Search

An ectopic pregnancy (EP) occurs when implantation of the embryo occurs outside of the uterus. If left untreated, the developing fetus will continue to grow, leading to life-threatening consequences for the mother. A major difficulty with the diagnosis of ectopic pregnancy is that methods of detection are limited, and some, such as ultrasound, are not very reliable in the earliest days of gestation. Currently, no effective serum test exists to distinguish an ectopic pregnancy from a normal intrauterine pregnancy. The incidence of ectopic pregnancy is increasing and has doubled in the last 20 years. It is now the second most common cause of maternal death in the first trimester of pregnancy. To address this issue, we initiated a project to identify serum markers of ectopic pregnancy. The subjects for these studies presented at the Hospital of the University of Pennsylvania. We obtained over 140 serum samples from women with suspected ectopic pregnancy: women presenting with pain and/or bleeding in the first trimester of pregnancy. The approximate racial breakdown of the subjects is as follows: African American, 36%; Caucasian, 3%; Asian, 2%; Hispanic, 1%; unknown, 58%. Serum samples from 139 women (62 with ectopic pregnancy and 77 with a normal intrauterine pregnancy) were applied to WCX2 (weak ion exchange) protein chip surfaces and analyzed for serum markers using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Several proteins in the 7500-18,000 Da mass range were identified that may discriminate an ectopic pregnancy from an intrauterine pregnancy. The most promising markers were analyzed using classification and regression tree analysis (CART) with and without clinical variables (serum hCG value, length of amenorrhea). Two different algorithms were developed that classify the patients on the basis of sensitivity (number of EPs who screen positive/# of EPs) or specificity (# of healthy patients who screen negative/# of healthy). Our current approach is to refine these two "rule sets" to segregate patients into three groups: those who need immediate intervention for a probable ectopic pregnancy, those who appear to have a normal pregnancy, and those who need further monitoring for diagnosis.

Although well-characterized in man, abnormal cornification secondary to heritable superficial keratin defects is rarely reported in animals. This report describes a mild cornification defect in seven related Norfolk terrier dogs. Lesions were present at birth and pedigree analysis suggested an autosomal recessive mode of inheritance. The affected dogs had hyperpigmented skin with scaling following mild trauma. The lesions were generalized but most prominent in the glabrous skin of the axillary and inguinal regions-areas where the epidermis is not protected by hair and is subject to frequent trauma. The most striking histological change was vacuolation in the upper epidermis, which often resulted in epidermolysis and blister formation. All of the affected dogs showed similar gross and histological changes. Ultrastructural changes included abnormal keratin filament clumping, prominent clear spaces in the cytoplasm of suprabasal keratinocytes, and abnormal keratohyaline granules. Immunohistochemical labelling for keratin 10 demonstrated a lack of expression in the superficial epidermis of affected dogs. All of the morphological changes noted in the Norfolk terriers were consistent with a mild form of a heritable defect in superficial keratin synthesis.

Objective: To perform a phase IV, open-label study of OVULE (Personal Care Products, Skillman, New Jersey) using magnetic resonance imaging (MRI) to describe the release, vaginal distribution and retention of a suspension containing miconazole nitrate in healthy female volunteers., Study Design: Three parous and 3 nulligravid women were enrolled. Each subject self-inserted the OVULE on the MRI table and remained recumbent after insertion. MRI images were obtained every 15 minutes for 1 hour and then at predetermined intervals following suspension insertion until the suspension was no longer identified., Results: Placement of the OVULE was in the upper vagina in 4 subjects and in the lower vagina in 1 subject, and the Ovule was expelled in 1 subject. Average time to capsule shell dissolution was 36 minutes. Average time to maximum vaginal mucosal coverage by the suspension was 49 minutes. The average time at which the suspension was last seen in the vagina was 5 hours and 53 minutes., Conclusion: MRI can be used to noninvasively quantify the intravaginal spread of a therapeutic antifungal agent. Understanding the intravaginal spread of antifungal agents may lead to further optimization of antifungal preparations as well as to better patient instructions.

Objective: The purpose of this study was to estimate whether the efficacy of treatment with intravaginal misoprostol for first-trimester pregnancy failure is enhanced by the addition of saline solution., Study Design: Eighty women with embryonic/fetal death or anembryonic pregnancy were assigned randomly to receive either 800 microg of misoprostol with saline solution (group I, 41 women) or without (group II, 39 women). Treatment was repeated on day 3 if the gestational sac remained. Curettage was performed if the gestational sac remained on day 8 or as necessary during at least 30 days of follow-up. Data were analyzed with the Student t test and the chi(2) or Fisher exact test., Results: By the first follow-up visit, 73% (group I) and 64% (group II) of women passed the gestational sac (P=.38). By the second follow-up visit, expulsion rates were 83% and 87%, respectively (P=.59). Five subjects in each group underwent curettage., Conclusion: Misoprostol is effective for the treatment of failed first-trimester pregnancy. The expulsion rate is not improved by adding saline solution.

The objective of our study was to compare to ability of collagen-treated membranes and bovine collagen gels to maintain murine preantral follicle growth and development in-vitro. To fulfill that objective, murine follicle and oocyte growth rates were followed for ten days in culture. Meiotic competence and the capacity to reach the two-cell stage after in-vitro maturation and fertilization, respectively, were then assessed. We used preantral follicles from 12 day-old CF-1 female mice that were isolated by enzymatic digestion from ovaries. Follicles were placed either on collagen-treated membranes or embedded in a bovine collagen matrix. The follicles were grown, changing the media and obtaining measurements every other day for ten days. Following culture, the granulosa-oocyte complexes were matured; the resultant metaphase II arrested oocytes were inseminated and cultured to the two-cell stage. The data was analyzed with significance considered for probability values of p < 0.05. We performed individual measurements on 650 follicles in seven separate experiments. Forty-eight hours after initial seeding and throughout the entire length of culture, both the follicles and oocytes grown in the collagen matrix were larger than follicles cultured on collagen-treated membranes (p < .0001). However, oocyte recovery rates were higher among follicles cultured on collagen-treated membranes (p < .01). Similar percentages of meiotically competent oocytes, fertilization and cleavage rates were observed in both groups. Our results show that mouse preantral follicles display a greater growth rate when grown embedded in a collagen gel matrix. This may be due to the maintenance of a normal three-dimensional organization of the follicle within the collagen matrix. However, this system does not enhance meiotic competency or fertilization rates in the mouse when compared to culture on collagen-treated membranes.

Objective: To compare signs and symptoms between patients with recurrent and single ectopic pregnancies., Design: Case-control study., Setting: University medical center., Patient(s): Three hundred six women with single ectopic pregnancies and 61 women with recurrent ectopic pregnancies., Intervention(s): None., Main Outcome Measure(s): Risk factors, historical factors, and findings at presentation that may predispose to recurrent ectopic pregnancy., Result(s): The risk of recurrent ectopic pregnancy was increased with a history of surgery, history of live birth, and history of spontaneous miscarriage and not with a history of gonorrhea, chlamydia, pelvic inflammatory disease, Caesarean section, or pregnancy termination. Patients with a recurrent ectopic pregnancy had a nonsignificant trend toward higher initial hCG values. The majority of ultrasounds in both groups were nondiagnostic on presentation. The patients with recurrent ectopic pregnancy were less likely to have bleeding on initial presentation, though both groups had similar complaints of pain., Conclusion(s): Secondary prevention of ectopic pregnancy is problematic because of the paucity of risk factors that can be modified to diminish the odds of recurrence. Clinicians' ability to both diagnose and counsel patients at risk for recurrent ectopic pregnancy can be optimized by awareness of the clinical features of this condition.

To provide effective contraception and protection against sexually transmitted disease, vaginal gels should maximally cover the cervical os and the vaginal epithelium before, during and after intercourse. To non-invasively monitor the intravaginal distribution of an applied intravaginal gel, we performed high-resolution magnetic resonance imaging (MRI) of the female pelvis before, during and after both real and simulated sexual intercourse. We sought to determine whether simulated intercourse with a plastic phallus could be used as a surrogate for real intercourse for such experiments. Dilute gadolinium chelate solution was mixed with Gynol-II gel and introduced intravaginally to volunteer female human subjects using a conventional applicator. MRI was performed at 1.5 Tesla with a surface coil. Imaging of the female pelvis was performed: (1) immediately after insertion of the gel; (2) during real intercourse with a male partner (2 subjects) or simulated intercourse with a plastic phallus (4 subjects); and (3) after completion of real or simulated intercourse. Subjects were studied after application of both 3 mL and 5 mL of vaginal gel. Measurements of gel thickness covering the vaginal mucosa were made digitally using electronic calipers. The bolus of gel is initially located in the upper vaginal canal, superior to the urogenital diaphragm. Both real and simulated intercourse dramatically increases the spread of gel to the lower vagina. The cervix appears to be adequately covered with gel both before and after intercourse. Increasing the volume of the gel increases initial vaginal mucosal coverage but also increases leakage from the introitus. No statistically significant differences in vaginal mucosal coverage were found between patients having undergone real vs. simulated intercourse, or on post-intercourse scans of 3 mL versus 5 mL. MRI is a sensitive, reproducible means of tracking the spread of intravaginal medications., (Copyright 2002 Elsevier Science Inc.)

Objective: To evaluate the consistency of the identification of abnormal findings on hysterosalpingogram (HSG) and compare the reliability of clinicians to that of radiologists., Design: Evaluation of reliability of diagnostic test., Patient(s): Women undergoing evaluation for infertility.INTEVENTION(S): Retrospective review of 50 HSG films by three reproductive endocrinologists and three radiologists. Each film was reread 30 days later in a blinded fashion., Main Outcome Measure(s): The consistency of each individual reader, the reliability of detecting specific abnormalities, and the consistency of clinicians compared with radiologists was evaluated with a kappa (K) statistic and interclass correlation coefficient (ICC)., Result(s): Average intrareader reliability was high for the detection of normal uterus, normal tubes, and tubal obstruction and low for the detection of hydrosalpinx, uterine adhesions, and pelvic adhesions. Inter-reader reliability was high in the detection of normal uterine contour, normal tubal patency, and uterine filling defect and lower for the detection of a hydrosalpinx. The reliability of detecting pelvic adhesion or salpingitis isthmica nodosa was poor., Conclusion(s): Intrareader reliability was generally good, especially for the detection of normal findings. Agreement among different readers is lower in detecting rare outcomes such as hydrosalpinx and pelvic adhesion and salpingitis isthmica nodosa. Clinicians more reliably diagnose hydrosalpinx and tubal obstruction, while radiologists more reliably detect the more subtle findings of salpingitis isthmica nodosa or uterine adhesions.

Objective: To investigate the IVF outcome for patients with endometriosis., Design: Meta-analysis., Setting: Academic research center., Patient(s): A MEDLINE search and review of the literature were performed. Patients were classified by level of endometriosis, and controls were classified according to the indication for IVF., Intervention(s): Bivariate analysis and multivariate logistic regression was used to estimate overall effect and control for confounding., Main Outcome Measure(s): Pregnancy rates, fertilization rate, implantation rates, and numbers of oocytes retrieved., Result(s): Twenty-two published studies were included in the overall analysis. The chance of achieving pregnancy was significantly lower for endometriosis patients (odds ratio, 0.56; 95% confidence interval, 0.44-0.70) when compared with tubal factor controls. Multivariate analysis also demonstrated a decrease in fertilization and implantation rates, and a significant decrease in the number of oocytes retrieved for endometriosis patients. Pregnancy rates for women with severe endometriosis were significantly lower than for women with mild disease (odds ratio, 0.60; 95% confidence interval, 0.42-0.87)., Conclusion(s): Patients with endometriosis-associated infertility undergoing IVF respond with significantly decreased levels of all markers of reproductive process, resulting in a pregnancy rate that is almost one half that of women with other indications for IVF. These data suggest that the effect of endometriosis is not exclusively on the receptivity of the endometrium but also on the development of the oocyte and embryo.

This study examined immunohistochemical staining patterns for several meningioma variants involving either the brain or spinal cord of dogs. Formalin-fixed, paraffin-embedded tissue from 15 tumors was obtained. The selected tumor group included seven meningothelial, three transitional, two malignant (anaplastic), one myxoid, one papillary, and one osteomatous meningiomas. Tumors were evaluated for reactivity to the following six immunohistochemical markers: vimentin, pancytokeratin, glial fibrillary acidic protein (GFAP), S100, neuron-specific enolase (NSE), and synaptophysin. Vimentin expression was detected in all meningiomas, and 14 of 15 tumors demonstrated intense vimentin staining in more than 50% of the neoplastic cells. Pancytokeratin expression was present in 11 of 15 neoplasms; however, positive staining frequently was focal and often involved a small percentage of the neoplastic cells. GFAP expression was detected in a single, anaplastic meningioma. Although expression of NSE and S100 was detected in 12 of 25 meningiomas, the intensity of the staining and the percentage of positive neoplastic cells was highly variable. Synaptophysin was uniformly negative. These results will help to establish immunohistochemical profiles for meningiomas that will improve our ability to correctly differentiate these neoplasms of meningeal origin from central nervous system tumors originating from other sites.

This study was performed to determine the location of a silicone rubber vaginal barrier contraceptive device, the Lea's Shield, in vivo. Two women, one parous and one nulligravid, were enrolled in the study. Surface coil, multiplanar Magnetic Resonance Imaging (MRI) was performed immediately following insertion of the contraceptive device, and was repeated following 35-40 min of normal ambulation by the participant.The contraceptive device was markedly hypointense to pelvic structures on both T1 and T2 weighted images. Its position within the vagina and relationship to the cervix were readily identifiable on MR images. The device was located in the upper vagina and completely covered the cervix in both patients. The valve appeared closed, and there was no apparent pressure on the urethra. The position was not altered by ambulation. In conclusion, MRI was a reproducible and rapid means for noninvasively determining the intravaginal location and orientation of a barrier contraceptive device. After insertion, the Lea's Shield occupies the upper vagina and completely covers the cervix.

We determined, by serial analysis of gene expression (SAGE) analysis of normal and DCIS (ductal carcinoma in situ) mammary epithelial cells, that psoriasin and several other genes implicated in psoriasis are aberrantly expressed in high-grade, comedo DCIS. Real-time PCR, mRNA in situ hybridization, and immunohistochemical analysis of breast carcinomas confirmed that psoriasin is frequently overexpressed in estrogen receptor-negative tumors. To gain insight into regulatory pathways that control psoriasin expression, we developed polyclonal and monoclonal antibodies and investigated mechanisms that may account for elevated levels of psoriasin in DCIS. Here, we report that loss of attachment to extracellular matrix, growth factor deprivation, and confluent conditions dramatically up-regulate psoriasin expression in MCF10A mammary epithelial cells. All of these conditions are characteristic of high-grade DCIS and psoriatic skin lesions; therefore, the same mechanisms may be responsible for increased expression of psoriasin in vitro and in vivo.

Background: A cycle day 3 FSH concentration is a popular screening tool for predicting success in achieving pregnancy after IVF. Difficulties interpreting this test have resulted from lack of consensus in defining an elevated FSH concentration, a change in the assays, and lack of controlling for factors which may confound the association between FSH concentration and pregnancy., Methods: Assessment was made of the ability of a moderately elevated (10-11.4 mIU/ml, World Health Organization 2nd International Standard (IRP 78/549) and elevated FSH (>11.4 mIU/ml, conversion factor to SI units, 1.00) in predicting ability to achieve pregnancy through IVF and embryo transfer, both independently, and after controlling for confounding variables such as age, diagnosis, and response to gonadotrophins., Results: A total of 293 IVF cycles were retrospectively reviewed. An FSH (>11.4) was strongly associated with inability to achieve pregnancy after IVF both independently (P < 0.01) and after multivariate analysis (P < 0.01), and had a strong predictive value (100%). A moderately elevated FSH (10-11.4) was not statistically associated with pregnancy outcome either independently or after multivariate analysis, and had a low predictive value (71%)., Conclusions: Much of the predictive value of an elevated FSH is confounded by poor response to gonadotrophin stimulation, which may be overcome in younger women.

The oral contraceptive pill is one of the most extensively studied medications ever prescribed. The health benefits are numerous and outweigh the risks of their use. Definitive evidence exists for protection against ovarian and endometrial cancers, benign breast disease, pelvic inflammatory disease requiring hospitalization, ectopic pregnancy, and iron-deficiency anemia. It has also been suggested that oral contraceptives may provide a benefit on bone mineral density, uterine fibroids, toxic shock syndrome, and colorectal cancer. Minimal supportive evidence exists for oral contraceptives protecting against the development of functional ovarian cysts and rheumatoid arthritis. Treatment of medical disorders with oral contraceptives is an "off-label" practice. Dysmenorrhea, irregular or excessive bleeding, acne, hirsutism, and endometriosis-associated pain are common targets for oral contraceptive therapy. Most patients are unaware of these health benefits and therapeutic uses of oral contraceptives, and they tend to overestimate their risk. Counseling and education are necessary to help women make well-informed health-care decisions and improve compliance.

Context: A large body of evidence documents the role of phytoestrogens in influencing hormone-dependent states. Infants fed soy formula receive high levels of phytoestrogens, in the form of soy isoflavones, during a stage of development at which permanent effects are theoretically possible. However, a paucity of data exists on the long-term effects of infant soy formulas., Objective: To examine the association between infant exposure to soy formula and health in young adulthood, with an emphasis on reproductive health., Design, Setting, and Participants: Retrospective cohort study conducted from March to August 1999 among adults aged 20 to 34 years who, as infants, participated during 1965-1978 in controlled feeding studies conducted at the University of Iowa, Iowa City (248 were fed soy formula and 563 were fed cow milk formula during infancy)., Main Outcome Measures: Self-reported pubertal maturation, menstrual and reproductive history, height and usual weight, and current health, compared based on type of formula exposure during infancy., Results: No statistically significant differences were observed between groups in either women or men for more than 30 outcomes. However, women who had been fed soy formula reported slightly longer duration of menstrual bleeding (adjusted mean difference, 0.37 days; 95% confidence interval [CI], 0.06-0.68), with no difference in severity of menstrual flow. They also reported greater discomfort with menstruation (unadjusted relative risk for extreme discomfort vs no or mild pain, 1.77; 95% CI, 1.04-3.00)., Conclusions: Exposure to soy formula does not appear to lead to different general health or reproductive outcomes than exposure to cow milk formula. Although the few positive findings should be explored in future studies, our findings are reassuring about the safety of infant soy formula.

Implantable contraception represents a safe, reversible form of long-term family planning with distinct advantages. It does not contain estrogen, thus avoiding many of it contraindications. Importantly, it is extremely effective, has high continuation rates, and is well tolerated. The main side effects of Norplant are irregular menstrual bleeding and the occasional difficult removal. Although recent high-profile litigation has severely diminished enthusiasm for Norplant, the FDA has reaffirmed its safety and efficacy. Norplant remains a viable family planning option for carefully selected women. New contraceptive implants may soon be available accentuating the advantages of Norplant while minimizing the disadvantages.

Ectopic pregnancy (EP) is a major cause of maternal morbidity and mortality. The treatment of this condition is primarily surgical, but medical management in selected cases is safe, effective, cost-effective and eliminates the morbidity of surgery. Methotrexate (MTX) is a folate antagonist that can be used for non-oncologic purposes including the treatment of EP. The dose and duration of MTX therapy for EP is much lower than that used in oncology cases, thus reducing side effects and increasing safety. MTX selectively acts on rapidly dividing cells, such as trophoblast cells which comprise the implantation site of the early gestation. The two most common methods of administering MTX to patients with EP are im. administration of a single-dose, based on body surface area and calculated by the equation 50 mg/m(2) (without the need for leucovorin rescue), or the multiple-dose regimen of 1 mg/kg of MTX, alternating with 0.1 mg/kg of leucovorin rescue. Both methods have a similar side effect profile, resulting in the rare occurrence of nausea, vomiting, stomatitis, elevated liver function tests, anorexia and diarrhoea. The two methods yield success rates similar to those of conservative surgical therapy with similar future fertility. The potential single- and multi-dose methods have never been directly compared, but it appears that the success of multiple dosing is more effective. As the efficacy of MTX therapy is not 100%, women must be followed clinically until there is compete resolution of human Chorionic Gonadotropin (hCG) titres from their serum.

Background: Previously we have shown that gene transfer to salivary gland epithelial cells readily occurs via recombinant adenoviruses, although the response is short-lived and results in a potent host immune response. The aim of the present study was to assess the feasibility of using cationic liposomes to mediate gene transfer to rat salivary cells in vitro and in vivo., Methods: Initially, for transfection in vitro, we used two cationic liposome formulations (GAP-DLRIE/DOPE and DOSPA/DOPE) complexed with plasmid encoding human growth hormone (hGH) as a reporter gene. Thereafter, using GAP-DLRIE/DOPE, plasmids were transferred to rat salivary glands in vivo, and hGH levels measured in saliva, serum and gland extracts., Results: Under optimal conditions, transfection of rat submandibular glands (SMGs) was consistently observed. Approximately 95% of the cells transfected with a plasmid encoding beta-galactosidase were acinar cells. Maximal hGH expression was obtained during the first 48 h post-transfection using a plasmid encoding the hGH cDNA and complexed with GAP-DLRIE/DOPE. hGH was detected in gland extracts and saliva, and occasionally in serum. No systemic or local gland pathology was consistently or significantly observed., Conclusions: The levels of the reporter gene product, hGH, obtained after GAP-DLRIE/DOPE-mediated gene transfer are considerably lower (<0.5%) than those achieved with adenoviral vectors (10(8) PFU). Nonetheless, cationic liposome-mediated gene transfer to salivary glands may be useful for potential therapeutic applications.

Objective: The aim of this study was to compare the perioperative morbidity associated with abdominal myomectomy with that of hysterectomy., Study Design: This was a retrospective cohort study of 394 women at an academic medical center. Main outcome measured was perioperative morbidity, with the following secondary outcomes: febrile morbidity, hemorrhage, unintended major surgical procedures, life-threatening events, and rehospitalization., Results: Morbidity was associated with myomectomy and hysterectomy in 39% and 40% of cases, respectively. The crude odds ratio for morbidity of myomectomy with respect to hysterectomy was 0.93 (95% confidence interval, 0.63-1.40). Women who underwent myomectomy were significantly younger, weighed less, and had a smaller preoperative uterine size. In a multivariable analysis that accounted for these differences the odds ratio increased to 1.46 (95% confidence interval, 0.77-2.77) but still was not statistically elevated. The study had >90% power to detect a clinically relevant 15% absolute difference in overall morbidity between the 2 groups., Conclusion: No clinically significant difference in perioperative morbidity between myomectomy and hysterectomy was detected. Myomectomy should be considered a safe alternative to hysterectomy.

Medical treatment of ectopic pregnancy with methotrexate has been shown to be effective and safe in appropriate patients. When considering medical management, the definitive diagnosis of an ectopic pregnancy and the assurance that the patient is a candidate are of paramount concern. An understanding of the mechanism of action and pharmacokinetics of methotrexate allows the clinician to better inform patients, recognize and treat side effects, and be cognizant when treatment is unsuccessful. Two common protocols, the "multi-dose" and the "single-dose" protocols, have excellent success rates; however, neither one of these is completely effective. The "multi-dose" protocol appears to have a higher success rate than the "single-dose" protocol.

Success in controlling hyperglycemia in type I diabetics will require a restoration of basal insulin. To this end, three plasmid DNAs (pDNA) encoding preproinsulin were compared for constitutive expression and processing to insulin in nonendocrine cells in vitro. The pDNAs were designed to express rat proinsulin I (VR-3501), rat proinsulin I with the B10 aspartic acid point mutation (VR-3502), and a derivative of VR-3502 with a furin cleavage site added at the B-chain and C-peptide junction (VR-3503). Cells transfected with VR-3501 or VR-3502 were able to secrete only proinsulin, whereas transfection with VR-3503 yielded 30-70% mature insulin, which could be increased to >99% by cotransfection with a furin expression plasmid (VR-3505). The insulin produced was biologically active. The bilateral injection of 100 microg of VR-3502 plasmid into the tibialis anterior muscles of mice on two consecutive days yielded, on average, several hundred picograms of heterologous proinsulin per milliliter of serum. In BALB/c mice, serum proinsulin peaked 7-14 days postinjection and declined to preinjection levels by days 21-28. In athymic nude mice, serum proinsulin was sustained for at least 6 weeks. The therapeutic efficacy of delivering insulin via muscle injection of pDNA was evaluated in athymic nude mice made diabetic with the beta cell toxin streptozotocin (STZ). All animals given control DNA died within 1 week of receiving STZ while 40% of the mice coinjected with plasmids VR-3503 and VR-3505 lived through the duration of the 4-week experiment. Muscles of the surviving animals contained 17-100 ng of immune-reactive insulin (IRI), 86-94% of which was mature insulin. The results suggest that heterologous insulin made in muscle increased the survival rate. We propose that insulin plasmid expression in skeletal muscle may be a valid approach to basal insulin delivery. The feasibility of plasmid DNA-based delivery of basal insulin was investigated. An expression system consisting of pDNAs encoding a selectively mutated rat preproinsulin and mouse furin was developed and characterized in vitro and in vivo. When injected with preproinsulin pDNA, the mouse tibialis anterior muscle expressed and released proinsulin into serum at levels comparable to normal basal insulin in rodents. These heterologous proinsulin levels were sustained for several weeks in immune-compromised nondiabetic mice. Mouse muscle coinjected with a pDNA encoding the endopeptidase furin and a pDNA encoding a pre-proinsulin modified to contain two furin cleavage sites produced fully processed insulin. This muscle-made insulin appears to have contributed to the survival of mice treated with a highly diabetogenic dose of streptozotocin, a beta cell toxin. The results demonstrate that skeletal muscle is able to express and deliver therapeutic insulin from plasmid DNA.

The antitumor effect of the type I IFN, IFN-omega, was evaluated in both in vitro and in vivo studies of human cancer. For these studies, the cDNA for human IFN-omega was cloned into a eukaryotic expression plasmid DNA (pDNA) driven by the cytomegalovirus promoter. Supernatants from UM449 cells transfected in vitro with IFN-omega pDNA had antiproliferative effects on 11 of 13 human tumor cell lines. For in vivo studies, nude mice were implanted s.c. with one of the following human tumors: NIH: OVCAR-3 ovarian carcinoma, A375 melanoma, or A431 epidermoid carcinoma. Direct intratumoral injection of 100 microg of a IFN-omega pDNA DMRIE/DOPE complex (1:1 DNA:DMRIE mass ratio) for 6 consecutive days resulted in a significant reduction in the tumor volume of NIH: OVCAR-3 ovarian carcinoma or A375 melanoma (P = 0.02). IFN-omega pDNA delivered by i.m. injection also had an antitumor effect. Nude mice bearing s.c. A431 epidermoid carcinoma and injected i.m. with 100 microg of IFN-omega pDNA, twice per week for 3 weeks, had a significant reduction in tumor volume (P = 0.009). These results demonstrate for the first time that IFN-omega can have in vivo antitumor effects in several models of human cancer.

This meeting collected experts from the field of clinical medicine and pharmaceutical companies to discuss various issues concerning vaginal drug delivery. The conference was divided into large sub-categories, such as the advantages of a vaginal system, including its potential use in providing both disease-specific and systemic drug administration. The specific need for topical microbicides and anti-HIV systems were highlighted. Discussions included the unique anatomical and physiological characteristics of the human vagina as well as the potential lack of an animal model to predict clinical results. Specific formulation techniques discussed included: polycarbophil, as a bio-adhesive; a hydrogel, Hycor-V; membrane lipids, such as Q-vail; and a hybrid emulsion system, such as the Site Release system. The potential development of vaginal vaccines and vaginal rings to provide systemic drug delivery were also discussed. Finally, there was discussion regarding the information needed to evaluate and develop novel actives and delivery systems. The general agreement from representatives of regulatory bodies was that, because of the uniqueness of this anatomical area and technology used, early participation of regulatory agencies to assist with development is highly recommended. The conference was chaired by Dr Ian Wilding (Chief Executive, Pharmaceutical Profiles, Nottingham, UK) and Professor SS Davis (Chairman, West Pharmaceutical Services, formerly DanBioSyst UK Ltd, Nottingham, UK). Forty participants from industry and academia were in attendance. Speakers included representatives of academic institutions from Europe and the US, the FDA, the National Institutes of Health as well as from numerous international pharmaceutical corporations.

A cancer treatment is described in which i.m. injection of plasmid DNA (pDNA) encoding murine interferon alpha (mIFN-alpha) leads to potent antitumor effects on primary and metastatic tumors in mice. Mice bearing s.c. B16F10 melanoma, Cloudman melanoma, or glioma 261 tumors were injected i.m. with mIFN-alpha pDNA. In all three tumor models, a significant reduction in tumor volume and enhancement of survival was found after IFN pDNA therapy. The mIFN-alpha pDNA could be injected as infrequently as once every other week and still produce a significant antitumor effect, and, in a metastatic tumor model, the therapy markedly reduced the number of lung tumor metastases. Depletion of immune cell subsets indicated that CD8(+) T cells were required for the antitumor response. These studies demonstrate that primary and metastatic tumors can be treated systemically by i.m. injection of a plasmid encoding a cytokine gene.

A DNA plasmid encoding the gene of the major outer membrane protein (MOMP) of the Chlamydia trachomatis mouse pneumonitis (MoPn) serovar and three plasmids containing the variable domains (VD) of the MOMP were constructed. Female mice were inoculated with the plasmids and 60 days later were challenged in the genital tract with C. trachomatis. Six weeks after challenge female mice were caged with male mice and the course of the mating followed. Mice immunized with the MOMP plasmids mounted weak humoral and cell mediated immune responses. However, following the genital challenge no significant differences in vaginal shedding were observed between the groups immunized with the MOMP and control plasmids. In addition, the fertility rates were similar in the experimental and negative control groups. In conclusion, vaccination with DNA plasmids encoding the MOMP elicited a modest immune response but did not protect against infection or disease.

Bacterial DNA contains immunostimulatory motifs that trigger an innate immune response characterized by the production of predominantly Th1-type cytokines. These motifs consist of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines. We examined whether synthetic oligodeoxynucleotides (oligos) expressing these motifs would act as adjuvants to boost the immune response to DNA- and protein-based immunogens. In vivo experiments demonstrate that CpG-containing oligos augment antigen-specific serum antibody levels by up to tenfold, and IFNgamma production by up to sixfold. These effects were optimized by physically linking the CpG-containing motifs to the immunogen.

Enhancers and promoters from various muscle-specific genes were substituted for or combined with the enhancer/promoter of the human cytomegalovirus (CMV) IE gene in a luciferase reporter gene plasmid in an effort to identify new promoter chimeras with increased expression activity after direct intramuscular injection. The regulatory sequence substitutions or additions varied in content, location, and orientation relative to the CMV regulatory sequences. The expression activities of the derivative and parent plasmids were compared quantitatively in vivo using a standard mouse intramuscular injection assay, and in vitro by transfection of differentiated C2C12 mouse myoblasts and BHK hamster kidney cells, to test whether cultured cell transfection could substitute for at least some animal experimentation. In vivo, 1 of 19 of the enhancer/promoter chimeras increased expression levels. In vitro, some chimeras showed significant expression augmentation in C2C12 cells, but not in BHK cells. We conclude that because of differences in plasmid expression profiles, these cell culture systems cannot readily substitute for in vivo testing of new plasmid constructs.

Using the murine parasite Plasmodium yoelii (Py) as a model for malaria vaccine development, we have previously shown that a DNA plasmid encoding the Py circumsporozoite protein (PyCSP) can protect mice against sporozoite infection. We now report that mixing a new plasmid PyCSP1012 with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) increases protection against malaria, and we have characterized in detail the increased immune responses due to GM-CSF. PyCSP1012 plasmid alone protected 28% of mice, and protection increased to 58% when GM-CSF was added (p < 0.0001). GM-CSF plasmid alone did not protect, and control plasmid expressing inactive GM-CSF did not enhance protection. GM-CSF plasmid increased Abs to PyCSP of IgG1, IgG2a, and IgG2b isotypes, but not IgG3 or IgM. IFN-gamma responses of CD8+ T cells to the PyCSP 280-288 amino acid epitope increased but CTL activity did not change. The most dramatic changes after adding GM-CSF plasmid were increases in Ag-specific IL-2 production and CD4+ T cell proliferation. We hypothesize that GM-CSF may act on dendritic cells to enhance presentation of the PyCSP Ag, with enhanced IL-2 production and CD4+ T cell activation driving the increases in Abs and CD8+ T cell function. Recombinant GM-CSF is already used in humans for medical purposes, and GM-CSF protein or plasmids may be useful as enhancers of DNA vaccines.

Intratumoral (i.t.) injection of a plasmid DNA vector encoding the murine interleukin-2 (IL-2) gene was used to treat established renal cell carcinoma (Renca) tumors in BALB/c mice. Tumor regression was observed in 60-90% of mice that were injected i.t. for 4 days with IL-2 plasmid DNA complexed with the cationic lipid DMRIE/DOPE ((+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propa naminium bromide/dioleoylphosphatidylethanolamine). The mice remained tumor-free until the conclusion of the study, which was 4 months after tumor challenge. In a rechallenge experiment, mice that were rendered tumor-free for 6 months by IL-2 plasmid DNA treatment rejected a subsequent challenge of Renca cells but could not reject a challenge with the unrelated, syngeneic CT-26 tumor. Spleen cells from cured mice contained Renca-specific cytotoxic T lymphocytes, and adoptive transfer of mixed lymphocyte cultures into naive mice at 2 days after challenge with Renca cells prevented tumor growth. In vivo depletion of T-cell subsets at the time of i.t. injection with IL-2 plasmid DNA demonstrated that CD8+ T cells, but not CD4+ T cells, were the primary effectors of the antitumor response.

Kaposi's sarcoma (KS) is the most frequent malignancy occurring in HIV-positive individuals. AIDS-KS is a more aggressive disease than the classical form, frequently having a rapid clinical course with numerous serious complications. Current systemic treatments for KS, such as chemotherapy and the administration of biological modifiers, are complicated by both the drug resistance of the tumor and the dose-limiting toxicity of the reagents. The relative accessibility of many KS lesions makes the disease a particularly attractive candidate for in vivo gene therapy protocols. In this regard, we are interested in delivering conditionally toxic suicide and/or antiangiogenic vectors to accomplish targeted cell death selectively in AIDS-KS cells. To this end, we examined both cationic lipid- and adenoviral-mediated DNA transfection methods. Using the firefly luciferase reporter gene, we optimized numerous variables known to be important in lipid-mediated DNA transfection, including lipid formulation, the amount of lipid and DNA, lipid/DNA ratio, and cell concentration. Under optimal transfection conditions, approximately 5-25% of KS cells expressed the introduced DNA sequences. Adenoviral-mediated DNA delivery was more efficient than lipid delivery in 4 of 5 primary KS cell lines. Two of the lines (RW248 and RW376) were transduced by adenovirus at frequencies approaching 100%; two cell lines (CVU-1 and RW80) gave efficiencies of 20-35%. Two immortalized KS cell lines (KS Y-1 and KS SLK) were poorly infected, giving a transduction efficiency of <5%. These findings demonstrate that gene transfer into AIDS-KS cells is feasible, and suggest that vector strategies may be permissive for translating gene therapy approaches for the disease.

Background: Medical management of ectopic pregnancy is effective in selected cases and is recommended to treat interstitial pregnancy to avoid surgery that may limit future fertility. Although successful medical management of interstitial pregnancy has been reported, evidence does not support success rates equal to that in other ectopic locations., Cases: Four cases of interstitial ectopic pregnancy were identified. Only one of the four was successfully treated with medical therapy. Case 1 was a confirmed, 2.5-cm interstitial pregnancy that failed a multiple-dose methotrexate protocol. Case 2 was an unrecognized interstitial pregnancy that failed single-dose therapy. Case 3 was a rupture of a medically managed interstitial pregnancy despite complete resolution of serum human chorionic gonadotropin. A literature review demonstrated an overall combined failure rate of 35% (7/20)., Conclusion: Compared with an isthmic or ampullary pregnancy, a disproportionate number of patients with interstitial pregnancy fail medical management and require emergency surgery. The presence of a documented interstitial pregnancy is not an absolute contraindication to medical management. Patients should be counseled that medical management of an interstitial pregnancy may not have the same rate of success as it does in other locations in the fallopian tube. Surgery in this case may be the preferred treatment option.

Objective: This study was undertaken to examine the accuracy of frozen section for the identification of products of conception on curettage of patients undergoing surgery for suspected ectopic pregnancy., Study Design: A retrospective analysis of 87 consecutive cases in which frozen-section assessment of an endometrial curettage specimen to identify products of conception was undertaken. A frozen-section diagnosis was considered correct if the final diagnosis concurred with the findings at frozen section., Results: Eighty-one of 87 (93.1%) specimens were correctly identified at frozen section. Frozen section had a sensitivity of 78.3% and a specificity of 98.4%. The positive predictive value of frozen section in this population was 94.7% and the negative predictive value was 92.6%. No significant correlation was found regarding the attending pathologist at frozen section, preoperative human chorionic gonadotropin, or time of day at frozen section., Conclusions: Frozen section is accurate for the identification of products of conception on endometrial curettage in the evaluation of a suspected ectopic pregnancy.

Objective: To evaluate the potential utility of endometrial stripe thickness in predicting pregnancy outcome in women with an hCG less than a discriminatory zone., Design: Retrospective case review., Setting: University emergency department., Patients: Women who presented with symptomatic early pregnancies after a spontaneous conception with an hCG level less than a discriminatory zone evaluated to rule out an ectopic pregnancy (EP)., Interventions: Measure endometrial stripe thickness by transvaginal ultrasound., Main Outcome Measures: Initial endometrial stripe thickness was correlated to eventual pregnancy outcomes., Results: The mean initial endometrial stripe thickness of patients eventually diagnosed with an intrauterine pregnancy (13.42 +/- 0.68 mm), spontaneous abortion (9.28 +/- 0.88 mm), and an EP (5.95 +/- 0.35 mm) were all statistically different from each other. Ninety-seven percent of pregnancies found to have an endometrial stripe thickness < or = 8 mm were abnormal (EP or spontaneous abortion)., Conclusions: This study suggest a role of the evaluation of the endometrial stripe thickness in the detection of abnormal pregnancies in patients presenting for evaluation of a symptomatic early pregnancy with an hCG below a discriminatory zone. Initial endometrial stripe thickness measured in patients with an EP is significantly thinner than in those with an intrauterine pregnancy.

In previous work, the direct injection of 50 micrograms of a plasmid DNA vector encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days postinjection. In this report, various elements of the VR1205 vector were modified to increase gene expression levels or to eliminate undesired viral sequences. Expression of the modified vectors was then compared to VR1205 using the intramuscular injection assay. In general, modifications to promoter, enhancer, and intronic sequences either decreased luciferase expression levels or had no effect. However, modifications to the polyadenylation and transcriptional termination sequences, plasmid backbone elements, and the luciferase gene itself each increased luciferase expression levels. The best-expressing vector, designated VR1255, contained a combination of these incrementally beneficial changes. A single intramuscular injection of 50 micrograms of VR1255 produced 300 ng of luciferase at 7 days postinjection, an expression level 46-fold higher than the VR1205 vector (or 22-fold higher, excluding modifications to the luciferase gene) and 154-fold higher than a commercially available luciferase expression vector. Thus, VR1255 represents an improved plasmid DNA vector that may be useful for gene therapy applications.

Many women have menstrual symptoms, but relatively few have severe PMS. PMS is a well-defined premenstrual cluster of predominantly affective symptoms that disrupt a woman's daily functioning. PMS is diagnosed with prospective charting of symptoms and should be differentiated from nondisruptive menstrual symptoms, major affective disorders, and other common medical and gynecologic conditions. Most women with PMS can be helped. The serotonin reuptake inhibitors are becoming the first line of therapy for PMS because they are effective, easily tolerated, and free of major side effects. There is also evidence supporting the role of other antidepressants, anxiolytics, and GnRH agonists in the treatment of PMS. Although increasing control of one's life, promoting a healthy diet, the avoidance of salt and caffeine, vitamin supplementation, and exercise have not been proved as effective treatment for PMS, they should be promoted for their obvious general health benefits. No one treatment fits the heterogeneous PMS population. A trial of medication should be continued for two or three menstrual cycles with appropriate dose adjustments. If relief is not sufficient, other agents or other treatments should be initiated.

Emergency contraception is the only form of contraception where implementation can occur after sexual relations or forced intercourse. Hormonal methods can be administered up to 72h after unprotected intercourse. Emergency contraception is safe, legal, and simple to administer. Widespread availability could dramatically decrease the number of unwanted pregnancies.

Objective: To evaluate quantitative hCG measurements and transvaginal ultrasound in the diagnosis of ectopic pregnancy in patients presenting to the emergency department., Methods: A discriminatory zone for detecting the presence or absence of an intrauterine pregnancy by transvaginal ultrasound was established prospectively. Women presenting to the emergency department were evaluated prospectively using a diagnostic algorithm consisting of clinical examination, quantitative serum hCG, and transvaginal ultrasound. Finally, ectopic pregnancies diagnosed over a 22-month period were evaluated prospectively., Results: All viable intrauterine pregnancies were identified in those subjects with hCG levels of 1500 mIU/mL (First International Reference) or greater. One thousand two hundred sixty-three subjects were evaluated prospectively; 59.8% were diagnosed with intrauterine pregnancy, 26.8% with spontaneous abortion, and 7.8% with ectopic pregnancy. At presentation, 13.2% of intrauterine pregnancies were diagnosed by clinical examination, whereas 82.9% were diagnosed by transvaginal ultrasound. Only 4% of normal intrauterine pregnancies were not confirmed on initial visit. Of 205 ectopic pregnancies diagnosed, 81.5% were hemodynamically stable; of these, 49.1% were diagnosed on initial presentation. Of all ectopics, 59% never reached an hCG level of 1500 mIU/mL and 35.8% had an hCG lower than the level at presentation. This protocol diagnosed ectopic pregnancies with a sensitivity of 100% and a specificity of 99.9%., Conclusion: A protocol of quantitative hCG levels (available within hours of presentation to an emergency department) combined with transvaginal ultrasound is effective in diagnosing ectopic pregnancy.

Sexually active couples need to be concerned with the risk of sexually transmitted diseases (STDs) and how their choice of contraception influences that risk. Condoms provide the best documented protection against such pathogens as: gonorrhea, herpes simplex virus (HSV), hepatitis B, HIV, and chlamydia. Female dependent barrier methods also provide protection against most STDs and also possibly HIV. Most hormonal non-barrier contraceptives, although providing excellent protection against unwanted pregnancies, provide little protection against STDs. Oral contraceptive pills (OCP) may increase the risk of infection with human papillomavirus (HPV) and cervical infections of chlamydia. Individuals at high risk for both an unwanted pregnancy and an STD should be counseled to use both a hormonal and barrier contraceptive. Recently, nonoxynol-9 (N-9) and OCP use have been associated with an increase in HIV infection in African women at high risk for HIV. This association has not been found in other studies and currently does not outweigh the proven benefits of these contraceptive methods.

Eighty-one patients with long-term tracheostomy tubes (mean duration, 4.9 months) were examined via fiberoptic bronchoscopy prior to decannulation. Obstructive airway lesions were observed in 54 patients (67 percent). All tracheal lesions were anatomically located proximal to the stoma. No cuff lesions were observed. The two most commonly observed lesions were tracheal granuloma (60 percent) and tracheomalacia (29 percent). Less frequently observed lesions were tracheostenosis (14 percent) and vocal cord and laryngeal dysfunction (8 percent). As a result of the high frequency of tracheal abnormalities, especially that for tracheal granuloma which has not been previously reported (to our knowledge), we recommend that all decannulation candidates undergo anatomic examination of the airways.

Bacteriophage T4 gene 32 protein (g32P) is a DNA replication accessory protein that binds single-stranded (ss) nucleic acids nonspecifically, independent of nucleotide sequence. G32P contains 1 mol of Zn(II)/mol of protein monomer, which can be substituted with Co(II), with maintenance of the structure and activity of the molecule. The Co(II) is coordinated via approximately tetrahedral ligand symmetry by three Cys sulfur atoms and therefore exhibits intense S(-)----Co(II) ligand to metal charge-transfer (LMCT) transitions in the near ultraviolet [Giedroc, D. P., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8452-8456]. A series of fluorescent 1,N6-ethenoadenosine (epsilon A)-containing oligonucleotides conforming to the structure (5'----3') d[(Tp)m epsilon A(pT)l-m-1] where 0 less than or equal to m less than or equal to l - 1 and length (l) six or eight nucleotides have been evaluated as dynamics probes and potential fluorescence energy transfer donors to Co(II) in mapping the spatial proximity of the (fixed) intrinsic metal ion and a variably positioned epsilon A-base in a series of protein-nucleic acid complexes. We provide spectroscopic evidence that the epsilon A-oligonucleotides bind to g32P-(A + B) with a fixed polarity of the phosphodiester chain. A Trp side chain(s) makes close approach to a epsilon A base positioned toward the 3' end of a bound l = 8 oligonucleotide. Six oligonucleotides of l = 8 and m = 0, 1, 3, 5, 6, or 7 were investigated as energy transfer donors to Co(II) at 0.1 M NaCl, pH 8.1, 25 degrees C upon binding to Co(II)-substituted or Zn(II) g32P-(A + B), i.e., in the presence and absence of an energy acceptor, respectively. Detectable quenching of the epsilon A-fluorescence by the Co(II)-LMCT acceptors was found to occur in all epsilon A-oligonucleotide-protein complexes, yielding energy transfer efficiencies (E) of 0.43, 0.31, 0.26, 0.26, 0.28, and 0.41 for l = 8 and m = 0, 1, 3, 5, 6, and 7 epsilon A-oligonucleotides, respectively. The two-dimensional distances R (in A) were found to vary as follows: d[epsilon A(pT)7] (m = 0), 16.0 (15.5-16.9); d[Tp epsilon A(pT)6] (m = 1), 17.7 (16.9-19.1); d[(Tp)3 epsilon A(pT)4] (m = 3), 20.7 (19.5-22.1); d[(Tp)5 epsilon A(pT)2] (m = 5), 20.5 (19.5-21.9); d[(Tp)6 epsilon ApT] (m = 6), 19.0 (18.0-20.4); and d[(Tp)7 epsilon A] (m = 7), 18.6 (17.8-19.8).(ABSTRACT TRUNCATED AT 400 WORDS)

Single i.p. injections of cisplatin followed by 12-O-tetradecanoylphorbol-13-acetate TPA promotion led to the formation of skin tumors in mice, as did topical applications of urethane with TPA promotion. This is the first example of the use of cisplatin in a single treatment as an initiating agent in the standard initiation-promotion regime. Treatment of the skin with TPA 24 h before initiation increased tumor yield, both in terms of the percentage of mice with tumors and the average number of tumors per mouse.