Distribution of histopathology and Ia positive cells in actively induced and passively transferred experimental autoimmune orchitis

Histopathology in testes from mice with actively induced experimental orchitis (EAO) (active EAO) and those from recipients of testis-sensitized lymphocytes (passive EAO) had different distributions. In passive EAO, maximum orchitis existed in the straight tubules, rete testis, and ductus efferentes, obstruction of which led to extreme dilatation of seminiferous tubules. Unusual intralymphatic granulomata also resulted in dilated testicular lymphatics. In active EAO, maximum orchitis affected seminiferous tubules under the testicular capsule, away from the rete testes. Vasitis was common and occurred in both active and passive EAO. In normal testes, IA+ F4/80+ cells were sparse but formed a cuff around the straight tubules. After immunization with testis in adjuvant or with adjuvant alone, the number, size, and staining intensity of IA+ cells increased dramatically beginning on day 5, 7 days before disease onset. Simultaneously, epithelial cells confined to the ductus efferentes became Ia+. Although recipients of sensitized lymphocytes also developed epithelial Ia in the ductus efferentes, they did not show changes in testicular interstitial Ia+ cells. Our findings indicate that testicular autoantigens are not completely sequestered, but are accessible to and can react with passively transferred immune lymphocytes in well-defined regions of the germ cell compartment. These regions coincided to a large extent with maximum expression of periductal or epithelial Ia. Changes in Ia+ cells in the testis, which are inducible by adjuvants and precede orchitis, may account in part for the different distribution of histopathology of active EAO.     

Adoptive transfer of murine autoimmune orchitis to naive recipients with immune lymphocytes

A protocol was developed for reproducibly transferring experimental autoimmune orchitis (EAO) to naive recipient mice. Cell donors were (C57BL/6 x A/J)F1 mice immunized about 14 days earlier with mouse testicular homogenate with Freund's adjuvant and an extract of Bordetella pertussis. Lymphocytes from lymph nodes and spleens were equally capable of transferring disease. As few as 5 X 10(6) cells were able to transfer EAO, which began on Day 5-7 after transfer. Infiltrate of lymphocytes and macrophages in the region of the rete testis and straight tubules was the most reproducible early lesion, suggesting that this is the initial site of T cell-antigen interaction. It was not necessary to use both Mycobacteria and B. pertussis adjuvants in donor immunization to achieve transfer of EAO. Disease transfer was antigen specific since only cells from donors immunized with TH could transfer disease. In vitro stimulation of the cells with testicular antigens and/or concanavalin A was a prerequisite to successful transfer of EAO, which was dependent on the presence of L3T4+ T cells since depletion of these cells greatly diminished EAO in recipients and the lymphocyte proliferation response to testicular antigens. Disease did not depend on an antibody response by the recipients. The results imply that effector cells, once generated by immunization and fully activated or selected by in vitro stimulation, can home to specific locations in the testis, locate relevant autoantigens, and cause disease.     

Pathogenesis of experimental allergic orchitis. III. T lymphocyte requirement in local adoptive transfer by peritoneal exudate cells.

In experimental allergic orchitis (EAO), a lesion characterized by mononuclear invasion of seminiferous tubules can be adoptively transferred within 1 to 4 days by testicular injection of peritoneal exudate cells (PEC) from syngeneic strain 13 guinea pigs (GP) immunized with homologous testicular antigens in complete Freund's adjuvant (CFA). This study examined the role of T lymphocytes, macrophages, and polymorphonuclear neutrophils (PMN) in the adoptive transfer. Guinea pig PEC contained 7% T lymphocytes, rare B lymphocytes, and over 90% of macrophages and PMN. After T lymphocytes were depleted by rabbit erythrocyte (E) rosette and Hypaque-Ficoll gradient centrifugation, cell preparations that contained 73% of original macrophages and 15% original T lymphocytes were obtained, and these cells did not transfer EAO (0 of 18 testes). In contrast, cell preparations enriched in T lymphocytes by nylon wool column or E rosette contained 1.5% of the original macrophages and 59% of the original T lymphocytes transferred EAO to 70% of the testes, starting at 1.5 x 10(6) T lymphocytes per testis. The number of T lymphocytes correlated with the incidence of adoptive transfer; the correlation existed regardless of the number of macrophages or PMN present. Finally, EAO was adoptively transferred to recipients that had total-body irradiation. The results indicate that (a) T lymphocytes are capable of transferring lesions of EAO, (b) in the transfer, the T lymphocytes did not function as helper T cells, since the transfer need not involve participation of host lymphoid cells, and (c) by inference, testis antigen-reactive T lymphocytes exist.     

Involvement of tumor necrosis factor-alpha in the pathogenesis of autoimmune orchitis in rats

We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-alpha (TNFalpha) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNFalpha concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNFalpha on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFalpha-positive testicular macrophages, the TNFalpha concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFalpha could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFalpha could trigger germ cell apoptosis. We also demonstrated that TNFalpha inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.     

Testosterone replacement effectively inhibits the development of experimental autoimmune orchitis in rats: evidence for a direct role of testosterone on regulatory T cell expansion

Despite the immune-privileged status of the male genital tract, infection and inflammation of the male genital tract are important etiological factors in male infertility. A common observation in clinical and experimental orchitis as well as in systemic infection and inflammation are decreased levels of testosterone. Emerging data point to an immunosuppressive role of testosterone. In our study, we substituted testosterone levels in experimental autoimmune orchitis (EAO) in rat by s.c. testosterone implants. EAO development was reduced to 17% when animals were treated with low-dose testosterone implants (3 cm long, EAO+T3) and to 33% when rats were supplied with high-dose testosterone implants (24 cm, EAO+T24) compared with 80% of animals developing disease in the EAO control group. In the testis, testosterone replacement in EAO animals prevented the accumulation of macrophages and significantly reduced the number of CD4(+) T cells with a strong concomitant increase in the number of regulatory T cells (CD4(+)CD25(+)Foxp3(+)) compared with EAO control. In vitro testosterone treatment of naive T cells led to an expansion of the regulatory T cell subset with suppressive activity and ameliorated MCP-1-stimulated chemotaxis of T lymphocytes in a Transwell assay. Moreover, expression of proinflammatory mediators such as MCP-1, TNF-α, and IL-6 in the testis and secretion of Th1 cytokines such as IFN-γ and IL-2 by mononuclear cells isolated from testicular draining lymph nodes were decreased in the EAO+T3 and EAO+T24 groups. Thus, our study shows an immunomodulatory and protective effect of testosterone substitution in the pathogenesis of EAO and suggests androgens as a new factor in the differentiation of regulatory T cells.     

Comparison of Water,Oils and Emulsifiable Adjuvant Oils as Formulating Agents for <Emphasis Type="Italic">Metarhizium Anisopliae</Emphasis> for Use in Control of <Emphasis Type="Italic">Boophilus Microplus</Emphasis>

Studies were conducted to identify oil-based formulating agents (paraffinic oil, palm oil and emulsifiable adjuvant oils (EAOs)) for Metarhizium anisopliae that were superior to water with simple surfactants using a germination test and a bioassay against Boophilus microplus. Germination of conidia in all formulations, except 10% coconut EAO, produced more than 68% germination at 24 h and nearly 100% at 48 h. Coconut oil (average survival time (AST)=4.6±0.28 days) and 10% liquid paraffin EAO (AST=4.4±0.15 days) enhanced the pathogenicity of M. anisopliae to B. microplus relative to water (AST=8.4±0.42 days). M. anisopliae in 10% liquid paraffin EAO was the most effective formulation having a moderately high germination after 24 h and a low AST as well as a high AST in the control. In the second experiment, germination of conidia in 2% liquid paraffin EAO and 2% Cropspray was higher than in 2% Codacide oil at 24 h, however, all treatments reached 100% germination after 48 h. The ASTs of the EAO based M. anisopliae formulations (Average AST=6.4±0.54 days) were similar but lower that the ASTs of the controls (Average AST=9.6±0.28 days).     

Inhibition of NOS-NO System Prevents Autoimmune Orchitis Development in Rats: Relevance of NO Released by Testicular Macrophages in Germ Cell Apoptosis and Testosterone Secretion

Background

Although the testis is considered an immunoprivileged organ it can orchestrate immune responses against pathological insults such as infection and trauma. Experimental autoimmune orchitis (EAO) is a model of chronic inflammation whose main histopathological features it shares with human orchitis. In EAO an increased number of macrophages infiltrate the interstitium concomitantly with progressive germ cell degeneration and impaired steroidogenesis. Up-regulation of nitric oxide (NO)-NO synthase (NOS) system occurs, macrophages being the main producers of NO.

Objective

The aim of our study was to evaluate the role of NO-NOS system in orchitis development and determine the involvement of NO released by testicular macrophages on germ cell apoptosis and testosterone secretion.

Method and Results

EAO was induced in rats by immunization with testicular homogenate and adjuvants (E group) and a group of untreated normal rats (N) was also studied. Blockage of NOS by i.p. injection of E rats with a competitive inhibitor of NOS, L-NAME (8mg/kg), significantly reduced the incidence and severity of orchitis and lowered testicular nitrite content. L-NAME reduced germ cell apoptosis and restored intratesticular testosterone levels, without variations in serum LH. Co-culture of N testicular fragments with testicular macrophages obtained from EAO rats significantly increased germ cell apoptosis and testosterone secretion, whereas addition of L-NAME lowered both effects and reduced nitrite content. Incubation of testicular fragments from N rats with a NO donor DETA-NOnoate (DETA-NO) induced germ cell apoptosis through external and internal apoptotic pathways, an effect prevented by N-acetyl-L-cysteine (NAC). DETA-NO inhibited testosterone released from Leydig cells, whereas NAC (from 2.5 to 15 mM) did not prevent this effect.

Conclusions

We demonstrated that NO-NOS system is involved in the impairment of testicular function in orchitis. NO secreted mainly by testicular macrophages could promote oxidative stress inducing ST damage and interfering in Leydig cell function.     

Identification of a dendritic cell population in normal testis and in chronically inflamed testis of rats with autoimmune orchitis

Experimental autoimmune orchitis (EAO) in the rat is the primary chronic animal model for the investigation of one of the main causes of male infertility, viz., testicular inflammation. Dendritic cells (DC) are potent antigen-presenting cells that play a fundamental role in autoimmune disease. We investigated the number of DC in normal testis and examined whether DC infiltrated the testis during the development of EAO. EAO was induced by active immunization with testis homogenate and adjuvants in two strains of rat (Wistar and Sprague Dawley). The presence of DC in testis was determined, 50 and 80 days after the first immunization, by immunohistochemical staining with specific antibodies (OX-62 and CD11c), and then the total number of DC was measured by stereological analysis. Labeled cells were found only in the interstitial compartment and within granulomas of EAO animals. The number of DC in EAO testes increased compared with control rats in both strains, whereas the number of OX-62+ and CD11c+ cells in adjuvant controls remained unchanged compared with untreated rats. Interspecies variations in the quantity of DC were found, with the total number of DC per testis in untreated and adjuvant control Sprague-Dawley rats being about three times higher than that seen in Wistar rats. Moreover, the increase in DC numbers at 80 days was less prominent in EAO testes of Sprague-Dawley rats than in the Wistar strain in which EAO was more severe and showed a higher number of granulomae. Thus, we have identified the DC population in normal and chronically inflamed testis. The increase in DC observed in EAO suggests that, under inflammatory conditions, the modified action(s) of these cells is a factor in the induction of the autoimmune response in testis.This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Germany (Me 1323/4–1 &4–2) and grants from Universidad de Buenos Aires (UBA) and Consejo Nacional de Investigaciones Científícas y Técnicas (CONICET, Argentina). The authors are Research Members (L.L.) or Fellows (C.R.) of CONICET and the UBA (V.A.G.).     

Immunopathology of murine experimental allergic orchitis

Experimental allergic orchitis (EAO) was induced consistently in BALB/c mice by immunization with homologous testicular tissue homogenate emulsified in complete Freund's adjuvant (CFA) providing that the animals had received simultaneously at least 1 microgram of an extract of Bordetella pertussis rich in pertussigen. All animals thus treated developed orchitis and serum antibody to testicular antigens within 20 days after immunization. The lesions were located in testis (100%), rete testis (37%), cauda epididymis (21%), and vas deferens (37%). Ductus efferentes and caput epididymis were only rarely affected. Early lesions in the seminiferous tubules were characterized by peritubular and/or intratubular accumulation of eosinophils, neutrophils, lymphocytes, and macrophages. This was followed by aspermatogenesis. Late lesions included massive necrosis and extensive fibrosis of the seminiferous tubules. Disruption of blood-testis barrier on day 20 was evidenced by the detection of 1) perfused lanthanum deposits between Sertoli cells and surrounding inflammatory cells inside the seminiferous tubules, 2) deposits of endogenous mouse IgG in germinal epithelium, and 3) probable immune complexes (granular C3) surrounding seminiferous tubules. Murine EAO differed from that of the guinea pig in the lack of involvement of the ductus efferentes, the extensive necrosis, the abundant polymorphonuclear eosinophils in the lesion, and the exquisite requirement of concomitant injection of B. pertussis extract.     

Experimental autoimmune orchitis: in vitro induction of an autoimmune disease.

Experimental autoimmune orchitis (EAO) can be induced in vitro. Normal lymph node lymphocytes cultured with autologous dissociated testis cells form rosette-like aggregates and later undergo blast transformation and proliferation. These stimulated lymphocytes cause in vivo EAO lesions, when injectd into syngeneic recipients. Moreover, their autoimmune reactivity can be monitored by an in vitro cytostasis assay. Density gradient analysis of the early lymphocyte-testis cultures reveals that the autoimmune reactive lymphocytes are enriched in the rosette populations. It therefore appears that testicular self-antigens are recognized by clonally preformed autologous lymphocytes.     

Actively induced experimental allergic orchitis in Lewis-resistant (Le-R) rats: reversibility of disease resistance by immunization with Bordetella pertussis

Differential susceptibility to the induction of experimental allergic orchitis (EAO) was examined in Lewis/NCr and Le-R subline rats. Lewis/NCr rats were found to be fully susceptible to the induction of EAO whereas Le-R subline rats were not. Disease resistance exhibited by Le-R rats could be overcome by including Bordetella pertussis in the immunization protocol. However, reversal of resistance with B. pertussis was dependent on the dose of rat testicular homogenate in the inoculum and found to be effective only at lower doses of antigen (10 mg/rat). Disease resistance in Le-R rats as well as B. pertussis-induced reversal of resistance did not appear to be associated with either (1) a significant difference in the number of mast cells in the ductus efferentes, the anatomic location of the earliest inflammatory infiltrates, or (2) an alteration in the phenotypic expression of either innate or B. pertussis-induced sensitivity to vasoactive amines. The results are discussed in the context of the role of B. pertussis in other animal models of organ-specific autoimmune diseases. It is proposed that the phenotypic expression of resistance to EAO in Le-R rats is a result of a mutation in a common regulatory locus affecting susceptibility to multiple autoimmune diseases and whose immunoregulatory action is normally exerted during the sensitization phase of the immune response.     

Contractile properties of the rat external abdominal oblique and diaphragm muscles during development.

We studied the in vitro contractile and fatigue properties of the rat external abdominal oblique (EAO) and costal diaphragm (DIA) muscles during postnatal development. Isometric twitch contraction (CT) and half-relaxation (RT1/2) times were longer in both the EAO and DIA muscles during the early postnatal period and decreased with age. In the first postnatal week, the CT and RT1/2 were longer in the EAO than the DIA muscle. At 14 days of age and thereafter, the CT and RT1/2 were shorter in the EAO than in the DIA muscle. Force-frequency relationships of the EAO and DIA muscles changed during postnatal development such that the relative force (percent maximum) generated at lower frequencies (less than 15 pulses/s) decreased with age. Moreover the relative force generated by the EAO muscle at lower frequencies was greater than that of the DIA muscle during the early postnatal period but less than that of the DIA muscle in adults. The specific force of both the EAO and DIA muscles increased progressively with age. There were no differences in specific force between the EAO and DIA muscles at any age. The fatigability of the EAO and DIA muscles was comparable during the early postnatal period and increased in both muscles with postnatal development. In adults the EAO muscle was more fatigable than the DIA muscle. We conclude that the contractile and fatigue properties of the EAO and DIA muscles undergo significantly different postnatal transitions, which may reflect their functional involvement in sustaining ventilation.     

Activation requirements of donor T cells and host T cell recruitment in adoptive transfer of murine experimental autoimmune orchitis (EAO)

The relative roles of donor and host T lymphocytes and the T cell activation requirements in adoptive transfer of experimental autoimmune orchitis (EAO) in (C57BL/6 x A/J)F1 mice were investigated in order to gain an understanding of the pathogenesis of this disease. Depletion of T cell subsets in recipients by adult thymectomy and treatment with monoclonal antibodies against CD4 or CD8 had no effect on the incidence of EAO following adoptive transfer of activated T cells from donors immunized with testis homogenate (TH) and adjuvants. In contrast, such depletion of CD4+ T cells inhibited development of EAO in actively immunized mice. Thus, CD4+ cells are required for induction of EAO, but donor CD4+ cells are sufficient by themselves without a comparable contribution from the recipient. Adoptive transfer of EAO required that donor splenic and lymph node T cells be activated in vitro before transfer. We found that exposure to antigen (TH) for as little as 4 hr allowed EAO to occur in 25% of recipients, and by 24 hr the cells were fully competent to induce disease. Proliferation of the cells could not be measured until 2 days later. In serial double-transfer experiments, it was found that the cells must be cultured with TH before each transfer in order for the secondary recipients to develop EAO. However, it was not necessary for the transferred T cells to "see" antigen in vivo in the primary recipients, since transfer to castrated primary recipients had no effect on EAO incidence in secondary recipients. Lymphocytes isolated from diseased testes of immunized donors were competent to transfer EAO without activation in vitro, suggesting that, unlike spleen and lymph node cells, these orchitic lymphocytes were already capable of trafficking to the testis.     

IL-18-binding protein protects against lipopolysaccharide- induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice.

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml). In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.     

Experimental allergic orchitis in mice. V. Resistance to actively induced disease in BALB/cJ substrain mice is mediated by CD4 + T cells

Previous studies have shown that differential susceptibility to actively induced experimental allergic orchitis (EAO) exists among various BALB/c substrains. Of 13 substrains studied, BALB/cJ mice consistently exhibit greater resistance to disease induction. Such resistance is associated with a single recessive genotypic difference in an immunoregulatory locus which is unlinked to any of the known alleles distinguishing the BALB/cJ substrain. In this study, gene complementation protocols were used to study the genetics of susceptibility and resistance to EAO. The results indicate that resistance in BALB/cJ mice is not due to a mutation in theH-2D ^d linked gene which governs the phenotypic expression of autoimmune orchitis. The mechanistic basis for disease resistance was examined using reciprocal bone marrow radiation chimeras generated between the disease-susceptible BALB/ cByJ (ByJ) substrain and BALB/cJ (Jax) mice. All constructs, including Jax - Jax and Jax - ByJ, developed severe EAO following inoculation with mouse testicular homogenate (MTH) and adjuvants whereas control chimeras immunized with adjuvants alone did not. These results suggest that an active immunoregulatory mechanism rather than a passive one, such as the lack of T cells and/or B cells with receptors for the aspermatogenic autoantigens relevant in the induction of EAO, is responsible for disease resistance in BALB/cJ mice. The role of immunoregulatory cells was examined by pretreating BALB/cJ mice with either cyclophosphamide (20 mg/kg) or low-dose whole body or total lymphoid irradiation (350 rads) 2 days prior to inoculation. BALB/cJ mice immunized with MTH plus adjuvants generate immunoregulatory spleen cells (SpCs) that, when transferred to naive BALB/cByJ recipients, significantly reduce the severity of autoimmune orchitis observed during actively induced EAO. Treatment of such cells with either cytotoxic monoclonal anti-Thy-1.2 or anti-CD4 plus C' before transfer abrogates the ability of BALB/cJ spleen cells to inhibit disease. In contrast, neither SpCs from adjuvantimmunized BALB/cJ nor MTH plus adjuvant-primed BALB/cByJ donors significantly influenced the severity of disease observed in recipients. Taken together, these results suggest that genetically controlled resistance to EAO in BALB/cJ mice is associated with a mutation in an immunoregulatory locus whose effects appear to be mediated through a cyclophosphamide and low-dose radiation-sensitive CD4⁺ T-cell population.     

Leydig cell involvement in the paracrine regulation of mast cells in the testicular interstitium of the rat

The accumulation of mast cells in the rat testicular interstitium was studied under different experimental conditions in order to correlate this accumulation with the alterations of specific testicular tissue compartments or cell types. Estrogen treatment was effective in inducing mast cell proliferation when administered on Day 1 or at higher doses at 10 days of age. Estrogens were ineffective beyond 20 days of age. Postnatal treatment of neonatal-estrogen-treated rats with FSH and LH prevented the appearance of mast cells. In contrast, treatment with the Leydig cell cytotoxic ethylene dimethane sulphonate (EDS) was effective in inducing mast cell accumulation only when administered to adult rats, inducing small numbers of mast cells at 45 days of age; it was ineffective on 30-day-old rats. Hypophysectomy alone did not determine the appearance of mast cells. However, when atrophic Leydig cells were destroyed with EDS, high numbers of mast cells accumulated in the testis. These results support the existence of Leydig cell-related inhibitory factors for mast cells in the rat testicular interstitium.     

Priming of the antitumor response promotes efficacy of interleukin-2 therapy

 We have studied the effect of active specific immunization (ASI) on the antitumor response induced by locoregional, low-dose interleukin-2 (IL-2) therapy. On day 0, mice were injected i.p. with viable, syngeneic tumor cells and with irradiated tumor cells (ASI). Low-dose IL-2 treatment was given i.p. for 5 consecutive days. ASI led to extended survival in two out of seven models tested. In these two models, enhanced efficacy was observed when both ASI and IL-2 were administered. In the five models in which ASI had no therapeutic value, ASI+IL-2 treatment was no more effective than IL-2 therapy. In the SL2 lymphoma model, use of ASI prior to IL-2 therapy given as early as days 1–5 led to at least 60% cure, whereas IL-2 therapy without ASI was only effective when administered after day 9. In the P815 mastocytoma model, however, ASI, IL-2, and the combination caused negative (suppressive) effects when administered on days 6–10. IL-2 administered on days 6–10 was therapeutically effective in this model when mice were treated with cyclophosphamide on day 6. In both the SL2 and the P815 tumor models, cured mice were specifically immune. The positive and negative effects observed were not due to the increased number of cells injected (non-specific inflammation) nor to possible antigenic alteration of the ASI cells by irradiation, as ASI with fragmented tumor cells was also effective in inducing synergy. Investigations into the underlying mechanism indicated that CD4⁺ cells play an important role. In total, the results indicate that ASI may be a good supplement to locoregional IL-2 treatment if care is taken to alleviate immunosuppressive activities. Received: 6 February 1997 / Accepted: 6 March 1997     

Suppression of testicular maturation and fertility following androgen administration to neonatal male rats

Administration of supraphysiologic doses of androgen to male rats within the first few neonatal days markedly suppresses subsequent testicular maturation; this effect diminishes as androgen is injected on succeeding postnatal days. Testosterone propionate (TP) administered neonatally at dosages up to 3.5 mg appreciably diminished postnatal testicular growth; postpubertal androgen secretion, as assessed by accessory sex organ weights and serum testosterone concentrations and as reflected by a castrationlike developmental pattern of the hepatic enzyme, histidase; spermatogenesis; and fertility. Beyond three mo of age testicular growth rates and androgen secretion--but not fertility--tended to be restored. These effects of neonatal androgen do not require aromatization to estrogen; indeed 5 alpha-dihydrotestosterone elicited more profound testicular suppression than TP, which was sustained until at least 100 days of age. Testes of neonatally androgenized rats were capable of responding to gonadotropins administered at three wk of age with increases in weight and androgen secretion. These findings suggest that a developmental event, suppressible by pharmacologic doses of androgen, occurs at a nontesticular site during the first few post partum days in the male rat; this event programs subsequent testicular maturation.     

Protective Effect of Selenium on Nicotine-Induced Testicular Toxicity in Rats

Effect of exogenous selenium at a dose of 10 mug/kg body weight on the testicular toxicity induced by nicotine in rats was investigated. Male albino rats were maintained for 60 days as follows: (1) control group (normal diet), (2) nicotine group (0.6 mg /kg body weight), (3) selenium (10 microg/kg body weight), and (4) nicotine (0.6 mg/kg body weight) + selenium (10 microg/ kg body weight). Administration of nicotine caused reduction in sperm count and sperm motility. Activity of HMG CoA reductase and concentration of cholesterol were increased in the testes of the nicotine administered group. Activities of testicular enzymes 3beta hydroxysteroid dehyrogenase (3 betaHSD), 17beta hydroxysteroid dehyrogenase (17 betaHSD) were decreased. Levels of testosterone in the serum were also reduced. However, the extent of these alterations was lesser in the group administered with nicotine along with selenium. Analysis of plasma revealed reduced quantity of cotinine in the group co-administered with nicotine along with selenium in comparison with the nicotine group. Nondetectable levels of nicotine were present in the co-administered group. This indicates altered metabolism of nicotine when administered along with selenium.