Testis-specific antigen (TSA-1) is expressed in murine sperm and its antibodies inhibit fertilization

PROBLEM: We recently cloned and sequenced a sperm-specific antigen, designated as testis-specific antigen-1 (TSA-1), from human testis. The present study was conducted to examine its expression and function in murine sperm, in order to find out whether or not the mouse can provide a suitable model for examining its immunocontraceptive effects. METHOD OF STUDY: The antibodies (Ab) were raised against purified human rTSA-1 in virgin female rabbits. The rTSA-1 was run in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the gel containing the approximately 18 kDa band was cut, minced and used for immunization to obtain the specific Ab. The immunoglobulins from preimmune bleed and from animals injected with adjuvant alone served as control. These Ab were analysed in enzyme-linked immunosorbent assay (ELISA), Western blot procedure, immunoprecipitation procedure, immunocytochemical technique (ICT), immunobead binding technique (IBT), acrosome reaction and sperm-zona binding assay. RESULTS: Active immunization of female rabbits with purified rTSA-1 protein of 18 kDa, produced high titer Ab against the recombinant antigen. These Ab to rTSA-1 were used in the present study. In Western blot procedure, rTSA-1 Ab recognized a specific protein band of approximately 24 +/- 3 kDa in murine sperm extract, the band similar to found in human sperm extract. In the immunoprecipitation procedure, rTSA-1 Ab immunoprecipitated the protein band of similar size from extracts of murine sperm and murine testis. The ICT and the IBT studies revealed the subcellular localization of TSA-1 on the surface of acrosome and tail regions of the non-capacitated and capacitated murine sperm cells. In functional bioassays, rTSA-1 Ab inhibited the acrosome reaction and sperm-egg binding in vitro. CONCLUSIONS: These data indicate that the TSA-1 is expressed in murine sperm and may have a biological role in sperm function and sperm-egg binding. In vitro inhibition of capacitation/acrosome reaction and sperm-zona binding suggests that the mouse can provide a suitable model to examine the immunocontraceptive effects of TSA-1 in actively immunized animals.     

Effect of timing of chest tube removal on development of pericardial effusion following cardiac surgery

BACKGROUND: There are no standard criteria for the timing of drain removal. The objective of this study was to determine whether the macroscopic appearance of chest tube drainage fluid to serosanguineous may be used as a criteria for drain removal. METHODS: 2,359 patients were assessed retrospectively and 80 randomized patients were followed prospectively who underwent cardiac surgery. In both parts of the study, patients were divided into two groups according to the timing of drain removal. Group I consisted of patients whose chest tubes were removed as soon as the macroscopic appearance of the drainage fluid turned to serosanguineous. Group II consisted of patients whose chest tubes were removed at the second postoperative day when the drainage output declined to less than 50 mL in a five-hour period. In the retrospective part, cases of hemodynamically significant pericardial effusion observed within seven days postoperatively were reviewed. In the prospective part, just before the drain removal, the fluid sample hematocrit obtained from the drain lines and patients' blood hematocrit were measured and recorded. Patients were evaluated with echocardiography for pericardial effusion. RESULTS: No statistically significant difference was detected in the frequency of hemodynamically significant pericardial effusion and incidence or amount of pericardial effusion between the two study groups. The drain hematocrit to blood hematocrit ratios before drain removal showed a significant correlation with pericardial effusion.The strength of correlation between the drain hematocrit to blood hematocrit ratios before drain removal and pericardial effusion was also studied using receiver operating characteristic curve, which suggests that a drain hematocrit to blood hematocrit ratio of < or = 0.3 is strongly predictive that pericardial effusion would be absent or mild between the fifth and seventh postoperative days. CONCLUSIONS: It is safe to remove the chest tubes as soon as the macroscopic appearance of the drainage fluid turns to serosanguineous since this practically indicates cessation of active bleeding.     

Inhibition of murine sperm-oolemma binding by antibodies to an oocyte membrane (OM) antigen: implication in contraceptive vaccine development

PROBLEM: The present study was conducted to investigate the oocyte membrane protein(s) that is involved in sperm binding in the mouse, and whether or not it can be used for the development of a contraceptive vaccine. METHOD OF STUDY: The zona-free oocytes were treated with Triton X-100 and the extract was analyzed for homogeneity in the sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) after staining with silver nitrate. The appropriate band of 50+/-4 kD, designated as OM antigen, was excised from the gel and used for the immunization. The female rabbits were immunized with the excised band per se and the female mice were immunized with the OM antigen after conjugation to keyhole limpet hemocyanin (KLH). The affinity-purified antibodies were analyzed in the enzyme-linked immunosorbent assay (ELISA), immunoprecipitation procedure, western blot procedure, indirect immunofluorescence technique (IFT), and spermoolemma binding assay. Actively-immunized mice were analyzed for in vivo fertility. RESULTS: The Triton X-100 extract of zona-free oocytes predominantly showed a single protein band of 50+/-4 kD in the SDS-PAGE. Active immunization of female rabbits and of female mice with OM antigen raised high antibody titers (ELISA titer > 1:4096) that specifically recognized the OM antigen in the immunoprecipitation and Western blot procedures, and reacted with the oocyte in the IFT. These antibodies demonstrated a significant (P < 0.05) up to a complete block of sperm-oolemma binding in the in vitro binding assay. Binding of both the acrosome-intact and acrosome-reacted sperm was inhibited. Mice actively immunized with OM antigen also showed a significant reduction in vivo fertility as seen by the 9-day implants in uteri. Preliminary data indicate that the antibodies to OM antigen were tissue-specific and did not react with the specific band in any tissue extract in the western blot procedure. CONCLUSIONS: These results indicate that the OM antigen is involved in spermoolemma binding and constitute an attractive molecule that needs further investigation for examining its utility in the contraceptive vaccine development.     

Low Density Granulocytes and Dysregulated Neutrophils Driving Autoinflammatory Manifestations in NEMO Deficiency

Journal of Clinical Immunology - NF-κB essential modulator (NEMO, IKK-γ) deficiency is a rare combined immunodeficiency caused by mutations in the IKBKG gene. Conventionally, patients are...     

The adenosine transporter, mENT1, is a target for adenosine receptor signaling and protein kinase Cepsilon in hypoxic and pharmacological preconditioning in the mouse cardiomyocyte cell line, HL-1

Brief exposure of the heart to hypoxia results in less cellular damage after subsequent hypoxia, an effect known as preconditioning (PC). PC has been widely studied but is still not fully understood. Adenosine (Ado), adenosine receptors, and protein kinase C (PKC) have been implicated as integral components of PC. Adenosine (nucleoside) transporters (NTs) facilitate flux of Ado across cell membranes, but their role in PC is unknown. Therefore, we used the murine cardiomyocyte cell line, HL-1, and asked if there was feedback regulation of NTs by Ado, Ado receptors, and PKC following either hypoxic or pharmacological PC. Activation (by specific agonists) of A1 or A3 Ado receptors or PKC resulted in PC in HL-1. The A1 (but not A3) receptor is coupled to PKCepsilon, and activation of PKCepsilon (by specific peptide agonist) resulted in PC. Moreover, PKCepsilon stimulates Ado uptake via the predominant NT in HL-1, mouse equilibrative nucleoside transporter 1 (mENT1). Studies in primary neonatal mouse cardiomyocytes confirmed our observations in HL-1 cells. Hypoxic challenge led to a rapid increase in, and efflux of, intracellular Ado from cells, which was blocked by NT inhibitors (dipyridamole/nitrobenzylthioinosine). Moreover, NT inhibition during hypoxia or PC was highly protective, suggesting that Ado loss contributes to decreased cell viability. Our data suggest that hypoxic challenge causes an efflux of Ado via ENTs, activation of A1 and/or A3 receptors, signaling through PKCepsilon, and activation of ENT1. Since Ado is required for ATP synthesis on reperfusion, this feedback regulation of mENT1 would promote reuptake of Ado.