LPS receptor expression and lps binding capacity of monocytes and macrophages in swine]

The expression of the lipopolysaccharide (LPS) receptor CD14, being of importance for an effective immune response, and the direct measurable binding of LPS molecules by alveolar macrophages (AM), peritoneal macrophages (PM) and peripheral blood mononuclear cells (PBMC) of swine were determined. Additionally, the influence of an experimental intrabronchial infection with Pasteurella multocida on these parameters was investigated. Whereas the AM and the PM differed only negligibly regarding their share of CD14 bearing and LPS binding cells, in contrast to AM the intensity of the LPS-binding of PM was significantly higher. Among the PBMC, two populations could be detected which expressed the CD14 with different intensity, whereas the LPS-binding of both populations was similarly pronounced. Following infection of the animals, AM showed an unchanged percentage of CD14 positive cells and simultaneously an increased CD14 receptor expression. On the other hand, the percentage of the LPS-binding cells rose, while no changes of the intensity of LPS-binding were observable. The studies revealed that intrabronchial infection results in local and systemic alterations of CD14 expression and LPS-binding capacity of different cell populations. However, a direct correlation between the appearance of CD14- and LPS-binding does not seem to inevitably exist, indicating probably CD14-independent LPS-binding.     

Effect of growth hormone on estrogen receptor binding properties in the chick oviduct in vivo

The equilibrium dissociation constant (K_d) and the maximum binding capacity (B_max) of estrogen receptor in cytosolic and nuclear fractions in oviduct of pullets following various daily injections (1–10 times) of growth hormone (GH: 50 µg/chick) were examined by Scathchard analysis of specific [³H]estradiol‐17β (E₂) binding. The K_d values of receptor in both fractions decreased after three times of GH‐injection. In the B_max values, three times of the injection caused a marked decrease in that value in the cytosolic fraction with a concomitant increase in that in the nuclear fraction, whereas the total B_max (sum of B_max in the cytosolic and nuclear fractions) did not change. A similar relationship between the K_d values in the binding of the two fractions was also observed in 4–10 times of GH‐injection. However, in 4–10 times of GH‐injection the B_max of the both fractions and total B_max was greater than that in vehicle‐injection (control). When the chicks were injected with 6–10 times of GH‐injection, the weight of oviduct was increased. No change in the plasma concentrations of E₂ was found following GH‐ and vehicle‐injections into the chicks. The results suggest that growth hormone stimulates the growth of the chick oviduct by increasing the binding affinity and capacity of estrogen receptor.     

Signal transduction systems mediated by protein kinase-C and estradiol receptors in cow placenta and caruncle.

Characteristics of signal transduction systems mediated by protein kinase-C (Ca2+/phospholipid-dependent protein kinase) and estradiol receptors in cow placenta and caruncle were conducted by evaluating protein kinase-C activity and estradiol receptor concentrations and by exploring substrate proteins for the enzyme. The enzyme activity was detected in cytosolic and total particulate fractions of both tissues. The activity levels in these fractions were comparable to those in other cow tissues such as liver and mammary gland. The enzyme activity was inhibited by palmitoylcarnitine, gossypol and adriamycin, known phospholipid-interacting inhibitors of the enzyme. Phosphorylation by the enzyme and subsequent autoradiography revealed that only 125K protein in placental cytosol and two low molecular weight proteins in caruncular cytosol were found to be substrates for protein kinase-C. Ca2+ acts as inhibitor of the phosphorylation of several phosphoproteins other than the substrates. Estradiol receptor concentrations in cytosolic and nuclear fractions were similar in both tissues, and the cytosolic concentrations were also comparable to those in pregnant uterus. However, the nuclear concentrations were extremely low when compared to those in the uterus. The signal transduction systems mediated by protein kinase-C and by estradiol receptors seem to be concertedly suppressed in cow placenta and caruncle.     

Effects of prenatal stress on corticosteroid receptors and monoamine concentrations in limbic areas of suckling piglets (Sus scrofa) at different ages

The present study was conducted in order to reveal the effects of prenatal stress on the central stress regulation in domestic pigs by measuring changes in corticosteroid receptor binding and monoamine concentrations in different limbic brain regions. Pregnant sows were subjected to a restraint stress for 5 min daily during the last 5 weeks of gestation. Maternal stress resulted in a significantly higher number of glucocorticoid receptors in the hippocampus, but decreased glucocorticoid receptors in the hypothalamus of the offspring at the first postnatal day. No alterations of hippocampal mineralocorticoid receptors were found. There was also no significant effect of prenatal stress on the brain monoamine concentrations. Prenatally stressed piglets showed lower basal plasma cortisol and increased corticosteroid binding globulin concentrations at the third postnatal day indicating decreased free cortisol concentrations after birth. Morbidity and mortality during the suckling period were significantly increased in prenatally stressed litters, as shown by a higher frequency of diseased and died piglets per litter. In conclusion, the results indicate that in pigs restraint stress during late gestation affects the ontogeny of the foetal neuroendocrine feedback system with consequences for the regulation of the hypothalamo-pituitary-adrenal function and the vitality of the offspring.     

Diurnal ACTH and plasma cortisol variations in healthy dogs and in those with pituitary-dependent Cushing's syndrome before and after treatment with retinoic acid

Daytime variations in ACTH and plasma cortisol were studied in healthy dogs and in dogs with pituitary-dependent hypercortisolism (PDH), before and after treatment with retinoic acid. In control dogs ACTH showed a higher concentration at 8.00 AM and between 2.00 and 6.00 PM, with the lowest concentration registered at 10.00 AM (p < 0.05 vs. 8.00 AM and 2.00 PM and p < 0.01 vs. 4.00 PM). Cortisol did not show significant differences. In dogs with PDH, ACTH was lower at 8.00 AM (ACTH: p < 0.01 vs. 2.00 and 4.00 PM; and p < 0.05 vs. 6.00 PM). The lowest cortisol concentration was registered at 8.00 AM and 8.00 PM and the highest at 4.00 PM (p < 0.05 vs. 8.00 AM and p < 0.01 vs. 8.00 PM). After treatment, the lowest ACTH concentration was registered at 10.00 AM (p < 0.01 vs. 2.00 and 4.00 PM). To conclude, the adrenal is desensitized in PDH possibly showing negative in diagnostic tests.     

Pharmacologic characterization of novel adenosine A2A receptor agonists in equine neutrophils

OBJECTIVE: To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes. SAMPLE POPULATION: Neutrophils isolated from 8 healthy horses. PROCEDURES: Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A(1), A(2A), and A(3) receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrophils and roles of specific adenosine receptor subtypes and cAMP production in mediating these effects were determined. RESULTS: Radioligand binding experiments yielded a ranked order of affinity for the brain equine A(2A) receptor on the basis of 50% inhibitory concentrations (IC(50)) of the agonists as follows: ATL307 (IC(50) = 1.9nM) and ATL313 > ATL309 and ATL310 > ATL202 > 2-([p-2- carboxyethyl] phenylethylamino)-5'-N-ethylcarboxyamidoadenosine > 5'-N-ethylcarboxamidoadenosine. Furthermore, ATL313 had approximately 100-fold greater selectivity for A(2A) over A(1) and A(3) receptors. In functional assays with equine neutrophils, the compounds inhibited endotoxin-induced ROS production and stimulated production of cAMP with the same ranked order of potency. Results of experiments performed with selective adenosine receptor antagonists indicated that functional effects of ATL313 were via stimulation of A(2A) receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that activation of A(2A) receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A(2A) receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.     

Effect of transportation on cortisol concentrations and on the circadian rhythm of cortisol in gilts

A study was conducted to determine whether the circadian rhythm of cortisol in gilts is disrupted or altered by transport. Sixteen ovariectomized gilts with indwelling jugular catheters were individually penned in an enclosed building (location 1). Blood samples were collected at 0700 and 1900 hours for 6 days. On day 7, gilts in groups of 4 were transported 5.6 km to environmentally controlled chambers (25 C) and were individually penned (location 2). On the day of transport, samples were collected at 0700 hours at location 1, immediately before and after transport in a trailer, after unloading at location 2, and at 1900 hours at location 2. For the first 6 days at location 2, blood samples were collected daily at 0700 and 1900 hours. For the 6 days at location 1, circadian rhythm was evidenced by higher cortisol concentrations in the AM hours than in the PM hours. During transport, serum cortisol concentrations increased (P less than 0.01). Highest concentrations developed at 0.5 hour after unloading; concentrations declined thereafter. During the first 6 days at location 2, circadian rhythm was evidenced by higher serum cortisol concentrations in the AM hours than in the PM hours. Therefore, the transportation of gilts 5.6 km to new pens was a transient stress causing a temporary increase in serum cortisol concentrations, but did not cause a disruption in the endogenous rhythm of cortisol.     

Effect of silage chop length on feed intake and feeding behaviour of finishing feedlot steers

This study determined the effects of silage chop length (20?mm; long or 10?mm; short) and diurnal time on feeding behaviour of 80 feedlots steers. Feed intake and feeding behaviour of each steer in each pen were measured continuously from 0:00 to 12:00?h (AM) and from 12:00 to 24:00?h (PM). Chop length had no effect on feed intake or the growth performance of steers, but longer chop length increased the duration of feeding and reduced the rate of intake. Head down time and head down time per feeding activities were greater during the PM than AM. There was a chop length?×?diurnal time interaction for feeding frequency with the number of bunk visits being greater in the PM than AM for both diets. Feeding activities were 50% higher during the PM than AM when steers were fed either long- or short-chopped silage diets twice daily.     

Muscarinic Receptors in Equine Airways

The distribution of muscarinic receptors in equine airways was investigated using autoradiography. Frozen sections of tissue from six different levels in the bronchial tree, from the trachea to the distal bronchioles, were incubated in vitro with 1.5 nmol/L of the muscarinic receptor antagonist 1-[N-methyl-³H]scopolamine methyl chloride (³H-NMS). In addition, the subtype pattern of muscarinic receptors was investigated in equine tracheal smooth muscle using radioligand binding with methoctramine, tripinamide, 4-DAMP-methiodide and pirenzipine as competitors against the binding of 1.3 nmol/L ³H-NMS. The autoradiograms showed specific labelling indicating a high density of muscarinic receptors in smooth-muscle tissue in all levels of the airway tree investigated. Besides muscle tissue, subepithelial glands were the only structures specifically labelled. The dominating subtypes in tracheal smooth muscle investigated with radioligand binding studies were found to be M₂ and M₄, as both methoctramine (pK _d = 8.5) and tripinamide (pK _d = 8.6 and 6.7 for two different sites) showed high affinity. The density of the M₃-muscarinic receptor subtype was low, but this subtype could be detected with statistical significance when methoctramine was used as the competitor against ³H-NMS binding.     

Serial determination of thyroxine concentrations in hyperthyroid cats

Serum thyroxine (T4) concentrations of 10 hyperthyroid cats were measured at hourly intervals between 9 AM and 4 PM. In 5 cats, blood samples were obtained by jugular venipuncture; the remaining 5 cats had blood samples obtained from jugular catheters. Over the 7-hour period, a significant temporal (diurnal) variation was not identified in the serum T4 concentrations of the cats (P greater than 0.01). The lowest mean serum T4 concentration (9.1 micrograms/dl) was measured at 3 PM and was 14.2% less than the highest mean serum T4 concentration (10.6 micrograms/dl) measured at 9 AM. Though there were fluctuations in serum T4 concentrations during the 7-hour period, the differences were not systematic. The maximal variation in serum T4 concentrations over the 7-hour period averaged less than 21%. Despite the random variations during the 7-hour period, none of the measured serum T4 concentrations was in the normal range. Measurement of serum T4 concentration from randomly obtained blood samples was determined to be reliable for diagnosing feline hyperthyroidism.     

Adenosine A2A receptor agonists inhibit lipopolysaccharide-induced production of tumor necrosis factor-alpha by equine monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes by activating one or more adenosine receptor subtypes, namely A1, A2A, A2B and A3. The results of previous studies indicate that adenosine analogues inhibit lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) by equine neutrophils primarily through activation of A2A receptors. Because peripheral blood monocytes produce cytokines that are responsible for many of the deleterious effects of LPS, the current study was performed to evaluate the effects of an array of novel adenosine receptor agonists on LPS-induced production of tumor necrosis factor-alpha (TNF-alpha), and to assess the selectively of these agonists for equine adenosine A2A over the A1 receptor. Radioligand binding studies performed with equine tissues expressing adenosine A1 and A2A receptor subtypes yielded a rank order of affinity for the equine A2A receptor of ATL307>ATL309 approximately ATL310 approximately ATL313>ATL202 approximately ATL361 approximately ATL376>ATL372>CGS21680>NECA. Co-incubation of equine peripheral blood monocytes with LPS and these agonists resulted in inhibition of TNF-alpha production with a rank order of potency that strongly correlated with their binding affinities for equine adenosine A2A receptors. Results of experiments performed with one of the adenosine receptor agonists (ATL313) and selective adenosine receptor antagonists confirmed that inhibition of LPS-induced production of TNF-alpha occurred via stimulation of A2A receptors. Although incubation of monocytes with IB-MECA, a compound purported to act as an adenosine A3 receptor agonist, reduced LPS-induced TNF-alpha production, this effect of IB-MECA was inhibited by the A2A selective antagonist ZM241385 but not by the A3 receptor antagonist MRS1220. These results indicate that the adenosine receptor subtype responsible for regulation of LPS-induced cytokine production by equine monocytes is the A2A receptor. To address the signal transduction mechanism responsible for the anti-inflammatory effects of ATL313 in equine monocytes, production of cAMP was compared in the presence and absence of either the adenosine A2A receptor antagonist ZM241385 or the adenosine A2B receptor antagonist MRS1706. In the absence of the antagonists, ATL313 increased production of cAMP; ZM241385 inhibited this effect of ATL313, whereas MRS1706 did not. Furthermore, incubation of monocytes with either the stable analogue of cAMP, dibutyryl cAMP, or forskolin, an activator of adenylyl cyclase, also inhibited LPS-induced production of TNF-alpha production by equine monocytes. Collectively, the results of the current study indicate that adenosine analogues inhibit LPS-induced production of TNF-alpha by equine monocytes primarily via activation of adenosine A2A receptors and do so in a cAMP-dependent manner. The results of this study indicate that stable adenosine analogues that are selective for adenosine A2A receptors may be suitable for development as anti-inflammatory drugs in horses.     

Antagonism of adenosine receptors by caffeine and caffeine metabolites in equine forebrain tissues

OBJECTIVE: To determine the presence of adenosine receptor subtypes A1 and A2a in equine forebrain tissues and to characterize the interactions of caffeine and its metabolites with adenosine receptors in the CNS of horses. SAMPLE POPULATION: Brain tissue specimens obtained during necropsy from 5 adult male research horses. PROCEDURE: Membrane-enriched homogenates from cerebral cortex and striatum were evaluated by radioligand binding assays with the A1-selective ligand [3H]DPCPX and the A2a-selective ligand [3H]ZM241385. Functional responses to adenosine receptor agonists and antagonists were determined by a nucleotide exchange assay using [35S]-guanosine 5'-(gamma-thio) triphosphate ([35S]GTPgammaS). RESULTS: Saturable high affinity [3H]DPCPX binding (A1) sites were detected in cerebral cortex and striatum, whereas high-affinity [3H]ZM241385 binding (A2a) sites were detected only in striatum. Caffeine and related methylxanthines had similar binding affinities at A1 and A2a sites with rank orders of drug binding affinities (theophylline > paraxanthine > or = caffeine > theobromine) similar to other species. [35S]GTPgammaS exchange revealed that caffeine and its metabolites act as pure adenosine receptor antagonists at concentrations that correspond to A1 and A2a receptor binding affinities. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study affirm the presence of guanine nucleotide binding protein linked adenosine receptors (ie, high-affinity A1 and A2a adenosine receptors) in equine forebrain tissues and reveal the antagonistic actions by caffeine and several biologically active caffeine metabolites. Antagonism of adenosine actions in the equine CNS by these stimulants may be responsible for some central actions of methylxanthine drugs, including motor stimulation and enhanced racing performance.     

Infection of central nervous system endothelial cells by cell-associated EHV-1

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late-term abortions and equine herpesvirus myeloencephalitis (EHM). Our understanding of EHM pathogenesis is limited except for the knowledge that EHV-1 infected, circulating peripheral blood mononuclear cells (PBMC) transport virus to the central nervous system vasculature causing endothelial cell infection leading to development of EHM. Our objective was to develop a model of CNS endothelial cell infection using EHV-1 infected, autologous PBMC. PBMCs, carotid artery and brain endothelial cells (EC) from 14 horses were harvested and grown to confluency. PBMC or ConA-stimulated PBMCs (ConA-PBMCs) were infected with EHV-1, and sedimented directly onto EC monolayers ('contact'), or placed in inserts on a porous membrane above the EC monolayer ('no contact'). Cells were cultured in medium with or without EHV-1 virus neutralizing antibody. Viral infection of ECs was detected by cytopathic effect. Both brain and carotid artery ECs became infected when cultured with EHV-1 infected PBMCs or ConA-PBMCs, either in direct contact or no contact: infection was higher in carotid artery than in brain ECs, and when using ConA-PBMCs compared to PBMCs. Virus neutralizing antibody eliminated infection of ECs in the no contact model only. This was consistent with cell-to-cell spread of EHV-1 infection from leucocytes to ECs, demonstrating the importance of this mode of infection in the presence of antibody, and the utility of this model for study of cellular interactions in EHV-1 infection of ECs.     

Tachykinin receptors in the equine pelvic flexure

Tachykinins, of which substance P (SP) is the prototype, are neuropeptides which are widely distributed in the nervous systems. In the equine gut, SP is present in enteric nerves and is a powerful constrictor of enteric muscle; in other species, SP is also known to have potent vasodilatory and pro-inflammatory effects. The specific effects of SP are determined by the subtype of receptor present in the target tissue. There are 3 known subtypes of tachykinin receptors, distinguished by their relative affinities for SP and other tachykinins. The distribution of SP binding sites in the equine pelvic flexure was determined using 125I-Bolton Hunter SP (I-BHSP) autoradiography. Most I-BHSP binding sites were determined to be saturable and specific, therefore presumably representing tachykinin receptors. The greatest degree of I-BHSP binding occurred over very small vessels, and over the muscularis mucosae; I-BHSP binding was also intense over the circular muscle of the muscularis externa and mucosa, and present, although less intense, over the longitudinal muscle of the muscularis externa. Competition of I-BHSP with specific receptor agonists for binding sites in the equine pelvic flexure were used to determine the subtypes of tachykinin receptors present. The neurokinin-1 receptor subtype predominated in the equine pelvic flexure, followed by the neurokinin-3 receptor subtype.     

Does cortisol bias cytokine production in cultured porcine splenocytes to a Th2 phenotype?

Glucocorticoids are reported to bias the production of cytokines from a type 1 to a type 2 phenotype. However, this dogma has been advanced largely from studies utilizing potent glucocorticoid analogs, particularly dexamethasone (DEX). Although studies utilizing DEX certainly have clinical and pharmacological relevance, DEX is probably not the best glucocorticoid for studies designed to evaluate the interaction and regulation of endogenous corticosteroids with immune cells in vivo in the domestic pig. Functional measures of immune suppression suggest that the pig is relatively resistant to DEX. Furthermore, type II corticosteroid receptors exclusively bind DEX with high affinity, whereas type I receptors, the so-called mineralocorticoid receptors, have a higher affinity for cortisol. In addition, DEX is not bound by serum binding proteins as are endogenous corticosteroids.These issues prompted us to revisit glucocorticoid regulation of type 1 and type 2 cytokines in cultured pig splenocytes and to test the broad hypothesis that cortisol biases cytokine production in favor of a Th2 response. We evaluated interferon gamma (IFNgamma) (also interleukin 2 (IL-2) in one experiment) and interleukin 10 (IL-10) as representative Th1 and Th2 cytokines, respectively. Furthermore, we evaluated macrophage migration inhibitory factor (MIF) because it is reported to be an essential factor in T cell activation; it is also upregulated by glucocorticoids and reported to be a product of Th2 lymphocytes. In general, both IFNgamma and IL-10 were sensitive to cortisol inhibition early in culture. However, IFNgamma ultimately escaped cortisol inhibition, whereas IL-10 continued to be substantially suppressed by high physiological concentrations of cortisol. Similarly, MIF mRNA could be suppressed by cortisol, but only when cortisol was added to cultures after ConA (concanavalin A) stimulation of splenocytes. So, taken together, our studies do not support the hypothesis that cortisol favors a Th2 cytokine profile in cultured pig splenocytes.     

Monitoring the circadian rhythm of serum and salivary cortisol concentrations in the horse

Daily fluctuations of cortisol concentration in the blood or saliva have been repeatedly reported. However, several contradictions in the existing literature appear on this subject. The present study was performed to definitively establish options for testing adrenocortical function. To the best of our knowledge, this is the first study to evaluate parallel circadian rhythms in salivary and serum cortisol concentrations during a 24-h period. Twenty horses were examined under the same conditions. Blood and saliva samples were taken every 2 h for 24 h to determine the daily changes in cortisol concentrations of saliva and serum at rest and to determine the relationship between salivary and serum cortisol levels. Cosinor analysis of group mean data confirmed a significant circadian component for both serum and salivary cortisol concentrations (P < 0.001 in both cases). The serum cortisol circadian rhythm had an acrophase at 10:50 AM (95% CI, 10:00 AM–11:40 AM), a MESOR of 22.67 ng/mL, and an amplitude of 11.93 ng/mL. The salivary cortisol circadian rhythm had an acrophase at 10:00 AM (95% CI, 9:00 AM–11:00 AM), a MESOR of 0.52 ng/mL, and an amplitude of 0.12 ng/mL. We found a significant but weak association between salivary and serum cortisol concentrations; the Pearson correlation coefficient was 0.32 (P < 0.001). The use of salivary cortisol level as an indicator of hypothalamic-pituitary-adrenal axis activity may be warranted. However, the salivary cortisol levels are more likely to be correlated with free plasma cortisol than with the total plasma cortisol concentration.     

海北高寒草甸矮嵩草光合日变化及与环境因子的关系

对海北高寒草甸矮嵩草光合放氧以及光照、温度和湿度等环境因子的测定结果表明,矮嵩草从早晨８：００便表现出明显的光合作用之后并缓慢上升。净光合速率至中午１２：００时达到最大,１４：００时有所下降,出现“午休”（Ｍｉｄｄａｙｄｅｐｒｅｓｉｏｎ）现象,而后又逐渐上升。表观光合量子效率在１４：００有所下降;暗呼吸速率在一天当中保持上升趋势,只是下午１８：００有所下降,与温度和相对湿度分别呈现正、负相关关系。净光合速率、表观光合量子效率和暗呼吸的速率日变化与环境因子（光照、空气温度和湿度等）有密切的关系。     

Equine herpesvirus-1 infected peripheral blood mononuclear cell subpopulations during viremia

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for β-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.     

Expression of 11β‐Hydroxysteroid Dehydrogenase Type 1 and Glucocorticoid Receptors in Reproductive Tissue of Male Horses at Different Stages of Sexual Maturity

Glucocorticoids (GCs) as mediators of the stress response may affect Leydig cell function by inhibiting either luteinizing hormone receptor expression or testosterone biosynthesis. The isozymes 11β‐hydroxysteroid dehydrogenase (11βHSD) 1 and 11βHSD2 control the intracellular cortisol levels. Little is known about the effects of stress on fertility in the equine. The objective of the present study was to determine the presence and cellular localization of glucocorticoid receptors (GCR) and glucocorticoid‐metabolizing enzymes (11βHSD1 and 11βHSD2) in equine epididymal and testicular tissue with special regard to sexual maturation. Testicular and epididymal tissue was collected from 21 healthy stallions, and four age groups were designed: pre‐pubertal, young, mature and older horses. Immunohistochemistry (IHC) analysis and quantitative real‐time PCR (qRT‐PCR) were used. Pre‐pubertal horses showed higher testicular gene expression of 11βHSD1, 11βHSD2 and GCR than horses of all other groups (p < 0.05). A positive intranuclear immunoreaction for GCR was seen in epithelial cells of caput, corpus and cauda epididymidis and in Leydig cells. Significant differences (p < 0.05) between age groups occurred. The number of Leydig cells staining positive for GCR was highest in immature stallions (p < 0.05). The enzyme 11βHSD1 was localized in epithelial cells of the caput and corpus epididymidis and in Leydig cells. As determined by enzyme assay, nicotinamide adenine dinucleotide (NAD)‐dependant dehydrogenase (oxidation) activity was not detected in testicular tissue from immature stallions but in all other age groups (n = 3 per group). Results of this study suggest a contribution of GCs to maturation of male reproductive tissue in horses. In mature stallions, expression of 11βHSD enzymes and the oxidative 11βHSD activity in Leydig cells and epididymal basal and principal cells suggest a protective role on these tissues contributing to physiological intracellular glucocorticoid concentrations.     

Increased nuclear type II estradiol receptor concentrations in rat spleen during the course of pregnancy.

Estradiol receptors are classified into type I and type II receptors by their affinity and capacity for estradiol binding. The type II receptors are thought to be significant in the suppression of immune response, such as in the pregnancy-associated immunosuppression. The present study was undertaken to show the presence of type II receptors in rat spleen and to examine the change of receptor distribution after estradiol administration and during pregnancy. Scatchard analysis revealed that the type II receptors were present in rat spleen, and dissociation constants were estimated to be 3.23 x 10(-9) M for cytosol and 4.29 x 10(-9) M for nuclei. The receptors possessed specificity for estradiol and diethylstilbestrol, but not for promegestone, methyltrienolone and dexamethasone. Administration of estradiol to rats resulted in the increase of nuclear receptor concentrations with concomitant decrease of cytosolic concentrations. During pregnancy, the receptor concentrations were increased in the nuclear fraction, but were not significantly changed in the cytosolic fraction. The dissociation constants of the receptors in pregnant rat spleen (4.77 x 10(-9) M for cytosol and 7.20 x 10(-9) M for nuclei) were similar to those in the non-pregnant control, suggesting the quantitative change of the receptors during pregnancy.