Experimental Biology and Medicine

The ovine uterus provides an excellent in vivo model system for the study of the cellular and molecular mechanisms of Type I IFN and steroid hormone regulation of hormone receptor gene expression in an epitheliomesenchymal organ. Future experiments will be directed toward determining the signal transduction pathway activated by IFN-τ and mechanisms involved in regulation of gene expression. These experiments include (i) cloning the Type I IFN receptor found on the endometrial luminal and superficial glandular epithelium; (ii) eluciding the signal transduction pathway activated by IFN-τ binding to the Type I IFN receptor; (iii) cloning and structural analysis of the ovine ER and OTR genes; (iv) determining mechanisms regulating transcription of the ovine ER and OTR genes, particularly those involving both IFN-τ and progesterone; (v) determining the role(s) of IFN-τ-induced proteins (e.g., Mx protein) in establishment of pregnancy; and (vi) investigating conceptus-endometrial and epithelial-stromal interactions that integrate and coordinate endometrial physiology during the estrous cycle and pregnancy.

The mesangial cell occupies a central position in the genesis of the pertubations occurring during the pathogenesis of glomerulonephritis. In vitro studies have shown that this cell is a metabolically active cell producing a variety of cytokines which act as autocoids; such cytokines are also liberated by the monocytes/macrophages which infiltrate the glomerulus in nephritis. This review summarizes the evidence for the participation of these cytokines in animal models of nephritis and in human renal disease, focusing on the roles of basic fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, colony-stimulating factor-1, tumor necrosis factor, interleukin-1, and interleukin-6.

Investigators have traditionally thought of the class of inflammation- and injury-associated cytokines in large part as “free” entities in the peripheral circulation. In the case of interleukin-6 (IL-6), the cytokine can be found in blood in complexes of molecular mass 400-500, 150-200, and 2535 kDa in association with binding proteins that can include soluble IL-6 receptor (sIL-6R), anti-IL-6, and anti-slL-6R IgG, and others. Sustained high levels of different particular IL-6 complexes are observed in the human circulation in cancer patients subjected to particular active anticancer immunotherapy regimens. In the “chaperoned” state, circulating IL-6 complexes display differential immunoreactivity in different ELlSAs and possess differential biological activity as assayed ex vivo. The discovery of “chaperoned” circulating IL-6 in humans points to a new level of modulation of cytokine function, that of regulated bioavailability of IL-6 in vivo.

Our understanding of the impact of opioid compounds on the function of the immune system has expanded greatly over the past 5 years. It is now clear that several cell populations serve as targets for the effects of the opioids, and this includes T cells, macrophages, and NK cells. The mechanism(s) of immunomodulation are now being described in greater detail on both a cellular and biochemical level. Indeed, the finding that the production of lymphokines and cytokines may be altered following opioid treatment may be particularly important since all immune responses are dependent to some degree on the synthesis of these protein mediators.

The opioid receptors have now been successfully cloned from cells of the immune system. There is no longer serious doubt about the presence of opioid receptors expressed by these cell populations. Extremely valuable information regarding the role of the opioid receptors in the function of the cells of the immune system should be obtained using molecular methods. Clearly, the molecular basis for the effect of the opioid compounds on the immune response represents a critical area of research in the immediate years ahead.

It is not surprising that opioid compounds have been found to alter resistance to infectious agents, since a great deal of evidence shows that these compounds modulate the immune response. The significance of the drugs of abuse in the host-parasite interaction for a number of microorganisms, including HIV, remains a critical area for additional research. In addition, because of the importance of opportunistic infections in the AIDS patients, the impact of opioids on the resistance to these infectious agents is also a matter of great concern. It is possible that combinations of certain drugs of abuse may serve to alter resistance to some, but not all, of these infectious diseases. In any case, answers to these questions will most certainly come only once a greater understanding of the basic mechanisms of immunomodulation is achieved.

We have shown that polarization of an electrogenic H⁺/K⁺ATPase pump located in the secretory (luminal) membrane of the frog gastric mucosa is the major factor contributing to the change in open circuit potential difference (OCPD) induced by voltage clamping. This transmucosal polarization was markedly reduced by H₂ blockers famotidine and cimetidine, and by the H⁺/K⁺-ATPase inhibitors omeprazole and SCH 28080. SCN^-, a nonspecific H⁺ secretion inhibitor, did not affect the polarization. In the present experiments, the effects of two other inhibitors of H⁺ secretion were examined, namely, acetazolamide (AA), a carbonic anhydrase inhibitor, and melittin (MEL), an inhibitor of the H⁺/K⁺-ATPase enzyme. When AA 10^-3 M or MEL 10^-5 M was added to the nutrient solution, H⁺ secretion was completely inhibited. While MEL markedly reduced the polarization induced by voltage clamp, AA did not affect the polarization. These data support the concept that MEL directly affects the electrogenic H⁺/K⁺-ATPase pump while the inhibition of H⁺ secretion by AA is by an indirect mechanism. The data further support the electrogenicity of the H⁺/K⁺-ATPase.

Thermogenesis in brown adipose tissue (BAT) is believed to be mediated mainly by p₃ adrenergic receptors. We previously demonstrated that the specific p₃ adrenergic agonist CGP-12177 increases whole body oxygen consumption and BAT GDP binding to a greater extent in young than in senescent rats. In contrast, the forskolin-induced increases were maintained with age, suggesting that early events in p₃ adrenergic signal transduction are impaired with age. To investigate whether β₁ or p₃ adrenergic function is decreased with age, we assessed β₁ and β₃ adrenergic receptor mRNA levels and the ability of β₁ and β₃ adrenergic receptors to activate adenylyl cyclase in BAT membranes from 4- and 24-month-old F-344 rats. Both β₁ and p₃ adrenergic receptor mRNA levels decreased by 50% with age. Adenylyl cyclase stimulated by the nonspecific agonist, isoproterenol, and by the specific β₃ agonist, BRL 37344, also declined by 50% with age, whereas glucagon stimulation decreased by more than 70%. The isoproterenol-stimulated adenylyl cyclase activation curves were resolved by two-site regression analysis to determine the contribution of β₁ and β₃ adrenergic receptors. The V_max for both β₁ and β₃ adrenergic receptors decreased by 50% with age. However, stimulation of adenylyl cyclase by NaF and forskolin was also diminished by the same amount as β adrenergic stimulation, suggesting that the activation with age may be limited by the amount of adenylyl cyclase catalytic unit rather than by receptor number. These data suggest both β₁ and β₃ adrenergic receptors and adenylyl cyclase catalytic units are deficient with age in rodent BAT.

Rats treated on Day 5 of life with testosterone propionate (TP) were tested for their ovulatory response to pregnant mare's serum gonadotropin (PMSG) on the 30th day of life with and without cardiac puncture under ketamine/xylazine anesthesia. TP without cardiac puncture in doses of 0.625, 1.25, or 2.5 μg did not inhibit ovulation, but 5.0 μg of TP inhibited ovulation in 15 of 16 rats. When cardiac puncture was performed at 1900 hr in rats not given TP, seven of eight ovulated on Day 33. However, none of the rats that received either 1.25 or 2.5 μg of TP ovulated after cardiac puncture at 1900 hr, and only one of seven given the 0.625-μg dose ovulated. Thus, the stress of bleeding greatly enhanced the inhibitory effect of otherwise ineffective doses of TP. The preovulatory luteinizing hormone (LH) surge was delayed and diminished by neonatal TP in a dose-related manner in animals from which blood was drawn from a previously inserted catheter. When blood drawn from similarly TP-treated animals by cardiac puncture, serum levels of LH were further reduced. Progesterone, given at 1100 hr on Day 32, was capable of partially overcoming the inhibitory effects of the combined treatments on both ovulation and serum LH levels at all but the highest dose of TP tested (5.0 μg). We conclude that neonatal exposure to androgen sensitizes rats to ovulation-inhibiting factors, such as bleeding, in a manner which appears to delay as well as inhibit the preovulatory release of LH. Such early exposure to androgen, therefore, may determine the susceptibility of individuals to reproductive failure in adult life.

Thyroid hormones are transported across the placenta. Thyroid hormone receptors are present in the midgestation rat fetus and fetal tissues selectively accumulate thyroid hormones prior to the onset of fetal thyroid function. It has not been demonstrated adequately that maternal thyroid hormones are essential for early fetal physiologic functions and neurological development. The present study compares the effects of maternal thyroid hormone versus fetal thyroid hormone on the regulation of rRNA and ribosomal protein synthesis in the developing fetus. This was accomplished by first comparing 16-day gestation (just prior to the onset of fetal thyroid function) fetuses of control and hypothyroid mothers. Then 19-day gestation (fetal thyroids are functional) fetuses of control and hypothyroid mothers were compared as development of fetal thyroid function lags in hypothyroid mothers. Rats made hypothyroid (Tx) by radiothyroidectomy were given replacement doses of thyroxine (T₄) until the day that they were placed with a male for mating. Control, Tx and growth hormone (GH)-treated Tx dams and their fetuses were sacrificed on either the 16th or 19th day of gestation. Ribosomes (r) were isolated from placentas and from fetal brains and livers and rRNA, total ¹⁴′C-leucine incorporation, and ¹⁴C-leucine incorporation into ribosomal protein per microgram of rRNA were determined. On the 16th day of gestation, prior to the onset of fetal thyroid function, all three of the above metabolic parameters were reduced significantly below control levels in the placentas of Tx rats and in the brains and livers of their fetuses. This was true also for the fetal brains and livers of GH-treated Tx mothers. Development of fetal thyroid function lags in the Tx mother. rRNA, total ¹⁴C-leucine incorporation, and ¹⁴C-leucine incorporation into ribosomal protein per microgram of rRNA continue to be significantly depressed in these fetal tissues and placentas of Tx rats on the 19th day of gestation. In fact, brain protein synthesis falls further behind in fetuses of Tx dams at this gestational age when compared with control fetal brains. These data support the hypothesis that maternal thyroid hormones are important, by at least midgestation, for normal fetal physiologic development and support the concept that appropriate maturation of the fetal pituitary-thyroid plays a role in fetal brain protein synthesis and therefore neurological development.

Muscle damage is often associated with an influx of extracellular fluid containing albumin into the muscle. Muscles affected by muscular dystrophy undergo severe muscle damage; therefore, the hypothesis was tested that muscles of dystrophic (mdx) mice contain elevated levels of albumin. Albumin levels in diaphragm (DIA) and soleus (SOL) muscles of control and mdx mice were measured at 3 months and 1 year of age. Albumin in mdx DIA at 1 year of age was twice that of control. In mdx SOL at 1 year of age albumin was increased 25% compared with control. The increase in albumin correlates well with the decline in function in mdx DIA and SOL muscles. Electron microscopy of muscles suggests that albumin is co-localized with transverse tubules of muscle fibers and thus may be mainly located in extracellular fluid.

We conclude that albumin is elevated in muscles affected by muscular dystrophy and suggest that this may be of clinical importance in view of substances bound to albumin under physiological conditions.

Nonobese diabetic (NOD) mice develop type I diabetes spontaneously and have been utilized as a model for human autoimmune insulin-dependent diabetes. The disease is caused by the destruction of insulin-producing β cells in the pancreatic islet of Langerhans by infiltrating inflammatory cells, which are primarily T lymphocytes. The incidence of diabetes in NOD mice is increased in females compared with males, suggesting that sex steroid hormones play an important role in the development of the disease. We therefore investigated the effect of a male steroid, 5-α-dihydrotestosterone (5DHT), on disease development, T-cell phenotype, T-cell proliferation, and cytokine profiles in this model. None of the mice that received 5DHT for 120 days (n = 7) developed insulitis, whereas all control mice (n = 8) developed the disease. The percentage of CD4⁺ T cells in peripheral blood mononuclear cells was markedly decreased in the 5DHT-treated females compared with those in controls (37.1 ± 4.8 vs 51.3 ± 9.3, P < 0.02), whereas no significant differences in the percentage of CD8⁺ T cells were observed between treated and control female mice. Results of a syngeneic mixed lymphocyte reaction (SMLR) also suggested that T cells are major target cells of 5DHT administration. An increased expression of IL-4 mRNA, representing T helper 2 (Th2) T cells, was observed in the SMLR. On the basis of these results, a systemic administration of 5DHT appears to have direct effects on the expansion of Th2 cell populations with subsequent restoration of normal immune responses.

Immunization of CBA/J mice with thryoglobulin (Tg) emulsified in complete Freund's adjuvant induces experimental thyroiditis (EAT), a well-characterized model of Hashimoto's disease. Recent studies have suggested that dietary factors play a role in the modulation of the immune response and that diet can have a profound effect on the induction of autoimmune diseases. In this study, we examined the influence of diet on autoimmune thyroiditis in mice. EAT was induced in mice fed ad libitum one of the three diets, a standard maintenance chow (Agway H1000), Purina 5020 Breeding Chow, and Purina 5010 Autoclavable (unautoclaved) Diet.

Tg-immunized mice fed the Agway 1000 diet were found to be resistant to the development of autoimmune thyroid disease, with only 4 out of 25 mice developing mild thyroiditis. In contrast, 16 out of 25 mice fed the Purina 5010 diet developed moderate to severe thyroiditis. Mice fed the 5020 diet were partly susceptible: 7 out of 25 developed a mild to moderate thyroiditis. Histologic examination of thyroid glands of diseased mice fed the 5010 and 5020 diets showed marked lymphocytic infiltration with destruction of follicles, compared with mice fed the Agway diet, the latter showing only mild infiltration with preservation of thyroid follicles. Titers of antibody to Tg did not differ among the groups, and there was no significant difference in the IgG isotype subclass usage.

The results demonstrate that diet can markedly affect the severity of autoimmune disease in the EAT model. In contrast, diet has little effect on the humoral autoimmune response in this system. These results implicate diet as a factor in the severity of cell-mediated autoimmune destruction and suggest that dietary modification could decrease pathology in some forms of autoimmune disease.