A male genital tract-specific carbohydrate epitope on human CD52: implications for immunocontraception.

The identification of unique sperm surface epitopes that are not expressed or exposed in the female reproductive tract is a key element in the development of antibody-based contraceptives. Western blotting and immunohistochemistry were performed to define the tissue distribution of the S19 epitope, which has been proposed as a target for immunocontraception. S19 is an IgG1 murine monoclonal antibody (mAb) directed to an N-linked carbohydrate epitope on a 15-25 kDa glycoprotein, sperm agglutination antigen-1 (SAGA-1), containing a peptide core identical to that of the lymphocytic surface protein CD52. In this study, the S19 epitope was shown to be absent from human lymphocytes, demonstrating a distinction between this epitope and the CAMPATH epitope that is recognized by an antibody against the terminal tripeptide and GPI-anchor of CD52. Further tissue specificity analysis identified the S19 epitope in the epithelium of the human epididymis and vas deferens, as well as on both epididymal and ejaculated spermatozoa. In contrast, the S19 epitope was absent in the five human female reproductive tract and 18 other somatic tissues tested. These results support the use of the S19 epitope as a contraceptive immunogen and the suitability of the S19 mAb as an intravaginal contraceptive. To test the agglutinating activity of the S19 mAb in a formulation designed for vaginal use, S19 mAb were bound to the surface of Novasomes, a multilamellar liposome delivery vehicle. S19-Novasome formulations agglutinated human spermatozoa and were as effective as unbound S19 mAb, demonstrating the feasibility of spermistatic contraceptives targeted to the male reproductive tract specific carbohydrate epitope.

Hazardous air pollutant emissions from gas-fired combustion sources: emissions and the effects of design and fuel type.

Air emissions from gas-fired combustion devices such as boilers, process heaters, gas turbines and stationary reciprocating engines contain hazardous air pollutants (HAPs) subjected to consideration under the federal clean air act (CAA). This work presents a recently completed major research project to develop an understanding of HAP emissions from gas-fired boilers and process heaters and new HAP emission factors based on field emission tests of gas-fired external combustion devices used in the petroleum industry. The effect of combustion system design and operating parameters on HAP emissions determined by both field and research tests are discussed. Data from field tests of gas-fired petroleum industry boilers and heaters generally show very low emission levels of organic HAPs. A comparison of the emission data for boilers and process heaters, including units with and without various forms of NOx emission controls, showed no significant difference in organic HAP emission characteristics due to process or burner design. This conclusion is also supported by the results of research tests with different burner designs. Based on field tests of units fired with natural gas and various petroleum industry process gases and research tests in which gas composition was intentionally varied, organic HAP emissions were not determined to be significantly affected by the gas composition. Research data indicate that elevated organic HAP emission levels are found only under extreme operating conditions (starved air or high excess air combustion) associated with poor combustion.

Inactivation of the small GTPase Rho disrupts cellular attachment and induces adhesion-dependent and adhesion-independent apoptosis.

Rho small GTPases regulate a variety of cellular signaling pathways involved in cell growth and transformation. In this study, we examined potential roles for Rho in adhesion-dependent and -independent pathways regulating apoptosis. Rho GTPases are specifically inactivated by exoenzyme C3 (C3) of Clostridium botulinum. Using a novel Sindbis virus-based gene expression system, we created a double subgenomic recombinant (dsSIN:C3) capable of expressing active C3 in intact cells. Infection of L929 fibroblasts with dsSIN:C3 caused essentially complete ADP-ribosylation of intracellular Rho within 1 h. dsSIN:C3-infected cells also became rounded within 1-2 h and detached by 5 h post-infection. Infection of L929 in suspension with dsSIN:C3 disrupted the ability for normal cellular attachment and spreading. Infection of primary cell explants of chicken embryo fibroblasts (CEF) and rat aortic smooth muscle cells (RSM) with dsSIN:C3 caused cytoskeletal effects similar to those seen in L929. We also observed that C3 markedly decreased the basal phosphorylation state of focal adhesion kinase (FAK). Most intriguingly, we found that dsSIN-based expression of C3 or loss of function mutants of Rho could each induce apoptosis and, in RSM, this effect was observed to be adhesion-independent. Rho GTPases, therefore, appear to regulate signal pathways that are required for cell survival and growth that are separate from, but likely overlap with, Rho-dependent pathways involved in cellular adhesion.

Assessment of cellular viability in cardiovascular tissue as studied with 3Hproline and 3Hinulin.

STUDY OBJECTIVE: The aim of the study was to develop a quantifiable assay for the assessment of cellular viability in cardiovascular tissue. DESIGN: Radiolabelled proline and inulin were used to assess metabolic viability of the cellular component of cuspal tissue from porcine heart valves. EXPERIMENTAL MATERIALS: Pig hearts were removed within 20 min of death and transported on ice in tissue culture medium. Cuspal tissues were dissected rapidly and held on ice in physiological medium until assayed. MAIN RESULTS: Radiolabelled inulin was shown to be a useful marker for determining the amount of radiolabelled proline present in the extracellular tissue volume, permitting calculation of the amount of radiolabelled proline accumulated by the cellular component. Proline accumulation by the cellular component was affected by concentration of proline, time allowed for proline accumulation, in vitro cold ischaemia, and metabolic poisons. Based on mg tissue protein, proline and inulin accumulations were equivalent for aortic, pulmonary, mitral, and tricuspid valve tissues, suggesting that these valve tissues may be used interchangeably in assessment of metabolic viability of cellular components of cardiovascular tissue. CONCLUSION: Radiolabelled proline and inulin transport assays allow a quantitative estimate of total cellular viability.

Effects of antibiotics on cellular viability in porcine heart valve tissue.

The purpose of this study was to find an antibiotic disinfecting solution for cardiovascular tissues which would allow maximal cellular viability while maintaining antibacterial and antifungal activity. Cellular viability of porcine heat valves sterilised using antibiotics was assessed by radiolabelled proline transport assays. The results show that storage of valve tissues in RPMI 1640 tissue culture medium (4 degrees C) alone decreases the cellular viability by 50%, 60% and 90% within 12, 24, and 72 h, respectively. Amphotericin B was shown to be the toxic component of the antibiotic sterilising solution recommended by Strickett and coworkers, accounting for all the observed antibiotic toxicity. It reduced the cellular viability by 100% within 12 h of storage at 4 degrees C. Our study also showed that streptomycin is responsible for the loss of cellular viability in the antibiotic medium utilised by O'Brien's group. The results show that after storage of valve tissues for 12 h in O'Brien's antibiotic solution, the cellular viability was reduced by 60%, and after 24 h by 90%. Cefoxitin, lincomycin, polymyxin B, vancomycin and penicillin had no apparent effects on viability of cells in the cardiovascular tissues utilised in this study. In conclusion, heart valve tissue may be sterilised effectively using selective antibiotics without causing significant damage to cellular viability.