall informed consent process.
Obtaining proper informed consent in the case of HIV screening is not a discrete action, but a process that can be enhanced through effective communication, repetition and reflection. Although the intent is to increase knowledge, information regarding informed consent may lose its meaning when administered within certain populations. The time and the technicalities of the process itself may intimidate women in societies where literacy and awareness about medical and legal rights is low. It has been argued that complicated concepts conveyed in a consent form, often to fulfill the requirements of funding agency for institutional and policy purposes, may in themselves be unethical and, indeed, pose the biggest barrier to the informed consent process \[[@B15]\]. It should also be recognized that implementing counseling and informed consent procedures is considerably more difficult in certain settings in India where facilities, supplies, personnel, and time are at a premium. In addition, educational levels for women in this population are low, where 36% have a primary school education or less and another 33% are illiterate \[[@B16]\].
Generally, for HIV screening, an individual reads or is read a prepared statement that includes detailed scientific and legal information on the aims and biological significance of the test, the risks and benefits of testing, and the individual\'s rights. The participant is expected to understand the main components of what is written within the document and make an autonomous decision on whether or not to be tested for HIV. This is further complicated by the fact that, in some studies, the original consent document is composed in English and then translated nearly verbatim into the local language, making the communication of already complex topics even more difficult.
Typically, informed consent for pregnant women in most Indian hospitals and clinics is for operative procedures such as cesarean section or laparotomy. It is usual for the doctor or resident on duty to put down in his or her own handwriting the text of the consent on a patient\'s case papers. The content of this consent gives blanket permission to the hospital and doctors to undertake all procedures on the patient that are indicated in order to maintain the good health of the mother and her fetus, while at the same time absolving the attending physician and hospital of any blame in the event of a mishap. This is signed (or a thumbprint given) by the patient, her husband or an accompanying relative, and is generally considered to serve as legal consent; therefore, it is not interpreted as voluntary. Most often, due to time constraints, very little is explained to the patient about the procedure, risks, and benefits, or what her signature actually means. As found in other regions in India, there is a general perception by clinicians and other healthcare workers that women are \"unable\" to understand any of the procedures even if explained, because they are illiterate or have no medical background \[[@B17],[@B18]\]. It is implicit in the physician-patient relationship that any treatment or procedures recommended by the physician will benefit the patient \[[@B19]-[@B21]\]. By de-mystifying the content and process of informed consent through standardization with structured visual cues and reiteration, we feel that these difficulties could be overcome.
In creating the modifications to the GEC, we focused on improving communication of those concepts that were the most unfamiliar to these women. For example, for most women in India, there is relatively little sense of autonomy \[[@B22]\]. For many, the woman\'s role is defined first by her father; after marriage, her husband\'s decision-making and the wishes of his parents or the elders in his home prevails. For such a woman, when an incurable disease like HIV/AIDS is presented to her in terms of her \"autonomy\" and \"power\" with reference to the disease, this could be confusing and even frightening. The prevailing practice of obtaining familial input on such decisions was demonstrated through women\'s knowledge of this topic despite the fact that it was hardly mentioned during the counseling sessions. On the other hand, our data show that by enhancing the GEC and reinforcing its messages through individual counseling, a significantly greater number of women can correctly understand the idea of their \"right to refuse\", indicating that even complex constructs such as autonomy can be conveyed. The understanding of the \"meaning of the signature\" was clearly enhanced with the use of visuals, because the improvements in women\'s knowledge directly followed the increase in coverage during the counseling sessions.
Clearly, some of the concepts related to informed consent may already be understood by these women. Women\'s understanding of \"consequences of refusal\" showed marked improvements in the second group of women studied. Although some of this improvement may be attributed to the individual counselling and visuals, the fact that only 55% of the GEC sessions actually adequately covered this topic indicates that other factors may have contributed to the women\'s knowledge of this topic.
This process of refining and evaluating the informed consent process can benefit the clinic and research settings in both developed and developing countries. Data from clinics in the USA, Belgium, and France report that even under well suited environments, informed consent for HIV screening was generally only obtained in about 70--85% of cases, and documentation of consent was substantially less \[[@B23],[@B24]\]. In research settings, studies suggest that, despite having signed a consent form, participants may not fully understand critical aspects of research participation or their individual rights \[[@B25],[@B26]\].
We have modified the standard model of informed consent by adapting it to suit the population that was served, and have documented subsequent improvements in patient understanding of the informed consent process. This study shows that culturally appropriate enhancement of the standard informed consent process moves the process towards its goal of being one that is truly informed and voluntary. Thus it not only fulfills the ethical requirements, but, most importantly, helps to assure that women\'s rights are preserved. This last point is critical because previous research has pointed out that, although women may be fully informed, they still may not feel their choice is fully voluntary \[[@B14]\]. We suggest that the current requirements of informed consent procedures are inadequate and that it should be a process that communicates information in an effective manner, allows for reiteration of information and includes an evaluation of the woman\'s knowledge prior to signing the informed consent document.
In an effort to allow all interested organizations involved in HIV counseling and testing to use or modify our visuals for their own programs, we are have created a downloadable version available for public access at our website <http://www.bjjhumit.org>. It is hoped that these types of visuals will become an integral part of all voluntary counseling and testing programs throughout India and elsewhere.
Competing interests
===================
None declared.
Author\'s contributions
=======================
JS was involved in developing the research and visuals, implementing the study and preparing the manuscript. HP participated in the design of the visuals and in the analysis and coordination of the study. SS and AJ were involved in data collection and analysis. NK-K participated in the design of the visuals and in the analysis of the study. NS participated in the design of the study and the visuals. KEB, AS and MAP participated in the design and assisted in the implementation of the study. RCB assisted in all aspects of the study design and implementation. AVS participated in the design, conducted the analysis and wrote the manuscript. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1741-7015/2/28/prepub>
Acknowledgements
================
The investigators are grateful to the participants for giving their time and information. A special thanks goes to Abhay Kadam for his photographic and computer work on the visuals. We are thankful to all members of the study team at BJ Medical College, especially the counseling staff, physicians and nurses at the Sassoon General Hospital, Pune, for their assistance and cooperation in conducting this work. This research was supported by the National Institutes of Health, grant number 1R01 AI45462-04.
Figures and Tables
==================
::: {#T1 .table-wrap}
Table 1
::: {.caption}
######
Coverage of selected components of informed consent during counseling sessions, with and without the use of visuals
:::
**Topic covered** Coverage in 30 group education sessions **without**visuals (Group A) Coverage in 40 group education sessions **with**visuals (Group B) Chi square ***χ***^2^statistic and significance level
--------------------------------------- ---------------------------------------------------------------------- ------------------------------------------------------------------- ------------------------------------------------------- ----- ------------
**Mother-to-infant HIV transmission** 30 100 40 100 \_\_\_
**Confidentiality** 30 100 38 95 0.2
**Right to refuse** 29 97 39 98 0.0
**Consequences of refusal to test** 12 40 22 55 1.4
**Social risk** 2 7 9 23 4.0\*
**Benefits of HIV testing** 30 100 39 98 0.0
**Meaning of signature** 21 70 36 90 6.8\*\*
**Right to consult** 0 0 14 35 12.6\*\*\*
**80% coverage in sessions** 9 30 27 68 13.4\*\*\*
\* = P \< 0.05, \*\* = P \< 0.01, \*\*\* = P \< 0.001
:::
::: {#T2 .table-wrap}
Table 2
::: {.caption}
######
Relative improvement in women\'s understanding of components of informed consent after use of visuals in group education and counseling (GEC) and individual counseling (IDC)
:::
**Topic covered** Group A: Women\'s understanding after GEC **without**visuals (posters) (*N = 136*) Group B: Women\'s understanding after GEC **with**visuals (posters) (*N = 224*) Chi square ***χ***^2^statistic Group A *vs*Group B Group B: retested after IDC **with**visuals (flipcharts) (*N = 224*) Z Score statistic Group B (GEC) *vs*Group B (GEC+IDC)
------------------------------------------------------------- ------------------------------------------------------------------------------------ --------------------------------------------------------------------------------- ---------------------------------------------------- ---------------------------------------------------------------------- ------------------------------------------------------- ----- ---- ------------
**Mother-to-infant HIV transmission** 128 94 213 95 0.03 221 99 2.17\*\*
**Confidentiality** 130 76 174 78 0.09 219 98 6.47\*\*\*
**Right to refuse** 73 54 176 79 23.43\*\*\* 215 96 5.52\*\*\*
**Consequences of refusal to test** 26 19 167 75 102.35\*\*\* 214 96 6.22\*\*\*
**Social risk** 86 63 153 68 0.76 194 87 4.63\*\*\*
**Benefits of HIV testing** 39 29 88 39 3.72 155 69 6.35\*\*\*
**Meaning of signature** 80 59 169 75 10.19\*\*\* 215 96 6.21\*\*\*
**Right to consult** 123 90 203 91 0.02 216 96 2.49\*\*
**Overall adequate understanding of women (6/8 questions)** 52 38 162 72 39.38\*\*\* 216 96 7.02\*\*\*
\* = P \< 0.1, \*\* = P \< 0.05, \*\*\* = P \< 0.01
:::
| PubMed Central | 2024-06-05T03:55:46.850717 | 2004-8-2 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509426/",
"journal": "BMC Med. 2004 Aug 2; 2:28",
"authors": [
{
"first": "J",
"last": "Sastry"
},
{
"first": "H",
"last": "Pisal"
},
{
"first": "S",
"last": "Sutar"
},
{
"first": "N",
"last": "Kapadia-Kundu"
},
{
"first": "A",
"last": "Joshi"
},
{
"first": "N",
"last": "Suryavanshi"
},
{
"first": "KE",
"last": "Bharucha"
},
{
"first": "A",
"last": "Shrotri"
},
{
"first": "MA",
"last": "Phadke"
},
{
"first": "RC",
"last": "Bollinger"
},
{
"first": "AV",
"last": "Shankar"
}
]
} |
PMC509427 | Background
==========
A role for immune responses, involving antigen presentation and immune-response-generating cytokines, in neurodegenerative diseases such as Alzheimer\'s disease was recognized for a decade before the term neuroinflammation came into widespread use \[[@B1],[@B2]\]. A PubMed search using \"neuroinflammation\" as the only key word yields some 300 papers, none before 1995 \[[@B3]\]. While some chronic/remitting neurological diseases, such as multiple sclerosis, have long been recognized as inflammatory, the term neuroinflammation has come to denote chronic, CNS-specific, inflammation-like glial responses that do not reproduce the classic characteristics of inflammation in the periphery but that may engender neurodegenerative events; including plaque formation, dystrophic neurite growth, and excessive tau phosphorylation. In this way, neuroinflammation has been implicated in chronic unremitting neurodegenerative diseases such as Alzheimer\'s disease -- diseases that historically have not been thought of as inflammatory diseases. This new understanding has come from rapid advances in the field of microglial and astrocytic neurobiology over the past fifteen to twenty years. These advances have led to the recognition that glia, particularly microglia, respond to tissue insult with a complex array of inflammatory cytokines and actions, and that these actions transcend the historical vision of phagocytosis and structural support that has long been enshrined in the term \"reactive gliosis.\" Microglia are now recognized as the prime components of an intrinsic brain immune system \[[@B4]\], and as such they have become a main focus in cellular neuroimmunology and therefore in neuroinflammation. This is not the inflammation of the adaptive mammalian immune response, with its array of specialized T-cells and the made-to-order antibodies produced through complex gene rearrangements. This is, instead, the innate immune system, upon which adaptive immunity is built \[[@B5]\].
Many researchers now consider this innate immune response in the brain to be a potentially pathogenic factor in a number of CNS diseases that lack the prominent leukocytic infiltrates of adaptive immune responses, but that do have activated microglia and astrocytes, i.e., neuroinflammation.
The idea that neuroinflammation is detrimental implies that glial cell activation precedes and causes neuronal degeneration \[[@B2]\], a sequence of events that appears to be at odds with experimental models of neurodegeneration in which glial cell activation occurs secondary to neuronal damage. What is missing from this simple linear model is the understanding that chronic neurological diseases are just that -- chronic, and that this chronicity introduces complex interactions and feedback loops between neurons and glia that render attempts to construct simple, linear cascades of cause and effect inelegant.
In the following, we provide some basic definitions and discussion to more precisely define the idea of neuroinflammation as a CNS tissue response to injury, and the notion of neuroinflammation as a pathogenic factor in neurodegenerative diseases.
Some basic definitions
----------------------
Inflammation is a reaction of living tissues to injury \[[@B6]\]. The discipline of pathology makes a fundamental distinction between acute and chronic inflammation. Acute inflammation comprises the immediate and early response to an injurious agent and is basically a defensive response that paves the way for repair of the damaged site. Chronic inflammation results from stimuli that are persistent. In the periphery, inflammation consists of leukocytic infiltrates characterized by polymorphonuclear cells (neutrophils) in acute inflammation and mononuclear cells (macrophages, lymphocytes, plasma cells) in chronic inflammation. In order to validate these principles of general pathology within the context of neuroinflammation, one must obviously consider both acute and chronic neuroinflammation and, therefore, these are addressed separately in the following sections.
Acute neuroinflammation
-----------------------
Before \"neuroinflammation\" became a commonly used term, neuroscientists spoke of \"reactive gliosis\" in describing endogenous CNS tissue responses to injury. Reactive gliosis specifically referred to the accumulation of enlarged glial cells, notably microglia and astrocytes, appearing immediately after CNS injury has occurred. In contrast to glial reactivity, which suggests a largely passive response to injury; glial activation implies a more aggressive role in responding to activating stimuli: activated glial cells release factors that act on and engender responses in target cells analogous to the responses of activated immune cells in the periphery. Activation of immune cells in the periphery leads to leukocyte infiltration of tissues, but this is notably absent in the brain unless there has been destruction or compromise of the blood brain barrier \[[@B7],[@B8]\]. In the presence of such destruction or compromise, peripheral leukocytes do enter the brain producing a scenario similar to that seen in inflammatory responses in the periphery.
In limited, acute reactions to injury, in the absence of blood-brain barrier breakdown, there is the subtler response of the brain\'s own immune system, composed largely of rapid activation of glial cells. These responses represent the other end of the spectrum of CNS injury, where limited neuronal insults trigger glial cell activation without breakdown of the blood brain barrier and without concomitant leukocytic infiltration. This form of \"pure\" glial response occurs in neuronal injury caused by either loss of afferents \[[@B9]\] or loss of efferents \[[@B10]\]. Axotomy, for instance, results in neuronal chromatolysis, the classic example of potentially reversible neuronal injury \[[@B9]\]. It is in these situations that microglial and astrocytic responses (like their peripheral counterparts) fulfill their evolutionarily programmed functions of a reparative response to the benefit of the organism as a whole.
Although such specific responses might, in a strict sense, be included in the term \"neuroinflammation,\" neuroinflammation as generally used and understood applies to more chronic, sustained cycles of injury and response, in which the cumulative ill effects of immunological microglial and astrocytic activation contribute to and expand the initial neurodestructive effects, thus maintaining and worsening the disease process through their actions.
Chronic neuroinflammation
-------------------------
The concept of chronic inflammation (as opposed to acute inflammation) is more relevant in the context of understanding CNS disease (as opposed to CNS injury), as the very term \"disease\" implies chronicity. Chronic multiple sclerosis is, of course, an unequivocal and long-recognized example of an inflammatory brain disease. Although the underlying cause(s) of multiple sclerosis have not been elucidated, it is probably safe to say that the persistent injurious stimulus that accounts for neuroinflammation in multiple sclerosis is a myelin-related protein that has escaped self-tolerance and become an autoimmunogen. Consistent with the chronic persistence of this autoimmunogen is a persistent accumulation of blood-derived mononuclear leukocytes in the CNS parenchyma, a phenomenon that is similar to what is found in other autoimmune diseases such as rheumatoid arthritis or polymyositis.
Infections are another group of diseases that are classically recognized as inflammatory in nature, with meningeal, perivascular, or even parenchymal infiltrates of peripheral leukocytes. There are, however, exceptions. Rabies is a disease in which the peripheral immune response is slow and inadequate, and in which classic inflammatory changes are less striking than those found in other viral encephalidites. Babes, in 1897 \[[@B11]\], described microglial activation in rabies infection, although he did not recognize the nodules he found as clusters of activated microglia. Similar small collections of activated microglia were subsequently found to occur in a wide variety of viral brain infections.
Today, the most important example of a chronic brain infection is human immunodeficiency virus (HIV). Chronic HIV encephalitis is characterized by the same nodules of activated microglia that Babes described in rabies. HIV enters and persists in the CNS *via*myelomonocytic cells: monocytes, perivascular cells, and microglia \[[@B12]\]. HIV infection is uniquely different from most other infectious diseases affecting the CNS in that the virus targets and disables precisely those cells that are key players in neuroinflammation; microglia in the brain and T lymphocytes in the periphery. It therefore comes as no surprise that prominent T cell infiltrates do not occur in HIV encephalopathy.
Prion diseases represent another chronic infectious CNS disease that is not accompanied by leukocytic infiltrates. Microglial activation, again, appears to be the most prominent inflammatory component of prion diseases \[[@B13],[@B14]\], although there are a few reports describing T cell infiltration as well \[[@B15],[@B16]\]. Prion diseases share interesting parallels to rabies infection in that infected cells are unrecognized by peripheral immune responses. This may explain in part the unusual patterns of neuroinflammation in prion diseases -- manifest not only in atypical cellular infiltrates but also in unusual cytokine profiles \[[@B17]\]. Both HIV and prion infections probably produce an altered microglial physiology that is likely to translate into cycles of neurodegeneration, which could be a contributing factor in the development of dementia that occurs in these conditions.
Chronic microglial neuroinflammation in neurodegenerative diseases
------------------------------------------------------------------
Neurodegenerative diseases -- particularly Alzheimer\'s disease, but also amyotrophic lateral sclerosis, Parkinson\'s disease, and Huntington\'s disease -- lack the prominent infiltrates of blood-derived mononuclear cells that characterize autoimmune diseases. On the other hand, there is abundant evidence that many substances involved in the promotion of inflammatory processes are present in the CNS of patients with such neurodegenerative diseases. By far the bulk of this body of evidence is related to studies in Alzheimer\'s disease \[[@B18]\]. What distinguishes Alzheimer\'s disease from other neurodegenerative diseases is the conspicuous presence of extracellular deposits of amyloid in senile plaques. Senile plaques in Alzheimer brain are present in different stages of maturity, ranging from diffuse to neuritic to dense core, but they all contain the amyloid beta protein (Aβ). Aβ is a peptide that forms insoluble and pathological extracellular aggregates that seem to attract microglial cells, as suggested by the clustering of microglia at sites of Aβ deposition (see \[[@B19]\] for a review). There is evidence from experimental studies in animals to support the idea that microglia can phagocytose and degrade amyloid \[[@B20],[@B21]\], but such phagocytosis is apparently either ineffective or inadequate in Alzheimer\'s disease. A key question within the current context is: \"Does the amyloid in Alzheimer brain by itself represent a persistent injurious stimulus that causes neuronal injury, or are additional factors involved in eliciting this outcome?\" Direct injection of Aβ into the brain produces activation of microglia and loss of specific populations of neurons \[[@B21]\]. Furthermore, transgenic mice that overexpress human, mutant β-amyloid precursor protein (βAPP) do develop Aβ deposits with associated evidence of neuritic injury (although they do not develop Alzheimer-type neurofibrillary tangles unless they are also transgenic for human tau protein) \[[@B22]\]. These Aβ deposits, born of transgenic overexpression of mutant human amyloid precursor protein, invariably contain activated microglia \[[@B22],[@B23]\].
β-Amyloid precursor protein βAPP functions as a neuronal acute-phase, injury-response protein. For instance, there is excessive expression of βAPP, accompanied by microglial activation and cytokine expression, after traumatic head injury \[[@B24]\]. With head injury, there is also Aβ deposition, both in experimental animals \[[@B25]\] and in humans -- particularly in individuals genetically susceptible for AD (i.e. ApoE ε4-positive) \[[@B26]\]. These observations emphasize the complex interactions that underlie neurodegeneration in Alzheimer\'s disease.
Conclusions
===========
Chronic microglial activation is an important component of neurodegenerative diseases, and this chronic neuroinflammatory component likely contributes to neuronal dysfunction, injury, and loss (and hence to disease progression) in these diseases. The recognition of microglia as the brain\'s intrinsic immune system, and the understanding that chronic activation of this system leads to pathologic sequelae, has led to the modern concept of neuroinflammation. This vision of microglia-driven neuroinflammatory responses, with neuropathological consequences, has extended the older vision of passive glial responses that are inherent in the concept of \"reactive gliosis.\"
Abbreviations
=============
Aβ: β-amyloid peptide
βAPP: Aβ precursor protein
CNS: central nervous system
HIV: human immunodeficiency virus
MS: multiple sclerosis
Competing interests
===================
None declared
Authors\' contributions
=======================
WJS conceived this review, wrote the initial draft, modified this with the comments of REM and WSTG, and wrote the final draft. REM and WSTG contributed particularly to the sections on infections and on Alzheimer\'s disease. All authors read and approved the final version.
Acknowledgments
===============
Supported in part by NIH R21 NINDS 049185, NIH PO1 AG 12411, NIH P30 AG 19606, NIH RO1 AG 37989, and the McKnight Brain Research Foundation at the University of Florida.
| PubMed Central | 2024-06-05T03:55:46.854469 | 2004-7-30 | {
"license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/",
"url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC509427/",
"journal": "J Neuroinflammation. 2004 Jul 30; 1:14",
"authors": [
{
"first": "Wolfgang J",
"last": "Streit"
},
{
"first": "Robert E",
"last": "Mrak"
},
{
"first": "W Sue T",
"last": "Griffin"
}
]
} |