Physiological Characterization of IF Bioactivity
Testicular IF induced a biphasic response of [3H]thymidine incorporation by PHA-stimulated rat thymocytes, i.e., stimulation at lower doses (0.15-2.5 |a1) and inhibition at higher doses (1.25-10 (xl/well) (Fig. 1 and Table 1). Similar results were observed using the mouse thymocyte assay (data not shown). An overlap in the sample dose ranges providing either stimulation or inhibition was clearly observed between samples and between assays. However, repeated assay of a reference pool of normal rat IF yielded a mean sample dose, necessary to inhibit PHA-stimulated rat thymocyte proliferation by 50% (ID50), of 2 |x 1/sample well with a relative CV (i.e., interassay variation) of only 8% (Table 1). buy asthma inhalers
K562 erythroid cell proliferation assay. Human K562 erythroleukemia cells were maintained in DMEM/5% FCS in log growth phase at 37°C. At the start of the assay, cells were plated in a 96-well plate (10 000/well) in DMEM/5% FCS and incubated with or without testicular IF for 4 h at 37°C, before addition of [3H]thymidine (0.5 (xCi/50 |л1) to each well. The cells were incubated a further 20 h at 37°C and harvested as described above for the rat thymocyte bioassay.
Rat Splenocyte Proliferation Assay
Spleens were collected from adult DA rats and dissociated by mechanical dissection. Isolated cells were cultured (1 X 106 cells per well) in DMEM with 1% FCS without stimulus, or in the presence of PHA (2 (xg/ml) or the T-cell receptor-activating monoclonal antibody, R73 (1:100 final dilution). Testicular IF samples were diluted in DMEM/0.01% BSA and added at the beginning of the culture. Cells were cultured for 48 h at 37°C, labeled with [3H]thymidine (16-20 h), and harvested as described above for the rat thymocyte bioassay. buy ampicillin
Relative IL-1 bioactivity data were obtained by comparison of the log-log transformed hrIL-l|3 standard dose-re-sponse and sample dose-response curves over the range of stimulatory IF doses, using parallel-line bioassay statistics. Specificity of IL-1 bioactivity was established by use of human recombinant IL-lra, which specifically blocks human and rat IL-1 bioactivity in vitro.
Thymocyte Proliferation Assays
The standard mouse thymocyte assay for IL-1 (IL-1 bioassay) has been described in detail previously. Briefly, thymocytes were collected from adult endotoxin-resistant C3H/HeJ mice and cultured in the presence of 1 (jig/ml PHA with hrIL-la, hrIL-ip, or samples diluted with Dulbecco’s Modified Eagle’s medium (DMEM)/0.01% BSA. Cells were cultured for 72 h at 37°C, [3H]thymidine (0.5 jxCi/50 |xl) was added to each well, and the cells were incubated a further 16-20 h at 37°C. The cells were harvested onto glass filters using an automated cell harvester (Packard micromate 196; Packard Instrument Company, Downers Grove, IL) and counted by liquid scintillation in a [3-counter (Packard 1900 TR). buy levaquin online
Collection of Samples
Testes were obtained from adult Sprague-Dawley rats under ether anesthesia or after C02 overdose. Testicular IF was collected via an incision in the posterior pole of the testes over 16-20 h at 4°C, as previously described. In general, IF samples from between 12 and 30 animals were pooled for subsequent studies, although some paired testes samples from individual animals were also retained for study. Aliquots of some IF pools were dialyzed (3500 Mt cutoff) against three 600-ml changes of Dulbecco’s PBS, pH 7.4 (DPBS), over 24 h at 4°C. flovent inhaler
Animals and Reagents
These studies were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals of the Society for the Study of Reproduction. buy diabetes drugs
Adult male Sprague-Dawley rats (80-120 days old), male Dark Agouti inbred (DA) rats (70-90 days old), and C3H/HeJ inbred mice (50-80 days old) were obtained from the Monash University Central Animal House.
The transforming growth factor (3 (TGF6) proteins are a family of related cytokines (TGFp 1-3) with potent immunosuppressive activity that, like IL-1, are widely distributed. The aqueous humour of the eye contains TGF(32, which may contribute to the immunologically privileged status of this site for grafts. Studies on the experimentally cryptorchid rat have identified a protein in extracts of the abdominally located testes that inhibits T-cell proliferation in vitro and is blocked by antisera specific for TGF(31 and 2. However, while mRNA and protein of all three mammalian TGFp forms are produced by the immature rat testis, the levels of both mRNA and protein appear to decline during development. The physiological importance of TGFp in the adult testis, and its contribution as a lymphocyte-inhibiting factor in IF from normal scrotal testes, have yet to be established.
Although the rat testis is supposedly an “immunologically privileged” site, T cells are not excluded from the testicular interstitial tissue. Several studies have indicated the presence of immunosuppressive activities that have the potential to down-regulate the activity of T cells within the testicular environment. As the primary interface between the testis and the immune system, the testicular interstitial fluid (IF) should contain the responsible lymphocyte-inhibiting factors. However, conflicting reports have been published of both stimulation and inhibition of T-cell activation or proliferation in vitro by IF collected from normal adult rats. asthma inhalers
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