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The use of agents that can boost host immunity to combat infections may be of considerable benefit in the treatment of animals' infectious outbreaks and complement the available modes of various treatments. This report deals with the role of beta androstenes as agents that up-regulate host immune response to a level that enables the host to resist lethal infection by viruses, bacteria, and parasites. The agents reviewed consist of a specific subgroup of androstene steroids that increase the levels of the TH1 cytokines such as, IL-2, IL- 3, and IFNγ. Similarly to hydrocortisone, they suppress inflammation, but do not suppress immunity and function in the maintenance of the TH1/TH2 balance and immune homeostasis. We report that DHEA, and AED up-regulate immune resistance and protect the host from lethal infection by RNA and DNA viruses, Gram positive and Gram negative bacteria, parasitic infections, and stress mediated immune suppression. These agents provide a unique new avenue for the control, mitigation, and prevention of animal diseases by viral, bacterial and parasitic infections. Moreover, immune up-regulation may have a significant role in limiting antibiotic resistant and stress mediated infection. [ABSTRACT FROM AUTHOR]

The ionizing radiation-induced hemopoietic syndrome is characterized by defects in immune function and increased mortality due to infections and hemorrhage. Since the steroid 5-androstene-3beta, 17beta-diol (5-androstenediol, AED) modulates cytokine expression and increases resistance to bacterial and viral infections in rodents, we tested its ability to promote survival after whole-body ionizing radiation in mice. In unirradiated female B6D2F1 mice, sc AED elevated numbers of circulating neutrophils and platelets and induced proliferation of neutrophil progenitors in bone marrow. In mice exposed to whole-body (60)Co gamma-radiation (3 Gy), AED injected 1 h later ameliorated radiation-induced decreases in circulating neutrophils and platelets and marrow granulocyte-macrophage colony-forming cells, but had no effect on total numbers of circulating lymphocytes or erythrocytes. In mice irradiated (0, 1 or 3 Gy) and inoculated four days later with Klebsiella pneumoniae, AED injected 2 h after irradiation enhanced 30-d survival. Injecting AED 24 h before irradiation or 2 h after irradiation increased survival to approximately the same extent. In K. pneumoniae-inoculated mice (irradiated at 3-7 Gy) and uninoculated mice (irradiated at 8-12 Gy), AED (160 mg/kg) injected 24 h before irradiation significantly promoted survival with dose reduction factors (DRFs) of 1.18 and 1.26, respectively. 5-Androstene-3beta-ol-17-one (dehydroepiandrosterone, DHEA) was markedly less efficacious than AED in augmenting survival, indicating specificity. These results demonstrate for the first time that a DHEA-related steroid stimulates myelopoiesis, and ameliorates neutropenia and thrombocytopenia and enhances resistance to infection after exposure of animals to ionizing radiation.

Androstene-3beta, 17alpha-diol (17alpha-AED) inhibits DNA synthesis and induces apoptosis in several myeloid cancer cell lines. The purpose of this study was to determine if 17alpha-AED inhibition of human breast carcinoma cell proliferation is dependent on the estrogen or androgen receptor. At concentrations of 12.5 to 50 x 10(-9) M 17alpha-AED inhibited the proliferation of ZR75-1, estrogen receptor-positive (ER+) cells, by 54% to 68%. Further, 17alpha-AED inhibited MDA-MB231, estrogen receptor-negative (ER-) cells, by 33.6% to 56.0%. The inhibitory effect was dose dependent with a minimal effective inhibitory dose at 12.5x10(-9) M for both cell lines. Both 17beta-AED and estradiol potentiate the inhibitory effect of 17alpha-AED on ER+ cells at lower doses (3.13 to 6.25 x 10(-9) M) where 17alpha-AED alone was not inhibitory. The inhibitory action of 17alpha-AED on human mammary carcinomas appears to be independent of either the alpha estrogen or the androgen receptors.

The 17 keto steroid, Dehydroepiandrosterone (5-androsten-3 beta-17-one, DHEA) has been shown to protect mice from a variety of lethal infections. This includes, but is not limited to, infection with viruses (herpesvirus type 2, coxsackievirus B4-CVB4),bacteria (Enterococcus faecalis, Pseudomonas aeruginosa), and a parasite (Cryptosporidium parvum). We have reported that androstenediol (5-androsten-3 beta-17 beta-diol, beta AED), which is derived from DHEA, is at least 100x more effective in up-regulating systemic resistance against CVB4-infection than its precursor. Furthermore, androstenetriol (5-androstene-3 beta-7 beta-17 beta-triol beta AET) which is formed by 7 beta hydroxylation of beta AED, was more effective against CVB4-infection than its precursor beta AED. Neither steroid however has shown any significant direct antiviral effects. The in-vitro influences of DHEA, beta AED, and beta AET on a mitogen-induced mixed splenocyte proliferation assay were determined. The results showed that DHEA suppressed the proliferation of concanavalin A (Con A) or lipopolysaccharide (LPS) activated cultures in a dose dependent manner. beta AED had little influence on the activation response. However, beta AET potentiated the response to both mitogens significantly above control. The regulation of interleukin-2 and interleukin-3 secretion from Con A-activated lymphocytes was analogous to these observations. These functions were suppressed by DHEA, unaffected by beta AED, and potently increased by beta AET. Moreover, the classic immuno-suppressive effects of hydro-cortisone on Con A-induced lymphocyte proliferation, as well as IL-2 and IL-3 production were unaffected by co-cultured with DHEA and only minimally counteracted by beta AED. In contrast, beta AET significantly counteracted the effect of hydrocortisone when co-cultured together. These results show that while in-vivo, DHEA, beta AED, and beta AET each function in a similar manner. In-vitro, their effects are dramatically different from one another with only beta AET potentiating the cellular response by increasing lymphocyte activation and counteracting the immuno-suppressive activity of hydrocortisone.

In these studies, the in vitro influences of dehydroepiandrosterone (DHEA), androstenediol (AED), and androstenetriol (AET) on proinflammatory cytokine production from macrophages was examined. From physiologic to pharmacologic doses, DHEA suppressed secretion of each pro-inflammatory cytokine while AED had little influence on the responses. In sharp contrast, AET augmented TNF-alpha and IL-1 secretion while not influencing IL-6 production. Furthermore, the antiglucocorticoid activity of DHEA, AED, and AET was also investigated. Co-culture with AET counteracted the down-regulatory effect of hydrocortisone on LPS-induced TNF-alpha and IL-1 secretion. These data imply that AET is capable of regulating cytokine secretion from macrophages and may function to counterbalance glucocorticoid function.

The in vitro effects of 17 alpha AED, the isomer of 5-androstene-3 beta,17 beta diol (17 beta AED) on the basal growth of murine RAW 264.7, P388D1, and human HL-60 cells were investigated. 17 alpha AED treatment of RAW cells for 48 h reduced total cell number without increasing cell death as detected by trypan blue exclusion. At these doses, DNA synthesis as measured by [3H]thymidine incorporation was suppressed by as much as 65%, P < 0.05. This effect was time- and dose-dependent and reversible on removal of the steroid. Similar results were obtained with P388D1 and human HL-60 cell lines. At 50 nM or above, 17 alpha AED induced apoptosis in RAW cells and HL-60 as detected by transmission electron microscopy and TUNEL assays. By contrast, treating cells with the isomer 17 beta AED had no such effect. These data suggest that the balance between the anti-proliferative effect of 17 alpha AED and the proliferative effects of 17 beta AED may determine the overall level of myelopoiesis.

The anti-inflammatory and immunosuppressive functions of corticosteroids have been well established and characterized. In contrast, a different group of native steroids, which include dehydroepiandrosterone (DHEA) and two of its metabolites, androstenediol (5-androstene-3 beta-17 beta-diol, AED) and androstenetriol (5-androstene-3 beta-7 beta-17 beta-triol, beta AET), function in vivo to up-regulate host immune response against infections and counteract stress-induced immunosuppression. Indeed, DHEA and particularly, AED and beta AET, have been shown to protect mice from viral, bacterial, and parasitic infections. In vivo, these three hormones are in opposition to the widely demonstrated immunosuppressive action of glucocorticoids, suggesting a possible new immune regulation mechanism. The individual activity in vitro of each of these steroids, i.e. DHEA, AED, and beta AET, on a mitogen-induced mixed splenocyte proliferation assay were determined. The results showed that DHEA suppressed the proliferation of cultures activated with concanavalin A (ConA) or lipopolysaccharide (LPS) in a dose-dependent manner. AED had little influence on the activation response. However, beta AET potentiated the response to both mitogens significantly above control. The regulation of the cytokine secretion, of both interleukin-2 (IL-2) and interleukin-3 (IL-3), from ConA-activated lymphocytes was affected in the same manner. These functions were depressed by DHEA, unaffected by AED, and potently increased by beta AET. Moreover, the classic immunosuppressive effects of hydrocortisone on ConA-induced lymphocyte proliferation, as well as on IL-2 and IL-3 production, were unaffected by being co-cultured with DHEA and only minimally counteracted by AED at high doses. In contrast, co-culturing with beta AET significantly counteracted the immunosuppressive effects of hydrocortisone on lymphocyte proliferation and cytokine production. These data show that in-vivo, DHEA, AED, and beta AET may have some similar functions, while in vitro, their effects are dramatically different from one another. Only beta AET could markedly potentiate the cellular response by increasing lymphocyte activation and counteracting the immnosuppressive activity of hydrocortisone on lymphocyte proliferation and cytokine production.

This laboratory reported that a single subcutaneous (SC) injection of the natural steroid hormone dehydroepiandrosterone (DHEA) resulted in significant protection against a lethal herpes virus type 2 encephalitis or a systemic coxsackievirus B4 infection. Our previous results have shown that SC injection of DHEA resulted in upregulation of the specific host immune response resulting in protection against a lethal infection. This hormone did not have any direct antiviral effects in vitro. Furthermore, results indicate that, in vivo, DHEA is not the agent directly mediating the upregulation of the immune response. In the skin, DHEA is converted to androstenediol (AED) and it, in turn, is converted to androstenetriol; this is a metabolic process which appears unique to the skin. This report demonstrates that SC injection of AED results in markedly greater resistance against both viral and bacterial infection. Both DHEA and AED appear to function by facilitating and upregulating host immune responses via mobilization of cutaneous immunity to obtain systemic protection against infections. Because these steroids are native to the host and are regulated by the central nervous system, it is suggested that they may be an integral element of neuroimmunomodulation.

To determine whether the clinical phenotype of ALD correlates with the extent of metabolic abnormality, we investigated VLFA metabolism in cultured fibroblasts from patients with the clinically severe childhood from of ALD and the milder AMN variant. No differences were seen in the content of neutral lipids or phospholipids, in incorporation of [1-14C]lignocerate into cellular lipids, or in the fatty acid composition of fibroblasts from patients with childhood ALD or AMN. [1-14C]Lignocerate oxidation was deficient to a similar extent (35-40% of normal) in both intact fibroblasts and cell homogenates from patients with childhood ALD and AMN. With the use of fibroblast homogenates, oxidation of lignocerate was partially inhibited by various long-chain fatty acids, and residual activity in ALD homogenates was more susceptible to inhibition by palmitate than normal. In the presence of competing palmitate, residual lignocerate oxidative activity in fibroblast homogenates was reduced to 20 +/- 4% of normal in childhood ALD and 24 +/- 2% of normal in AMN. These results indicate that residual VLFA oxidative activity, fatty acid composition, VLFA metabolism, and lipid content of cultured fibroblasts do not correlate with the clinical expression of the ALD gene.

Experiments were designed to determine whether genetic predisposition to diabetes or overt diabetes in the identical genotype had an effect on the level of neutralization antibodies to coxsackievirus B4 (CB4). Quantitation of neutralization antibody (NT) levels against CB4 was performed using both the 50% endpoint procedure and the multivariate Wilcoxon rank sum test. The results of these experiments demonstrate that the use of the multivariate procedure for the analysis of neutralization antibody levels provides both quantitative and qualitative information not evident when only the classical 50% end point procedure is used. Moreover, when comparison on antibody levels between different groups is being considered, the power of the comparison is markedly greater using the multivariate Wilcoxon rank sum test results. The present report provide an illustration of the difference in the qualitative and quantitative information obtained by the end-point procedure and the more comprehensive multivariate procedure for the analysis of neutralization antibody levels in diabetic mutant mice infected with coxsackievirus B4.

The inbred diabetic mutant mouse, C57BL/KsJ db +/db + (db +/db +), spontaneously develops diabetes mellitus when allowed food ad libitum. However, restriction of food intake prevents the expression of this genetic predisposition for diabetes. This experimental design has been used previously to demonstrate a deficient neutralizing antibody response to coxsackievirus B4 (CB4) in mutants with the genetic predisposition only. These observations demonstrate that in the genetically predisposed diabetic mutant, deficient humoral immunity extends further to a general impairment in both total IgM and IgG production after CB4 infection. Furthermore, these mice are unable to produce a virus-specific IgG response but do show a high level of nonspecific antibody suggesting a polyclonal activation following CB4 challenge. In addition, we observed an increase in the number of spleen IgM antibody-forming cells to sheep erythrocytes (SRBC) in the overtly diabetic animal following CB4 infection with little change apparent in the genetically predisposed animal after infection. These results were identical to the changes seen in total spleen cell numbers. Our animal model provides an opportunity to distinguish between the genetic predisposition to diabetes and the overt disease and suggests that some of the immune impairment found prior to diabetes onset may be partially diminished afterwards.

Experiments were performed to determine whether genetic predisposition to diabetes mellitus (DM) or clinical DM or both exert an influence on the production of neutralization antibodies to coxsackievirus B4 (CB4). The homozygous diabetic mutant mouse db+/db+, on the inbred C57BL/KsJ genetic background, develops a diabetes-like disease when maintained on ad libitum diet but restriction of excess food intake prevents overt disease. The doubly heterozygote db+/+m or the homozygote +m/+m misty coat color mutant, on the C57BL/KsJ genetic background, do not develop DM and served as controls. Animals infected with one-half a previously determined LD50 of CB4 were bled prior to infection and at 3, 5, 7, 14, 21 days and at 1, 2, 3, 4 and 5 months after infection. Serum neutralization antibody (NA) levels were determined from the percent CB4 plaque reduction. Until 2 months following infection, NA levels were not significant in either of the homozygous diabetic mutant groups, db+/db+. In the diabetic mutant group db+/db+, without overt disease, neutralization of CB4 when observed, was low, short-lived, and apparently not specific. However, in the homozygous diabetic mutants with spontaneous diabetes, CB4 NA became evident at 2 months after infection. By 3 months post-infection, serum NA levels were sufficient to cause 90% virus plaque reduction. These observations demonstrate that hereditary DM as characterized by the mutation diabetes, db, in the C57BL/KsJ mouse, is associated with a marked impaired humoral immune response to a diabetogenic human CB4. Specifically, there is an inability to develop an adequate level of anti-CB4 antibodies. The type and degree of immunological impairment are apparently different prior to and after onset of diabetes mellitus.

A significant protective effect of a native adrenal steroid, dehydroepiandrosterone (DHEA), was demonstrated in studies of two lethal viral infection models in mice: systemic coxsackievirus B4 and herpes simplex type 2 encephalitis. The steroid was active either by long-term feeding or by a single subcutaneous injection. A closely related steroid, etiocholanolone, was not protective in these models. Histopathological analysis, leukocyte counts, and numbers of spleen antibody forming cells in the coxsackievirus B4 model suggests that DHEA functions by maintaining or potentiating the immune competence of mice otherwise depressed by viral infection. DHEA was not effective in genetically immunodeficient HRS/J hr/hr mice and did not demonstrate antiviral activity in vitro. While the molecular basis for DHEA's effect on the immune system is not known, studies by others suggest that it may counteract the stress related immunosuppressive effects of glucocorticoids stimulated by viral infection. Because DHEA is a native steroid that has been used clinically with minimal side effects, the utility of DHEA in the therapeutic modulation of acute and chronic viral infections including the acquired immune deficiency syndrome deserves intensive study.

Our findings reveal that intestinal infection with coxsackie B5 results in decreased intestinal epithelial cell division in association with an increase in carbohydrate (glucose) and amino acid (leucine) absorption in the small intestine. These findings are contrasted with those occurring during Salmonella infection, which results in increased intestinal cell division rate but decreased carbohydrate (glucose) absorption. The changes in intestinal function and physiology that have been described occurred during an asymptomatic viral infection characterized by normal intestinal histology. A reasonable hypothesis is that these pathophysiological changes may be due not only to a variety of local factors but also to hormonal effects induced by systemic spread of coxsackievirus B.

This study investigates whether experimental diabetes alters the ease with which platelet aggregation can be initiated in pial and mesenteric microvessels of the mouse. Aggregation was elicited by exposing microvessels to radiant energy from a mercury lamp in the presence of sodium fluorescein. The time required for this noxious stimulus to initiate aggregation was similar in fed or fasted alloxan diabetics and their controls, and in fed streptozotocin diabetics and their controls, but was significantly shortened in streptozotocin mice fasted for 18 to 24 hours when these animals were compared with either fed or fasted controls. Aggregation was also elicited by puncture of microvessels or by micropunture plus locally applied adenosine 5'-diphosphate. No differences in aggregability were found between either fed or fasted diabetics and their respective controls. In the light plus dye model of injury, the capacity to enhance aggregation at will by fasting streptozotocin diabetics may provide a means by which some of the factors controlling aggregation in this model of diabetes can be identified, enhanced aggregation in other species or in other types of diabetes.

Studies on the pathogenic potential of the human cardiotropic enterovirus, coxsackievirus B5, show that this agent localizes and replicates in the aorta of mice. Nutritionally-induced hypercholesterolemia leads to an increased replication and persistence of virus in tissues, specifically the aorta. Coxsackievirus B cardiopathy is markedly augmented in the hypercholesterolemic host, resulting in a persistent cardiomyolysis which is not evident in virus-infected animals with normal cholesterol levels. Pathological changes in the aorta become evident only months after the acute infection, and only in hypercholesterolemic animals previously infected with coxsackievirus B5. Our findings of coxsackievirus B-induced angiopathy and cardiopathy in the hypercholesterolemic host extend the known pathogenic range of these human viruses, and further emphasizes their potential as etiological agents of cardiovascular disease.

Inbred mice with the mutation diabetes C57BL/KsJ db+/db+ and the mutation obese C57BL/6J ob/ob displayed a total liver mitochondrial capacity to oxidize glutamate or succinate which was approximately eight times greater than the capacity of the C57BL/6J +/+ control mice. This increase in oxidation capacity was estimated by multiplying the observed twofold increase in each of the following components: total liver weight, the mitochondrial protein content per gram of liver, and glutamate or succinate respiration activity per milligram of liver mitochondrial protein. No significant difference in liver mitochondrial function and capacity for oxidation was observed between db+/db+ and ob/ob mutants, which indicated that these results may be primarily mediated by the genetic factors responsible for obesity and hyperphagia in these mutants, and not by the genetic traits associated with diabetes. These findings may provide a biochemical foundation in support of the thrifty gene hypothesis.

The diabetogenic potential of the human isolate, Coxsackievirus B4 (CB4) (Edwards) was studied in three inbred mice strains, SWR/J, DBA/2, and C57BL/6. The mice were infected with this agent and evaluated for mortality, pancreatic histopathology, and glucose tolerance. Results showed that the mortality induced by CB4 in these inbred strains differed considerably. There was no evidence of a correlation between virus-induced mortality and virus-induced pancreopathy. Although CB4 (Edwards) was most lethal to C57BL/6 mice, based on the infecting 50 per cent lethal dose (LD50), this mouse strain developed no pancreatic pathology. The most severe pancreopathy, i.e., acinar necrosis with acute interstitial inflammation and islet atrophy, was observed in SWR/J mice, which had an intermediate susceptibility to virus-induced mortality. DBA/2 mice, which displayed the lowest susceptibility to virus-induced lethality, showed less pancreatic pathology (i.e., acute and chronic interstitial inflammation) than SWR/J mice. IN SWR/J mice, virus-mediated alteration in glucose homeostasis was expressed by an increase in glucose tolerance 7 and 21 days after infection. In contrast, C57BL/6 mice showed a tendency toward chemical diabetes at 21 days postinfection. This study suggests that CB4-induced mortality and pancreatic pathology are independent parameters and do not necessarily determine the glucose tolerance of a given host genotype.

The use of the standard chemical technic for measuring intestinal glucose absorption was compared with a new double-label radioisotope technic. Glucose absorption and water flux in the mouse small intestine were measured by both methods. The results indicated that the 2 approaches yield almost identical values. The radioisotope technic utilized [14C] polyethylene glycol to measure water flux and [3H] glucose. The technic was found to be reliable, rapid, and applicable to small samples and other substrates. It is particularly suited to absorption studies in small animals such as the mouse, where sample size is limited. The study also showed that the polyethylene glycol recovery rate from the mouse intestine was 97.8%, indicating that it is a valid absorption marker in this species.