A combinatorial phage display library for the generation of specific Fab fragments recognizing human spermatozoa and inhibiting fertilizing capacity in vitro

To select a source of lymphocytes for the generation of an anti-sperm-biased combinatorial phage display library, venous blood was obtained from 34 vasovasostomy (vasectomy reversal) patients approximately 3 mo after surgery. Using a variety of immunoassays, serum was analyzed for antibodies against human spermatozoa, and a patient was selected on the basis of high titer of antibodies that recognized the equatorial region of the sperm head and inhibited sperm fertilizing capacity in vitro. Total RNA isolated from the stored lymphocytes of this individual was reversed transcribed, and gamma1 (Fd) region and kappa chains were amplified by polymerase chain reaction for the successful construction of an antibody phage display library. The library was panned against human spermatozoa to isolate sperm-specific phage that recognized the equatorial region of the sperm head. Three preparations of Fab were tested via the hamster egg penetration test. Each preparation significantly (p < 0. 005) inhibited sperm-egg binding and fusion, with one preparation (designated Fab-G) causing complete inhibition. Sequence analysis of the kappa light gene encoding Fab-G revealed a 93% homology with the light chain of human anti-human immunodeficiency virus gp120 p35 variable region. This technology may have a practical application in characterization of the immune response to spermatozoa and for the design of sperm-based contraceptive vaccines.     

Molecular basis and species specificity of high affinity binding of vasoactive intestinal polypeptide by the rat secretin receptor

The affinity and specificity of the binding interaction between ligands and their receptors are key for appropriate hormonal regulation of target tissues. However, it is now apparent that vasoactive intestinal polypeptide (VIP) binds to the rat secretin receptor with similar affinity to that for its natural ligand, secretin (Holtmann et al., 1995). In this report, we establish that this is not a characteristic of the human secretin receptor, and use rat-human secretin receptor chimeras, site mutants and truncated receptor constructs to establish the molecular basis for this unusual binding interaction. Of note, isolated N-terminal domains of the rat secretin and the VIP receptors are capable of high affinity binding of VIP. In the recently recognized secretin family of receptors, this domain has six conserved cysteine residues and disulfide bonds that are likely important to achieve the complex conformation critical for this binding. A single acidic residue (Asp98) present in the rat secretin receptor appears to be critical, because a site-mutant changing this to the polar, but uncharged residue present in that position in the human receptor (Asn) eliminates the high affinity binding of VIP. Of interest, a previously identified critical basic residue in VIP (Lys15) provides a candidate for charge-pairing with this residue, potentially aligning the peptide ligand in a nonproductive orientation within this receptor.     

Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.     

Treatment of spondylolysis with external electrical stimulation and bracing in adolescent athletes: a report of two cases

BACKGROUND: An epidemic of bilateral optic neuropathy is affecting large numbers of people aged between 10 and 40 in Dar es Salaam, the capital city of Tanzania. The disease is characterised by acute onset of bilateral visual impairment, bilateral impairment of colour vision, and a characteristic temporal pallor of the optic discs. The disease often occurs in association with peripheral neuropathy and sensorineural hearing loss. This report presents the first data on disease prevalence in adolescents, based on a rapid assessment of schoolchildren. METHODS: Three schools in Dar es Salaam were visited and all children aged between 10 and 16 were screened for the disease. RESULTS: The prevalence of bilateral optic neuropathy among the schoolchildren is estimated to be 1.0% (95% CI 0.5-1.4%). This suggests that 5000 children (95% CI 2600-7300) aged 10-16 in Dar es Salaam may have the disease. CONCLUSION: The results of this rapid assessment indicate this epidemic is a major public health problem. The prevalence of the disease in the community is likely to be far higher than found in this survey because children with the disease may have withdrawn from school. As the disease predominantly affects young adults, resulting in impaired vision and hearing, the economic and social consequences are enormous.     

The role of type III collagen in spontaneous cervical arterial dissections

OBJECTIVE: To determine the efficacy of lymphadenectomy after nephroureterectomy in patients with transitional cell carcinoma (TCC) of the upper urinary tract. PATIENTS AND METHODS: Between January 1986 and December 1995, 72 patients (mean age 67 years, range 45-82) underwent nephroureterectomy for primary TCC of the upper urinary tract. In 35 patients, a lymphadenectomy was also performed. The clinicopathological data were analysed retrospectively, focusing on the significance of lymphadenectomy. RESULTS: Lymph vessel invasion was found in 28 patients and its incidence was closely correlated with both tumour grade and pathological stage. Of the 35 patients who underwent lymphadenectomy, lymph node metastases were found in 13 patients, all of whom had lymph vessel invasion. There was no significant difference in the survival rate between patients with and without lymphadenectomy; however, among the 44 patients with no lymph vessel invasion, the survival rate of those with lymphadenectomy was significantly higher than in those without (P<0.05). CONCLUSION: Lymphadenectomy may provide a therapeutic advantage in patients with upper urinary tract TCC and no lymph vessel invasion. However, patients with lymph vessel invasion seem to have systemic disease; therefore, aggressive systemic adjuvant therapies rather than regional lymphadenectomy should be applied in these patients.     

Studies on the efficacy of hyperbaric rendering procedures in inactivating bovine spongiform encephalopathy (BSE) and scrapie agents

The efficacy of the procedures in use at the two rendering plants in the Netherlands was assessed on a laboratory-scale using procedures that simulated the pressure cooking part of the rendering process. A pool of bovine spongiform encephalopathy (BSE)-infected brainstem from the United Kingdom and a pool of scrapie-infected brainstem from Dutch sheep were used to spike the rendering materials. The mixtures were subjected to various time-temperature combinations of hyperbaric heat treatment related to the conditions used in Dutch rendering plants in the early 1990s, and to the combination of 20 minutes at 133 degrees C required by the EU Directive on rendering of 1996. The efficacy of the procedures in inactivating BSE or scrapie infectivity was measured by titrating the materials before and after heat treatment in inbred mice, by combined intracerebral and intraperitoneal inoculations at limiting dilutions. Two independent series of experiments were carried out. The design of the study allowed for minimum inactivations of up to 2.2 log (2.0 in the second series) to be measured in the diluted infective material and 3.1 log in the undiluted material. After 20 minutes at 133 degrees C there was a reduction of BSE infectivity of about 2.2 log in the first series (with some residual infectivity detected), and in the second series more than 2.0 log (with no residual infectivity detected). With undiluted brain material there was an inactivation of about 3.0 log (with some residual infectivity detected). With the same procedure, scrapie infectivity was reduced by more than 1.7 log in the first series and by more than 2.2 log in the second series. With undiluted brain material there was an inactivation of more than 3.1 log. In each case no residual scrapie infectivity was detected. The BSE agent consistently appeared to be more resistant to heat inactivation procedures than the scrapie agent, particularly at lower temperatures and shorter times.     

Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5

PURPOSE: To determine the polarity of fibroblast growth factor 5 (FGF5) secretions from retinal pigment epithelium (RPE) cells and to examine the viability and utility of the ARPE-19 cell line as a model for the study of RPE polarity. METHODS: Influenza infection and adenovirus-mediated gene transfer were used to deliver and express genes encoding influenza hemagglutinin (HA), p75-NTR (a neurotrophin receptor), low-density lipoprotein (LDL) receptor (LDLR), and FGF5 in confluent monolayers of ARPE-19 cells. The localization of HA, p75-NTR, and LDLR was determined by confocal microscopy. Domain selective biotinylation assays were used to quantitatively determine the polarities of p75-NTR and LDLR. The secretion of FGF5 into the apical and basal media of ARPE-19 cultures was examined by immunoblot analysis of conditioned media. RESULTS: Hemagglutinin and p75-NTR were found to be localized on the apical surface of infected and transduced ARPE-19 cells. In contrast, LDLR was associated preferentially with the basolateral membrane of ARPE-19 cells. Biotinylation studies indicated that 84% of p75-NTR was present on the apical surface, and 79% of LDLR was basolaterally polarized. Over the course of 6 hours, more than 90% of the total secreted FGF5 protein accumulated in the basolateral media. CONCLUSIONS: ARPE-19 cells exhibit a polarized distribution of cell surface markers when examined by either confocal microscopy or surface-labeling assays. This indicates that the ARPE-19 cell line is a valid model for studies of RPE cell polarity. FGF5, a secreted protein normally produced by RPE cells, is accumulated preferentially in the basal media after only 6 hours, suggesting that it is vectorially secreted from the basolateral surface of ARPE-19 cells.     

Cytomegalovirus retinitis and viral resistance: 3. Culture results. CMV Retinitis and Viral Resistance Study Group

PURPOSE: The Strong Heart Study is a study of cardiovascular disease and its risk factors among American Indian men and women aged 45-74 yr representative of 13 communities from Arizona (AZ), Oklahoma (OK), and North/South Dakota (N/SD). This investigation sought to characterize the amount and type of physical activity and to determine the association between activity and lipids in this population. METHODS: Total physical activity (occupational plus leisure) was assessed with a validated questionnaire. RESULTS: Men and women from OK (21 +/- 19 and 16 +/- 15 h.wk-1; respectively) and N/SD (23 +/- 21 and 17 +/- 17 h.wk-1; respectively) had activity levels that were similar if not lower than the U.S. population with the AZ communities (17 +/- 21 and 10 +/- 14 h.wk-1; respectively) being substantially lower than the other two communities. Total cholesterol (TC), high density lipoprotein cholesterol (HDL-c), and low density lipoprotein cholesterol (LDL-c) levels were lower than the U.S. population. CONCLUSIONS: For most of the population (diabetic men and nondiabetic men and women), activity was significantly associated (P < 0.05) with apolipoprotein (apo) AI after controlling for covariates. With levels of activity as low if not lower than the general U.S. population coupled with high prevalence of obesity and diabetes, efforts to increase physical activity in American Indians are warranted. Hopefully these increases in activity will result in favorable lipid changes as well as decreasing the risk of diabetes which is epidemic in these populations.