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Vernekar VJ, Bandivdekar AH, Raghavan VP, Kamada M, Koide SS. Studies with synthetic peptides of 80 kDa human sperm antigen (80 kDa HSA). AJRI 2004; 51:106–111 © Blackwell Munksgaard 2004 The 80 kDa human sperm antigen (HSA) is a sperm-specific and conserved antigen, capable of inducing immunological infertility. Partial N-terminal amino acid sequences of 80 kDa HSA (Peptide NT) and its peptides obtained by digestion with endoproteinase Lys-C (peptides 1–4) and endoproteinase Glu-C (peptides 5–6) did not show any sequence homology with reported known proteins deposited in the Gen-Bank. These sequenced peptides were synthesized and conjugated to key hole limpet haemocyanin (KLH) and evaluated for its antifertility effects. The present communication describes the characterization of these peptides and their antibodies. Peptides NT, 1, 2, 3 and 4 were synthesized and conjugated to KLH. Antibodies to KLH conjugated peptides were raised in rabbits by active immunization and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) using sperm extract coated wells. The binding specificity of the synthetic peptides or purified 80 kDa HSA to their antibodies was assessed in the presence of various doses of respective synthetic peptides or 80 kDa HSA. The binding specificity was further confirmed by Western blot analysis. Antipeptide antibodies were also checked for sperm agglutinating activity, in-vitro. Active immunization of rabbits elicited significant antibody titers against the synthetic peptides, except for peptide 3. Antipeptide antibodies specifically recognized the native protein in an ELISA and induced in-vitro agglutination of human, rat and monkey sperm. In addition, Western blot analysis showed that these antipeptide antibodies specifically bind to the 80 kDa HSA band of the sperm extract. Synthetic peptides of 80 kDa HSA are immunogenic and antibodies raised against these peptides recognize the native protein detected by ELISA, Western blot analysis. In addition, they possess sperm agglutinating activity. These findings suggest that they are promising candidates in the development of immunocontraceptives. [ABSTRACT FROM AUTHOR]

Summary Background Gelatin-specific cell-mediated immunity develops in subjects inoculated with gelatin containing DTaP vaccine. However, it is not yet known whether such established sensitization to gelatin disappears or persists with time. Objective The aim of this study was to follow the patients with gelatin sensitization elicited by DTaP vaccination for their lymphocyte responsiveness and IgE, IgG antibody specific to gelatin over several years and to compare the activities with those at the time of enrolment into the study. Methods We studied 28 subjects who developed positive lymphocyte proliferation test (LPT) after receiving gelatin containing DTaP vaccine and eight subjects who had a negative LPT after inoculation of non-gelatin DTaP. Determination of IgE, IgG antibodies and specific lymphoproliferative response directed against gelatin were performed at enrolment and on follow up. Results None of the subjects had antibody to gelatin at enrolment and none developed gelatin IgE or IgG during follow-up. There was no significant difference in the SIs of the subjects receiving gelatin DTaP (P = 0.150, 95% CI, -0.198–0.032), whereas lymphocyte activity to gelatin increased between enrolment and follow-up in the subjects with non-gelatin DTaP ( P = 0.011, 95% CI, 0.063–0.338). Conclusion Gelatin-specific lymphocyte activity persists at comparable levels for more than 3 years in subjects who acquire a positive LPT response to gelatin after receiving primary DTaP vaccine containing gelatin. Furthermore, five out of eight subjects initially with negative LPT to gelatin have been shown to acquire specific LPT with time. [ABSTRACT FROM AUTHOR]

Background This study was designed to evaluate the immunogenic characteristics of enzymatically digested gelatin, ‘FreAlagin’, employing the lymphoproliferative response in subjects with gelatin hypersensitivity. Our purpose was to assess the response of primed lymphocytes to the newly developed FreAlagin and compare it to the response to conventional gelatin. Methods A gelatin-specific lymphocyte proliferation test (LPT) was performed in 110 children with adverse reactions to gelatin-containing vaccines, who showed positive gelatin-specific cell-mediated immunity and were thus diagnosed as having gelatin hypersensitivity. Gelatin-specific IgE was measured in all subjects. The antigenic activity of FreAlagin to lymphocytes was compared with that of conventional bovine gelatin. Positive and negative control specimens were obtained from the patients with anaphylaxis and from subjects inoculated with gelatin-free vaccine who showed no adverse reactions in order to establish the fluorometric ELISA system to determine IgE antibody to gelatin and LPT. Results The lymphocyte activity against FreAlagin was much less than that to Wako gelatin and more than half of the subjects who reacted positively to Wako gelatin had a negative LPT to FreAlagin. Although 47% of the subjects had positive LPTs to FreAlagin, all but two still had lower SIs to FreAlagin compared with Wako gelatin. Conclusion We conclude that the antigenic activity of FreAlagin as measured by the cell-mediated immune response is significantly less than that of conventional bovine gelatin. However, it is still necessary to perform clinical trials to show a reduced or absent clinical reactivity to FreAlagin in sensitized patients to conventional gelatin. [ABSTRACT FROM AUTHOR]

Background: Few data exist on the association between strength training and mortality rates. We sought to examine the association between strength training and all-cause, cardiovascular disease, and cancer mortality., Methods and Results: Beginning in 2001 to 2005, 28 879 women throughout the United States (average baseline age, 62.2 years) from the Women's Health Study who were free of cardiovascular disease, diabetes mellitus, and cancer reported their physical activities, including strength training. During follow-up (average, 12.0 years) through 2015, investigators documented 3055 deaths (411 from cardiovascular disease and 748 from cancer). After adjusting for covariables, including aerobic activity, time in strength training showed a quadratic association with all-cause mortality ( P =0.36 for linear trend; P <0.001 for quadratic trend); hazard ratios across 5 categories of strength training (0, 1-19, 20-59, 60-149, and ≥150 min/wk) were 1.0 (referent), 0.73 (95% confidence interval, 0.65-0.82), 0.71 (0.62-0.82), 0.81 (0.67-0.97), and 1.10 (0.77-1.56), respectively. A significant quadratic association was also observed for cardiovascular disease death ( P =0.007) but not cancer death ( P =0.41). Spline models also indicated a J-shaped nonlinear association for all-cause mortality ( P =0.020); the point estimates of hazard ratios were <1.00 for 1 to 145 min/wk of strength training, compared with 0 min/wk, whereas hazard ratios were >1.00 for ≥146 min/wk of strength training. However, confidence intervals were wide at higher levels of strength training., Conclusions: Time in strength training showed a J-shaped association with all-cause mortality in older women. A moderate amount of time in strength training seemed beneficial for longevity, independent of aerobic activity; however, any potential risk with more time (≈≥150 min/wk) should be further investigated., (© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Cantu syndrome is an autosomal dominant overgrowth syndrome associated with facial dysmorphism, congenital hypertrichosis, and cardiomegaly. Some affected individuals show bone undermodeling of variable severity. Recent investigations revealed that the disorder is caused by a mutation in ABCC9, encoding a regulatory SUR2 subunit of an ATP-sensitive potassium channel mainly expressed in cardiac and skeletal muscle as well as vascular smooth muscle. We report here on a Japanese family with this syndrome. An affected boy and his father had a novel missense mutation in ABCC9. Each patient had a coarse face and hypertrichosis. However, cardiomegaly was seen only in the boy, and macrosomia only in the father. Skeletal changes were not evident in either patient. Craniosynostosis in the boy and the development of aortic aneurysm in the father are previously undescribed associations with Cantu syndrome., (© 2013 Wiley Periodicals, Inc.)

We describe two new cases of pure 1q terminal deletions. BAC FISH analysis precisely defined the size of deletions. The first is a girl with 10.3-Mb deletion showed typical features of 1q43 deletion as well as a simplified gyral pattern, which was rarely found in 1q43 deletion. The other boy also presented with most of 1q43 deletion features but several atypical symptoms were noted including hydrocephalus, adducted thumbs, and flexion restriction of proximal interphalangeal joints in left hand. A concomitant novel missense mutation in L1CAM was identified in addition to 11.5-Mb deletion. Reviewing all the cases of pure 1q terminal deletion in the literature suggests that it is a clinically recognizable syndrome., (2008 Wiley-Liss, Inc.)

Many pathogens colonize host tissues by binding to the extracellular matrix via their cell surface adhesion molecules, which are called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). Staphylococcus aureus expresses several of these adhesion molecules, some of which bind to fibronectin. Of these adhesion molecules, fibronectin-binding proteins play a role in the pathogenicity of S. aureus, although it is not yet clear whether they enhance its virulence. We have previously shown that fibronectin-bound S. aureus is efficiently phagocytosed by thioglycolate-induced mouse peritoneal macrophages. Bacterial ingestion is mediated by Very Late Antigen-5 (VLA-5; alpha5beta1 integrin) and is accompanied by the formation of adhesion complexes. Here we show that the expression of VLA-5 is restricted to thioglycolate-induced inflammatory macrophages and is not found in the resident macrophages. When cells were in suspension, alpha5 integrin was not expressed on the surface of either resident or inflammatory macrophages, whereas in adherent cells, this integrin was distributed on the surface of inflammatory but not resident macrophages. A high level of this integrin was present in the cytoplasmic region only in inflammatory macrophages. In agreement with this, fibronectin-mediated phagocytosis of S. aureus was observed only in the inflammatory macrophages. In inflammatory macrophages ingesting fibronectin-bound S. aureus, alpha5 integrin was concentrated close to the phagocytosed bacteria. This change in distribution was not found in macrophages ingesting untreated bacteria. Together with our previous work, these results indicate that, upon ingestion of fibronectin-bound S. aureus, VLA-5 accumulates in the area of phagocytosis in inflammatory macrophages, where it forms adhesion complexes.

Problem: A human sperm antigen of molecular size of about 80 kDa (80 kDa HSA) has been reported to be sperm-specific, conserved and responsible for inducing immunological infertility. The partial N-terminal amino acid sequence of 80 kDa HSA (peptide NT) and its peptides obtained by enzymatic digestion with endoproteinase Lys-C (peptides 1-4) and with endoproteinase Glu-C (peptides 5 and 6) did not show sequence homology with any of the proteins of the GenBank. The peptides NT, 1, 2, 3 and 4 were synthesized, conjugated to keyhole limpet hemocyanin and used as an immunogen to raise the antibodies in rabbits. Peptide 3 did not elicit significant antibody titer while peptides NT, 1, 2 and 4 elicited significant antibody titer and immunobiologically mimicked the native protein., Method of Study: Effects of passive administration of two injections each of 200 microL of antibodies or 10 and 40 microg purified immunoglobulins to 80 kDa HSA, peptides NT, 1, 2 and 4 on fertility in male and female rats were investigated., Results: Passive administration of antibodies to 80 kDa HSA and its peptides NT, 1, 2 and 4 resulted in agglutination of epididymal spermatozoa with loss of motility but had no effect on sperm count or weights of the reproductive organs. These animals failed to impregnate normal female rats. Passive administration of these antibodies to female rats also resulted in infertility. The presence of antibodies was detected by enzyme-linked immunosorbent assay in uterine secretions of animals treated with antipeptide antibody. The presence of agglutinated spermatozoa was observed in the post-coital vaginal smears of these animals. The immunized females were found to be ovulating normally and the number of corpora lutea were unaltered. Of the four antipeptide antibodies studied, antibodies to peptides NT and 1 were most effective in inhibiting fertility both in male as well as female rats. Hence, the antifertility studies were further confirmed by passive administration of 10 and 40 microg of purified immunoglobulins of antibodies to NT and 1, which resulted in a dose-dependent inhibition of fertility in male and female rats., Conclusions: The study demonstrated that the synthetic peptides of 80 kDa HSA immunobiologically mimicked the native protein and impaired fertility following passive administration of antipeptide antibodies and hence, suggested the suitability of synthetic peptides of 80 kDa HSA as candidates for development of antifertility vaccine.

Organotypic slice culture preserves the morphological and physiological features of the hippocampus of live animals for a certain time. The hippocampus is one of exceptional regions where neurons are generated intrinsically and spontaneously throughout postnatal life. We investigated the possibility that neurons are generated continuously at the dentate granule cell layer (GCL) in slice culture of the rat hippocampus. Using 5-bromodeoxyuridine (BrdU) labelling and retrovirus vector transduction methods, the phenotypes of the newly generated cells were identified immunohistochemically. At 4 weeks after BrdU exposure, BrdU-labelled cells were found in the GCL and were immunoreactive with a neuronal marker, anti-NeuN. There were fibrils immunoreactive with anti-glial fibrillary acidic protein (GFAP), an astrocyte marker, in the layer covering the GCL and occasionally encapsulated BrdU-labelled nuclei. When the newly divided cells were marked with the enhanced green fluorescent protein (EGFP) using a retrovirus vector, these cells had proliferative abilities throughout the following 4-week cultivation period. Four weeks after the inoculation, the EGFP-expressing cells consisted of various phenotypes of both early and late stages of differentiation; some were NeuN-positive cells with appearances of neurons in the GCL and some were immunoreactive with anti-Tuj1, a marker of immature neurons. Some EGFP-expressing cells were immunoreactive with anti-GFAP or anti-nestin, a marker of neural progenitors. The present study suggests that slice cultures intrinsically retain spontaneous neurogenic abilities for their cultivation period. The combination of slice culture and retrovirus transduction methods enable the newly divided cells to be followed up for a long period.

Previous studies have demonstrated that the murine monoclonal antibody (MoAb) NM-01 activates the human complement classical pathway resulting in lysis of human immunodeficiency virus (HIV). The present study was performed to determine the availability of the V3-loop of gp120 relative to the complement regulatory proteins, CD55 (DAF) and CD59 (HRF20) molecules on HIV. The results demonstrate that CD55 and CD59 exist on HIV virions, along with gp120 molecules. These findings suggest that activation of human complement on free viral particles is induced by MoAb NM-01 and that this occurs regardless of the presence of CD55 and CD59 molecules. The destruction of viral particles was demonstrated by a decrease in infectivity. The involvement of human complement in this process was confirmed with an immunoelectron microscopy technique by the presence of a human C9 to prove membrane attack complex (MAC). The results indicate that NM-01 can induce complement activation because of the ratios of CD55 and CD59 to gp120 molecules on HIV virions. The availability of the gp120 V3 domain on the virion is sufficient for binding of NM-01 and thereby the formation of MAC that results in virolysis.

Problem: The physiological significances of the two known subtypes of interleukin-1 (IL-1) in amniotic fluid (AF) were examined by measurements of their concentrations and detection of their location., Method: Each IL-1 subtype in AF collected from 61 patients were examined by newly developed enzyme-linked immunosorbent assay with high sensitivity. Placenta and umbilical cord from 17 patients were examined immunohistochemically for localization of IL-1., Results: Different types of IL-1 were predominant in AF; the mean (+/- SE) concentration of IL-1 beta (412.8 +/- 105.4 pg/ml) was significantly higher (P < 0.01) than that of IL-1 alpha (116.4 +/- 26.7 pg/ml) in cases with spontaneous vaginal delivery, whereas the concentration of IL-1 alpha (243.1 +/- 81.4 pg/ml) was significantly higher (P < 0.01) than that of IL-1 beta (139.7 +/- 59.4 pg/ml) in cases with premature rupture of the membranes (PROM) without intrauterine infection. In these cases, immunohistochemical studies revealed the presence of IL-1 alpha in the trophoblastic layer of the placenta and epithelium of the umbilical cord., Conclusion: Amniotic IL-1 alpha was detected predominantly in cases with PROM, and it is thought to be produced in placenta and fetal skin keratinocyte.

Problem: Autoantibodies to zona pellucida (ZP) have been implicated as a cause of infertility in woman by three lines of clinical and laboratory evidence., Method: First, positive anti-zona activities, as assessed by the passive hemagglutination reaction (PHAR) using bovine red blood cells sensitized with porcine zona antigen, were detected in 45 of 1,872 serum samples collected from infertile women, but in only three (two nonpregnant and one pregnant) of 592 serum samples from control subjects. Their incidence in 320 women with unexplained infertility (5.6%) was much higher than that in 1,552 women with infertility of known cause (1.7%, P < 0.01), which was comparable to the incidence observed in 193 age-matched fertile nonpregnant and pregnant women (1.5%, P < 0.05). None of the serum samples from 292 age-matched adult men and 100 children of between 5 and 10 years old gave a positive PHAR. Second, follow-up study for a minimum of 2 years with treatment revealed that no pregnancy occurred in 11 infertile women with a consistently positive PHAR, and only three pregnancies in 19 infertile women with a fluctuating positive PHAR. Third, three of seven serum samples from women who gave a consistently positive PHAR produced strong immunofluorescence reactions on human ZP even after adsorption with porcine and human AB blood cells. Pre-exposure of human ZP to the sera showing positive immunofluorescence, including that of one patient undergoing an in vitro fertilization program, greatly diminished the number of spermatozoa of normal quality that bound to and penetrated across human ZP., Conclusions: The observed high incidence of anti-zona activities and long-term resistancy to treatment in women with unexplained infertility may be closely correlated with inhibition of sperm-egg interaction by anti-zona autoantibodies produced in these women.