Lymphocyte reduction induced by hindlimb unloading： distinct mechanisms in the spleen and thymus

Hindlimb unloading (HU) in rodent is a well-accepted ground-based model used to simulate some of the condi-tions of space flight and reproduce its deleterious effects on the musculoskeletal, cardiovascular and immune systems. In this study, the effects of HU on lymphocyte homeostasis in the spleen and thymus of mice were examined. HU was found to drastically deplete various cell populations in the spleen and thymus. These changes are likely to be mediated by apoptosis, since DNA strand breaks indicative of apoptosis were detected by terminal deoxynucleotidyl transferase-mediated nick end-labeling in both splenocytes and thymocytes. Surprisingly, administration of opioid antagonists or interference with the Fas-FasL interaction was able to block HU-induced reductions of splenocytes, but not thymocytes. On the other hand, steroid receptor antagonists blocked the reduction of lymphocyte numbers in both spleen and thymus. Therefore, the effects of HU on the homeostasis of splenocytes and thymocytes must be exerted through distinct mechanisms.     

五步蛇毒对小鼠免疫功能的影响

目的 探索五步蛇毒对正常小鼠免疫功能的影响.方法 将KM小鼠随机分为5组,分别为空白组、五步蛇毒安全范围内低剂量组(30 mg/kg)、五步蛇毒安全范围内中剂量组(60 mg/kg)、五步蛇毒安全范围内高剂量组(120 mg/kg)(以下简称五步蛇毒低、中、高剂量组)以及西洋参组(30 mg/kg),每组10只.各组小...     

Melatonin-induced suppression of the pinealectomy-stimulated rat adrenal cortex mitotic activity

Pineal involvement in the regulation of adrenocortical mitotic activity has recently been suggested. It has been shown that melatonin (Mel) decreased the mean mitotic activity rate (MMAR) of the adrenal cortex both in vivo and in organ culture. The goal of the present study was to test the influence of pinealectomy (PX) and/or Mel-treatment on the MMAR of adrenocortical cells, as well as on the adrenal weight in rats. The stathmokinetic method was used in the study. It was found that PX significantly increased the MMAR of the adrenocortical cells. Moreover, Mel suppressed the proliferogenic effect of PX on the rat adrenocortical cells. Melatonin alone did not significantly affect the mitotic activity of the adrenal cortex. None of the three experimental procedures, i.e. Mel, PX and Mel-treatment of pinealectomized animals significantly affected the adrenal weight. The present data suggest that Mel may be involved in the inhibitory control of adrenocortical cell proliferation.     

Characterization of protective immunity induced against Schistosoma mansoni via DNA priming with the large subunit of calpain (Sm-p80) in the presence of genetic adjuvants

Despite advances in control via snail eradication and large-scale chemotherapy using praziquental, schistosomiasis continues to spread to new geographic areas particularly in sub-Saharan Africa. Presently, there is no vaccine for controlling this disease. We have concentrated on a functionally important schistosome antigen Sm-p80 as a possible vaccine candidate for schistosomiasis. Here we report the proliferation of spleen cells in response to the recombinant Sm-p80 protein and cytokine (IFN-gamma and IL-4) production by the splenocytes. These spleen cells were obtained from groups of mice that were vaccinated with a DNA vaccine formulation containing Sm-p80 and one of the Th-1 (IL-2 or IL-12) or Th-2 (GM-CSF, IL-4) enhancer cytokines. The splenocytes from the groups of mice vaccinated with Sm-p80 DNA in the presence of Th-2 enhancer cytokines showed moderate but detectable proliferation. The splenocytes obtained from mice vaccinated with Sm-p80 DNA with Th-1 enhancer cytokines IL-2 and IL-12 provided the highest proliferation. The IFN-gamma production by splenocytes was found to follow the similar pattern [(Sm-p80) < (Sm-p80 + IL-4) < (Sm-p80 + GMCSF) < (Sm-p80 + IL-12) < (Sm-p80 + IL-2)], as has been observed for the proliferation and protection data. However, the elevated IL-4 production was inversely correlated to Sm-p80-induced splenocyte proliferation or the protection. These results show again that protective immune response induced by Sm-p80 is of Th-1 type.     

Trichinella spiralis: the influence of short chain fatty acids on the proliferation of lymphocytes, the goblet cell count and apoptosis in the mouse intestine

This study was carried out to determine the influence of short chain fatty acids (SCFA) on spleen and mesenteric lymph node lymphocyte proliferation, goblet cells and apoptosis in the mouse small intestine during invasion by Trichinella spiralis. BALB/c mice were infected with 250 larvae of T. spiralis. An SCFA water solution containing acetic, propionic and butyric acids (30:15:20 mM) was administered orally starting 5 days before infection and ending 20 days post infection (dpi). Fragments of the jejunum were collected by dissection 7 and 10 dpi, and were examined for apoptotic cells in the lamina propria of the intestinal mucosa, and for goblet cells. The proliferation index of the cultured spleen and mesenteric lymph node lymphocytes with MTT test was also determined. The orally administered SCFA solution decreased the proliferation of mesenteric lymph node lymphocytes in the mice infected with T. spiralis at both examination times, but did not influence the proliferative activity of the spleen cells. Seven dpi, both in the spleen and mesenteric lymph nodes, the highest proliferation index of concanavalin A (Con A)-stimulated lymphocytes was found in the group of uninfected animals receiving SCFA animals. This tendency could still be seen 10 dpi in the mesenteric lymph nodes but not in the spleen, where the proliferation index in this group had significantly decreased. In vitro studies revealed, that butyric and propionic acids added to the cell cultures suppressed the proliferation of Con A-stimulated mesenteric lymph nodes and spleen lymphocytes taken from uninfected and T. spiralis-infected mice. Acetic acid stimulated proliferation of splenocytes taken from uninfected mice but did not affect lymphocyte proliferation in mesenteric lymph nodes from uninfected or infected mice. Orally administered SCFA increased the number of goblet cells found in the epithelium of the jejunum 7 dpi, but this number had decreased 10 dpi. The number of apoptotic cells in the lamina propria of the intestinal mucosa of animals infected with the T. spiralis and receiving SCFA was also lower, particularly 10 dpi. The above results show that SCFA can participate in the immune response during the course of trichinellosis in mice.     

Cancer cachexia is mediated in part by the induction of IL-6-like cytokines from the spleen

The development of cancer cachexia has been linked to cytokines related to interleukin6 (IL-6). We examined the kinetics of IL-6, IL-11, oncostatinM (OSM) and leukaemia inhibitory factor (LIF) induction in the splenocytes of tumour-bearing mice. Using a lung carcinoma line, which grows in C57BL/6J mice, we observed that when the tumour grew and cachexia was observed, the splenocytes produced IL-6, IL-11, and OSM, but not LIF. Cytokine expression was observed within 1 week (day 3 for IL-6 and IL-11, and day 1 for OSM) of administration of tumour cells, and was observed in splenocytes without tumour metastases to the spleen. Cytokine expression preceded cachexia (determined by changes in serum triglyceride levels and decrease in epididymal fat-pad weights) development by over 1 week. Exogenous administration of IL-11 resulted in the accelerated onset of cachexia, compared to control protein treatment, but without an effect on the tumour burden. In vivo treatment with a neutralizing dose of anti-OSM antibody inhibited the triglyceride dysregulation only until the synthesis of IL-6 and IL-11 began in the spleen (day 3). Afterward, IL-6 and IL-11 induced lipid catabolism in the absence of functional OSM. We conclude from the data described above that cachexia developed due to a systemic cytokine response induced by a tumour burden, and that IL-6-like cytokines contributed independently to lipid hypercatabolism in the aetiology of cancer cachexia.     

Cytogenetic studies of mice exposed to styrene by inhalation.

The data for the in vivo genotoxicity of styrene (STY) are equivocal. To evaluate the clastogenicity and sister-chromatid exchange (SCE)-inducing potential of STY in vivo under carefully controlled conditions, B6C3F1 female mice were exposed by inhalation for 6 h/day for 14 consecutive days to either 0, 125, 250 or 500 ppm STY. One day after the final exposure, peripheral blood, spleen, and lungs were removed and cells were cultured for the analysis of micronucleus (MN) induction using the cytochalasin B-block method, chromosome breakage, and SCE induction. Peripheral blood smears were also made for scoring MN in erythrocytes. There was a significant concentration-related elevation of SCE frequency in lymphocytes from the spleen and the peripheral blood as well as in cells from the lung. However, no statistically significant concentration-related increases were found in the frequency of chromosome aberrations in the cultured splenocytes or lung cells, and no significant increases in MN frequencies were observed in binucleated splenocytes or normochromatic erythrocytes in peripheral blood smears.     

Inhibitory effect of thyrotropin releasing hormone on spontaneous proliferation of mouse spleen lymphocytes in vitro.

The effects of thyrotropin releasing hormone (TRH) and of TRH-like tripeptide pGlu-His-Gly-OH (colon mitosis inhibitor, CMI) on spontaneous proliferation of murine splenocytes were investigated in vitro. The 3H-thymidine incorporation into splenocyte DNA was used as an index of proliferation. It was found that TRH suppressed the proliferation of splenocytes. In contrast, CMI was ineffective by itself but used together with TRH blocked the effect of the latter.     

Variations of prolactin content in human cerebrospinal fluid after metoclopramide and morphine

Serum and cerebrospinal fluid (CSF) prolactin (PRL) concentrations were determined in fourteen patients of both sexes suffering from hydrocephalus, in basal conditions and after i.m. administration of 10 mg metoclopramide or 10 mg morphine. A significant increase in both serum and CSF hormone values was found after administration of both drugs. Serum and CSF PRL values after metoclopramide administration increased earlier and to a greater extent than after morphine. Furthermore, the metoclopramide induced CSF PRL increase immediately followed the serum peak, whereas after morphine administration an evident delay in the CSF hormone peak with respect to the serum increase was found. These data suggest that PRL entry in the CSF compartment is subject to a controlling mechanism which acts at the blood/brain barrier.     

Splenocytes from NZB mice exhibit spontaneously high rates of fusion compared to control strains

A previous report (G. E. Woloschak and D. Senitzer, submitted for publication) has demonstrated that mitogen-stimulated splenocytes fuse at a much higher frequency than untreated splenocytes as measured by the fusion index (a calculation of the number of nuclei in fused cells vs the total number of nuclei). A measurement of the fusion indices of NZB spleen cells provides results markedly different from those observed with splenocytes from control strains of mice—NZB spleen cells exhibit a spontaneously high fusion index. In this assay, they spontaneously display the same fusing capacity as that observed in cultures of mitogen-treated spleen cells from control strains of mice. This elevated fusion index is not affected by treatment with anti-Thy-1.2 and complement, but is abrogated by treatment with anti-immunoglobulin serum and complement. This suggests that a B cell is responsible for the high fusion index of NZB splenocytes. This high fusion index is present when using splenocytes from both male and female mice in the fusion assay, and can be observed using spleen cells from NZB mice as young as 12 days. This appears to be the result of a spontaneous polyclonal B-cell activation in NZB mice.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

A novel nervous system-induced factor inducing immune responses in the spleen

This study relates to a novel mediator signaling between the nervous system and the spleen following an immune challenge. Using enzyme-linked immunospot and cell proliferation assays, we found that supernatants of cultured splenocytes prepared from subcutaneously trypanosome-inoculated rats and mice spleens obtained immediately after inoculation and added to naive cells significantly stimulate interferon-gamma production and cell proliferation compared to phosphate-buffered saline-inoculated animals. This action was abrogated by surgical denervation of the spleen. Using the fluorescent differential display technology, the gene involved in this process was identified and further cloned and its sequence was mapped to chromosome 14 (GenBank accession number: EU552928). Protein expression revealed approximately 15 kDa molecule with biological activities similar to the cultured supernatants of splenocytes obtained directly from parasite-inoculated animals. Antibodies raised against the protein blocked the activities of both the protein and the supernatant and also recognized a band in the active supernatant with the same molecular mass as the protein. Furthermore, the protein was able to reactivate experimentally immunosuppressed cells by regaining their ability to proliferate, suggesting that such a nervous system-induced immune system-released activating agent (ISRAA) may have a potential therapeutic benefit in immunocompromised situations and in further understanding the mechanism for innate immunity commencement and action.     

Disorders in the immune responses of T- and B-cells in mice administered intravenous verotoxin 2

To obtain a better insight into the pathogenesis of verotoxin-producing Escherichia coli (VTEC)-associated diseases, we explored the effect of verotoxin 2 (VT2) on the immune response in mice. The distribution of lymphocyte phenotypes and the lymphocyte immune response were examined after intravenous administration of VT2 to mice. Among the peripheral lymphocytes and splenocytes of 4-week-old C57BL/6 mice, there was first of all a decrease in T-cells, which began 24 h after intravenous administration of VT2 (50 ng/kg, lethal dose). The CD4+ cell subpopulations of the peripheral blood and spleen were significantly decreased at 24 h, while the B220+ splenocyte subpopulation was markedly decreased at 45 h after VT2 administration. In the thymus, a decrease in CD4+CD8+ cells was predominantly observed near death. Interestingly, in E. coli lipopolysaccharide (LPS)-responder mouse strains (C57BL/6 and C3H/HeN) cotreated with LPS, the susceptibility to VT2 was enhanced, and the increase in B220+ cells induced by LPS alone was suppressed. Furthermore, splenocytes from C57BL/6 mice treated with VT2 (50 ng/kg) 6-24 h earlier reduced LPS-induced proliferative responses to 50-52% of that in control cells, indicating that the effect of VT2 on the immunoresponse seen in vivo may be negatively exerted on the proliferation of the cells. In addition, the number of splenocytes that produced anti-sheep red blood cell antibody was decreased in mice treated with VT2. These results suggest that VTEC infection may eliminate CD4+ and CD8+ T-cells and B-cells by affecting their survival and proliferative responses, leading to reduced antibody production.     

The effects of corticosteroid hormones and thyroid hormones on lymphocyte viability and proliferation during development and metamorphosis of Xenopus laevis

Abstract. Metamorphosis in the South African clawed frog, Xenopus laevis , is characterized by a striking loss of lymphocytes in the thymus, liver, and spleen. Changes in the proliferative responses of splenocytes and thymocytes to T cell mitogens and semi-allogeneic cells are also observed at metamorphosis. Because the levels of circulating thyroid hormones (TH) and corticosteroid hormones (CH) increase dramatically during the climax of metamorphosis, we have investigated the possible role of TH and CH as mediators of the changes in lymphocyte numbers or lymphocyte function. Here we report on the in vitro effects of CH and TH on lymphocyte viability and on phytohemagglutinin-P (PHA)-stimulated lymphocyte proliferation at prometamorphosis and climax of metamorphosis. We have observed consistently significant inhibition of proliferation by corticosterone. In contrast, we have observed inconsistent inhibition of proliferation by both thyroxine (T₄) and triiodothyronine (T₃). In short-term studies, the viability of thymocytes and splenocytes was reduced in the presence of CH but not TH.
These observations are consistent with a hypothesis that loss of larval lymphocytes and changes of lymphocyte function at metamorphosis may be due to elevated concentrations of CH rather than TH.
Because CH have been shown to enhance TH-induced effects during metamorphosis, we looked at the combined effects of these agents on PHA-stimulated lymphocyte proliferation. While each agent was inhibitory in several experiments, there was no significantly greater inhibition when splenic lymphocytes were cultured with both.     

A correlation between the cellular composition of the spleen and the change in splenocyte chemiluminescence after laser irradiation]

Chemiluminescence (CL) of splenocytes of A/Sn mice (1.5-9-month old) was recorded after irradiation of the cells with lambda semiconductor laser at 820 nm (dose 1.1 x 10(3) J/m2, pulse repetition rate 292 Hz). Laser radiation was found to stimulate or suppress the spontaneous CL (SCL) of splenocytes, the amplitude and its sign depending on cellular composition of the spleen. Direct correlations between effect of laser radiation (per cent in changes in SCL) and per cent of plasmatic cells (r = 0.743, p < 0.001), neutrophils (r = 0.650, p < 0.001) and myelocytes and metamyelocytes (r = 0.507, p < 0.01) were established. The correlation with per cent of lymphocytes (r = -0.590, p < 0.001) was found to be a reverse one.     

The in vivo distribution of murine lymphokine activated killer cells in splenectomized host

The in vivo distribution of intravenously injected lymphokine activated killer (LAK) cells, generated in vitro with rIL-2 from normal murine splenocytes, was studied in BALB/c mice and compared with that of normal splenocytes. Both normal splenocytes and LAK cells were labeled with 51Cr, and the results were analyzed at 6, 24, and 48 hours after injection by localization index as the parameter. After injection through tail veins of mice, LAK cells were found to migrate to the spleen, lungs, liver, lymph nodes, bones and the kidneys. The apparent increased distribution pattern of LAK cells to the lung at 6 and 24 hours after injection was not detected when normal splenocytes were injected. Since almost one third of the injected LAK cells were found to localize in the spleen, it was postulated that splenectomy would affect the in vivo organ distribution of LAK cells. Accordingly, the in vivo distribution of LAK cells in splenectomized mice was further investigated. Results indicated that splenectomy enhanced the convergence of LAK cells to the lungs, liver, lymph nodes and bones. Therefore, splenectomy may augment the therapeutic effect of the adoptive transfer of LAK cells in pulmonary, hepatic, lymph node and bony metastases.     

Effect of monthly administration of GHRH (fragment 1-29) with osmotic pump on the rat anterior pituitary

Growth Hormone-Releasing Hormone (GHRH) is the main factor, which regulates GH secretion and somatotrope proliferation. However, its chronic effect on anterior pituitary gland is still unknown. It is known that excessive GHRH secretion in patients with gastroenteropancreatic tumors secreting GHRH results in acromegaly and somatotrope hyperplasia. In mice transgenic for GHRH somatotrope tumors develop. Thus, the aim of this paper was to examine the effect of GHRH chronic administration on somatotrope secretion, their percentage and cell proliferation in anterior pituitary gland in rats. The experiment was performed on male Fischer 344 rats weighing 200+/-20 g. The animals were divided into two groups: group I-controls (13 rats) received solvent for GHRH (5% ethanol in demineralized water); group II (10 rats) received GHRH (Growth Hormone Releasing Factor, fragment 1-29 amide) at a dose of 5 microg/day. The substances were given for 1 month via osmotic pump (ALZET), which were implanted subcutaneously in the dorsal region under ketamin anesthesia. After 4 weeks all rats were decapitated and the blood was collected. In the microscopic preparations of anterior pituitary gland the morphology of pituitary (Herlant staining) and the percentage of somatotrope cells and proliferation index based on PCNA staining were assessed. It was found that the chronic treatment with GHRH caused a statistically significant increase in serum rGH concentration and in percentage of somatotropes, but did not change proliferation index and did not induce pathological changes in the morphology of the anterior pituitary gland when compared to the control group. Summing up, monthly GHRH administration did not induce somatotrope adenomas but it caused serum GH level elevation, what seems to depend partially on the increase of somatotrope number.     

Mechanism of the central effects of dopamine and metoclopramide on aldosterone regulation in the rat

To investigate the mechanism of the central action of dopamine and its antagonist, metoclopramide, on the regulation of aldosterone, studies were performed in 54 conscious rats with and without bilateral nephrectomy. In normal and sham-operated rats, intracerebroventricular injection of dopamine resulted in a significant suppression of plasma renin activity and plasma aldosterone at 30 min, and intracerebroventricular injection of metoclopramide resulted in a significant elevation of plasma renin activity and plasma aldosterone at 30 min without altering the plasma corticosterone and potassium levels. In bilaterally nephrectomized rats, the plasma renin activity was significantly reduced and it did not respond to dopamine or metoclopramide. In these rats, intracerebroventricular injection of metoclopramide exerted no effect on the plasma aldosterone, but intracerebroventricular injection of dopamine increased the plasma aldosterone slightly. However, this increase was not statistically significant. These findings suggest that the dopaminergic system in the brain is involved in the regulation of aldosterone secretion, mainly with changes in the peripheral renin-angiotensin axis in rats.     

Controlling effect of T lymphocyte suppressors on the proliferation of cells in various tissues

The changes in mitotic index of mouse bone marrow, spleen, liver, kidneys, small intestine, cornea were studied during alterations in the number of T-suppressors. It was found that mitotic activity in all tissues investigated enhanced significantly after a decrease in the number of T-suppressors caused by single injection of an antiserum against T-suppressors. On the contrary, the mitotic index diminished significantly after the transfer of tolerant spleen lymphoid cell suspension enriched by T-suppressors to normal syngeneic mice. These data indicate that T-suppressors are responsible for the control over cell proliferation in nonlymphoid tissues.     

Melatonin ameliorates oxidative stress and induces cellular proliferation of lymphoid tissues of a tropical rodent, Funambulus pennanti, during reproductively active phase

Effect of melatonin treatment on free radical production was assessed with simultaneous investigation of hormonal level (melatonin and testosterone), blastogenic response, stimulation index, and histological observation of lymphoid organs (spleen, thymus, and bone marrow) in male Indian palm squirrel (Funambulus pennanti) during reproductively active phase (RAP). Low endogenous melatonin and high testosterone level were noted during RAP. Daily subcutaneous injection of melatonin (25 μg/100 g B wt.) at 17.30–18.00 h to squirrels for 60 consecutive days during May–June significantly decreased the thiobarbituric acid reactive substances (TBARS) level compared to control squirrels. Melatonin treatment significantly increased % stimulation ratio (%SR) of splenocytes and thymocytes against T cell mitogen concanavalin A. Pinealectomy (Px) led to a significant increase in TBARS level whereas a significant decrease was observed in blastogenic response and stimulation index was noted. Melatonin injection to Px squirrels showed restoration in %SR of thymocytes and splenocytes with a significant decrease in the TBARS level of the lymphoid tissues. Further, free radical load was induced by lipopolysaccharide (LPS; 400 μg/ml) in lymphatic tissue homogenates and noted that melatonin supplementation (2 mM/ml) led to a significant decrease in TBARS level compared to the control and LPS-supplemented groups. Histological observation showed dense cellularity of thymocytes and splenocytes. Acridine orange staining technique shows a significant increase in thymocyte apoptosis Px squirrels when compared with melatonin-treated squirrels. These findings suggest that endogenous and exogenous melatonin might be responsible for the maintenance of immune system to adapt this seasonal breeder for the rigors of the environmental changes.