Splenic Lymphocyte Populations Research Articles

Whole blood transfusion improves vascular integrity and increases survival in artemether-treated experimental cerebral malaria

Pathological features observed in both human and experimental cerebral malaria (ECM) are endothelial dysfunction and changes in blood components. Blood transfusion has been routinely used in patients with severe malarial anemia and can also benefit comatose and acidotic malaria patients. In the present study Plasmodium berghei-infected mice were transfused intraperitoneally with 200 μL of whole blood along with 20 mg/kg of artemether. ECM mice showed severe thrombocytopenia and decreases in hematocrit. Artemether treatment markedly aggravated anemia within 24 h. Whole blood administration significantly prevented further drop in hematocrit and partially restored the platelet count. Increased levels of plasma angiopoietin-2 (Ang-2) remained high 24 h after artemether treatment but returned to normal levels 24 h after blood transfusion, indicating reversal to quiescence. Ang-1 was depleted in ECM mice and levels were not restored by any treatment. Blood transfusion prevented the aggravation of the breakdown of blood brain barrier after artemether treatment and decreased spleen congestion without affecting splenic lymphocyte populations. Critically, blood transfusion resulted in markedly improved survival of mice with ECM (75.9% compared to 50.9% receiving artemether only). These findings indicate that whole blood transfusion can be an effective adjuvant therapy for cerebral malaria.

The Early Changes in Splenic Lymphocyte and Macrophage Populations Following Major Liver Resection in Rats.

Background and aim of the work: Splenic hypertrophy occur after major hepatectomy (HTX). The spleen was suggested to inhibit hepatocyte proliferation and consequently, splenectomy was considered. The present study aims to examine the influence of 70% partial hepatectomy (HTX) on the splenic histological structure, T, B lymphocyte and macrophage populations and to discuss the functional correlation.Methods: The rats were assigned to two groups; Sham group, and 70% Partial hepatectomy (HTX) group; which was further subdivided into 2 subgroups; sacrificed 24 or 48h, after HTX; Groups (HTX 24h) and (HTX 48h); respectively. H & E as a routine stain, iron staining by Prussian blue, as well as immunohistochemical detection of splenic CD3; a marker for T lymphocyte; CD20; a marker for B lymphocyte and CD68; a marker for macrophage; were done.Results: HTX 24h and HTX 48h groups exhibited enlargement of splenic follicles, no expansion of red pulp , no increase in apoptosis, mild increase in the number of melanomacrophages. CD3 expression increased significantly in HTX groups as compared to Sham group. However, CD3 expression in HTX 24h and HTX 48h groups exhibited insignificant difference. CD20 expression showed no significant difference among the studied groups. CD68 expression and Prussian blue staining showed a non-significant increase in HTX 24h group and significant increase in HTX 48h group.Conclusion: During the first two days after HTX, there was a rapid increase in splenic T- but not B-lymphocytes with subsequent increase in splenic macrophages. The splenic changes may explain a role of in liver regeneration.

C9–13 chlorinated paraffins cause immunomodulatory effects in adult C57BL/6 mice

Short-chain chlorinated paraffins (SCCPs, C10–13) were listed as persistent organic pollutants (POPs) by the Stockholm Convention in 2017 and pose extensive exposure risks to humans. To our knowledge, there have been no studies reporting the immmunomodulatory effects of SCCPs until now. C9-CPs have also been shown to be present in the environment. In this study, adult male C57BL/6 mice were exposed to 1, 10, or 100 mg/kg/d C9–13-CPs by gavage for 28 d. The results showed that compared to those of the controls, exposure to C9–13-CPs led to increased spleen weight, delimited germinal centers, enhanced energy metabolism, and elevated glutathione content, but no variation in the malonaldehyde level in the spleen was observed. Exposure to C9–13-CPs also increased the populations of splenic lymphocytes, T lymphocytes, NK cells, and the ratio of the CD3+/CD19+ subsets and CD4+/CD8+ subsets compared to those of the controls. RNA-seq revealed 424 differentially expressed genes (DEGs) (fold change ≥ 1.5, FDR < 0.05) in the spleen between the control group and the 100 mg/kg/d C9–13-CPs-treated group. KEGG analysis demonstrated that folate biosynthesis, pathways in cancer and thyroid hormone signaling were the three most significantly enriched pathways, and despite not reaching statistical significance, some immune-related pathways were also enriched in the KEGG functional enrichment analysis, including the chemokine signaling pathway (FDR < 0.0584), the NF-κB signaling pathway (FDR < 0.0663), Th17 cell differentiation (FDR < 0.0839), and the Jak-STAT signaling pathway. Moreover, compared to those of the controls, exposure to C9–13-CPs enhanced the Concanavalin A (Con A)-stimulated cultured splenocyte proliferation, while the exposure showed no effect on the splenocyte proliferation that was stimulated by lipopolysaccharides (LPS). Taken together, these results demonstrated that subacute exposure to C9–13-CPs could have immunomodulatory effects in mice. The present study helps to provide an understanding of the comprehensive health risks posed by C9–13-CPs.

In vitro effect of 4-pentylphenol and 3-methyl-4-nitrophenol on murine splenic lymphocyte populations and cytokine/granzyme production

Gasoline exhaust particles (GEP) and diesel exhaust particles (DEP) are considered to be some of the most important air pollutants. Among the many constituents in these pollutant particles, 4-pentylphenol (PP) and 3-methyl-4-nitrophenol (PNMC) are considered important phenolics in GEP and DEP, respectively. The aim of this study was to investigate the effect of in vitro exposure to commercially-supplied PP and PNMC on populations of, and production of interleukin (IL)-2, IL-4 and granzyme-B by, mouse splenic lymphocytes. After in vitro exposure to PP or PNMC for 48 h, splenocyte viability was measured, cell phenotypes, e.g. B-cell (CD19), T-cells (CD3), T-cell subsets (CD4 and CD8), were quantified by flow cytometry and production of IL-2, IL-4 and granzyme-B was assessed via ELISA. The oxidative toxicity of PP and PNMC toward the splenocytes was also evaluated using measures of hydroxyl radical and malondiadehyde production and changes in glutathione peroxidase and superoxide dismutase activities. Results showed that in vitro exposure to PP and PNMC inhibited splenic cell parameters in a dose-related manner. Exposure to PP and PNMC decreased splenic T-lymphocyte populations and splenocyte production of cytokines and granzyme B, as well as induced oxidative stress in the splenocytes. The results also showed that the percentages of CD3+ T-cells overall and of CD4+ and CD8+ T-cells therein, among exposed splenocytes, were reduced; neither compound appeared to affect levels of CD19+ B-cells. Overall, the suppressive effects of PP were stronger than PNMC. The data here provide support for the proposal that PP-/PNMC-induced toxicity in splenocytes may be due at least in part to oxidative damage and that PP and PNMC – as components of GEP and DEP – might significantly impact on splenic T-cell formation/release of cytokines/granzymes in situ.

The In Vivo Granulopoietic Response to Dexamethasone Injection Is Abolished in Perforin-Deficient Mutant Mice and Corrected by Lymphocyte Transfer from Nonsensitized Wild-Type Donors.

Exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone-marrow. A hematological response dependent on endogenous glucocorticoids underlies bone-marrow eosinophilia induced by trauma or allergic sensitization/challenge. We detected a defect in granulopoiesis in nonsensitized, perforin-deficient mice. In steady-state conditions, perforin- (Pfp-) deficient mice showed significantly decreased bone-marrow and blood eosinophil and neutrophil counts, and colony formation in response to GM-CSF, relative to wild-type controls of comparable age and/or weight. By contrast, peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency. Dexamethasone enhanced colony formation by GM-CSF-stimulated progenitors from wild-type controls, but not Pfp mice. Dexamethasone injection increased bone-marrow eosinophil and neutrophil counts in wild-type controls, but not Pfp mice. Because perforin is expressed in effector lymphocytes, we examined whether this defect would be corrected by transferring wild-type lymphocytes into perforin-deficient recipients. Short-term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild-type donors. Transfer of the same amount of splenic lymphocytes from perforin-deficient donors was ineffective. This demonstrates that the perforin-dependent, granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations.

Polyamine supplementation in infant formula: Influence on lymphocyte populations and immune system-related gene expression in a Balb/cOlaHsd mouse model

The aim of this work was to study whether the proportion of polyamine found in human milk, administered with a commercial infant formula, affected the maturation of the immune system in a BALB/cOlaHsd mouse model. Forty-eight pups (14-days old) were randomly assigned to four-day intervention groups: 1) breast-fed (normal lactation); 2) fed infant formula; and 3) two different groups fed with infant formula supplemented with two different amounts of polyamines. The influence of polyamine administration on lymphocyte populations in the blood, spleen, and mesenteric lymph nodes, as well as on the modulation of immune system-related gene expression in the small intestine, was analyzed. The results demonstrated that polyamine supplementation induced an increase in splenic B cells to levels observed during normal lactation when compared with formula without supplementation. The correlation coefficients for the splenic lymphocyte populations increased with polyamine supplementation, with a dose-dependent effect. Our results demonstrate that polyamines influence gene expression profile, mainly Cd1d1, Cd40, Hdac5, Hdac7, Clcf1 and Tlr4 compared with normal lactation. In general, the gene expression results verified that the expression of genes associated with immune system was similar in the group with high polyamine supplementation to that observed in the group with normal lactation.

Immunomodulatory and Antidiabetic Effects of a New Herbal Preparation (HemoHIM) on Streptozotocin-Induced Diabetic Mice.

HemoHIM (a new herbal preparation of three edible herbs: Angelica gigas Nakai, Cnidium officinale Makino, and Paeonia japonica Miyabe) was developed to protect immune, hematopoietic, and self-renewal tissues against radiation. This study determined whether or not HemoHIM could alter hyperglycemia and the immune response in diabetic mice. Both nondiabetic and diabetic mice were orally administered HemoHIM (100 mg/kg) once a day for 4 weeks. Diabetes was induced by single injection of streptozotocin (STZ, 200 mg/kg, i.p.). In diabetic mice, HemoHIM effectively improved hyperglycemia and glucose tolerance compared to the diabetic control group as well as elevated plasma insulin levels with preservation of insulin staining in pancreatic β-cells. HemoHIM treatment restored thymus weight, white blood cells, lymphocyte numbers, and splenic lymphocyte populations (CD4+ T and CD8+ T), which were reduced in diabetic mice, as well as IFN-γ production in response to Con A stimulation. These results indicate that HemoHIM may have potential as a glucose-lowering and immunomodulatory agent by enhancing the immune function of pancreatic β-cells in STZ-induced diabetic mice.

The combined effects of reduced weightbearing and ionizing radiation on splenic lymphocyte population and function

Purpose: The effects of radiation +/− hypogravity on immunologic function were investigated using the Partial Weight Suspension (PWS) model ().Materials and methods: Mice were exposed to 0.5, 1, or 2 Gray (Gy) dose of gamma radiation and then placed in the PWS system for 4, 24, 48 hours, or 4 days. Spleens were excised and white blood cells were prepared for flow cytometry analyses.Results: The combination of PWS + radiation (1 and 2 Gy doses only) resulted in decreased cell viability at the 24 h (∼16% decrease), 48 h (∼20% decrease), and 4 day (∼20% decrease) time points, compared to the PWS (no radiation) and no treatment (non-suspended, non-irradiated) groups. The T lymphocyte (thymus-derived) population increased by ∼10% (24 h, 48 h, and 4 day time points), while the B lymphocyte (bursal or bone marrow-derived) population decreased by ∼10% (at all time points examined), when mice were exposed to PWS + radiation (2 Gy dose only), compared to the PWS or no treatment groups. T cell activation was observed in the PWS group and the 0.5 Gy +/− PWS groups at the 4 and 24 h time points, compared to the no treatment group. However, T cell activation was significantly suppressed (∼85%) at the acute time points in the 2 Gy +/− PWS groups, comparable to the no treatment group.Conclusions: Ionizing radiation in the absence and presence of simulated hypogravity results in acute lymphocyte dysfunction and compromised immune response.

Aberrant expression of costimulatory molecules in splenocytes of the mevalonate kinase‐deficient mouse model of human hyper‐IgD syndrome (HIDS)

We sought to determine the activation status and proliferative capacities of splenic lymphocyte populations from a mevalonate kinase-deficient mouse model of hyper-IgD syndrome (HIDS). We previously reported that murine mevalonate kinase gene ablation was embryonic lethal for homozygous mutants while heterozygotes (Mvk (+/-)) demonstrated several phenotypic features of human HIDS including increased serum levels of IgD, IgA, and TNFα, temperature dysregulation, hematological abnormalities, and splenomegaly. Flow cytometric analysis of cell surface activation markers on T and B lymphocytes, and macrophage populations, demonstrated aberrant expression of B7 glycoproteins in all splenic cell types studied. Differences in expression levels between Mvk (+/-) and Mvk (+/+) littermate controls were observed in both the basal state (unstimulated) and after Concanavalin A (Con-A) stimulation in vitro of whole splenocyte cultures. In Mvk (+/-) CD4 and CD8 T cells, alterations in expression of CD25, CD80, CD152, and CD28 were observed. Mvk (+/-) splenic macrophages expressed altered levels of CD80, CD86, CD40, and CD11c while Mvk (+/-) B lymphocytes had differential expression of CD40, CD80, and CD86. Mvk (+/-) splenocyte subpopulations also exhibited altered proliferative capacities in response to in vitro stimulation. We postulate that imbalances in the expression of cell surface proteins necessary for activation, proliferation, and regulation of the intensity and duration of an immune response may result in defective T cell activation, proliferation, and effector functions in our model and potentially in human HIDS.

Toxicologic and immunologic effects of perinatal exposure to the brominated diphenyl ether (BDE) mixture DE‐71 in the Sprague‐Dawley rat

Brominated diphenyl ethers (BDEs) are persistent environmental contaminants found in human blood, tissues, and milk. To assess the impact of the commercial BDE mixture DE-71 on the developing immune system in relation to hepatic and thyroid changes, adult (F0) rats were exposed to DE-71 by gavage at doses of 0, 0.5, 5, or 25 mg/kg body weight (bw)/d for 21 weeks. F0 rats were bred and exposure continued through gestation, lactation and postweaning. F1 pups were weaned and exposed to DE-71 by gavage from postnatal day (PND) 22 to 42. On PND 42, half of the F1 rats were assessed for toxicologic changes. The remaining F1 rats were challenged with the T-dependent antigen keyhole limpet hemocyanin (KLH) and immune function was assessed on PND 56. Dose-dependent increases in total BDE concentrations were detected in the liver and adipose of all F0 and F1 rats. In F0 rats, increased liver weight, hepatocellular hypertrophy, and decreased serum thyroxine (T4) were characteristic of DE-71 exposure. In F1 rats perinatal DE-71 exposure caused a nondose-dependent increase in body weight and dose-dependent increases in liver weight and hepatocellular hypertrophy. Serum T3 and T4 levels were decreased. In spleen from DE-71 exposed rats the area occupied by B cells declined while the area occupied by T cells increased; however, cellular and humoral immune responses to KLH challenge were not altered. Thus hepatic and thyroid changes in rats exposed perinatally to DE-71 were associated with altered splenic lymphocyte populations, an effect which has been linked to hypothyroidism.

The Splenic Response to Ischemic Stroke: What Have We Learned from Rodent Models?

The majority of promising experimental compounds have failed in clinical trials, highlighting the need for novel approaches to treat stroke. Much research has been devoted to elucidating the signaling pathways involved in delayed neuroinflammation that can be targeted at clinically relevant time points. The field of stroke research has benefited from experiments characterizing the temporal expression profiles of candidate cytokines, chemokines, matrix metalloproteinases, and other putative pro-inflammatory molecules. Yet, these data have offered only a glimpse into the complex pathological sequelae and have not advanced the treatment of neuropathies. Upon recognition that peripheral immune cell activation is involved in penumbral expansion, the spleen has emerged as a novel target that mediates the peripheral immune response and promotes pro-inflammatory injury. Although the precise mechanisms have yet to be elucidated, accumulated evidence demonstrates that focal cerebral ischemia alters cytokine, chemokine, and immune cell profiles in the spleen. Additionally, removal of this peripheral lymphoid organ is neuroprotective, and the efficacy of several protective therapies has been linked to actions at the level of the spleen. Future experiments aimed at identifying the splenic lymphocyte populations that respond to ischemic stroke, as well as their signaling mechanisms, are critical in developing novel therapies.

Abstract 466: Does body weight influence tumor-infiltrating B cells in murine pancreatic cancer

Abstract Background: We have developed a murine model of pancreatic cancer in congenitally obese (LepOb and LepDb) mice where tumors grow larger, metastasize, and decrease survival. Obese mice also had altered splenic T and B cells as well as downregulation of B cell and immunoglobulin genes and reduced numbers of B cells in the tumors. The question remained, however, whether alterations in tumor-infiltrating lymphocytes were a reflection of body weight or leptin metabolism. Therefore, we designed an experiment to determine whether diet-induced weight gain would alter systemic and tumor-infiltrating lymphocytes. Methods: Thirty lean C57 female mice were fed either a control 10% fat (n=10) or a 60% fat diet (n=20) starting at 6 weeks of age. At 11 weeks, all mice were inoculated with 2.5x105 PAN02 murine pancreatic cancer cells. After 6 weeks tumors that developed in 18 mice, the spleen (n=12), and the sera were harvested. Mice were categorized as Lean or Overweight if they weighed less or more than the mean final weight of the 60% fat animals (23.1g). Tumors in Lean (n=10) and Overweight (n=8) mice were sectioned and stained for B and T cells. Two observers blinded to animal weight graded cell density on a scale of 0-4 in 10 high-powered fields. Flow cytometry was performed on the spleen from Lean and Overweight mice for B, T, activated T and B (ACT), natural killer (NK) cells, and macrophages (MACRO). Serum leptin was measured. ANOVA and Student's t-test were applied. Results: Tumors were larger (1.33 vs. 0.54g, p=0.03) and leptin levels were higher (3.1 vs. 1.4ng/ml, p=0.05) in Overweight mice. Mean tumor-infiltrating lymphocyte (TIL) scores and spleen lymphocyte data are presented in the table. Conclusion: The data suggest that 1) both B and T cells are present in the pancreatic cancer microenvironment and 2) TIL and splenic lymphocyte populations do not differ between lean and overweight mice. We conclude that obesity, as opposed to overweight, is required to alter systemic and tumor microenvironment lymphocyte populations. B Cell TILT Cell TILB Cell (%) SpleenT Cell (%) SpleenACT (%) SpleenNK (%) SpleenMACRO (%) SpleenLean0.7±0.12.2±0.264±244±32.3±0.72.3±0.71.6±0.2Overweight0.7±0.12.0±0.258±139±51.5±0.31.4±0.41.4±0.3 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 466.

Citalopram Enhances B Cell Numbers in a Murine Model of Morphine‐Induced Immunosuppression

Patients with chronic pain are often challenged with depression stemming from the long-term psychophysiological effects of their condition. Consequently, patients with chronic pain are often treated with morphine, which can induce immunosuppression, along with an antidepressant. The antidepressant citalopram (CTP; Sigma-Aldrich Chemical, St. Louis, MO, U.S.A.) is a serotonin reuptake inhibitor that is reported to have immunomodulatory effects. Thus, we investigated whether CTP administration impacted immunity in morphine-treated animals. Adult mice were pretreated for 7 days with either saline or CTP (10 or 30 mg/kg intraperitoneal injections twice daily), followed by subcutaneous implantation of a 25 mg morphine pellet for 48 hours. Spleen, thymus, and lymph nodes were harvested to analyze total cell numbers, relative lymphocyte populations, and lymphocyte function. In this study, CTP had no effect on either total cell counts or lymphocyte populations in the thymus. However, in the spleen, total splenocyte numbers in all CTP-treated animals displayed an increasing trend over saline-treated animals. Interestingly, although more cells were found in the spleen, distribution of splenic lymphocyte populations did not differ between treatments. Despite no increase in total cell number, a high dose of CTP (30 mg/kg) resulted in a significantly higher B cell population in the lymph nodes, while T cell and NK cell numbers were not different. CTP did not significantly reverse morphine-induced weight loss or splenic B cell antibody secretion in vitro. Additionally, CTP treatment demonstrated a slight but not significant increase in both splenic B and T cell mitogen-induced proliferation in vitro. In summary, CTP may have a specific potential in the attenuation of morphine's immunosuppressive effect by enhancing splenocyte numbers and lymph node B cell populations.

Reduced Fas ligand-expressing splenic CD5+ B lymphocytes in severe collagen-induced arthritis

IntroductionThe objective was to study immune regulation in a mouse model of rheumatoid arthritis that exhibits considerable heterogeneity of disease activity.MethodsT-cell receptor transgenic mice, in which nearly all CD4+ T cells recognize a single peptide of type II collagen, were immunized with collagen and observed for development of arthritis for 4 weeks. At 28 days post-immunization, splenocytes were analyzed by flow cytometry and in vitro assays for markers of immune activation and regulation.ResultsDisease severities ranging from 0 to 12 (on a 12-point scale) were observed. Among splenic lymphocyte populations, only the CD5+ B-cell subset displayed a decrease in relative numbers as arthritis severity increased. Splenic CD5+ B cells expressed higher levels of Fas ligand (FasL) than did CD4+ T cells or CD5- B cells in all mice, and antigen-dependent T-cell death correlated with higher levels of CD5+ B cells in cocultures. Ratios of interleukin (IL)-17 to interferon-gamma production were higher in antigen-driven cultures of splenocytes from severely arthritic mice compared to mildly or nonarthritic mice. A correlation was established between the reduced production of IL-17 in antigen-driven T-cell/B-cell cocultures and FasL, but not IL-10. Confirmation of the direct killing effect of B cells on T cells was demonstrated using an antigen-specific T hybridoma cell line.ConclusionsReduced numbers of splenic FasL+ CD5+ B cells correlated with increasing arthritis severity and decreased T-cell death in a T-cell receptor transgenic mouse model of collagen-induced arthritis. These 'killer' B cells may provide a novel mechanism for inducing T-cell death as a treatment for arthritis.

A potential role for Citalopram as an immunoenhancer on morphine-induced immunosuppression (101.9)

Abstract Chronic pain patients are often challenged with depression stemming from the long-term physiopsychological effects of their condition. Consequently, chronic pain patients are often treated with morphine, which can induce immunosuppression, along with an antidepressant. The antidepressant, Citalopram, is a serotonin reuptake inhibitor that may have immunomodulatory effects. We investigated whether Citalopram has immunoenhancing effects on morphine-induced immune suppression. Adult mice were pretreated for 7 days with either saline or Citalopram (10 or 30 mg/kg, twice daily intraperitoneal injections), followed by immunosuppression by subcutaneous implantation of a 25 mg morphine pellet for 48 h. Spleen, thymus, and lymph nodes were harvested to analyze total cell numbers. Total splenocyte numbers in all Citalopram-treated animals were significantly higher than saline-treated animals, while no differences were found in the thymus or lymph nodes. More specifically, a high dose of Citalopram (30 mg/kg) resulted in significantly higher B cell numbers in the lymph nodes, where T cell and NK cell numbers were not different. Interestingly, splenic lymphocyte populations did not differ between treatments. In summary, Citalopram may have a specific potential in the attenuation of morphine’s immunosuppressive effect by enhancing splenocyte numbers and lymph node B cell populations.

Physiological responses of a natural antioxidant flavonoid mixture, silymarin, in BALB/c mice: III. Silymarin inhibits T-lymphocyte function at low doses but stimulates inflammatory processes at high doses.

Silymarin is a mixture of bioactive flavonoids isolated from Milk Thistle (Silybum marianum). Crude extracts from this plant have been used for centuries as a natural remedy and silymarin is now effectively used in the treatment of inflammatory liver toxicity and disease in humans. In vitro studies show that silymarin can inhibit the production and damage caused by tumor necrosis factor alpha (TNFalpha) and is a potent antioxidant both in vitro and in vivo. Such findings suggest silymarin may impact the immune system but little information exists following in vivo exposure. Therefore, we tested the hypothesis that exposure to silymarin will modulate the inflammatory immune response. Male BABL/c mice (6/group) were treated intraperitoneally once daily for five days with 0, 10, 50 or 250 mg/kg of silymarin. Silymarin exposure did not produce any signs of overt toxicity or any changes in relative organ weights. Flow cytometric examination of splenic lymphocyte populations showed that the absolute number of CD3+ T-lymphocytes was reduced in the 10 and 50 mg/kg groups although significance was evident only in the 10 mg/kg group. Concomitant decreases in CD4+ and CD8+ T-cell populations were observed but only the CD4+ population in mice treated with 10 mg/kg of silymarin was significantly different from control. Functional examination of secondary lymphoid cells revealed that phytohemagglutinin-induced T-lymphocyte proliferation was increased in the lowest dose group only. B-lymphocyte blastogenesis induced by lipopolysaccharide was increased following exposure to 10 and 50 mg/kg of silymarin. Similarly, expression of TNFalpha, inducible nitric oxide synthase, IL-1beta and IL-6 mRNA were increased dose-dependently. The expression of IL-2 and IL-4 were reduced in mice treated with 10 and 50 mg/kg of silymarin although only the 10 mg/kg group was significantly different from control. The results indicate that in vivo parenteral exposure to silymarin results in suppression of T-lymphocyte function at low doses and stimulation of inflammatory processes at higher doses. Further studies investigating the effects of silymarin on the immune system are warranted.

Transgenic anti-CD4 monoclonal antibody secretion by mouse segmental pancreas allografts promotes long term survival

To compare the effectiveness of transgenic and systemic monoclonal antibody therapy for pancreas transplantation, vascularised segmental pancreas allografts from wild-type or transgenic pancreatic tissue that secreted monoclonal anti-CD4 were placed in CBA recipients in which diabetes had been induced chemically by streptozotocin (STZ, non-autoimmune diabetes). In untreated CBA recipients, wild-type BALB/c or C57BL/6 bm1 pancreas transplants were rejected in a mean survival time (MST) of 27 and 30 days, respectively. BALB/c and C57BL/6 graft survival improved when recipients were given a short course of T cell depleting monoclonal anti-CD4 antibody, (GK 1.5, 2 mg total on days –1, 0, 1, 2 with grafting on day 0) with MST±S.D. of 71±29 and 44±36 days, respectively. Thus, transient depletion of CD4 was effective in delaying pancreas allograft rejection in these strain combinations. The use of C57BL/6 bm1 mice transgenic for a rat anti-CD4 antibody (GK5 mice) as pancreas donors provided allografts that secreted sufficient anti-CD4 antibody to cause CD4 T cell depletion in the recipients (CD4 cells decreased from 30 to <5% of small lymphocytes). This degree of depletion was not sustained and the CD4 recovery inversely correlated with graft survival. Mice with >20% CD4 cells in the splenic lymphocyte population 4 weeks post-transplant rejected their grafts (3 of 10 mice). However, in 7 of 10 mice CD4 cells remained low (<15%) and allografts survived for >80 days. The GK5 allografts survived significantly longer than those from non-transgenic bm1 controls (MST 83±32 days, compared with 30 days, P<0.0005). This survival time was similar to that of BALB/c allografts in CBA recipients treated with a high dose of anti-CD4 antibody. Thus, transgenic secretion of anti-CD4 antibody by the pancreas allograft was very effective in prolonging its survival.

Enhancement of MHC class I-stimulated alloresponses by TNF/TNF receptor (TNFR)1 interactions and of MHC class II-stimulated alloresponses by TNF/TNFR2 interactions.

In vivo TNF inhibition has been observed to ameliorate the disease process attributed to T cell-dependent immune responses such as those generated during graft-vs.-host disease. The present studies were designed to evaluate whether TNF/TNF receptor (TNFR)1 and TNF/TNFR2 interactions were involved in the generation of allospecific T cell responses. Splenic lymphocyte populations were obtained from TNFR1- or TNFR2-deficient B6 mice and from control B6 mice. These responder cells were cultured with irradiated MHC class II-disparate B6.C-H-2bm12 (bm12) or MHC class I-disparate B6.C-H-2bm1 (bm1) or irradiated syngeneic stimulator cells for 3 days before assay of [3H]thymidine incorporation. IL-2 levels of the mixed lymphocyte culture (MLC) supernatants were assessed by enzyme-linked immunosorbent assay. With MHC class II-disparate bm12 stimulator cells, a significant reduction in T cell proliferation was observed utilizing TNFR2-deficient CD4+ responder T cells, but not when using TNFR1 -deficient CD4+ responder T cells. A significant decrease in proliferation of TNFR1-deficient CD8+ responder cells, but not of TNFR2-deficient CD8 responder T cells was observed after stimulation with MHC class I-disparate bm1 stimulator cells. IL-2 levels were lower in MLC utilizing MHC class I stimulators and TNFR1-deficient responders or MHC class II stimulators and TNFR2-deficient responders. These results indicate that TNF/TNFR2 interactions promote MHC class II-stimulated alloresponses, while TNF/TNFR1 interactions promote MHC class I-stimulated alloresponses.

CHARACTERIZATION OF AN APPROACH TO DEVELOPMENTAL IMMUNOTOXICOLOGY ASSESSMENT IN THE RAT USING SRBC AS THE ANTIGEN

Although developmental immunotoxicology is an area of emerging importance, few data exist that compare profiles of activity in neonatal and adult rodents. One of the factors contributing to the paucity of comparative results is that it is unclear whether immunotoxicological test procedures optimized to evaluate the immune system of adults can be integrated into studies of younger animals. Therefore,the following objectives were addressed in this study for both male and female Crl:CD(SD)BR rat pups and weanlings: (1) to quantify baseline values for absolute and relative splenic lymphocyte populations using flow cytometry in nonimmunized animals; (2) to optimize and establish baseline values for the primary humoral immune response to sheep red blood cells (SRBC) using either an enzyme-linked immunosorbant assay (ELISA) or the plaque-forming cell (PFC) assay; and (3) to determine the impact of SRBC immunization on the histology of the spleen. Responses for each age group were compared both withinandbetween l...

PHASE TWO OF AN INTERLABORATORY EVALUATION OF THE QUANTIFICATION OF RAT SPLENIC LYMPHOCYTE SUBTYPES USING IMMUNOFLUORESCENT STAINING AND FLOW CYTOMETRY

In phase one of an interlaboratory study, baseline values for rat splenic lymphocyte populations were established. In phase two, rat splenic lymphocyte populations were evaluated using immunofluorescent staining and flow cytometry following exposure to the immunosuppressive agent cyclophosphamide (CY). The study involved four independent facilities employing a common protocol. All laboratories purchased animals and reagents from the same sources. The objective of phase two was to determine whether each laboratory could detect a significant change in the same splenic lymphocyte population(s) at the same or similar CY dose levels. Crl:CD R BR male rats were dosed by the intraperitoneal route with 1, 3, or 10 mg / kg CY for 4 days. On day 5, spleen cell number and weights were obtained and splenic lymphocytes were evaluated following the lysis of red blood cells with ammonium chloride. Splenic lymphocyte populations were enumerated with monoclonal antibodies using the dual labeling of T-cell subpopulations a...