Expression of lymphocyte subsets after exercise and dexamethasone in high and low stress responders.

PURPOSE: Recent work indicates that among the normal population, persons can be classified as low (LR) or high (HR) stress responders based on hypothalamic-pituitary-adrenal (HPA) axis responses to high-intensity exercise. We studied whether differential activation of the HPA axis affected cytokine production and expression of selected lymphocyte subsets in HR and LR in response to high-intensity exercise after placebo and dexamethasone (DEX; 4 mg). METHODS: Healthy HR (N = 12) and LR (N = 10) underwent two exercise tests at 90% of VO2max, 8 h after placebo or DEX. Expression of lymphocyte surface markers (CD3+, CD4+, CD8+, CD56+), adhesion molecule markers (intercellular adhesion molecule-1/ICAM-1: CD54+ and L-selectin: CD62L+), and concentrations of plasma interleukin 6 (IL-6) were examined before and after exercise. RESULTS: Baseline percentages of CD8+ and CD56+ cells were significantly higher, and concentrations of IL-6 and percentages of CD4+ cells were significantly lower in HR as compared with LR. The percentage of CD54+ and CD62L+ cells was not significantly different in HR and LR. DEX significantly reduced the percentage of CD3+ and CD4+ and increased the percentage of CD8+ and CD56+ subsets; the percent of cells expressing CD54+ increased, whereas CD62L+ decreased. Exercise-induced changes in the percentage of lymphocyte subsets were similar to those induced by DEX. CONCLUSION: In summary, HR and LR have different baseline patterns of IL-6 and lymphocyte subsets, which may reflect differential sensitivity to endogenous glucocorticoids. However, exogenous glucocorticoids induced similar patterns of lymphocyte expression in HR and LR.

Using of thin-layer chromatography for identification and quantitative determination of benzalkonium chloride in eye drops.

Conditions for thin-layer chromatographic separation of components (therapeutic and auxiliary substances) in selected eye drops have been established and next used for the densitometric determination of benzalkonium chloride content in the drugs.

Lymphocyte subpopulation expression in women: effect of exercise and circadian rhythm.

It is well established that exercise affects immune responses in men, but little information is available for women. Whether exercise-induced immune responses are affected by circadian rhythm has not been determined. This study examined lymphocyte responses of women to exercise in the morning (AM) and evening (PM). Lymphocytes expressing markers for T, B, and natural killer (NK) cells were identified by FACScan in blood samples collected before (baseline), immediately after, and 40 min after exercise (recovery). Absolute counts of all lymphocyte subpopulations increased immediately after exercise, but at recovery values were below baseline counts. Circadian variations were noted in absolute counts for total T, helper/inducer T cells, and B cells. Although these counts were significantly higher in the PM, the magnitude of the immune response to exercise was the same in the AM and PM. In contrast, natural killer cells, both the relative counts and the magnitude of immune response were higher in the AM. Thus, it appears that despite differences in baseline cell counts the overall exercise-induced response in most lymphocyte subpopulations is similar in the AM and PM.

Lymphocyte subset responses to exercise and glucocorticoid suppression in healthy men.

It is well established that in vivo changes in ratios of lymphocyte phenotype subsets is altered by glucocorticoid administration. To determine whether the lymphocyte response would be further affected by strenuous exercise, since glucocorticoids are released during exercise, 14 physically fit men were randomly given placebo (P), 4 mg of dexamethasone (DEX), or 100 mg of hydrocortisone (HCO) 4 h before high-intensity treadmill running. Blood was drawn pre- and immediately post-exercise; lymphocyte subsets (CD3, CD4, CD8, CD19, and CD56) were determined by flow cytometry. Pre-exercise CD3 and CD4 percentages were lower, whereas CD8 and CD56 were higher with DEX and HCO as compared to P. Exercise induced a lymphocytosis after all treatments, but subsets did not change proportionally. With P, DEX, and HCO, the magnitudes of change were comparable: CD3 and CD4 decreased and CD8 and CD56 increased. Notably, for all treatments exercise induced in approximately 2-fold increase in the percentage of cells expressing CD56 (natural killer cells). Thus, a single oral dose of DEX or HCO did not alter the direction or magnitude of immediate post-exercise changes in lymphocyte subset expression. Whether glucocorticoid pretreatment influences lymphocyte responses during recovery from strenuous exercise remains to be determined.

Inflammatory responses to carrageenan injection in LEW/N and F344/N rats: LEW/N rats show sex- and age-dependent changes in inflammatory reactions.

We studied the inflammatory responses of LEW/N and F344/N inbred rat strains after peripheral injection of carrageenan. The inflammatory responses were assessed in terms of volume, relative and total white blood cell counts of the exudates. Moreover, in both strains, blood CD4, CD8, CD25, naive CD4 (CD4/CD45RC) cell and B (CD45R) cell counts and plasma corticosterone levels, constituents of systemic inflammatory responses to carrageenan were evaluated. In general, LEW/N rats are highly responsive to challenge with carrageenan, whereas F344 rats are not. The strong local inflammatory responses to carrageenan are primarily exhibited by female LEW/N rats. The intensity of local inflammatory responses of LEW/N rats changes with the rat age, the highest exhibited by LEW/N rats up to 3 months of age, thereafter the carrageenan-induced inflammatory responses decline. Our results indicate that peripheral injection of carrageenan induces strong systemic immune component. After carrageenan injection, increases in CD8 and naive CD4 blood lymphocytes are seen. Although the carrageenan challenge does not change CD4 blood lymphocytes in both LEW/N and F344/N rat strains, LEW/N rats exhibit higher levels of CD4 cells than F344/N rats. Additionally, LEW/N rats demonstrated lower levels of B cells and higher naive CD4 lymphocytes. Carrageenan challenges induce significant increases in plasma corticosterone response in F344/N rats, as well as increases in LEW/N rats 1 h after injection. Our data stress the importance of rat age and gender in experiments studying inflammatory responses.

The estrogen antagonist tamoxifen inhibits carrageenan induced inflammation in LEW/N female rats.

Carrageenan induces a measurable inflammatory response in susceptible animals, and mature females are more responsive to carrageenan, than males. In the present study, we tested whether the estrogen antagonist tamoxifen influences carrageenan-induced inflammatory responses. Female LEW/N rats were treated with tamoxifen and compared to a control group of animals injected with vehicle. Tamoxifen significantly reduced estrous phase of estrous cycle during treatment, consistent with its functional anti-estrogen effects. Moreover, tamoxifen significantly decreased exudate volume but did not significantly influence relative white blood cell counts in the exudate. Interestingly, tamoxifen induced differential dose-dependent alterations in peripheral blood lymphocyte subpopulations. Low dose of tamoxifen increased CD25 cells. The high tamoxifen dose significantly increased CD8 blood lymphocytes counts. Our data indicate that tamoxifen treatment decreases carrageenan-induced inflammatory response in female LEW/N rats and suggest therefore that this inflammatory response is, at least in part, estrogen related. Moreover, our results suggest a possible role for tamoxifen in treatment of inflammatory disorders.

Differential mineralocorticoid (type 1) and glucocorticoid (type 2) receptor expression in Lewis and Fischer rats.

Lewis (LEW/N) and Fischer (F344/N) rats represent two extremes of the spectrum of corticosterone responses to stressful stimuli, from the chronical hyporesponsiveness of LEW/N to the chronical hyperresponsiveness of F344/N. It might be expected that the amount of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) binding, and the levels of their corresponding mRNAs in various tissues in LEW/N and F344/N rats might reflect the overall integrated levels of corticosterone to which these receptors have been exposed. We have found that while the binding affinity (Kd) of MR and GR varies between tissues, there was no strain difference in any tissue. Receptor binding number (Bmax), however, varied not only between tissues, but also between strains. MR Bmax in the hippocampus and pituitary was lower in LEW/N than in F344/N, whereas the GR Bmax in the LEW/N thymus was greater than that found in F344/N rats. The hippocampal levels of MR mRNAs in Adx LEW/N and F344/N rats were in good agreement with, and paralleled, the functional levels of these receptors as determined by binding assays. On the other hand, the number of hippocampal GR binding sites and the level of GR nRNA while similar were not identical in the two strains: the hippocampal GR Bmax did not differ between strains, while the hippocampal GR mRNA level was slightly, but significantly, lower in Adx LEW/N compared to F344/N rats.

Release of hypothalamic corticotropin-releasing hormone and arginine-vasopressin by interleukin 1 beta and alpha MSH: studies in rats with different susceptibility to inflammatory disease.

The susceptibility of Lewis rats is related to blunted hypothalamic-pituitary-adrenal (HPA) axis responsiveness to a variety of inflammatory and neuroendocrine stimuli. In contrast resistance to inflammatory disease of histocompatible Fischer rats is associated with their intact HPA axis responses to the same stimuli. We have examined the contribution of IL-1 beta to in vitro corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) release from hypothalamic explants derived from LEW/N and F344/N rats. The same animal model has been used to investigate the regulatory effect of alpha MSH, an immunosuppressive neurohormone, on IL-1 beta stimulated CRH and AVP secretion. CRH basal release in both strains was similar. However, LEW/N hypothalamic AVP basal secretion was significantly elevated. CRH relative response of LEW/N hypothalamic explants to IL-1 beta stimulation was lower compared to Fischer, which is consistent with their hyporesponsiveness to inflammatory mediators. AVP secretion however, was significantly decreased in hypothalamic explants from both strains after 40 min exposure to IL-1 beta. alpha MSH suppressed basal CRH and AVP release in both LEW/N and F344/N rats and prevented IL-1 beta stimulated CRH secretion in these strains. AVP was further diminished in F344/N explants following incubation with alpha MSH + IL-1 beta, while LEW/N level was significantly elevated. However, AVP levels remained significantly below baseline in explants from both strains after final incubation with IL-1 beta. Although our findings indicate a modulatory action of alpha MSH in HPA axis regulation in vitro, the physiological importance of this phenomenon in Lewis and Fischer rats requires further investigation.

Nonidentity of subunits of human kidney arginase A1 and human liver arginase A5.

The main forms of arginase A1 from human kidney and A5 from human liver were purified to homogeneity. Molecular weight of both forms of enzyme approximates 120,000. In the presence of EDTA these arginases dissociate into single type distinct subunits. M(r) of both kinds of subunits is 30,000. Similarly as native arginase forms, they differ in electric charge and display complete immunological incompatibility.

Alkaline phosphatase from adult rat femur.

Four alkaline phosphatase forms from adult rat femur were distinguished on polyacrylamide gel electrophoresis: two soluble forms of Mr 165,000 and 110,000 in the water extract, and three membrane-bound forms of Mr 130,000, 110,000 and 100,000 extractable with deoxycholate. Alkaline phosphatase after SDS-treatment disintegrated into three kinds of monomers: of Mr 80,000, 65,000 and 50,000. The soluble fraction (extract I) contained subunits of Mr 80,000 and 55,000--whereas the pellet fraction (extract II), subunits of Mr 65,000 and 50,000. Since for native forms only three types of subunits were found it seems that, apart from homodimers, there are also some heterodimers composed of the Mr 65,000 and 50,000 subunits forming the native enzyme of Mr 110,000-115,000. Two denatured monomers: of Mr 80,000 and 50,000 may form two native homodimeric forms of Mr 165,000 and 100,000 while in the pellet two monomers: of Mr 65,000 and 50,000 may correspond to three native alkaline phosphatase forms: of Mr 130,000, 110,000-115,000 and 100,000. Probably the Mr 110,000-115,000 form is a heterodimer composed of subunits of Mr 65,000 and 50,000.

Changes of femoral alkaline phosphatase activity in adult rats treated by sorbitol enriched or vitamin D3 deficient diet.

The effect of vitamin D3-deficiency and dietary sorbitol on serum calcium level, the activity and alkaline phosphatase (AP) pattern in femoral epiphysis were studied. Rats fed a diet supplemented with sorbitol or vitamin D3 showed the same serum calcium concentration and AP activity in serum and femur. Rats fed a vitamin D3-deficient diet displayed decreased serum calcium concentration and increased AP activity both in serum and femur. Four forms of AP were isolated from the femur of these rat groups: of Mr 100,000, 110,000, 130,000 and 165,000. Rats receiving the diet supplemented with sorbitol showed a marked rise in the activity of the Mr 165,000 form, and appearance of a new monomer of 100,000, never formed in two remaining groups.

Differential effect of ethanol on formation of phosphorylated intermediates of the jejunal brush border alkaline phosphatase Mr 90,000 and 65,000 subunits.

The influence of ethanol on formation of phosphorylated intermediates of different forms of alkaline phosphates in brush border of adult rat was studied. Ethanol enhanced phosphorylation of the Mr 65,000 subunit of jejunal enzyme but had no effect on the Mr 90,000 subunit. Commercial highly purified alkaline phosphate in the presence of ethanol also displayed a two times higher phosphorylation ability.

The effect of 1,25 dihydroxycholecalciferol on the ratio of two alkaline phosphatase subunits of Mr 90,000 and 65,000 in adult rat intestine.

The effect of vitamin D3 on the activity of different forms of alkaline phosphatase as well as on the ability of their subunits to form phosphorylated intermediates in the presence of [gamma-32P]ATP was studied. In experiments with rats fed a diet enriched in vitamin D3 phosphatase F3 activity was doubled and the radioactivity of the phosphorylated intermediate of the F3 subunit was greatly enhanced. In contrast, in the case of phosphatase F1 the enzymic activity and the radioactivity of F1 subunit phosphorylated intermediate remained unchanged. In rats fed a vitamin D3-deficient diet, the phosphatase F3 activity was greatly reduced and there were only traces of radioactivity of F3 subunit phosphorylated intermediate.

Melatonin-induced suppression of human lymphocyte natural killer activity in vitro.

One of the important immune functions influenced by neuroendocrine factors is natural killer (NK) activity, which is directed against neoplastic and virus-infected cells. The effects of melatonin (Mel) and N-acetylserotonin (NAc-5HT) on NK activity of human peripheral blood lymphocytes were investigated. Leukocytes of healthy human subjects were used in the experiment. NK activity was estimated by measurement of radioactive chromium (51Cr) release from human leukemia cells K 562 (target cells). The previous exposure of human lymphocytes (effector cells) to Mel in concentrations of 10(-6) M and 10(-10) M resulted in an inhibition of NK activity (P less than 0.01) for all the examined effector-target cell ratios (10:1; 20:1, 40:1). NK activity was also suppressed by Mel (10(-8) M), but only if effector-target ratio equal to 20:1 was used (P less than 0.02), and by Mel (10(-12) M) for effector-target ratio equal to 40:1 (P less than 0.05). In none of the examined concentrations did NAc-5HT inhibit NK activity of human lymphocytes. On the basis of the data reported above, a direct suppressive effect of Mel (but not of NAc-5HT) on NK activity of human peripheral blood lymphocytes can be assumed.

Histamine levels and activity of histidine decarboxylase (HD) and histamine-methyltransferase (HMT) in neonate and adult human brain.

The adult human brain contained histamine (HI), and showed well-expressed histidine decarboxylase (HD), histamine-methyltransferase (HMT) and monoamine oxidase (MAO) activity. The neonate human brain contained HI, and showed HMT and MAO activity; there was no HD activity. Neither the adult nor neonate brains possessed diamine oxidase activity.

Molecular forms of alkaline phosphatase in particular segments of the small intestine of adult rat. Separation of three enzyme forms from jejunum.

Rat duodenum and jejunum were found to contain three forms of alkaline phosphatase, F1, F2 and F3, and ileum two forms of this enzyme, F1 and F2. The procedure for separation of phosphatase F1, F2 and F3 from jejunum is presented.