bluuebunny/crossref_metadata_2025 huggingface dataset:
Laparoscopic Technique for Secondary Vaginoplasty in Male to Female Transsexuals Using a Modified Vascularized Pedicled Sigmoid
Authors: Wedler V. , Meuli-Simmen C. , Guggenheim M. , Schneller-Gustafsson M. , Künzi W.
Published: January 2004

Between 1995 and 2002, 53 primary sex reassignments in male to female transsexuals were performed at our division. The objective of this procedure is to mimic the female external and part of the internal genitalia both esthetically and functionally. In 11 of the 53 patients, a secondary vaginal lengthening had to be performed due to a short neovagina. This was achieved using a pedicled sigmoid segment, with an open approach in the first 2 patients and using a laparoscopic method in the following 9. This paper focuses on the laparoscopic technique, its benefits and potential complications. Where a primary vaginoplasty, combining inversion of the penile and scrotal skin flaps, yields unsatisfactory functional results, a secondary vaginoplasty using the pedicled sigmoid represents an elegant means to achieve functional improvement. Furthermore, we report a modified surgical approach to the conventional sigmoid transition.
Effects of Diazeniumdiolates (NONOates) and Methylene Blue on the Reduction of Postoperative Adhesion in Rats
Authors: Cetin Meral , Duran Bulent , Demirkoprulu Nihal , Guvenal Tevfik , Erden Omur , Cetin Ali
Published: January 2004

We investigated the effects of spermine NONOate (SPER/NO), diethylenetriamine NONOate (DETA/NO) and methylene blue for preventing postoperative adhesion in a rat uterine horn model. Before operations, rats were randomly assigned into 6 groups, each composed of 12 rats. These were the sham, control, normal saline, SPER/NO, DETA/NO and methylene blue groups. Each rat was anesthetized with ketamine hydrochloride (40 mg/kg i.v.). The abdominal wall was shaved; the surgical site was scrubbed with povidone iodine and rinsed with sterile saline 3 times before surgery. Under sterile conditions, a 3-cm vertical midline incision was made in all groups. In the sham group, we closed the abdominal wall without any procedure. In the other groups, a 2-cm segment of each uterine horn was injured in 10 spots on the antimesenteric surface using unipolar cautery. Before the final abdominal closure, no adjuvant therapy was administered intraperitoneally to the rats in the control group; 2 ml of normal saline solution, 1% methylene blue solution, SPER/NO (0.5 mg/ml) and DETA/NO (0.1 mg/ml) were instilled into the uterine horns of the rats in the respective groups. The incision was closed with a running 4-0 monofilament delayed absorbable suture in a single layer of muscle and fascia in a running pattern, excluding the peritoneum, and in the covering layer of skin in an interrupted pattern, in all groups. Two weeks after the surgery, all animals were killed, second laparotomies were performed, and the extent and severity of adhesions were determined by a blinded examiner. The adhesion scores of the sham group were significantly lower than those of the other groups (p < 0.05). In the methylene blue, SPER/NO and DETA/NO groups, adhesion scores were significantly lower than in the normal saline and control groups (p < 0.05). However, there were no significant differences related to the extent and severity scores of adhesions between the methylene blue, SPER/NO and DETA/NO groups (p > 0.05). This study showed that SPER/NO, DETA/NO and methylene blue administered at the end of surgery reduced the adhesion formation in a rat uterine horn model.
Effect of Puberty on the Relationship between Bone Markers of Turnover and Bone Mineral Density in Turner’s Syndrome
Authors: Gallicchio Carla T. , Figueiredo-Alves Solange T. , Prendin Tórtora Rosângela , Mendonça Laura Maria , Farias Maria Lucia F. , Guimarães Marília M.
Published: January 2004

It has been suggested that the appropriate timing of puberty is necessary for normal bone mineral acquisition which may not be achieved amongst patients with Turner’s syndrome (TS). The aim of this study was to assess bone mineral density (BMD) and bone turnover in 34 patients with TS (age range 2.2–39.0 years). The areal BMD (aBMD) was determined by dual-energy X-ray absorptiometry, and the volumetric BMD was calculated. Blood and second voided urine samples were taken the morning after an overnight fast for evaluation of the biochemical markers of bone turnover: bone-specific alkaline phosphatase (BAP) and N-telopeptides of type I collagen (NTX), respectively. Both were determined by enzyme-linked immunosorbent assay. The patients were divided into three groups: group 1 (n = 13; prepubertal; age range 2.2–19.0 years), group 2 (n = 10; teenagers; age range 12.4–19.0 years), and group 3 (n = 11; adults; chronological age >20 years). They were also grouped by breast development according to Tanner stage into B1 (n = 12), B2–3 (n = 9), and B4–5 (n = 13). The aBMD was significantly lower in group 1 and was higher at Tanner stages 4 and 5 as compared with patients at Tanner stage 1. The bone turnover markers were significantly higher in group 1 (NTX: p = 0.002; BAP: p = 0.0005) and declined, as puberty progressed. A negative correlation was observed between aBMD and biochemical bone markers at the lumbar spine (NTX: r = –0.54, p = 0.05; BAP: r = –0.44, p = 0.01) and in the whole body (NTX: r = –0.60, p = 0.0008; BAP: r = –0.19, p = 0.002). We conclude that the negative relationships between aBMD and biochemical markers suggest a high bone turnover, mainly in prepubertal patients and that the results observed in relation to aBMD and puberty are imputed to the delayed puberty which occurs amongst TS patients.
Tall Stature, Insulin Resistance, and Disturbed Behavior in a Girl with the Triple X Syndrome Harboring Three SHOX Genes: Offspring of a Father with Mosaic Klinefelter Syndrome but with Two Maternal X Chromosomes
Authors: Kanaka-Gantenbein Christina , Kitsiou Sophia , Mavrou Ariadni , Stamoyannou Lela , Kolialexi Aggeliki , Kekou Kyriaki , Liakopoulou Magda , Chrousos George
Published: January 2004

Aims: To describe the tall stature and its possible underlying mechanism in a Caucasian girl (age 12 years and 10 months) with 46,XX (28%)/47,XXX (72%) mosaicism and to identify the parental origin of her extra X chromosome. Methods: The fasting glucose-to-insulin ratio was studied. The karyotypes of the girl and her parents as well as the presence of SHOX copies and the parental origin of her extra X chromosome were assessed. Results: Clinical examination revealed a tall stature and severe acne, and endocrinological/metabolic assessment revealed insulin resistance. Fluorescence in situ hybridization cytogenetic analysis depicted the presence of three SHOX genes in the 47,XXX cell line of the patient. Karyotyping of her parents showed a normal 46,XX karyotype in the mother and 46,XY(93%)/47,XXY(7%) Klinefelter mosaicism in the father. However, DNA analysis unequivocally showed maternal origin of the extra X chromosome of the patient. Conclusions: This report suggests that SHOX gene triplication may produce a tall stature, even in the presence of preserved ovarian function. X triplication might predispose to insulin resistance and behavioral disorders.
Charcot and Art: From a Hobby to Science
Authors: Bogousslavsky Julien
Published: January 2004

Jean-Martin Charcot not only was one of the founders of modern neurology, but he displayed an exceptionally developed visual perception and memory, with special artistic gifts, which he used first as a hobby and subsequently as a tool in his profession. Previously unpublished drawings emphasize Charcot’s talents in caricature, including autoderision. One of the best achievements of Charcot in correlating the clinic with art includes his thorough study of artistic representations of ‘possessed states’, which allowed him to refine his work on hysteria. The artist and the scientist are two unique facets of Charcot, whose permanent coexistence help to understand his legacy.
Neurosarcoidosis: Clinical Experience and Diagnostic Pitfalls
Authors: Kellinghaus C. , Schilling M. , Lüdemann P.
Published: January 2004

Objective: To describe a group of patients with neurosarcoidosis and to highlight diagnostic difficulties based on current diagnostic criteria. Methods: The patient database of a general neurological department was searched for patients with established or suspected diagnosis of neurosarcoidosis. Twenty-four patients were identified with definite (n = 3), probable (n = 10) and possible neurosarcoidosis (n = 10). History and clinical, laboratory and imaging data of patients with definite and probable neurosarcoidosis were analyzed. Results: Cranial nerve symptoms were a dominant clinical feature, with the optic nerve being affected most frequently. Cerebrospinal fluid pleocytosis was found in more than half of the patients. Intrathecal IgG synthesis and oligoclonal bands were less frequent. There was a wide array of MRI lesions in both groups. Chest X-ray was false negative in 2 of 5 patients who also underwent a thoracic CT. Therapy with prednisolone was initiated in all patients. After a median of 36 months, 6 of 8 patients with follow-up data of >24 months were still in remission. Aggravation of symptoms required therapy escalation in 2 patients. Conclusion: There is a wide range of clinical symptoms and test results in patients with ‘definite’ or ‘probable’ neurosarcoidosis. Because systemic involvement is a crucial diagnostic criterion, extensive medical work-up may be necessary. Prognosis under corticosteroid treatment may be better than previously thought.
Effect of Growth Hormone Replacement Therapy on Pituitary Hormone Secretion and Hormone Replacement Therapies in GHD Adults
Authors: Hubina Erika , Mersebach Henriette , Rasmussen Åse Krogh , Juul Anders , Sneppen Simone Bjerregaard , Góth Miklós I. , Feldt-Rasmussen Ulla
Published: January 2004

Objective: We tested the impact of commencement of GH replacement therapy in GH-deficient (GHD) adults on the circulating levels of other anterior pituitary and peripheral hormones and the need for re-evaluation of other hormone replacement therapies, especially the need for dose changes. Methods: 22 GHD patients were investigated in a double-blind randomized study and 90 GHD patients in an open study at baseline and after 6 and 12 months of GH replacement therapy. Results: In the placebo-controlled trial, the FT3 levels increased after 6 months in the GH-treated group, and in the open study the FT3 levels tended to increase. Other hormone concentrations did not change in either part of the study. Four patients required an increase in thyroxine dose, while 2 patients needed dose reduction. One originally euthyroid patient required thyroxine replacement. Two patients with originally conserved pituitary-adrenal function developed ACTH insufficiency. The hydrocortisone dose was increased in 1 and decreased in 1 of the 66 patients with secondary hypocortisolism. None of the females required any adjustment of sex hormone replacement therapy. Two of 37 males needed dose increase of testosterone, while 1 needed dose reduction. Conclusion: GH replacement therapy required dose adjustments regarding other hormone replacement therapies in 12.2% (n = 11), while initiation of new hormone replacement was performed in 3.3% (n = 3) of the 90 patients during the 1-year follow-up. Monitoring of pituitary hormone axes is advisable after commencement of GH replacement therapy, since changes of hormone replacement therapy was observed in a small but clinically significant number of patients.
EUK-134 Reduces Renal Dysfunction and Injury Caused by Oxidative and Nitrosative Stress of the Kidney
Authors: Chatterjee Prabal K. , Patel Nimesh S.A. , Kvale Espen O. , Brown Paul A.J. , Stewart Keith N. , Mota-Filipe Helder , Sharpe Martyn A. , Di Paola Rosanna , Cuzzocrea Salvatore , Thiemermann Christoph
Published: January 2004

Background/Aims: Oxidative and nitrosative stress plays important roles in the pathogenesis of renal ischemia/reperfusion (I/R) injury. Here we investigate the effect of EUK-134, a synthetic superoxide dismutase and catalase mimetic, (i) on renal dysfunction and injury caused by I/R in vivo and (ii) on proximal tubular cell (PTC) injury and death caused by oxidative and nitrosative stress. Methods: Rats, subjected to bilateral renal ischemia (45 min) followed by reperfusion (6 h), were administered EUK-134 (0.3 and 3 mg/kg, i.v.) prior to and during reperfusion, after which biochemical and histological indicators of renal dysfunction and injury were measured. The expression of poly(ADP-ribose) (PAR) and inducible nitric oxide (NO) synthase (iNOS) and nitrotyrosine formation were determined immunohistochemically and used as indicators of oxidative and nitrosative stress. Primary cultures of rat PTCs, isolated and cultured from the kidney cortex, were incubated with hydrogen peroxide (H2O2; 1 mM for 2 h) in the presence of increasing concentrations of EUK-134 (1–100 µM) after which PTC injury and death were measured. The effects of EUK-134 on serum levels of NO in rats subjected to renal I/R or on NO production by PTCs incubated with interferon-γ (IFN-γ, 100 IU/ml) and bacterial lipopolysaccharide (LPS, 10 µg/ml) in combination for 24 h were also measured. Results: EUK-134 produced a significant reduction in renal dysfunction and injury caused by I/R. Specifically, serum creatinine levels, an indicator of renal dysfunction, were reduced from 227 ± 11 (n = 12, I/R only) to 146 ± 9 µM (n = 12, I/R +3 mg/kg EUK-134). Urinary N-acetyl-β-D-glucosaminidase activity, an indicator of tubular damage, was reduced from 42 ± 5 (n = 12, I/R only) to 22 ± 3 IU/l (n = 12, I/R +3 mg/kg EUK-134). EUK-134 significantly reduced renal injury caused by oxidative stress in vivo (reduction in PAR formation), and in vitro EUK-134 reduced PTC injury and death caused by H2O2. However, EUK-134 also reduced nitrosative stress caused by I/R in vivo (reduction of iNOS expression and nitrotyrosine formation), which was reflected by a significant reduction in serum NO levels in rats subjected to renal I/R. Specifically, serum NO levels were reduced from 57 ± 12 (n = 12, I/R only) to 23 ± 3 mM (n = 12, I/R +3 mg/kg EUK-134). In vitro, EUK-134 significantly reduced NO production by PTCs incubated with IFN-γ/LPS. Conclusion: We propose that EUK-134 reduces renal I/R injury not only via reduction of oxidative stress, but also by reducing nitrosative stress caused by renal I/R.
Cardiovascular Events in Diabetic and Nondiabetic Adults With or Without History of Myocardial Infarction
Authors: Lee Chong Do , Folsom Aaron R. , Pankow James S. , Brancati Frederick L.
Published: February 2004

Background—

Whether diabetic patients without a history of myocardial infarction (MI) have the same risk of coronary heart disease (CHD) events as nondiabetic patients with a history of MI remains controversial. We compared risks of CHD and stroke events and mortality from cardiovascular disease (CVD) in diabetic and nondiabetic men and women with and without a history of MI.

Methods and Results—

We followed a total of 13 790 African American and white men and women ages 45 to 64 years who participated in the Atherosclerosis Risk in Communities study, beginning in 1987 to 1989. There were 634 fatal CHD or nonfatal MI events, 312 fatal or nonfatal strokes, and 358 deaths from CVD during an average of 9 years of follow-up (125 998 person-years). After adjustment for age, sex, race, Atherosclerosis Risk in Communities field center, and multiple baseline risk factors, patients who had a history of MI without diabetes at baseline had 1.9 times the risk of fatal CHD or nonfatal MI (95% CI, 1.35 to 2.56;
P
<0.001) compared with diabetic patients without a prior history of MI. The nondiabetic patients with MI also had 1.8 times the risk of CVD mortality compared with diabetic patients without MI (95% CI, 1.22 to 2.72;
P
=0.003). However, stroke risk was similar between diabetic patients without MI and nondiabetic patients with MI (RR, 1.05; 95% CI, 0.61 to 1.79;
P
=0.87). We also observed that nondiabetic patients with MI had a carotid artery wall thickness similar to diabetic patients without MI (
P
=0.77).

Conclusions—

Diabetic patients without MI had lower risk of CHD events and mortality from CVD compared with nondiabetic patients with MI, but stroke risk was similar between these 2 groups.
Atrial Fibrillation Originating From Persistent Left Superior Vena Cava
Authors: Hsu Li-Fern , Jaïs Pierre , Keane David , Wharton J. Marcus , Deisenhofer Isabel , Hocini Mélèze , Shah Dipen C. , Sanders Prashanthan , Scavée Christophe , Weerasooriya Rukshen , Clémenty Jacques , Haïssaguerre Michel
Published: February 2004

Background—

The left superior vena cava (LSVC) is the embryological precursor of the ligament of Marshall, which has been implicated in the initiation and maintenance of atrial fibrillation (AF). Rarely, the LSVC may persist and has been associated with some organized arrhythmias, though not with AF. We report 5 patients in whom the LSVC was a source of ectopy, initiating AF.

Methods and Results—

In 5 patients (4 men; age, 46±11 years) with symptomatic drug-refractory AF, ectopy from the LSVC resulting in AF was observed after pulmonary vein isolation. The ectopics were spontaneous in 2 and induced by isoproterenol in the others and preceded P-wave onset by 67±13 ms. During multielectrode or electroanatomic mapping, venous potentials were recorded circumferentially at the proximal LSVC near its junction with the coronary sinus (CS), but at the mid-LSVC level, they were recorded only on part of the circumference. The LSVC was electrically connected to the lateral left atrium (LA) and through the CS to the right atrium, with 4.1±2.3 CS-LSVC and 1.6±0.5 LA-LSVC connections per patient. Catheter ablation in the LSVC targeting these connections resulted in electrical isolation in 4 of the 5 patients without complications. After 15±10 months, the 4 patients with successful isolation, including 1 who had successful reablation for LA flutter, remained in sinus rhythm without drugs.

Conclusions—

The LSVC can be the arrhythmogenic source of AF with connections to the CS and LA. Ablation of these connections resulted in electrical isolation.
Severity of depressive episodes according to ICD-10: prediction of risk of relapse and suicide
Authors: Kessing Lars Vedel
Published: January 2004

Background

The ICD–10 categorisation of severity of depression into mild, moderate and severe depressive episodes has not been validated.

Aims

To validate the ICD–10 categorisation of severity of depression by estimating its predictive ability on the course of illness and suicidal outcome.

Method

All psychiatric in-patients in Denmark who had received a diagnosis of a single depressive episode at their first discharge between 1994 and 1999 were identified. The risk of relapse and the risk of suicide were compared for patients discharged with an ICD–10 diagnosis of a single mild, moderate or severe depressive episode.

Results

At their first discharge, 1103 patients had an ICD–10 diagnosis of mild depressive episode, 3182 had a diagnosis of moderate depressive episode and 2914 had a diagnosis of severe depressive episode. The risk of relapse and the risk of suicide were significantly different for the three types of depression – increasing from mild to moderate to severe depressive episode.

Conclusions

The ICD–10 way of grading severity is clinically useful and should be preserved in future versions.
Postnatal depression across countries and cultures: a qualitative study
Authors: Oates Margaret R. , Cox John L. , Neema Stella , Asten Paul , Glangeaud-Freudenthal Nine , Figueiredo Barbara , Gorman Laura L. , Hacking Sue , Hirst Emma , Kammerer Martin H. , Klier Claudia M. , Seneviratne Gertrude , Smith Mary , Sutter-Dallay Anne-Laure , Valoriani Vania , Wickberg Birgitta , Yoshida Keiko ,   TCS–PND Group
Published: January 2004

Background

Postnatal depression seems to be a universal condition with similar rates in different countries. However, anthropologists question the cross-cultural equivalence of depression, particularly at a life stage so influenced by cultural factors.

Aims

To develop a qualitative method to explore whether postnatal depression is universally recognised, attributed and described and to enquire into people's perceptions of remedies and services for morbid states of unhappiness within the context of local services.

Method

The study took place in 15 centres in 11 countries and drew on three groups of informants: focus groups with new mothers, interview swith fathers and grandmothers, and interviews with health professionals. Textual analysis of these three groups was conducted separately in each centre and emergent themes compared across centres.

Results

All centres described morbid unhappiness after childbirth comparable to postnatal depression but not all saw this as an illness remediable by health interventions.

Conclusions

Although the findings of this study support the universality of a morbid state of unhappiness following childbirth, they also support concerns about the cross-cultural equivalence of postnatal depression as an illness requiring the intervention of health professionals; this has implications for future research.
Medical records: Doctors' and patients' experiences of copying letters to patients
Authors: Nandhra Harpal , Murray Graham , Hymas Nigel , Hunt Neil
Published: January 2004

Aims and Method

We conducted a pilot study to determine patients' views on receiving a copy of the assessment letter sent to their general practitioner and to determine how psychiatrists' letter writing practice would be altered in the knowledge that patients would receive copies of such letters. Seventy-six consecutive new outpatients received copies of the initial assessment letter sent to general practitioners. Patients were asked to complete a short questionnaire on how the practice affected them. For each letter, psychiatrists were asked to provide details of anything of importance that had been omitted from the letter that in their normal practice they would have included.

Results

There was a broad range of responses on how patients felt about the letters. Only two patients found the letters unhelpful, and 83% expressed a positive desire to continue receiving letters, even though initially 18% found the letter distressing. For 56 out of 76 patients, psychiatrists stated that they composed and sent out the letter to the GP in accordance with their usual practice and copied the letter to the patient in an unaltered form. For 17 patients, the psychiatrist stated that some information he/she would usually have included in the GP letter was omitted in the copy the patient received. In a further 3 cases, the psychiatrist sent no letter to the patient.

Clinical Implications

Patients found it helpful to receive copies of their assessment letters. Psychiatrists might require training and reassurance about this policy before implementation.
Children of adults with severe mental illness: mental health, help seeking and service use
Authors: Cowling Vicki , Luk Ernest S. L. , Mileshkin Cristea , Birleson Peter
Published: January 2004

Aims and Method

We aimed to determine the prevalence of childhood mental health problems in children of parents registered with an Australian area mental health service, and to study the parents' help-seeking and service use for their children. Parents were recruited through their case managers, and asked to complete the Strengths and Difficulties Questionnaire (SDQ), the Service Utilisation Questionnaire and the Help-seeking Questionnaire.

Results

A quarter of the children were in the clinical range of the SDQ total scores, with high sub-scale scores. However, 63% of the parents reported reluctance to seek help, and 19% reported not using services.

Clinical Implications

Children of parents with mental illness are at higher risk of childhood psychiatric disorders. Assessment of the child at the time of referral of the parent is an opportunity for problem identification, parental education, and early intervention.
Treatment preferences of opiate-dependent patients
Authors: Luty Jason
Published: January 2004

Aims and Method

To assess the preferences of people attending a substance misuse facility towards the treatment options available for opiate dependency. Interviews were conducted using a card sorting technique.

Results

The majority (60%) of the 101 participants believed that detoxification was superior to maintenance in preventing illicit heroin use. The preferred treatment options were oral methadone, buprenorphine, drug-free rehabilitation, in-patient detoxification and prescription of injectable drugs.

Clinical Implications

Both pharmacological and psychosocial options, including in-patient detoxification and rehabilitation, are among the treatments preferred by clients of substance misuse services. There is also a significant demand for both injectable drugs and dihydrocodeine.
Medical records: Patient-held records in mental health
Authors: Laugharne Richard , Henderson Claire
Published: January 2004

‘But don't they get lost?’ This is usually the first comment made when the authors mention the use of patient-held records (PHRs) to colleagues. Nevertheless, PHRs have been used in mental health care as well as several other settings, including services for diabetes, cancer, maternity and child health. In some of these services, including mental health, PHRs have been an addition to clinician held standard notes, whereas in others the patient holds the only record for their care. The main purposes of introducing PHRs have been to empower patients with a sense of ownership of their care and to improve communication, between both patients and clinicians, as well as between different clinicians involved in that person's care (Laugharne & Stafford, 1996).
Medical records: Disclosing confidential clinical information
Authors: Dolan Bridget
Published: January 2004

Although our courts remain reluctant to create any general right to privacy, whether under common law or even by virtue of statutes such as the Human Rights Act 1998, one important aspect of privacy is recognised in common law – that of the confidentiality of medical information.
Medical Records: Copying letters to patients
Authors: Lloyd Geoffrey
Published: January 2004

Access to medical information is going to be extended by recent Government proposals that patients who agree are sent copies of correspondence relevant to their illness and medical treatment. The National Health Service (NHS) Plan for England (Department of Health, 2000) has stated unequivocally that letters between clinicians about an individual patient's care will be copied to the patient as of right. No exceptions have been made and the plan did not suggest that patients suffering from a psychiatric illness are to be treated differently from any other group of patients. However, the Department of Health has recently stated its intention to fund a series of pilot projects to test some key concepts before the policy is fully implemented in 2004. A number of areas to be informed by pilot work have been identified. These include the style and content of letters, testing formats and language that patients find acceptable and particular issues concerning mental health, children and carers (www.doh.gov.uk/patientletters).
Reading, Writing, and Modulating Genetic Information with Boranophosphate Mimics of Nucleotides, DNA, and RNA
Authors: SHAW BARBARA RAMSAY , DOBRIKOV MIKHAIL , WANG XIN , WAN JING , HE KAIZHANG , LIN JIN‐LAI , LI PING , RAIT VLADIMIR , SERGUEEVA ZINAIDA A. , SERGUEEV DMITRI
Published: January 2004

Abstract:TheP‐boranophosphates are efficient and near perfect mimics of natural nucleic acids in permitting reading and writing of genetic information with high yield and accuracy. Substitution of a borane (−BH3) group for oxygen in the phosphate ester bond creates an isoelectronic and isosteric mimic of natural nucleotide phosphate esters found in mononucleotides, i.e., AMP and ATP, and in RNA and DNA polynucleotides. Compared to natural nucleic acids, the boranophosphate RNA and DNA analogs demonstrate increased lipophilicity and resistance to endo‐ and exonucleases, yet they retain negative charge and similar spatial geometry. Borane groups can readily be introduced into the NTP and dNTP nucleic acid monomer precursors to produce α‐P‐borano nucleoside triphosphate analogs (e.g., NTPαB and dNTPαB). The NTPαB and dNTPαB are, in fact, good to excellent substrates for RNA and DNA polymerases, respectively, and allow ready enzymatic synthesis of RNA and DNA withP‐boranophosphate linkages. Further, boranophosphate polymer products are goodtemplatesfor replication, transcription, and gene expression; boronated RNA products are also suitable for reverse transcription to cDNA. Fully substituted boranophosphate DNA can activate the RNase H cleavage of RNA in RNA:DNA hybrids. Moreover, certain dideoxy‐NTPαB analogs appear to be better substrates for viral reverse transcriptases than the regular ddNTPs, and may offer promising prodrug alternatives in antiviral therapy. These properties make boranophosphates promising candidates for diagnostics; aptamer selection; gene therapy; and antiviral, antisense, and RNAi therapeutics. The boranophosphates constitute a versatile family of phosphate mimics for processing genetic information and modulating gene function.
The Effect of Hydroxyurea and Trichostatin A on Targeted Nucleotide Exchange in Yeast and Mammalian Cells
Authors: PAREKH‐OLMEDO HETAL , ENGSTROM JULIA U. , KMIEC ERIC B.
Published: January 2004

Abstract: Targeted nucleotide exchange (TNE) is a process by which a synthetic DNA oligonucleotide, partially complementary to a site in a chromosomal or an episomal gene directs the reversal of a single nucleotide at a specific site. To protect against nuclease digestion, the oligonucleotide is modified with derivative linkages among the terminal bases. We have termed these molecules modified single‐stranded oligonucleotides (MSOs). Current models suggest that the reaction occurs in two steps. The first, DNA pairing, involves the alignment of the MSO with the target site and its assimilation into the target helix forming a D‐loop. The second phase centers around the repair of a single base mismatch formed between the MSO and its complementary strand in the D‐loop. Nucleotide exchange is promoted in all likelihood by the mismatch repair system. A critical feature of successful TNE is the accessibility of the target site for the MSO and the factors that increase the dynamic nature of the chromatin that will likely increase the frequency. Here, we report that two factors, trichostatin A and hydroxyurea, elevate gene repair of a mutant hygromycin gene in Saccharomyces cerevisiae and a mutant eGFP gene in a mammalian cell line, MCF‐10AT1 cells. Trichostatin A (TSA) acts by preventing the deacetylation of histones while hydroxyurea (HU) reduces the rate of replication. Both of these activities, by their very nature, create a more open configuration of the MSO into the target site.
Short Interfering RNA (siRNA)—Mediated RNA Interference (RNAi) in Human Cells
Authors: CAPLEN NATASHA J. , MOUSSES SPYRO
Published: January 2004

Abstract: Transient gene silencing in mammalian cells can be mediated by double stranded RNA (dsRNAs) molecules of ∼20–25 nucleotides termed short interfering (siRNAs). Naturally occurring siRNAs in lower eukaryotes have characteristic structural elements; however, little is known about what features are critical for an exogenous siRNA to mediate RNAi in mammals. We have recently determined some of the critical parameters that influence the efficiency of siRNA‐mediated RNAi in mammalian cells and have been considering the use of RNAi as a functional genomics tool, particularly for high throughput analysis, and the potential use of RNAi as a therapeutic tool.
Antigene Radiotherapy
Authors: PANYUTIN I. G. , SEDELNIKOVA O. A. , KARAMYCHEV V. N. , NEUMANN R. D.
Published: January 2004

Abstract: Antigene radiotherapy is based upon damaging selected genes by a high dose of radiation from radionuclides delivered to this gene by a sequence‐specific DNA‐binding molecule. Here we describe our recent trials of antigene radiotherapy using the human mdr1 gene over‐expressed in KB‐V1 cells as a model. As a delivery molecule, we used a triplex‐forming oligonucleotide (TFO) with a binding site in intron 14 of mdr1. This TFO was labeled with an Auger‐electron‐emitting radionuclide 125I. Decay of 125I releases a shower of low energy electrons that produce DNA strand breaks mostly within 10 bp from the decay site. Targeting in situ was assessed by restriction enzyme digestion of the DNA recovered from the TFO‐treated cells followed by Southern hybridization with DNA probes flanking the target sequence. Double‐strand breaks in the target sequence were detected in purified nuclei and digitonin‐permeabilized cells, but not in the intact cells when TFO were delivered with liposomes. On the basis of these observations we hypothesized that there are cytoplasmic factors that bind such TFO and deliver them into the nucleus, but do not release them inside the nucleus, thus preventing TFO from binding their genomic targets. To test this hypothesis we (i) delivered TFO along with an excess of unlabeled oligonucleotide with an arbitrary sequence (“ballast”) and (ii) conjugated TFO with a nuclear localization sequence peptide (NLS). We have found that TFO/NLS conjugates cleaved the target in a concentration‐dependent manner regardless of the presence of the “ballast” oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the “ballast.” These results may provide a new insight into the mechanism of intracellular transport of oligonucleotides.
Potent Inhibition of HhaI DNA Methylase by the Aglycon of 2‐(1H)‐Pyrimidinone Riboside (Zebularine) at the GCGC Recognition Domain
Authors: MARQUEZ VICTOR E. , ERITJA RAMON , KELLEY JAMES A. , VANBEMMEL DANA , CHRISTMAN JUDITH K.
Published: January 2004

Abstract: A short oligodeoxynucleotide (ODN) with 2‐(1H)‐pyrimidinone at the HhaI DNA methyltransferase target site (GCGC) is shown to induce a level of inhibition of methyl transfer and thermal stability of the complex with the enzyme identical to that achieved with a similar ODN substituted with 5‐azacytosine. The drugs responsible for these effects—zebularine and 5‐azacytidine/2′‐deoxy‐5‐azacytidine—are contrasted in terms of chemical stability and possible metabolic activation by a brief structure‐activity analysis.
Custom Cancer Therapies
Authors: SUMMERTON JAMES E.
Published: January 2004

Abstract: Gene Tools, LLC has embarked on a program to develop safe and effective custom therapies for essentially all cancers. This entails: 1) identifying in each patient's cancer multiple molecular targets which are absent from that patient's normal cells, but are present in and essential to that patient's cancer; and 2) treating that patient with a custom cocktail of therapeutic agents effective for specifically inhibiting those selected targets in that patient's cancer.
Therapeutic Potentialities of EWS‐Fli‐1 mRNA‐Targeted Vectorized Antisense Oligonucleotides
Authors: MAKSIMENKO A. , LAMBERT G. , BERTRAND J. R. , FATTAL E. , COUVREUR P. , MALVY C.
Published: January 2004

Abstract: We have used structured antisense oligonucleotides (AON), which are protected against extra and intracellular degradation by their internal structure. We have shown that if correctly designed this structure does not prevent them from hybridizing to the mRNA target. This concept allows reducing the number of thioate groups in the oligonucleotide and therefore the potential toxicity. Junction oncogenes are found in cancers such as certain leukemias, Ewing sarcoma, and thyroid papillary carcinomas. Ewing sarcoma is a cancer of children and young adults with bone metastasis. It is caused by a chromosomic translocation t(11;22) (q24;q12) creating a fusion gene between the genes EWS and Fli‐1 giving rise to a chimeric protein which is an unnatural transcription factor. Immortalized NIH/3T3 cells transfected by the EWS‐Fli‐1 cDNA under the control of the LTR retroviral promoter—which do not undergo apoptosis and which became tumoral—were used for this study. As a model of Ewing sarcoma in nude mice, we have used permanently expressing human EWS‐Fli‐1 cells grafted to nude mice. The nanospheres or nanocapsules have been used to deliver two different AON: a phosphorothioate, and a structured chimeric AON, both targeted toward the junction area of EWS‐Fli‐1. Both types of AON‐loaded nanoparticles inhibited the growth of the xenografted tumor after intratumoral injections into nude mice, whereas similar nanoparticles with control oligonucleotides had no effect. With AON in nanospheres, we have shown after 24 hours that the mRNA of EWS‐Fli‐1 was specifically down‐regulated, confirming the antisense activity of the targeted AON.
HER‐2‐Targeted Antisense Oligonucleotide Results in Sensitization of Head and Neck Cancer Cells to Chemotherapeutic Agents
Authors: RAIT ANTONINA S. , PIROLLO KATHLEEN F. , ULICK DAVID , CULLEN KEVIN , CHANG ESTHER H.
Published: January 2004

Abstract: Existing HER‐2 targeted therapies for human head and neck cancers, usually administered in combination with chemotherapeutic drugs or irradiation, include monoclonal antibodies to HER‐2, receptor tyrosine kinase inhibitors and HER‐2 specific immunotoxins. Instead of targeting the existing protein, interference with HER‐2 mRNA translation by antisense oligonucleotides may be a more efficient method to downregulate levels of HER‐2 protein for combination therapy. To test this hypothesis we have used a phosphorothioate pentadecamer, complementary to the HER‐2 mRNA initiation codon region (AS HER‐2 ODN), to increase sensitivity to four chemotherapeutic agents in human head and neck cancer cell lines, all of which express low levels of the HER‐2 protein. To improve delivery into tumor cells, the AS HER‐2 ODN was complexed with our previously established folate‐liposome delivery system. Cell survival assays and Western blot analysis data demonstrated that folate‐liposome mediated AS HER‐2 oligonucleotide treatment inhibited cell growth and HER‐2 expression, and induced apoptosis in SCC‐25CP cells. Moreover, there was a synergistic effect on the percent of apoptotic cells. Additionally, the combination of folate‐liposome‐AS HER‐2 ODN and CDDP had a synergistic effect on the induction of apoptosis. Using confocal microscopy, FITC labeled ODN (FITC‐ODN) in complex with folate‐liganded, rhodamine (Rh) labeled, cationic liposomes was observed to enter SCC‐25CP head and neck tumor cells within 3 to 6 h. Intracellularly, the FITC‐ODN separated from the Rh‐folate‐liposomes, and FITC‐ODN accumulated in the nucleus while Rh‐liposomes remained in punctate cytoplasmic structures. Thus, folate‐liposome‐mediated delivery of AS HER‐2 ODN has potential as a new means of increasing the responsiveness of head and neck cancer to conventional chemotherapy.
Antisense to Apoptosis Inhibitors Facilitates Chemotherapy and TRAIL‐Induced Death Signaling
Authors: ZANGEMEISTER‐WITTKE UWE
Published: January 2004

Abstract: Impaired apoptosis signaling is associated with tumor development and confers resistance to chemotherapy and apoptosis triggered by the extrinsic death receptor pathway including Fas and TRAIL‐R1/R2. In addition to genetic and epigenetic alterations, such as mutational inactivation and silencing of potential tumor suppressor genes, the antiapoptotic proteins Bcl‐2, Bcl‐xL, and survivin are overexpressed in many human tumors, and targeted inhibition of their expression has potential to facilitate apoptosis induced by various stimuli. We have used antisense and RNAi technology to counteract the expression of these antiapoptotic proteins in various tumor cell types and investigated the effect of this intervention on apoptosis induction by chemotherapy and the tumor‐selective death ligand TRAIL. The oligonucleotide targeting Bcl‐2 and Bcl‐xL was used in the 2′‐MOE or LNA‐modified gapmer format, the survivin siRNA was derived from the sequence of an effective first generation antisense oligonucleotide. Modulation of gene expression was monitored by real‐time PCR and Western blotting, cell death was determined in cell growth and apoptosis assays. In the tumor cells tested, downregulation of Bcl‐2, Bcl‐xL or survivin expression facilitated apoptosis via the intrinsic and extrinsic signaling pathway and sensitized tumor cells to various chemotherapeutic agents and to TRAIL. All combinations of antisense and chemotherapy as well as of Bcl‐2/Bcl‐xL antisense and TRAIL resulted in more than additive cytotoxicity. Although survivin represents a promising target for antisense therapy owing to its tumor‐selective expression, its downregulation less effectively enhanced TRAIL‐induced apoptosis compared to the Bcl‐2/Bcl‐xL antisense approach. Our data suggest the use of Bcl‐2‐, Bcl‐xL‐ and survivin‐directed antisense therapy to improve the treatment options for apoptosis‐resistant cancer.
Clusterin and IGFBPs as Antisense Targets in Prostate Cancer
Authors: GLEAVE MARTIN , JANSEN BURKHARD
Published: January 2004

Abstract: The primary hurdle to improved survival of advanced prostate cancer is our failure to prevent or treat the tumor's progression to its lethal and untreatable stage of androgen independence. Novel treatment modalities designed to prevent androgen‐independent progression including prostate cancer metastasis are required. Accelerated identification and characterization of cancer‐relevant molecular targets has sparked considerable interest in the development of new generations of anticancer agents that specifically inhibit a progression‐relevant target. Antisense oligonucleotides, short synthetic stretches of chemically modified DNA capable of specifically hybridizing to the mRNA of a chosen cancer‐relevant target gene, promise to show enhanced specificity for malignant cells with a favorable side‐effect profile due to well‐defined and tailored modes of action. Although not all of the challenges have been met to date, emerging clinical evidence supports the premise that antisense oligonucleotides stand a realistic chance of emerging as major partners of rationally designed anticancer regimens. The rationale and status of antisense targeting of the treatment resistance factor clusterin and of insulin‐like growth factor binding protein (IGFBP) 2 and 5 are discussed.
Regulation of CpG‐Induced Immune Activation by Suppressive Oligodeoxynucleotides
Authors: KLINMAN DENNIS M. , ZEUNER RAINALD , YAMADA HIROSHI , GURSEL MAYDA , CURRIE DEBBIE , GURSEL IHSAN
Published: January 2004

Abstract: Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated “CpG motifs” stimulate an innate immune response characterized by the production of cytokines, chemokines, and polyreactive Igs that promote host survival following infectious challenge. Yet CpG‐driven immune activation can have deleterious consequences, such as increasing the host's susceptibility to autoimmune disease. The immunomodulatory activity of CpG DNA can be blocked by DNA containing “suppressive” motifs. This work explores the rules governing cellular recognition of stimulatory and suppressive motifs, and the resultant modulation of the immune system. Results suggest that both CpG and suppressive ODN may find use as therapeutic agents.
Antisense to the Early Growth Response‐1 Gene (Egr‐1) Inhibits Prostate Tumor Development in TRAMP Mice
Authors: BARON VÉRONIQUE , DUSS STEPHAN , RHIM JOHNG , MERCOLA DAN
Published: January 2004

Abstract: Egr‐1 is a transcription factor induced by stress or injury, mitogens, and differentiation factors. Egr‐1 regulates the expression of genes involved in growth control or survival. Expression of Egr‐1 results in either promotion or regression of cell proliferation, depending on cell type and environment. Egr‐1 acts as a tumor suppressor in many cell types and loss of Egr‐1 has been proposed to contribute to cancer progression. There is strong new evidence however suggesting that Egr‐1 overexpression is involved in prostate cancer progression. For example, Egr‐1 expression levels are elevated in human prostate carcinomas in proportion to grade and stage. Furthermore, prostate cancer progression was significantly delayed in two models of prostate cancer mice lacking Egr‐1. Our objective in the present study is to test whether inhibition of Egr‐1 function would block cell proliferation and inhibit the transformed phenotype of prostate cancer cells in vitro and in vivo. We describe the development of high affinity and high specificity antisense oligonucleotides that efficiently inhibit Egr‐1 expression. We show that inhibition of Egr‐1 expression in mouse or human prostate cancer cells decreased proliferation and reduced the capacity of these cells to form colonies and to grow in soft agar. Conversely, stable expression of Egr‐1 in normal human prostate epithelial 267B1 cells promoted transformation. In TRAMP mice, treatment with Egr‐1 antisense oligonucleotides delayed the occurrence of prostate tumors. Importantly, Egr‐1 antisense showed little or no toxicity when injected into animals. Finally, we identified a few genes such as cyclin D2, p19ink4d, and Fas that are directly regulated by Egr‐1 in prostate cancer cells and that control cell cycle and survival.
Chemosensitization and Radiosensitization of Human Cancer by Antisense Anti‐MDM2 Oligonucleotides
Authors: WANG HUI , OLIVER PATSY , ZHANG ZHUO , AGRAWAL SUDHIR , ZHANG RUIWEN
Published: January 2004

Abstract: MDM2 oncogene is overexpressed in many human cancers including breast, colon, and prostate cancer, and MDM2 levels are associated with poor prognosis in patients with cancer. Here, we summarize the investigation of the functions of MDM2 oncogene in human cancer growth and the value of MDM2 as a drug target for prostate cancer therapy by using antisense to inhibit MDM2 expression. Antisense anti‐human‐MDM2 oligonucleotides and mismatch controls were tested in in vitro and in vivo human cancer models for antitumor activity. Targeted gene products and related proteins were analyzed and the antitumor activity was determined when the oligonucleotides were used alone or in combination with cancer chemotherapeutics and radiation therapy. The antisense oligonucleotide specifically inhibited MDM2 expression in a dose‐ and time‐dependent manner, resulting in significant antitumor activity in vitro and in vivo. The antisense oligonucleotides also potentiated the effects of p53 activation and p21 induction by chemotherapeutic agents 10‐hydroxycamptothecin, adriamycin, 5‐fluorouracil, and paclitaxel. In a dose‐dependent manner, the antisense oligonucleotide showed antitumor activity in nude mice bearing human cancer xenografts and increased therapeutic effectiveness of the chemotherapeutic agents irinotecan, paclitaxel, and Rituxan and radiation therapy. These results indicate that MDM2 has a role in various tumor growth through both p53‐dependent and p53‐independent mechanisms, indicating that MDM2 inhibitors have a broad spectrum of antitumor activities in human cancers regardless of p53 status. These results provide a basis for clinical evaluation of antisense anti‐MDM2 oligonucleotides as chemosensitizer and radiosensitizer.