Mitogen-induced Lymphocyte Proliferation Research Articles

Early-life maternal deprivation affects the mother-offspring relationship in domestic pigs, as well as the neuroendocrine development and coping behavior of piglets.

Early-life adversity may have programming effects on the psychological and physiological development of offspring. Domestic pigs (Sus scrofa) are an excellent model species for studying these effects because of their many physiological similarities to humans. Piglets from 10 sows were subjected to daily 2-h maternal deprivation on postnatal days (PND) 2–15 alone (DA) or in a group of littermates (DG). Control piglets (C) from 10 sows stayed with their mothers. Mother-offspring interaction, milk oxytocin, and cortisol were analyzed. An open-field/novel-object (OF/NO) test was performed with piglets on PNDs 16 and 40. Plasma cortisol and immune parameters were determined on PND 5 and 16. Two piglets from each group and sow were sacrificed on PND 20 and stress-related gene expression in the limbic system and prefrontal cortex (PFC), as well as splenic lymphocyte proliferative abilities, were examined. The milk cortisol of sows increased during the first separation of mother and offspring on the second day of lactation, whereas milk oxytocin did not change. The increase in cortisol by the OF/NO test on PND 16 was greater in C piglets than in DA and DG ones. DA piglets showed less agitated behavior than DG and C piglets in the OF/NO test at PND 16, but appeared more fearful. On PND 40, DA piglets showed more arousal than DG and C piglets in the OF/NO test. Neither plasma IgA nor N/L ratios in blood nor mitogen-induced proliferation of spleen lymphocytes were affected by deprivation. We found a higher mRNA expression of CRHR1 in the hypothalamus and a higher expression of MR in the hippocampus in DA piglets than in DG ones. The expression of GR, MR, and CRHR1 genes in the PFC was reduced by maternal deprivation, however, the expression of arginine vasopressin and oxytocin receptors was not affected. Repeated maternal deprivation induces sustained effects on stress reactivity and behavior of domestic piglets. Some of these effects were buffered by the presence of littermates. In addition, we found sex-specific differences in behavior and gene expression.

Baicalin protected mice against radiation-induced lethality: A mechanistic study employing in silico and wet lab techniques

Baicalin is a main active ingredient of the dried root of Scutellaria and has been extensively employed in Traditional Chinese Medicine for the treatment of asthma, fever, and psoriasis. Based on the reports of antioxidant, anti-inflammatory, anti-infection, and anti-tumor activities of baicalin, we have explored its radioprotective efficacy using in vitro and in vivo experimental model systems. In the present study, we have investigated the radioprotective, immunomodulatory, and anti-inflammatory properties of baicalin using wet lab and in silico approaches. It was observed that pre-treatment of murine splenic lymphocytes with baicalin protected cells against radiation-induced cell death possibly by decreasing the cellular reactive oxygen species levels. Prophylactic oral administration of baicalin offered significant increase in endogenous spleen colony counts and an enhancement in the survival of mice. We have also observed that baicalin suppressed mitogen-induced splenic lymphocyte proliferation and IL-2 production. It also inhibited the production of nitric oxide in RAW 264.7 cells in response to elicitation of lipopolysaccharide. Further, in silico study was performed to evaluate the possible mechanism of radioprotection and immunomodulation by selecting different pro-inflammatory mediators such as COX2, Lck, NIK, and IKK-β which have a significant role in radioprotection, lymphocyte activation, and inflammation. Our molecular docking and molecular dynamics study show that baicalin has a significant predicted binding affinity with COX2, Lck, NIK, and IKK-β. These in silico results can explain the experimentally observed radioprotective, immunosuppressive, and anti-inflammatory properties of baicalin. Thus, radioprotection offered by baicalin may be because of its antioxidant, anti-inflammatory, and immunomodulatory properties.

Photoperiodic regulation of the splenocyte immune responses in the fresh water snake, Natrixpiscator

Photoperiod and melatonin are important regulators of immunity. We hypothesized that these two factors play an important role in the regulation of immune responses in the Natrix piscator. Animals were kept in either short or long days and splenocyte immune responses were studied. Respiratory burst activity of splenocytes was assessed through reduction of nitrobluetetrazolium salt while production of nitric oxide was assessed indirectly by nitrite assay. Density gradient centrifugation was used to isolate splenic lymphocytes which were utilized to study proliferation with and without mitogens. Super oxide production by splenocytes was reduced significantly in the cultures obtained from animals kept either in short or long days. Nitrite release was decreased when animals were subjected to long days. The photoperiodic alterations acted differentially on proliferations of the splenic lymphocytes. Spontaneous and mitogen-induced proliferation of splenic lymphocytes were enhanced in cultures obtained from snakes maintained in short days when compared with cultures from snakes obtained either from long day or natural day length conditions. In vitro melatonin significantly enhanced the splenic lymphocyte proliferation of the cultures obtained from animals kept in long days when compared with splenic lymphocyte proliferations of the cultures obtained from long day animals or the animals kept in natural day length conditions. We found evidence which suggest that photoperiod may influence seasonal energy budgets and induce adjustments which optimize energy allocation for costly physiological processes such as immune function. In seasonally breeding animals such as Natrix piscator, the pineal hormone melatonin assists in the suppression of reproduction and elevation of immunity, which are the crucial adaptation for perpetuation of species.

Analysis of suppressive effects of pesticide triazophos on leucocyte immune responses in a teleost, Channa Punctatus

Triazophos is a commonly used organophosphate insecticide, which inhibits the acetylcholinesterase enzyme and causes paralysis and death of insects. Impact of the pesticides on immunity has scarcely been investigated, especially in fishes. The present study was designed to analyze the immunotoxic role of in vitro triazophos exposure to the leucocytes in freshwater teleost, Channa punctatus. Triazophos, at in vitro concentrations of 0.1, 0.5, and 1 µg ml−1, was used to study leucocyte phagocytosis, superoxide production, nitrite release, and lymphocyte proliferation. Dose-dependent suppression of various immune responses was observed. Nitrite release and superoxide production by leucocytes were reduced in cultures incubated with triazophos. Mitogen-induced lymphocyte proliferation was significantly reduced at 0.5 and 1 µg ml−1 but not at 0.1 µg ml−1 concentration of pesticide. The biphasic suppressive effect was also discovered while evaluating phagocytic response. These investigations describe the effects of pesticide on immune responses in C. punctatus, which are helpful in understanding the immunotoxicity in fish. Substantially more researches are required to help design the measures to combat ecotoxicity in freshwater bodies.

Interkingdom Cross-Talk in Times of Stress: Salmonella Typhimurium Grown in the Presence of Catecholamines Inhibits Porcine Immune Functionality in vitro.

In stressful situations, catecholamines modulate mammalian immune function, and in addition, they can be sensed by many bacteria. Catecholamine sensing was also found in the zoonotic gut pathogen Salmonella Typhimurium, probably contributing to the stress-induced increased risk of salmonellosis. Virulence traits such as proliferation and invasiveness are promoted upon bacterial catecholamine sensing, but it is unknown whether S. Typhimurium may also inhibit mammalian immune function in stressful situations. We thus investigated whether supernatants from S. Typhimurium grown in the presence of catecholamines modulate porcine mitogen-induced lymphocyte proliferation. Lymphocyte proliferation was reduced by supernatants from catecholamine-exposed Salmonella in a dose-dependent manner. We further examined whether adrenaline oxidation to adrenochrome, which is promoted by bacteria, could be responsible for the observed effect, but this molecule either enhanced lymphocyte functionality or had no effect. We could thereby exclude adrenochrome as a potential immunomodulating agent produced by S. Typhimurium. This study is the first to demonstrate that bacteria grown in the presence of catecholamine stress hormones alter their growth environment, probably by producing immunomodulating substances, in a way that host immune response is suppressed. These findings add a new dimension to interkingdom signaling and provide novel clues to explain the increased susceptibility of a stressed host to Salmonella infection.

Photoperiodic manipulation modulates the innate and cell mediated immune functions in the fresh water snake, Natrix piscator

Objectives of the current work were to investigate the role of photoperiod and melatonin in the alteration of immune responses in a reptilian species. Animals were kept on a regimen of short or long days. Blood was obtained and leucocytes were isolated to study various innate immune responses. Lymphocytes were separated from blood by density gradient centrifugation and were used to study proliferation. Respiratory burst activity was measured through nitrobluetetrazolium reduction assay while nitric oxide production by leucocytes was assayed by nitrite assay. Lymphocytes were isolated and used to study proliferation with and without B and T cell mitogens. Photoperiodic manipulation acted differentially on leucocyte counts. Nitrite release was increased while superoxide production was decreased in cultures obtained from the snakes kept on the short day regimen. Significant enhancement of mitogen induced lymphocyte proliferation was observed in cultures from the animals kept in either long or short days compared to cultures from the animals kept in natural ambient day length. Use of in vitro melatonin showed that lymphocytes from the animals, kept in long days, were more reactive. Photoperiod induces changes in immune status which may permit adaptive functional responses in order to maintain seasonal energetic budgets of the animals. Physiological responses (like elevated immune status) are energetically expensive, therefore, animals have evolved a strategy to reduce immune functions at times when energy is invested in reproductive activities. Natrix piscator breeds from September to December and elevated pineal hormone in winter suppresses reproduction while immunity is stimulated.

Methyl mercury (MeHg) in vitro exposure alters mitogen-induced lymphocyte proliferation and cytokine expression in Steller sea lion (Eumetopias jubatus) pups

Steller sea lions (Eumetopias jubatus, SSLs) are managed as two distinct population segments within U.S. waters: the endangered western distinct population segment and the recently delisted eastern distinct population segment. Recent studies reported concentrations of mercury in several tissues collected from young SSLs in the Aleutian Islands that were at or above concentrations found to negatively impact health in other fish-eating mammals. However, there are limited studies which have investigated the range of mercury concentrations that may negatively influence the SSL immune system. This study assessed relationships between methyl mercury (MeHg+) concentrations and two immune functions, lymphocyte proliferation and cytokine expression. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved from pups on three rookeries within the western distinct population segment: Chiswell Island, Ulak, and Agattu Islands. Lymphocyte proliferation and cytokine expression were assessed in vitro using thawed PBMCs with exposure to MeHg+ (unexposed control, 0.001, 0.01, and 0.1 μg/ml). Lymphocyte proliferation was measured without and with stimulation with a T cell mitogen (ConA) and B cell mitogen (LPS) and the concentration of cytokines was measured in the cell culture supernatant (with and without ConA or LPS). Spontaneous lymphocyte proliferation was significantly increased at 0.01 and 0.1 μg/ml. T lymphocyte proliferation was significantly increased at 0.001 μg/ml and 0.1 μg/ml, while B lymphocyte proliferation was decreased at 0.1 μg/ml. Cytokine concentrations for INFγ, IL-10, IL-6, and TNFα were reduced at 0.1 μg/ml upon either T or B cell mitogen stimulation, with the exception for IL-10, where 0.1 μg/ml reduced IL-10 concentration compared to unstimulated cells. These data suggest immune functions were affected by MeHg+ exposure requiring in vivo follow up investigations. The observed modulation of immune functions is of concern as any toxicant-induced modulation may adversely affect the health of individuals, particularly younger animals undergoing periods of critical development.

Ouabain Decreases Regulatory T Cell Number in Mice by Reducing IL-2 Secretion

Background: Ouabain (OUA) is a cardiotonic glycoside originally extracted from African plants. It has also been described as an endogenous component in mammals, being released in stress situations mainly by the adrenal gland. OUA has been reported to be capable of inhibiting mitogen-induced lymphocyte proliferation and also affects B and T lymphocytes. Objectives: The aim of this work is to show the effects of OUA in peripheral T lymphocytes. Methods: In the in vivo experiments, mice were injected intraperitoneally for 3 consecutive days with RPMI medium (control group) or 0.56 mg/kg of OUA diluted in RPMI medium (OUA group). On the fourth day, spleen or mesenteric lymph nodes were removed. Results: OUA significantly reduced the number of CD4+ T lymphocytes in the spleen, especially regulatory T cells (Tregs). In vitro OUA did not inhibit the proliferation of CD4⁺T lymphocytes stimulated with anti-CD3 neither was able to induce the apoptosis of CD4⁺ nor Tregs. There was no increase in the number or percentage of T lymphocytes in the mesenteric lymph nodes, suggesting that there was no preferential accumulation of these cells in this organ. Secretion of IL-2 by activated T lymphocytes was decreased by the OUA, explaining at least in part the reduction of Tregs, since this cytokine is involved in the peripheral conversion and maintenance of Tregs. Conclusion: The impact of this reduction in autoimmune diseases, allergy and cancer as well as the potential use of OUA as a therapeutic approach in tumor treatment still needs more investigation.

Glucocorticoids and Catecholamines Affect in Vitro Functionality of Porcine Blood Immune Cells.

Simple SummaryIn modern livestock husbandry, animals may face stressful events like weaning, regrouping, or transportation, all of which can impair animal welfare and health. Research in model organisms has revealed that stress hormones, such as glucocorticoids and catecholamines, strongly modulate the immune system and thus the animals’ ability to fight infections. In the pig, knowledge about this relationship is rare, and results from rodents cannot readily be transferred due to some physiological differences. Therefore, the effects of glucocorticoids and catecholamines on porcine immune cell proliferation and the production of the pro-inflammatory cytokine TNFα were investigated in an in vitro study. Blood was obtained from catheterized pigs to exclude pre-exposure to stress hormones. Glucocorticoids exerted inhibitory effects on both investigated immune functions. Catecholamines, on the other hand, showed diverse effects on lymphocyte proliferation and TNFα production of particular immune cell types. This suggests that studies from model species are not entirely transferrable to pigs. Future research should extend the preliminary findings on cytokine production and focus on the molecular mechanisms and health impacts of stress hormones in pigs.Stress hormones exert important modulating influences on the functionality of immune cells. Despite its major role as a livestock animal and its increasing use as an animal model, knowledge about this relationship in the domestic pig is rare. This study therefore aimed to characterize the effect of glucocorticoids and catecholamines on the proliferation and cytokine production of porcine peripheral blood mononuclear cells (PBMC). Blood was obtained from donor pigs equipped with indwelling catheters to exclude stress hormone exposition before in vitro testing. PBMC were stimulated in the presence of cortisol, adrenaline or noradrenaline at concentrations resembling low to high stress conditions. Proliferation was determined via 3H-thymidine incorporation, and TNFα producers were quantified by intracellular cytokine staining. Cortisol led to a decrease in mitogen-induced lymphocyte proliferation and the number of TNFα producing cells. In contrast, catecholamines increased proliferation while exerting repressive or no effects on the number of cytokine producers. Remarkably, in concentrations presumably found in lymphatic tissue in stress situations, noradrenaline suppressed lymphocyte proliferation completely. The shown repressive effects might especially have implications on health and welfare in pigs. The obtained results provide a preliminary database for extended studies on the molecular mechanisms of glucocorticoid and catecholamine actions on porcine immune cells.

Sex steroids exert a suppressive effect on innate and cell mediated immune responses in fresh water teleost, Channa punctatus

The main objective of this study was to evaluate the potential role of two important sex steroids, crucial for reproductive success, on innate and cell mediated immune responses in a seasonally breeding, economically important fish, Channa punctatus. Intraperitoneal injections of testosterone and progesterone were given to different groups of fishes. Spleen and head kidney macrophages were isolated and studied for phagocytosis. Superoxide production and nitrite release by phagocytes were also investigated. Cell mediated immunity was measured by lymphocyte proliferation in presence of T and B cell mitogens. In vitro effect of steroids on mitogen induced lymphocyte proliferation was also analyzed. Results of the present investigation revealed the suppressive effects of testosterone and progesterone on immune responses of cells from spleen and head kidney. Concentration dependent effect of sex steroids were observed in vitro studies where phagocytosis and lymphocyte proliferation were suppressed. Immunosupression by these hormones may be the cost of reproduction and it is postulated that by suppressing immune responses, these steroids may, therefore, act as a physiological check regulating the relative amount of energy invested into either reproductive effort or immunocompetence.

Direct effects of poly(ε-caprolactone) lipid-core nanocapsules on human immune cells.

Aim: Poly(ε-caprolactone) lipid-core nanocapsules (LNCs) are efficient drug carriers and drug-free LNCs display therapeutic effects, inhibiting tumor growth and neutrophil activities. Herein, we investigated the direct actions of LNCs on human immune cells, to guide their therapeutic application. Materials & methods: LNC's uptake, cytokine release, cell migration, proliferation and intracellular pathways under inflammatory stimulation were investigated. Results & conclusion: LNCs quickly penetrated leukocytes without cytotoxicity; inhibited mitogen-induced lymphocyte proliferation, cytokine release and leukocyte migration under inflammatory stimulation, which were associated with inhibition of the MAP kinase pathway and intracellular calcium influx. Hence, we showed LNCs as a down-regulatory agent on immune cells, suggesting that either the particles themselves or their application as a drug carrier can halt non-desired inflammatory processes.

Clinical and Immunological Characterization of ICF Syndrome in Japan

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare autosomal recessive primary immunodeficiency. Hypogammaglobulinemia is a major manifestation of ICF syndrome, but immunoglobulin replacement therapy does not seem to be effective for some ICF patients. Therefore, we aimed to reassess the immunological characteristics of this syndrome. Eleven Japanese patients with ICF syndrome were enrolled. We performed whole-exome sequencing in four cases and homozygosity mapping using SNP analysis in two. We evaluated their clinical manifestations and immunological status. We newly diagnosed six ICF patients who had tentatively been diagnosed with common variable immunodeficiency. We identified two novel mutations in the DNMT3B gene and one novel mutation in the ZBTB24 gene. All patients showed low serum IgG and/or IgG2 levels and were treated by periodic immunoglobulin replacement therapy. Three of the six patients showed worse results of the mitogen-induced lymphocyte proliferation test. Analyses of lymphocyte subpopulations revealed that CD19+CD27+ memory B cells were low in seven of nine patients, CD3+ T cells were low in three patients, CD4/8 ratio was inverted in five patients, CD31+ recent thymic emigrant cells were low in two patients, and CD19+ B cells were low in four patients compared with those in the normal controls. ICF2 patients showed lower proportions of CD19+ B cells and CD16+56+ NK cells and significantly higher proportions of CD3+ T cells than ICF1 patients. T cell receptor excision circles were undetectable in two patients. Despite being treated by immunoglobulin replacement therapy, three patients died of influenza virus, fatal viral infection with persistent Epstein-Barr virus infection, or JC virus infection. One of three dead patients showed normal intelligence with mild facial anomaly. Two patients presented with autoimmune or inflammatory manifestations. Infectious episodes decreased in three patients who were started on trimethoprim-sulfamethoxazole and/or antifungal drugs in addition to immunoglobulin replacement therapy. These patients might have suffered from T cell immunodeficiency. These results indicate that patients with ICF syndrome have a phenotype of combined immunodeficiency. Thus, to achieve a better prognosis, these patients should be treated as having combined immunodeficiency in addition to receiving immunoglobulin replacement therapy.

Endometrial mesenchymal stem/stromal cell modulation of T cell proliferation.

Perivascular mesenchymal stem/stromal cells can be isolated from the human endometrium using the surface marker SUSD2 and are being investigated for use in tissue repair. Mesenchymal stem/stromal cells from other tissues modulate T cell responses via mechanisms including interleukin-10, prostaglandin E2, TGF-β1 and regulatory T cells. Animal studies demonstrate that endometrial mesenchymal stem/stromal cells can also modify immune responses to implanted mesh, but the mechanism/s they employ have not been explored. We examined the immunomodulatory properties of human endometrial mesenchymal stem/stromal cells on lymphocyte proliferation using mouse splenocyte cultures. Endometrial mesenchymal stem/stromal cells inhibited mitogen-induced lymphocyte proliferation in vitro in a dose-dependent manner. Inhibition of lymphocyte proliferation was not affected by blocking the mouse interleukin-10 receptor or inhibiting prostaglandin production. Endometrial mesenchymal stem/stromal cells continued to restrain lymphocyte proliferation in the presence of an inhibitor of TGF-β receptors, despite a reduction in regulatory T cells. Thus, the in vitro inhibition of mitogen-induced lymphocyte proliferation by endometrial mesenchymal stem/stromal cells occurs by a mechanism distinct from the interleukin-10, prostaglandin E2, TGF-β1 and regulatory T cell-mediated mechanisms employed by MSC from other tissues. eMSCs were shown to produce interleukin-17A and Dickkopf-1 which may contribute to their immunomodulatory properties. In contrast to MSC from other sources, systemic administration of endometrial mesenchymal stem/stromal cells did not inhibit swelling in a T cell-mediated model of skin inflammation. We conclude that, while endometrial mesenchymal stem/stromal cells can modify immune responses, their immunomodulatory repertoire may not be sufficient to restrain some T cell-mediated inflammatory events.

Effects of repeated social mixing on behavior and blood immune cells of group-housed pregnant sows (Sus scrofa domestica)

Commercial housing of sows often involves frequent regrouping (“mixing”) of animals that poses a potential welfare problem due to social stress. Although mixing is a common management practice worldwide, immunological consequences are poorly investigated in pregnant sows. This study combines both behavioral and endocrine data with detailed analyses of blood cellular immunity to examine whether repeated social mixing affects the immune system in pregnant sows, which none of the studies conducted so far could confirm. Pregnant multiparous sows (German Landrace, n = 40) were housed in groups of five animals. Sows were either assigned to a repeated social mixing treatment with a mutual exchange of two randomly selected sows of two specific groups (2 × 2) twice a week over a period of eight weeks, or remained undisturbed in their original group. Blood samples of all sows were collected at five time points before, during, and after the mixing period to evaluate the distribution of blood lymphocyte subpopulations, mitogen-induced lymphocyte proliferation and plasma cortisol concentrations. Mixing caused higher levels of aggressive behavior (p ≤ 0.05) during the entire mixing period. Compared to pre mixing baseline values, lymphocyte numbers were lower in mixed sows (p ≤ 0.05), which was primarily caused by lower antigen-experienced T helper cells, cytotoxic T cells and natural killer cells. Mixed sows showed a higher proliferative response of lymphocytes while granulocytes and cortisol concentrations were not affected (p > 0.05). Repeated social mixing thus not only resulted in an increase of aggressive behavior, but also in altered immune cell numbers of the adaptive immune system, even though sows became eventually familiar with each other. These findings suggest, in contrast to previous studies, an adverse influence of frequent mixing on the immune system of pregnant sows.

Comparative Analysis of Biological and Functional Properties of Bone Marrow Mesenchymal Stromal Cells Expanded in Media with Different Platelet Lysate Content

Due to their ability to induce immunological tolerance in the recipient, mesenchymal stromal cells (MSCs) have been utilized in the treatment of various hematological and immune- and inflammation-mediated diseases. The clinical application of MSCs implies prior in vitro expansion that usually includes the use of fetal bovine serum (FBS). The present study evaluated the effect of different platelet lysate (PL) media content on the biological properties of MSCs. MSCs were isolated from the bone marrow of 13 healthy individuals and subsequently expanded in three different culture conditions (10% PL, 5% PL, 10% FBS) during 4 passages. The cells cultured in different conditions had comparable immunophenotype, clonogenic potential, and differentiation capacity. However, MSC growth was significantly enhanced in the presence of PL. Cultures supplemented with 10% PL had a higher number of cumulative population doublings in all passages when compared to the 5% PL condition (p < 0.03). Such a difference was also observed when 10% PL and 10% FBS conditions were compared (p < 0.005). A statistically significant difference in population doubling time was determined only between the 10% PL and 10% FBS conditions (p < 0.005). Furthermore, MSCs cultured in 10% PL were able to cause a 66.9% reduction of mitogen-induced lymphocyte proliferation. Three chromosome aberrations were detected in PL conditions. Since two changes occurred in the same do nor, it is possible they were donor dependent rather than caused by the culture condition. These findings demonstrate that a 10% PL condition enables a higher yield of MSCs within a shorter time without altering MSC properties, and should be favored over the 5% PL condition.

Novel Mutations in RASGRP1 are Associated with Immunodeficiency, Immune Dysregulation, and EBV-Induced Lymphoma

RAS guanyl-releasing protein 1 (RASGRP1) deficiency has recently been shown to cause a primary immunodeficiency (PID) characterized byCD4+ T cell lymphopenia and Epstein-Barr virus (EBV)-associated B cell lymphoma. Our report of three novel patients widens the scope of RASGRP1 deficiency by providing new clinical and immunological insights on autoimmunity, immune cell development, and predisposition to lymphoproliferative disease. One patient of Turkish origin (P1) and two Palestinian patients (P2, P3) were evaluated for immunodeficiency. To decipher the molecular cause of disease, whole exome sequencing was conducted. Identified mutations were validated by immunological and biochemical assays. We report three patients presenting with similar clinical characteristics of immunodeficiency and EBV-associated lymphoproliferative disease. In addition, P2 and P3 exhibited overt autoimmune manifestations. Genetic screening identified two novel loss-of-function mutations in RASGRP1. Immunoblotting and active Ras pull-down assays confirmed perturbed ERK1/2 signaling and reduced Ras-GTPase activity in heterologous Jurkat cells with ectopic expression of RASGRP1 mutants. All three patients had CD4+ T cell lymphopenia. P2 and P3 showed decreased mitogen-induced lymphocyte proliferation, reduced T cell receptor excision circles, abnormal T cell receptor (TCR) Vβ repertoires, and increased frequencies of TCRγδ cells. TCR gamma repertoire diversity was significantly reduced with a remarkable clonal expansion. RASGRP1 deficiency is associated with life-threatening immune dysregulation, severe autoimmune manifestations, and susceptibility to EBV-induced B cell malignancies. Early diagnosis is critical and hematopoietic stem cell transplantation might be considered as curative treatment.

Immunoenhancement Effects of Glycosaminoglycan from Apostichopus japonicus: In Vitro and In Cyclophosphamide-Induced Immunosuppressed Mice Studies.

In this study, the immunomodulatory activities of Apostichopus japonicus glycosaminoglycan (AHG) on the nature killer (NK) cells, cytotoxic T lymphocytes (CTLs) and cyclophosphamide (CY)-treated mice were investigated. After stimulation with multiple concentrations of AHG (0–100 μg/mL), NK cells and CTLs displayed outperformance against YAC-1 and B16 cells, respectively. Furthermore, the mitogen-induced splenic lymphocyte proliferation in CY-induced immunosuppressed mice was significantly promoted by AHG. In addition, the administration of AHG dramatically increased the splenocytes Ca2+ concentration and the delayed-type hypersensitivity (DTH) reaction in a dose-dependent manner. Besides, AHG could strongly increase the total antioxidant capacity (T-AOC), the activities of superoxidase dismutase (SOD), catalase (CAT) as well as glutathione peroxidase (GSH-PX), and could decrease the malondialdehyde (MDA) level in the heart, kidney and liver. These findings indicated that AHG played an important role in the immune enhancement and protection against CY-induced immunosuppression and oxidative damage. Our findings provide experimental evidence for further research and possible immunostimulatory applications of AHG in clinical practice.

Fluoxetine Treatment in Rats Increases the Rate of Taurine Transport in Mononuclear Cells

Fluoxetine, a selective serotonin reuptake inhibitor antidepressant, modulates the mitogen-induced proliferation of lymphocytes. Lymphocytes contain taurine and express taurine transporter (TauT). Among the effects of taurine on lymphocytes are protection against oxidants and regulation of the inflammatory aspects of the immune response. Our aim was to determine the influence of fluoxetine treatment on taurine transport, and to determine the presence of TauT in the mononuclear cells of rats. Methods: Male adult Sprague-Dawley rats were treated with fluoxetine 10 mg/kg i.p. for 1, 2, and 3 weeks. The cells were obtained by density gradients. [³H]Taurine was used for transport assays. Amino acid levels were determined by high-performance liquid chromatography. Immunolabeling of CD4+, CD8+, and TauT was performed. The mRNA of TauT was evaluated by RT-PCR. Controls were included for each protocol. Results: The transport of taurine, after 1 week of treatment, was significantly augmented compared to controls. The affinity significantly increased at 1 and 2 weeks. While the percentage of CD4+ cells decreased and that of CD8+ cells increased, the percentage of TauT in CD4+ and CD8+ cells was not affected. Reduction of levels of aspartic acid, glutamic acid, threonine, alanine, glycine, and arginine occurred at 1 and 2 weeks. The taurine concentration significantly decreased after 2 and 3 weeks of treatment. The estimation of mRNA of TauT was not different. Conclusion: Taurine transport increases with fluoxetine treatment, and so this could be related to an immunomodulatory role of fluoxetine through TauT. Inhibition of serotonin reuptake might be involved in the regulation of taurine transport in mononuclear cells.

Spores of two probiotic Bacillus species enhance cellular immunity in BALB/C mice

Previous studies found that Bacillus subtilis BS02 and B. subtilis subsp. natto BS04 isolated in our laboratory could activate the immune response of murine macrophages in vitro. This study aims to investigate the effects of dietary supplementation with Bacillus species spores on the systemic cellular immune response in BALB/C mice. Results showed that both B. subtilis BS02 and B. subtilis natto BS04 enhanced the phagocytic function of the mononuclear phagocyte system (MPS) and the cytotoxicity of natural killer (NK) cells. In addition, B. subtilis BS02 could increase the respiratory burst activity of blood phagocytes. Furthermore, B. subtilis BS02 and B. subtilis natto BS04 increased the percentage of gamma-interferon-producing CD4+ cells and CD8+ T-cells, but only BS04 increased the percentage of CD3+ cells and CD3+ CD4+ cells in splenocytes. However, there were no effects on other subsets of splenic lymphocytes and mitogen-induced splenic lymphocyte proliferation. All data suggested that oral administration of B. subtilis BS02 or B. subtilis natto BS04 could significantly enhance cellular immunity in BALB/C mice by increasing phagocytic activity of MPS and cytotoxic activity of NK cells in a strain-specific manner.

Glycerol Monolaurate (GML) Inhibits Human T Cell Signaling, Metabolism, and Function By Disrupting Lipid Dynamics

Glycerol Monolaurate (GML) is a naturally occurring fatty acid widely utilized in food, cosmetics, and homeopathic supplements. GML is a potent antimicrobial agent that targets a range of bacteria, fungi, and enveloped viruses. Interestingly, GML suppresses mitogen induced lymphocyte proliferation and inositol triphosphate production, suggesting that GML has immunomodulatory functions. In this study, we have mechanistically examined if GML affects the signaling, metabolism, and functional output of human primary T cells. We found that GML potently altered lipid order and disorder dynamics in the plasma membrane that resulted in reduced membrane localized clustering of the proteins LAT, PLC-γ, and AKT, events integral for proper T cell receptor (TCR) signal propagation. Altered membrane signaling events induced selective inhibition of TCR-induced phosphorylation of SLP-76, regulatory P85 subunit of PI3K, and AKT as well as abrogated calcium influx. In addition to signaling defects, GML treated cells have profoundly altered metabolism profiles characterized by suppressed oxidative phosphorylation and increased glycolysis. Functionally, GML treatment potently reduced TCR-induced production of the cytokines IL-2, IFN-γ, TNF-α, and IL-10. Our data reveal that the widely used anti-microbial agent GML alters the lipid dynamics of human T cells, leading to their defective signaling, metabolism, and function.