bluuebunny/crossref_metadata_2025 huggingface dataset:
IgM‐enriched human intravenous immunoglobulin strongly inhibits complement‐dependent porcine cell cytotoxicity mediated by human xenoreactive antibodies
Authors: Roos Anja , Rieben Robert , Faber‐Krol Maria C. , Daha Mohamed R.
Published: October 2003

Abstract:  Treatment with intravenous immunoglobulin preparations consisting of human IgG (IVIgG) prevents hyperacute rejection of pig xenografts transplanted into primates by inhibition of the classical complement pathway. Recent studies indicate that IVIg preparations mainly consisting of human IgM (IVIgM) have a stronger capacity than IVIgG to inhibit the complement system. IVIg preparations also contain xenoreactive antibodies (XAb) binding to pig cells. In the present study, we compared IVIgG and IVIgM for their capacity to inhibit xenogeneic complement activation, with special reference to the roles of IgG and IgM XAb present in these preparations. Xenogeneic complement activation was studied by exposure of pig cells (PK15) to human serum. For some experiments, IVIgG and IVIgM were depleted from XAb by immune absorption. Exposure of PK15 cells to human serum induced surface deposition of C4 and C3 and cytotoxicity, which could be inhibited in a dose‐dependent fashion by both IVIgM and IVIgG. The efficacy of IVIgM was more than 10 times higher than that of IVIgG. IgG XAb were detected in both IVIgG and IVIgM whereas IgM XAb were only present in IVIgM. Depletion of XAb from the IVIg preparations did not modify the protective properties of IVIgG against cytotoxicity induced by human serum, whereas the IVIgM‐mediated protection against xenogeneic cytotoxicity was only slightly improved. IgM‐enriched IVIg is a potent inhibitor of xenogeneic complement activation and complement‐dependent cytotoxicity of human serum to pig cells, irrespective of the presence of cytotoxic xenoreactive IgM antibodies in this preparation. Therefore, IVIgM has a promising therapeutic significance for the treatment of (hyper)acute xenograft rejection.
Tacrolimus inhibits discordant islet xenograft rejection: a study in the pig‐to‐rat model
Authors: Song Z , Zhang J , Bennet W , Wennberg L
Published: October 2003

Abstract: The aim of the present study was to evaluate the immunosuppressive effect of tacrolimus (TAC) in discordant islet xenotransplantation. Fetal porcine islet‐like cell clusters (ICCs) were transplanted under the kidney capsule in normoglycemic rats treated with TAC monotherapy, TAC plus other immunosuppressive drugs or cyclosporin A (CsA) monotherapy. Twelve or 24 days after transplantation, the extent of a cellular infiltration in the xenografts was evaluated using immunohistochemistry. In some animals, the grafts were examined for antibody and complement deposition and the levels of xenoreactive antibodies in serum were determined. In untreated rats, the xenografts were completely rejected after 12 days and no intact ICCs remained. TAC monotherapy (at 0.5 and 1.0 mg/kg b.w.) almost completely inhibited rejection for up to 12 days. In animals treated with TAC monotherapy (at 0.5 mg/kg b.w.), rejection was markedly inhibited for up to 24 days. However, the effect after 24 days was not consistent and in some grafts there were signs of rejection. The protective effect of TAC observed in this study is in contrast to the findings in rats given CsA monotherapy in which no or only a marginal effect on islet xenograft rejection was observed. Only when CsA was given at 20 mg/kg b.w., an inhibitory effect could be observed. Immunosuppression with TAC at a suboptimal dose (0.3 mg/kg b.w.) plus 15‐deoxyspergualin or brequinar also had an inhibitory effect on the rejection. In animals given TAC plus mycophenolate mofetil, a protective effect was observed as well; however, this effect was not consistent.
Porcine pancreatic icosapeptide as a marker of graft survival and rejection in xenotransplantation
Authors: Amaratunga Anil , Khoury Pauline , Wang Liping , Williams Lindy , Tuch Bernard E.
Published: October 2003

Abstract:  The previous study showed that it is possible to monitor the viability of xenografted fetal pig pancreas in the first 3 weeks after transplantation in a normoglycemic immunoincompetent mouse [1]. This is achieved by measuring serum levels of porcine pancreatic icosapeptide (PI) in the host using a specific immunoassay. PI is secreted from the pancreatic polypeptide (PP) cell, the first mature endocrine cell of the fetal pig pancreas. Insufficient insulin is produced by the graft at this time to use it as a marker of graft viability. We have now examined the usefulness of PI to monitor graft survival in diabetic immunoincompetent mice and in immunosuppressed mice. Fetal porcine pancreatic tissue was transplanted beneath the renal capsule of streptozotocin‐diabetic non‐obese diabetic severe combined immunodeficient mice (NOD‐SCID) and BALB/c mice given cyclosporin (CsA) for 21 or 10 days, respectively. Blood and grafts were analyzed periodically over 15 and 3 weeks, respectively, for porcine PI and C‐peptide/insulin. In the diabetic mice, porcine PI was detected in sera up to 2 weeks following transplantation and was absent thereafter. Serum porcine C‐peptide was detectable at 3 weeks and reached maximum levels at 12 to 15 weeks. PI content of the grafts was highest at day 4 and 3 weeks and lowest from 9 weeks onwards while insulin levels in the graft were highest from 9 weeks onwards. In BALB/c mice immunosuppressed with CsA, serum PI was detectable for 21 days. In the mice given CsA only for 10 days, serum PI was detectable for that time, but not on day 14 although PI was detectable in the graft. In mice given no CsA, PI was undetectable in serum at any time although PI was present in the graft at day 4. The presence of porcine PI in sera is a marker of graft survival of fetal porcine pancreatic tissue in diabetic immunocompetent mice in the first 2 weeks after transplantation. Its absence in immunosuppressed mice in this period is an early indicator of graft rejection.
A manic‐depressive symptom self‐report in optical scanable format
Authors: Glick Henry A , McBride Linda , Bauer Mark S
Published: October 2003

Introduction: The Internal State Scale (ISS) is a self‐report instrument that allows the simultaneous assessment of both manic and depressive symptoms in individuals with manic‐depressive disorder. Prior work indicates that subscales are highly correlated with clinician ratings of mania and depression and provide a discriminant function that identifies individuals in manic/hypomanic, mixed, depressed, and euthymic mood states. A drawback to the ISS is that its items were developed in the visual analogue scale (VAS) format which is labor‐intensive to score, particularly with repeat (e.g. daily) administration. A Likert‐based format would allow quick and easy optical scanning for which scoring could be automated.

Methods: To compare discriminating properties in Likert versus VAS format we re‐analyzed previously collected data and collected new data: (a) VAS‐based ISS scores from 86 subjects from a prior four‐site study were re‐analyzed by collapsing scores into 20 and then 10 Likert‐based bins to assess loss of precision from collapsing scores, and (b) 24 additional subjects were administered the ISS in VAS and Likert formats to assess loss of precision due to instrument completion factors.

Results: Discriminant ability, including kappas and receiver operator characteristic curves, were unchanged across the two formats. Within‐subjects reliability was uniformally high across formats.

Conclusions: Likert‐based scoring of the ISS can be used without loss of precision, thus making automated scoring of the ISS feasible. This format will be particularly useful for studies that require processing of large numbers of ISSs, such as those that collect frequent ratings over long periods of time and/or those that utilize large samples.
Life events and onset of a new phase in bipolar affective disorder
Authors: Christensen Ellen Margrethe , Gjerris Annette , Larsen Jens Knud , Bendtsen Birgitte Bjerg , Larsen Birgitte Holt , Rolff Heidi , Ring Gudrun , Schaumburg Eric
Published: October 2003

Background: There is an increasing focus on the impact of psychosocial factors and stressors on the course of bipolar affective disorder. The life event research has revealed many biases and the results are conflicting. In a prospective study we examined the relationship between life events and affective phases in a group of bipolar patients with a long duration of the disease.

Methods: A group of patients with at least three admissions to hospital for bipolar disorder was followed every 3 months for up to 3 years. At each examination an evaluation of affective phase was made according to the Hamilton Depression Scale, the Newcastle Depression Rating Scale and the Bech‐Rafaelsen Mania Rating Scale. Moreover, the patients were rated according to the Paykel Life Events Scale. Their current medical treatment was noted.

Results: Fifty‐six patients (19 men and 37 women) were included in the study. Women experienced a significantly higher number of life events than men. In 21% of the 353 examinations of women, a new phase was preceded by life events whereas this was the case only in 8% of the 152 examinations of men. In 13% of the male examinations the patients were in a manic phase and in 5% in a depressive phase. In 5% of the female examinations the patients were in a manic phase and in 15% in a depressive phase. Half of the women's depressive phases were preceded by life events, but none of the depressive phases of men. The categories of life events preceding the depressive phases presented a significant overweight of somatic ill health and conflicts in the family.

Conclusion: We found a gender difference in the course of bipolar affective disorder, as women had a significantly higher number of depressive episodes than men and men had a higher number of manic episodes than women. In bipolar patients with long duration of disease a significant number of depressive episodes in women were preceded by negative life events. Somatic health problems and conflicts in the family were significant factors preceding new depressive phases.
Bipolar disorder. II: Personality and age of onset
Authors: Engström Christer , Brändström Sven , Sigvardsson Sören , Cloninger Robert , Nylander Per‐Olof
Published: October 2003

Objectives:The aim of this study was to examine whether personality i.e. temperament and character interacts with age of onset in bipolar disorder.

Methods:Bipolar patients were recruited among in‐ and outpatients from lithium dispensaries of northern Sweden. Patients were diagnosed according to DSM‐IV criteria for bipolar disorder type I and II. Temperament and Character Inventory (TCI) was used for measuring personality. TCI was administered to 100 lithium treated bipolar patients and 100 controls.

Results:Treatment response was significantly lower (p = 0.005) in patients with early onset compared with late onset. Family history (p = 0.013) and suicide attempts (p = 0.001) were also significantly more common in patients with early onset. Further, patients with early onset were significantly higher (p = 0.045) in the temperament factor harm avoidance (HA) than patients with late onset, but the difference was weak. Patients with early onset had more fear of uncertainty (HA2; p = 0.022) and were more shy (HA3; p = 0.030). Bipolar I patients showed similar results as those in the total bipolar group (I and II), with significantly higher HA (p = 0.019, moderate difference), HA2 (p = 0.015) and HA3 (p = 0.043) in patients with early onset compared with late onset. Bipolar II patients showed no differences between early and late age of onset but the groups are small and the results are therefore uncertain.

Conclusions:Early age of onset in bipolar disorder was correlated to an increase in severity, family history, poorer treatment response and poorer prognosis. Early onset was also correlated to personality.
Verbal memory in mania: effects of clinical state and task requirements
Authors: Fleck David E , Shear Paula K , Zimmerman Molly E , Getz Glen E , Corey Kimberly B , Jak Amy , Lebowitz Brian K , Strakowski Stephen M
Published: October 2003

Objectives: Manic patients exhibit impaired verbal learning and memory, particularly following longstanding illness. However, it is unclear whether recognition and recall performance are differentially influenced by a manic mood state.

Methods: To examine this issue, we administered the California Verbal Learning Test and symptom‐rating scales to inpatients with pure or mixed mania, euthymic outpatients, and healthy comparison subjects.

Results: An overall performance difference was identified between groups. Manic and euthymic patients performed more poorly than healthy subjects on recall. However, manic patients performed more poorly than euthymic patients and healthy subjects on recognition.

Conclusions: These results suggest that verbal retrieval deficits are stable vulnerability indicators in bipolar disorder, whereas verbal encoding deficits are manic episode indicators. The known subcortical dysfunction in this disorder may produce stable retrieval deficits while acute mood symptoms attenuate encoding during affective episodes only.
Trends in mortality from suicide, 1965–99
Authors: Levi F. , La Vecchia C. , Lucchini F. , Negri E. , Saxena S. , Maulik P. K. , Saraceno B.
Published: October 2003

Objective: To analyse trends in mortality from suicide over the period 1965–99.

Method: Data were derived from the WHO database, including data for 47 countries.

Results: In the European Union (EU), all age suicide mortality peaked at 16.1/100 000 in men in 1980–84, and declined thereafter to 14.4/100 000 in 1995–98. In females, the fall was 29% to reach 4.6/100 000. A similar pattern of trends was observed in several eastern European countries. In contrast, mortality from suicide rose substantially in the Russian Federation, from 37.7/100 000 in males in 1985–89 to 58.3/100 000 in 1995–98 (+55%), and to 9.5/100 000 (+12%) in females. In the USA and most other American countries providing data, no consistent pattern was evident for males, but falls were observed in females. Steady declines were registered for Japan, starting from the highest suicide rates worldwide in the late 1950s. Suicide rates were upwards in Ireland, Italy, Spain, the UK, Cuba, Australia and New Zealand. Substantial rises were observed in a few countries (Ireland, Cuba, Mexico, Australia and New Zealand) for young males.

Conclusion: In spite of mixed trends, suicide remains a significant public health problem worldwide.
No association between two polymorphisms at the5HT2Agene and bipolar affective puerperal psychosis
Authors: Robertson E. , Jones I. , Middle F. , Moray J. , Craddock N.
Published: October 2003

Objective:To examine whether variation at two common polymorphisms, T102C and −1438AG, of the serotonin 2A gene (5HT2A) are involved in the puerperal triggering mechanism of bipolar affective puerperal psychosis.

Method:A total of 242 parous women diagnosed with bipolar disorder were genotyped for the two polymorphisms. Of these, 165 women had experienced a manic or psychotic episode, according to DSM‐IV criteria, within 6 weeks of childbirth (the puerperal psychosis group).

The comparison group comprised of 77 parous women who had not experienced psychiatric disturbance following childbirth.

Results:No significant differences between genotype or allelic frequencies were found between the two groups for either polymorphism.

Conclusion:The results indicate that variation at two common polymorphisms of the5HT2Agene does not appear to play a major role in the development of bipolar affective puerperal psychosis.
Chlamydia trachomatis seropositivity is associated both with stillbirth and preterm delivery
Authors: Gencay MESUT , Koskiniemi MARJALEENA , ÄMmÄLÄ PIRKKO , Fellman VINETA , NÄRvÄNen ALE , WahlstrÖM TORSTEN , Vaheri ANTTI , Puolakkainen MIRJA
Published: October 2003

The cause of stillbirth and preterm delivery is often unknown. We studied the prevalence of Chlamydia trachomatis antibodies in mothers with stillbirth and preterm labor. Serum specimens from 72 mothers with stillbirth after the 21st gestational week, and from 48 mothers with preterm delivery between gestational weeks 23 and 29, both from the greater Helsinki area, and cord blood from 96 consecutive liveborn deliveries at the Department of Obstetrics and Gynecology, the University of Helsinki, were studied for antibodies to C. trachomatis immunotypes CJHI, GFK and BED by microimmunofluorescence test. The prevalence of C. trachomatis antibodies was highest, 33.3%, in mothers with stillbirth, 18.8% in mothers with preterm delivery, and 10.4% in cord blood. The IgM seropositivity rate was high among mothers with preterm delivery (8.3%). We conclude that C. trachomatis IgG antibodies are frequently detected in sera from mothers with stillbirth, suggesting past infection, while mothers with preterm delivery often have serum IgM antibodies, suggesting of acute infection.
A new DIG‐PCR‐EIA method for the detection of Chlamydia pneumoniae DNA in clinical samples
Authors: Gnarpe JUDY , Lindquist LINDA
Published: October 2003

Chlamydia pneumoniae, a common respiratory pathogen, may also play a role in the pathogenesis of other chronic conditions. For accurate detection of infected persons and verification of results obtained by other PCR methods, a DIG‐PCR‐EIA method was evaluated. In the DIG‐PCR‐EIA, a 437 bp DNA sequence was amplified and hybridized with a newly synthesized 229 bp biotin‐labeled probe. The end product was detected by an enzyme immunoassay. The sensitivity of DIG‐PCR‐EIA was compared with Southern blot hybridization and one‐step HR/HL PCR, which was the routine method used. DNA was detected to the level of 20 elementary bodies of DIG‐EIA‐PCR compared to less than 2 by Southern blot, and 200 by HR/HL PCR. Thus a 100‐fold increase in sensitivity could be expected by DIG‐EIA‐PCR compared to the routine method. Throat swabs and adenoid tissue from 22 children with otitis and middle ear secretions from 29 children, as well as throat swabs from 179 blood donors, were analyzed with DIG‐EIA‐PCR, HL/HR PCR and nested touchdown PCR. 32% of the ear secretions were positive by DIG‐EIA‐PCR as compared to 5% by the other two methods. Three adenoid tissue samples were positive by all methods applied. Among the child and adult throat samples, 18% and 32%, respectively, were positive by DIG‐EIA‐PCR and 5% and 10% by HR/HL‐PCR. The results indicate the suitability of DIG‐PCR‐EIA for verification of results of HR/HL PCR. DIG‐PCR‐EIA has a potential for increased sensitivity and adaptation for automation. It should be further evaluated using various types of tissue specimens and DNA extraction methods.
Erythema migrans ‐ influence of posture
Authors: Oksi J. , Hietarinta M. , Viljanen M. K.
Published: October 2003

Despite widespread awareness of the most classical clinical presentation with central clearing of erythema migrans, a pathognomonic sign of infection with Borrelia burgdorferi, diagnosis of other forms of erythema migrans remains more difficult. We describe a case of a patient with secondary lesions of erythema migrans that within three months formed a complicated pattern and affected at last nearly the entire lower limb of the patient. In addition, the erythema appeared to be posture‐dependent in the way that the lesion was with central clearing in the supine and with homogeneous appearance in the upright position. The borrelial infection was confirmed by PCR sequencing that detected DNA of B. afzelii in the skin biopsy specimen. The lesions disappeared during antibiotic therapy. This case shows how posture can be important in the examination of patients with a suspected erythema migrans.
Genomic diversity of oral Candida krusei isolates as revealed by DNA fingerprinting and electrophoreric karyotyping
Authors: Dassanayake R. S. , Samaranayake Y. H. , Samaranayake L. P.
Published: October 2003

Candida krusei is receiving increasing attention as an important human pathogen, especially in compromised patients, who frequently manifest with multiepisodes of candidosis. As there is scant information on the genetic diversity of this pathogen the present study was undertaken to establish its genetic profiles using three different typing methods: PFGE (pulsed‐field gel electrophoresis), RFLP (restriction fragment length polymorphism), RAPD (randomly amplified polymorphic DNA). When 11 oral isolates of C. krusei were molecular typed by PFGE, 3 to 5 chromosomes with sizes ranging from 1000 kb to 3000 kb per isolate were revealed. All isolates produced a single bright band at approximately 1,100 kb and two to three bands between 2,500 kb and 3,000 kb, demonstrating 5 different karyotypes. RFLP with Hinfl yielded 9 different genotypes, while DNA fingerprinting by RAPD with 3 primers (RSD6 (5′GCGATCCCCA3′), RSD7 (5′AGTGAATTCG CGGTGA‐GATGCC3′) and RSD12 (5′GCATATCAATAAGC GCAGGAAAAG 3′)), resulted in 8, 3 and 11 different genotypes, respectively. This study provides evidence hitherto unavailable on the genetic polymorphism of C krusei isolates colonizing the oral niche under different clinical conditions. Such genotypic polymorphism should help strain delineation in epidemiologic surveillance of either nosocomial or community outbreaks of C. krusei infections.
NO and de novo mammalian angiogenesis: Further evidence that NO inhibits bFGF‐induced angiogenesis while not influencing VEGF165‐induced angiogenesis
Authors: Näslund Ingrid , Norrby Klas
Published: October 2003

Using the non‐surgical rat mesenteric window angiogenesis assay, we studied the systemic effect of (i) the nitric oxide (NO)‐releasing vasodilator isosorbide‐5‐mononitrate (ISMN) and (ii) the NO‐synthase inhibitor L‐NAME on angiogenesis induced by the intraperitoneal injection of bFGF and VEGF165. The response was assessed objectively and quantitatively by microscopic morphometry and image analysis in terms of the vascularized area (VA; a measurement of microvessel spatial extension), the microvascular length (MVL; a composite measurement of microvessel density), the total microvascular length (TMVL=VAxMVL), the number of microvessel segments per unit tissue volume (No. MS), the length of the microvessel segments (Le. MS) and the degree of microvessel tortuosity (MVT). Additional architectural features of the network were assessed in terms of variables introduced here: the number of microvessel branching points per unit tissue volume (No. BP), the index of interconnecting microvessel loop formation (In. LF), the index of microvessel intersection (In. IS), the number of microvessel sprouts per unit tissue volume (No. SP) and their length (Le. SP). In bFGF‐mediated angiogenesis, L‐NAME significantly, augmented angiogenesis, whereas ISMN significantly inhibited angiogenesis. By contrast, neither L‐NAME nor ISMN affected the angiogenic response to VEGF165.
p53 and Bcl‐2 oncoprotein expression in placentas with hydropic changes and partial and complete moles
Authors: Al‐Bozom ISSAM A.
Published: October 2003

Forty molar and non‐molar placentas with hydropic changes (14 complete moles, 14 partial moles, and 12 hydropic non‐molar placentas) were examined immunohistochemically for expression of oncoproteins p53 and Bcl‐2. The data were evaluated to determine if p53 and Bcl‐2 expression could aid in differentiating molar from non‐molar pregnancies on the one hand and complete mole from partial mole on the other. Thirteen out of fourteen complete moles showed p53 expression (93%), 8 of the 14 partial moles expressed p53 (57%), and none of the non‐molar pregnancies expressed p53 in the extravillous intermediate trophoblasts. Regarding Bcl‐2, the syncytiotrophoblasts of most molar and non‐molar placentas showed strong and diffuse positivity. These results suggest that p53 expression can be used as a distinguishing parameter between complete moles and placentas with hydropic changes on the one hand and to a lesser extent between partial moles and placentas with hydropic changes on the other hand. Bcl‐2 cannot be used in the same way as p53.
Distribution and sequence variations of selected virulence genes among group A streptococcal isolates from western Norway
Authors: Mylvaganam H. , Bjorvatn B. , Osland A.
Published: October 2003

In order to compare the distribution of selected virulence genes among group A streptococci recovered from invasive disease and superficial infections, 42 isolates were screened for mga, speB, speA, ssa and ska, by PCR. The isolates were predominantly of the sequence types emm1, emm3 and emm6, but also included a few of the types emm22, emm28, emm75 and emm78. The phage‐mediated speA seemed to be prevalent in emm types 1 and 3, and its distribution was not related to disease severity. The other genes were present in all isolates. The mga, speB and speA were further studied by sequence analysis. Although allotypic associations with invasiveness were not found, allelic specificity to the emm sequence type was observed. In addition, the mga sequences indicated two lineages, related to opacity factor production. A possible recombination between these two main divergent mga genes was observed in isolates of the types emm22 and emm15. A logical nomenclature of the alleles of mga and speB is suggested.
Application of the polymerase chain reaction in determination of recombinant retrovirus titers as fifty percent endpoints
Authors: Husemoen Lise Lotte N. , Gram Gregers J. , Hansen John‐Erik S.
Published: October 2003

Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. One of the most laborious routine assays in the application of retroviral‐mediated gene transfer is the determination of viral titers of vector producer cell lines. Traditionally, determination of virus titer involves the testing of culture medium from individual packaging cell lines for the ability to transfer drug resistance to susceptible cells – a process that can easily take up to 14 days. It is generally agreed that this method is cumbersome. We sought to develop PCR‐based protocols that would significantly simplify and shorten this procedure. Using PCR and primers specific for the Neoregion of the MLV‐derived vector LeGSN, we determined 1. the proviral integration in target cells, and 2. the viral nucleic acid (RNA or DNA) content of the vector stock. Results were compared with those using the conventional method. We found that these specific PCR‐based procedures were indeed useful for rapid determination of viral titers as well as for quick screening for high‐titer vectorproducing cell clones and successful transduction of target cells.
Molecular epidemiology of viral infections.
Authors: Hungnes O. , Jonassen T. ø. , Jonassen C. M. , Grinde B.
Published: October 2003

Viruses evolve much faster than cellular organisms. Together with recent advances in nucleic acid sequencing and biocomputing, this allows us to distinguish between related strains of viruses, and to deduce the relationships between viruses from different outbreaks or individual patients. Databases of nucleotide sequences contain a large number of viral sequences with which novel sequences from local outbreaks can be compared. In this way the dissemination of viruses can be followed both locally and globally. We here review the biological and technological background to the use of virus nucleic acid sequences in epidemiological studies, and provide examples of how this information can be used to monitor human viruses. Molecular studies are particularly valuable for understanding the dissemination and evolution of viruses. The knowledge obtained is useful in epidemiological reconstructions, in real‐time surveillance, and may even enable us to make predictions about the future developments of viral diseases.
Evaluation of a commercial test for antibodies to the chlamydial lipopolysaccharide (MedacTM) for serodiagnosis of acute infections by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci
Authors: Persson Kenneth , Haidl Sven
Published: October 2003

A commercial test (rELISA) based on a recombinant chlamydial lipopolysaccharide (LPS) antigen has been evaluated for the diagnosis of acute infections caused by Chlamydia pneumoniae (TWAR) and Chlamydia psittaci. This test and a microimmunofluorescence test (MIF) were compared in 160 patients with community‐acquired pneumonia. Seventeen of nineteen cases with significant titre changes detected by rELISA were confirmed by MIF. The two remaining cases not confirmed by MIF were considered false‐positive reactions. One case positive by MIF only was judged not to be a true‐positive reaction. All three cases occurred in patients with Mycoplasma pneumoniae infection and may be the result of a mitogenic effect. High antibody titres have been used to indicate acute C. pneumoniae infection. We found high MIF or rELISA titres to be equally common in patients and controls; no association between the two tests was detected. An unexpected cross‐reactivity between the rELISA antigen and parvovirus was observed, which might have diagnostic implications. Both MIF and rELISA detected acute C. pneumoniae and C psittaci infection, and there was good agreement between the tests. Single serum diagnosis was generally not feasible with either MIF or rELISA.
Characterization of the genetic diversity in superficial and systemic human isolates of Candida parapsilosis by randomly amplified polymorphic DNA (RAPD)
Authors: Dassanayake R. S. , Samaranayake L. P.
Published: October 2003

The application of randomly amplified polymorphic DNA (RAPD) technology for strain delineation of medically important yeasts has proved to be a valuable tool in clinico‐epidemiological studies of Candida species. Candida parapsilosis, a form species of the fungi imperfecti, is an emerging pathogen gaining recognition as an opportunistic agent, especially in the immunocompromised. Therefore, 15 clinical isolates of C. parapsilosis obtained from oral, cutaneous and systemic Candida infections were typed by RAPD analysis using four different primers. The primers RSD6 and RSD9 elicited 7 geno‐types each, whereas primers RSD7 and RSD12 revealed 6 and 10 genotypes, respectively. When the data were correlated, a higher degree of genomic heterogeneity in systemic isolates was noted compared with the oral and cutaneous isolates, which shared somewhat similar RAPD profiles. However, a single oral isolate (P5) and two systemic isolates (P13 and P15) elicited radically divergent profiles, dissimilar to their counterparts. RAPD study of the latter two isolates with three additional primers (RSD8, RSD10 and RSD11) confirmed the observed genomic disparity. These data substantiate the previous observations on the genomic heterogeneity in C. parapsilosis and point to genetic shifts which may be associated with ecodiversity, as well as the possible existence of distinct genetic groups within this form species.
Identification of a Salmonella typhimurium genomic region involved in invasion of HeLa and Henle‐407 epithelial cells
Authors: Park Jeong U. , Smith Stuart C. , Coloe Peter J.
Published: October 2003

To identify the invasion determinant, a cosmid library was constructed by cloning a genomic library of Salmonella typhimurium 82/6915 into a cosmid vector, pLA2917. A genomic region involved in invasion of cultured HeLa and Henle‐407 cells was subcloned into plasmid pGEM‐7Z. E. coli strain DH1 carrying pSV6235 consisting of a S. typhimurium 4.6 kb genomic region in pGEM‐7Z showed invasion of cultured HeLa and Henle‐407 cells. Nested sequential deletions were introduced into the 4.6 kb genomic region of pSV6235. The E. coli recombinants which contained less than 1.5 kb deletions from the 5′ end (SmaI site) of the genomic region invaded the cells as effectively as DH1 (pSV6235). The invasion of the recombinants carrying over 2.0 kb deletions from the end of pSV6235 was significantly inactivated compared to DH1 (pSV6235). Restriction enzyme analysis showed that the 3.1 kb fragment from the 3′ end of the 4.6 kb genomic region was distinguished from the Salmonella pathogenicity I genes of S. typhimurium such as the inv, spa, and hil regions showing invasion of the cultured eukaryotic cells.
Absence of mutations in the kinase domain of the Met gene and frequent expression of Met and HGF/SF protein in primary gastric carcinomas
Authors: Park Won Sang , Oh Ro Ra , Kim Young Sil , Park Jik Young , Shin Min Sun , Lee Hun Kyoung , Lee Sug Hyung , Yoo Nam Jin , Lee Jung Young
Published: October 2003

Hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, are known to play important roles in tumor cell migration, invasion, and metastasis. We analyzed the expression of these genes and the mutations in the kinase domain of the Met gene in 43 gastric carcinomas. Of the 43 cases, the Met and HGF/SF protein were expressed in 29 (67%) and 22 (51%), respectively. All of the cases with HGF/SF immunopositivity also expressed Met. Of 22 cases with HGF/SF immunopositivity, 13 (59%) expressed HGF/SF in tumor cells as well as fibroblasts. We detected no aberrant single‐strand conformational polymorphism patterns, suggesting that there are no genetic alterations in the kinase domain of the Met gene. These results indicate that HGF/SF‐mediated autocrine and/or paracrine stimulation and overexpression rather than structural alteration of its receptor may contribute to the development and progression of gastric carcinoma, and that expression of Met and HGF/SF may confer a growth advantage to neoplastic cells.
The bactericidal/permeability‐increasing protein (BPI) is membrane‐associated in azurophil granules of human neutrophils, and relocation occurs upon cellular activation
Authors: Calafat Jero , Janssen Hans , Knol Edward F. , Malm Johan , Egesten Arne
Published: October 2003

Neutrophilic granulocytes contain the 55 kDa bactericidal/permeability‐increasing protein (BPI). BPI binds to lipopolysaccharides (LPS), and exerts bacteriostatic and bactericidal effects against a wide variety of Gram‐negative bacterial species. We have investigated the subcellular location of BPI in immature and mature neutrophils using cryotechnique for immunoelectron microscopy. BPI was found to colocate with myeloperoxidase (MPO), a marker for azurophil granules, and it also showed the same pattern of distribution as CD63, a transmembrane‐anchored protein. This suggests that BPI is membrane‐associated in the azurophil granules in neutrophils. Its presence in azurophil granules was further confirmed by the finding of BPI in the azurophil granules of neutrophil promyelocytes of the bone marrow. Induction of selective release of azurophilic granules by the Na‐ionophore monensin resulted in fusion of endosomes with azurophil granules, leading to the formation of large vacuoles containing MPO, CD63, and BPI. After phagocytosis of serum‐treated zymosan (STZ), BPI was detected in phagosomes, both in association with membranes as well as in the lumen, suggesting the release of BPI into activated compartments. The results show that BPI is present in azurophil granules, is probably primarily membrane‐associated, and is relocated after activation, following the same route as MPO and CD63.
Expression of topoisomerase II and Ki‐67 in cervical carcinoma — clinicopathological study using immunohistochemistry
Authors: Davidson Ben , Goldberg Iris , Lerner‐Geva Liat , Gotlieb Walter H. , Ben‐Baruch GIlad , Novikov Ilya , Kopolovic Juri
Published: October 2003

Aim. To study the correlation between the expression of topoisomerase II and Ki‐67 antigen and disease outcome in cervical squamous cell carcinomas.

Experimental design. Forty‐nine cervical carcinomas, 10 cases of high‐grade cervical intraepithelial neoplasia (CIN II‐III) and 5 control cervices were stained by monoclonal antibodies for topoisomerase II and Ki‐67 (MIB‐1 clone). Nuclear counts were correlated with patient age, tumor stage, histological grade and survival.

Results. Thirteen patients died of disease, 35 remained free of disease, and one patient was lost to follow up. Ki‐67 counts were higher in CIN lesions, when compared to both invasive carcinomas and control cervices. Topoisomerase II counts were comparable for CIN and invasive tumors. No immunoreactivity for topoisomerase was detected in control cases. Neither stage nor grade was associated with nuclear counts using either marker. In multivariate survival analysis, stage (p=0.001), grade (p=0.03) and older patient age (p=0.02) predicted poor survival. Ki‐67 counts predicted survival with borderline significance (p=0.07), while topoisomerase II counts were not related to survival.

Conclusion. Ki‐67 and topoisomerase II counts do not appear to have a significant role in the prediction of survival in cervical squamous cell carcinoma.
Pig endogenous retrovirus – a threat to clinical xenotransplantation?
Authors: Fiane Arnt E. , Mollnes Tom E. , Degre Miklos
Published: October 2003

Transplantation shows good results for patients with end‐stage disease, but there is an increasing lack of organs. Xenotransplantation, the transfer of live animal cells, tissues, or organs to another species, offers a potential solution to this shortfall. Pig is regarded as the animal of choice for this purpose. Meanwhile demonstration of pig endogenous retrovirus (PERV) in all porcine herds has caused serious concern with respect to a possible transmission of the virus to humans with a transplanted organ. Transmission to human cells has been documented under certain in vitro conditions. However, no such transmission has been demonstrated in vivo. The possible consequences of introducing PERV into immunocompromised human organisms are not known and it is necessary to collect more information. Novel and sensitive genomic assays to detect PERV infection are now available in addition to established virological, immunoserological and molecular methods. In order to minimise the risk of PERV transmission rigorous procedures should be established. International guidelines to reduce the risk should be followed. Although a number of immunological, physiological and virological questions need to be answered before the introduction of xenotransplantation as an alternative clinical treatment, some problems can only be solved by judicious clinical trials.
Disease‐dependent changes in T‐cell populations in patients with shigellosis
Authors: Islam Dilara , Christensson Birger
Published: October 2003

In shigellosis, bacterial infection is associated with an extensive inflammation of the rectal mucosa, resulting in bloody dysentery. The role of T‐cell‐mediated pro‐inflammatory mechanisms has been implicated in this process, but the specific role of T‐cell subsets is still not well understood. In this study we attempted to identify the changes in T‐cell populations in patients with shigellosis during the disease course. The T‐cell subset distribution was analyzed by immunohistochemistry in the rectal mucosa and by immuno‐flow cytometry in the peripheral blood. Blood and rectal biopsies were studied from patients with Shigella dysenteriae 1 (n=11) and S. flexneri (n=11) infection and 20 healthy age‐matched controls. We found an expansion of γ,δ+T cells in the rectal mucosa, but a decrease in the percentage of γ,δ+T cells in the blood in acute shigellosis. There was also a preferential increase in CD8+ T cells in the surface epithelium of rectal tissue in patients infected with S. dysenteriae 1, but not in patients infected with S. flexneri. Our findings suggest that the rectal mucosal inflammation in shigellosis is associated with an expansion of T cells, in particular CD8+ and γ,δ+T‐cell subsets in the gut mucosa, which may be of importance for the pathogenesis of shigellosis.
Detection of Mycobacterium‐specific interferon‐gamma‐producing human T lymphocytes by flow cytometry
Authors: Tilley Peter A. G. , Menon Juthika N.
Published: October 2003

Flow cytometry has proven to be a useful tool for the investigation of cytokine synthesis by selected cell subpopulations. While most reports have used mitogen stimulation or long‐term cultures with antigen, we describe here a novel method to allow the detection of rare mycobacterial antigen‐specific cytokine synthesizing cells within one day. The most important feature of this method is the use of an FITC‐conjugated isotype‐matched control antibody to identify and exclude cells which fluoresce non‐specifically. With this technique, we demonstrate interferon‐γ (IFN‐γ) staining in 785 cells per 1times105 T cells counted, in mycobacterial antigen‐stimulated peripheral blood mononuclear cells from a BCG‐vaccinated subject. In comparison, only 14 IFN‐γ‐staining T cells were seen in the cultures not stimulated by mycobacterial antigen. Less than 10 cells per 1times105 T cells are stained by an irrelevant control antibody. Specific responses are detectable after 12 h of in vitro culture, and peak at 24 h. In volunteer health care workers, IFN‐γ staining correlated with IFN‐γ production using a published ELISPOT assay (r=0.927). IFN‐γ staining was also higher in PBMC from mantoux skin test‐positive volunteers, compared to cells from skin test‐negative subjects (p=0.0045). Flow cytometry following short‐term culture can thus be used for enumeration of antigen‐specific IFN‐γ synthesizing cells.
Lipid profiles of Helicobacter pylori colony variants
Authors: Tannæs Tone , Grav Hans J. , Bukholm Geir
Published: October 2003

A phase variation in Helicobacter pylori has been previously described. In one phase the bacterium had a cell wall lipid content typical for gram‐negative bacteria (HpL), whereas in the other phase the bacterium was found to have a cell wall with increased amounts of lysophospholipids (HpS). The conversion is spontaneous, but could also be induced by acid (HpSind) and was associated with in vitro release of Vac A and urease. The purpose of the present study was to determine the full phospholipid content of the cell wall to indicate a molecular mechanism of the colony variation. There were no appreciable differences between the lipid profiles of HpS and HpSind, while there were major differences between HpL and the S‐variant. In the S‐variant, lysophospholipids constituted about 50% of the total phospholipids, as compared to less than 2% in the L‐variant. The proportion of total and individual cholesteryl glucosides also showed considerable changes. HpL was dominated by the phosphate‐linked cholesteryl glucoside (72%) while the acylated cholesteryl glucoside was the main cholesteryl glucoside of the S‐variant (65%). Our results demonstrate a dramatic change in cell wall properties after acid induction and spontaneously in vitro, and suggest some molecular mechanisms for this variation from an in vitro non‐virulent to a virulent variant.
Serological analysis of specific IgA to Chlamydia pneumoniae: increased sensitivity of IgA antibody detection using prolonged incubation and high antigen concentration
Authors: Gnarpe Judy , Sparr Annika , Nääs Jessica , Lundbäck Annika
Published: October 2003

The microimmunofluorescence technique (MIF) is recognized as the only test hitherto allowing discrimination between different Chlamydia species and is considered to be the reference method for serology. This method was developed for the detection of IgG and IgM antibodies only. We investigated the effects of some test parameters on the ability of MIF to detect Chlamydia pneumoniae IgA. These parameters were the time needed for binding of serum IgA to C. pneumoniae antigen and the effect of antigen concentration on the outcome of IgA antibody testing. It was found that the most sensitive MIF tests for the detection of serum IgA antibodies were those in which an overnight incubation of sera with antigen slides containing high concentrations of chlamydial elementary bodies was employed. The number of patients with chronic infections found to have elevated IgA titers was increased by 25% using longer incubation times for the antibody‐antigen reaction. Thirty‐two sera from patients with coronary artery disease and confirmed chronic C. pneumoniae infection were used to compare antigen slides with low and high concentrations of elementary bodies with respect to IgA levels; 31/32 patients were found to have specific IgA antibodies to C. pneumoniae using the high antigen concentration, as opposed to only 22/32 patients using the low antigen concentration.
Reduced phagocytic activity of polymorphonuclear leukocytes in α(1,3) fucosyltransferase VII‐deficient mice
Authors: BartŮNkovÁ Jir̂ina , MalÝ Petr , Smetana Karel , ŠEdivÁ Anna , Klubal Radek , MayerovÁ Dita , SedlÁČEk Aleš , ŠPlÍChalovÁ Vlasta
Published: October 2003

Deficiencies in adhesion molecules or their counter‐receptors in humans may have severe consequences as exemplified by leukocyte adhesion deficiency (LAD) I or II syndromes. Because such diseases occur with great rarity, animal models are valuable for studying the role of particular adhesion molecules and their natural ligands in immunity. We studied selected immune parameters and general health in mice with a defect in the sialyl‐Lewis X antigen (selectin ligand) caused by disruption of the gene encodingα(1,3)fucosyltransferase VII (Fuc‐TVII). Leukocytes from Fuc‐TVII –/– and control mice were tested for adherence to cellophane membranes or polymer particles in vivo and phagocytic activity in vitro. While no difference in adherence was found, the number of neutrophil granulocytes in exudate induced by intraperitoneal injection of polymer beads was reduced in knock‐out mice. Moreover, the phagocytic activity in Fuc‐TVII –/– mice was significantly reduced. These animals have splenomegaly due to increased hematopoiesis and reduced weight but do not exhibit clinical signs of immunodeficiency. In conclusion, the lack of Fuc‐TVII activity leads to several morphological and functional abnormalities without an impact on survival rate.
Strain differentiation in Bacteroides fragilis by ribotyping and computer‐assisted gel analysis
Authors: Eribe Emenike Ribs , Olsen Ingar
Published: October 2003

It is important to distinguish between Bacteroides fragilis strains as their virulence may vary and as B. fragilis seems to be a heterogeneous species. The aim of our study was to evaluate ribotyping for differentiation of 46 strains of B. fragilis and for assessment of strain heterogeneity within and between the two DNA‐DNA homology strain groups established in this species. Twenty‐seven strains belonged to Johnson's DNA homology group I and eight to group II. Eleven strains had not been assigned to any group (NI group). DNA from all strains was cut with BglI, EcoRI and HindIII. Restriction fragment length polymorphisms were investigated using a non‐radioactive digoxigenin‐labelled cDNA probe transcribed from Escherichia coli 16S+23S rRNA. Ribotyping with BglI was most discriminatory, revealing a total of 26 different patterns by visual inspection of gels. EcoRI followed with 20 patterns and HindIII with 13 patterns. The gels from ribotyping were processed using the Dendron computer‐assisted program. Strain clusters established using Dendron were not always in agreement with homology‐based strain groups. Strains of the NI group fell into both homology groups. Ribotyping, as it is based on a relatively small portion of the genome, is useful for strain distinction in epidemiological studies with B. fragilis, whereas DNA‐DNA homology, using the entire genome, is more reliable for taxonomy. The Dendron computer‐assisted program, which enabled objective assessment of multiple banding patterns, increased the reliability of ribotyping.