In vivo blockade of Ca(+2)-dependent nitric oxide synthases impairs expressions of L-selectin and PECAM-1

Interactions of leukocytes with endothelium play a role for the immune system modulated by endogenous agents, such as glucocorticoids and nitric oxide (NO). Glucocorticoids inhibit leukocyte-endothelial interactions whereas the role of NO is still controversial. In this study, the activity of Ca(+2)-dependent nitric oxide synthases was in vivo blocked in male Wistar rats by given L-NAME, 20 mg kg(-1) for 14 days dissolved in drinking water and expression of adhesion molecules involved in leukocyte-endothelial interactions was investigated. Expressions of L-selectin and PECAM-I in peripheral leukocytes and PECAM-1 in endothelial cells were reduced by L-NAME treatment. Only L-selectin expression was controlled at transcriptional levels. These effects were not dependent on endogenous glucocorticoids, as corticosterone levels were not altered in NAME-treated rats. Our results show that NO, produced at physiological levels, controls expression of constitutive adhesion molecules expressions in cell membranes by different mechanisms of action. Published by Elsevier Inc.

Soy lecithin supplementation alters macrophage phagocytosis and lymphocyte response to concanavalin A: a study in alloxan-induced diabetic rats

Dietary soy lecithin supplementation decreases hyperlipidemia and influences lipid metabolism. Although this product is used by diabetic patients, there are no data about the effect of soy lecithin supplementation on the immune system. The addition of phosphatidylcholine, the main component of lecithin, to a culture of lymphocytes has been reported to alter their function. If phosphatidylcholine changes lymphocyte functions in vitro as previously shown, then it could also affect immune cells in vivo. In the present study, the effect of dietary soy lecithin oil macrophage phagocytic capacity and on lymphocyte number in response to concanavalin A (ConA) stimulation was investigated in non-diabetic and alloxan-induced diabetic rats. Supplementation was carried Out daily with 2 g kg(-1) b.w. lecithin during 7 days. After that, blood was drawn from fasting rats and peritoneal macrophages and mesenteric lymph node lymphocytes were collected to determine the phospholipid content. Plasma triacylglycerol (TAG), total and HDL cholesterol and glucose levels were also determined. Lymphocytes were stimulated by Conk The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye reduction method and flow cytometry were employed to evaluate lymphocyte metabolism and cell number, respectively. Soy lecithin supplementation significantly increased both macrophage phagocytic capacity (+29%) in non-diabetic rats and the lymphocyte number in diabetic rats (+92%). It is unlikely that plasma lipid levels indirectly affect immune cells, since plasma cholesterol, TAG, or phospholipid content was not modified by lecithin supplementation. In Conclusion, lymphocyte and macrophage function were altered by lecithin supplementation, indicating ail immunomodulatory effect of phosphatidylcholine. Copyright (C) 2008 John Wiley & Sons, Ltd.

Induction of Lymphocyte Death by Short- and Long-Duration Triathlon Competitions

LEVADA-PIRES, A. C., M. F. CURY-BOAVENTURA, R. GORJAO, S. M. HIRABARA. E. F. PUGGINA, I. L. PELLEGRINOTTI, L. A. DOMINGUES FILHO, R. CURI, and T. C. PITHON-CURI. Induction of Lymphocyte Death by Short- and Long-Duration Triathlon Competitions. Med. Sci. Sporty Exerc., Vol. 4 1, No. 10, pp. 1896-1901, 2009. Purpose: The effect of triathlon competitions on death of lymphocytes from elite athletes was investigated. Material and Methods: Blood was collected from sedentary volunteers and triathletes at rest and after a short-duration triathlon (SDT) and after a long-duration triathlon (LDT-half Ironman) competitions. Results: The athletes had lowered lymphocyte proliferation capacity compared with sedentary volunteers either at rest or after the competitions. There was no difference in the parameters associated with lymphocyte death when sedentary volunteers were compared with triathletes at rest. Lymphocytes from triathletes after SDT competition showed an increase in DNA fragmentation, phosphatidylserine externalization, and mitochondrial transmembrane depolarization and did not alter membrane integrity when compared with cells from athletes at rest. In contrast, the LDT competition raised the proportion of lymphocytes with loss of membrane integrity when compared with cells from athletes at rest and did not change the apoptotic parameters. The LDT competition induced an increase of reactive oxygen species (ROS) production by lymphocytes compared with triathletes at rest. The SDT competition did not alter ROS production by lymphocytes when compared with cells from triathletes at rest. ROS production by lymphocytes after LDT competition was 60% higher than in SDT. Conclusions: Evidence is presented herein that an LDT competition caused lymphocyte death by necrosis, whereas an SDT induced lymphocyte apoptosis. The mechanism for lymphocyte death induced by the triathlon competitions may involve an increase in ROS production at different extents.

Acute exhaustive exercise regulates IL-2, IL-4 and MyoD in skeletal muscle but not adipose tissue in rats

Background: The purpose of this study was to evaluate the effect of exhaustive exercise on proteins associated with muscle damage and regeneration, including IL-2, IL-4 and MyoD, in extensor digitorum longus (EDL) and soleus muscles and mesenteric (MEAT) and retroperitoneal adipose tissues (RPAT). Methods: Rats were killed by decapitation immediately (E0 group, n = 6), 2 (E2 group, n = 6) or 6 (E6 group, n = 6) hours after the exhaustion protocol, which consisted of running on a treadmill at approximately 70% of VO(2max) for fifty minutes and then at an elevated rate that increased at one m/min every minute, until exhaustion. Results: The control group (C group, n = 6) was not subjected to exercise. IL-2 protein expression increased at E0 in the soleus and EDL; at E2, this cytokine returned to control levels in both tissues. In the soleus, IL-2 protein expression was lower than that in the control at E6. IL-4 protein levels increased in EDL at E6, but the opposite result was observed in the soleus. MyoD expression increased at E6 in EDL. Conclusion: Exhaustive exercise was unable to modify IL-2 and IL-4 levels in MEAT and RPAT. The results show that exhaustive exercise has different effects depending on which muscle is analysed.

Endurance training modulates lymphocyte function in rats with post-MI CHF

Purpose: Exercise training restores innate immune system cell function in post-myocardial infarction (post-MI) rats. However, studies of the involvement of lymphocyte (Ly) in the setting of the congestive heart failure (CHF) are few. To address this issue, we investigated the function of Ly obtained from cervical lymph nodes from post-MI CHF rats submitted to treadmill running training. Methods: Twenty-five male Wistar rats were randomly assigned to the following groups: rats submitted to ligation of the left coronary artery, which were sedentary (MI-S, N= 7, only limited activity) or trained (MI-T, N= 6, on a treadmill (0% grade at 13-20 m.m(-1)) for 60 min.d(-1), 5 d.wk(-1), for 8-10 wk); or sham-operated rats, which were sedentary (sham-S, N = 6) or trained (sham-T, N = 6). The incorporation of [2-C-14]-thymidine by Ly cultivated in the presence of concanavalin A (Con A) and lipopolysaccharide (LPS), cytokine production by Ly cultivated in the presence of phytohemagglutinin (PHA), and plasma concentration of glutamine were assessed in all groups, 48 h after the last exercise session. Results: Proliferative capacity was increased, following incubation with Con-A in the MI groups, when compared with the sham counterparts. When incubated in the presence of PHA, MI-S produced more IL-4 (96%) than sham-S (P < 0.001). The training protocol induced a 2.2-fold increase in the production of interleukin-2 (P < 0.001) of the cells obtained from the cervical lymph nodes of MI-T, compared with MI-S. Conclusion: The moderate endurance training protocol caused an increase in IL-2 production, and a trend toward the reversion of the Th-1/Th-2 imbalance associated with IL-4 production increased in the post-MI CHF animal model.

The role of hemocytes in the immunity of the spider Acanthoscurria gomesiana

Invertebrates protect themselves against microbial infection through cellular and humoral immune defenses. Since the available information on the immune system of spiders is scarce, the main goat of the present study was to investigate the role of hemocytes and antimicrobial peptides (AMPs) in defense against microbes of spider Acanthoscurria gomesiana. We previously described the purification and characterization of two AMPs from the hemocytes of naive spider A. gomesiana, gomesin and acanthoscurrin. Here we show that 57% of the hemocytes store both gomesin and acanthoscurrin, either in the same or in different granules. Progomesin labeling in hemocyte granules indicates that gomesin is addressed to those organelles as a propeptide. In vivo and in vitro experiments showed that lipopolysaccharide (LPS) and yeast caused the hemocytes to migrate. Once they have reached the infection site, hemocytes may secrete coagulation cascade components and AMPs to cell-free hemolymph. Furthermore, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore, the main reactions involved in the spider immune defense might be coagulation and AMP secretion. (C) 2007 Elsevier Ltd. All rights reserved.

Melatonin Protects CD4(+) T Cells from Activation-Induced Cell Death by Blocking NFAT-Mediated CD95 Ligand Upregulation

Over the past 20 y, the hormone melatonin was found to be produced in extrapineal sites, including cells of the immune system. Despite the increasing data regarding the biological effects of melatonin on the regulation of the immune system, the effect of this molecule on T cell survival remains largely unknown. Activation-induced cell death plays a critical role in the maintenance of the homeostasis of the immune system by eliminating self-reactive or chronically stimulated T cells. Because activated T cells not only synthesize melatonin but also respond to it, we investigated whether melatonin could modulate activation-induced cell death. We found that melatonin protects human and murine CD4(+) T cells from apoptosis by inhibiting CD95 ligand mRNA and protein upregulation in response to TCR/CD3 stimulation. This inhibition is a result of the interference with calmodulin/calcineurin activation of NFAT that prevents the translocation of NFAT to the nucleus. Accordingly, melatonin has no effect on T cells transfected with a constitutively active form of NFAT capable of migrating to the nucleus and transactivating target genes in the absence of calcineurin activity. Our results revealed a novel biochemical pathway that regulates the expression of CD95 ligand and potentially other downstream targets of NFAT activation. The Journal of Immunology, 2010, 184: 3487-3494.

Mycoplasmal lipid-associated membrane proteins and Mycoplasma arthritidis mitogen recognition by serum antibodies from patients with rheumatoid arthritis

Mycoplasmal lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. The objective of this work was to detect antibodies to MAM and LAMPs of Mycoplasma hominis and M. fermentans in the sera of patients affected by rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) to identify mycoplasmal products that can be involved in the etiopathogenesis of these autoimmune diseases. Serum samples from female RA and SLE patients and controls, recombinant MAM, and LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blot assays. A similar frequency of sera from patients and controls reactive to MAM was detected. A larger number of M. hominis and M. fermentans LAMPs were recognized by sera from RA patients than controls, but no differences were detected between sera from SLE patients and controls. Among the LAMPs recognized by IgG antibodies from RA patients, proteins of molecular masses in a range of < 49 and a parts per thousand yen20 KDa (M. hominis) and < 102 and a parts per thousand yen58 KDa (M. fermentans) were the most reactive. These preliminary results demonstrate the strong reactivity of antibodies of RA patients with some M. hominis and M. fermentans LAMPs. These LAMPs could be investigated as mycoplasmal antigens that can take part in the induction or amplification of human autoimmune responses.

Detection of the Tim-3 ligand, galectin-9, inside the allograft during a rejection episode

Introduction: Tim-3 is a Th1 lymphocytes membrane protein with inhibitory function. Its ligand, galectin-9, was recently identified and it is expressed in some lymphocyte subpopulation. In addition, endothelial cells and fibroblasts can also express galectin-9 according to the local cytokine milieu. Both molecules can act as important regulatory tools in the immune system. Aim: Evaluate the expression of these immunoregulatory molecules inside kidney allografts during acute rejection episodes. Methods: By using a quantitative polymerase chain reaction assay, we measured the levels of messenger RNA (mRNA) for galectin-9 and Tim-3 in 21 samples obtained at allograft nephrectomy. Five samples received the histological diagnosis of acute non-vascular rejection (ANVR), twelve of acute vascular rejection (AVR), and five of loss of non-immune cause (LNIC; as control). As cytolytic response markers we measured mRNA levels of granzyme B, interferon-gamma and perforin. The statistic analysis was performed using one way analysis of variance (ANOVA) and Pearson correlation. Results: The mean levels of Tim-3 mRNA expression were 13.99 +/- 6.99 for LNIC, 48.13 +/- 54.47 for RACNV and 238.63 +/- 333.14 for RAV (p = 0.004). For galectin-9, the mean values were 0.57 +/- 0.49 for LNIC, 0.66 +/- 0.36 for RACNV and 2.34 +/- 1.62 for RAV (p = 0.006). Furthermore, there was a positive correlation between both molecules (r = 0.526, p = 0.016). Also. granzyme B, perforin and interferon-gamma mRNA expression were different among the three groups. Conclusion: Messenger RNA level expressions of all the studied molecules were higher inside allografts with more severe rejection. Moreover, there was a positive correlation between galectin-9 and Tim-3 mRNA levels. The simultaneous expression of galectin-9 and Tim-3 may indicate an immunoregulatory function, during the ongoing cytotoxic response. (C) 2008 Elsevier B.V. All rights reserved.

Crucial cytokine interactions in nitric oxide production induced by Mycoplasma arthritidis superantigen

Mycoplasma arthritidis causes autoimmune arthritis in rodents. It produces a superantigen (MAM) that simultaneously activates antigen presenting cells and T cells inducing nitric oxide and cytokine release. Nitric oxide is a key inducer and regulator of the immune system activation. Here, we investigated nitric oxide and cytokine production and interactions of these molecules in MAM-stimulated co-cultures of macrophages (J774A.1 cell line) with spleen lymphocytes. We found that: a) MAM-induced nitric oxide, interferon-gamma, membrane-associated tumor necrosis factor and interleukin-2 production in co-cultures of macrophages with lymphocytes from BALB/c and C3H/HePas but not from C57B1/6 mice; b) production of nitric oxide was dependent on interferon-gamma whereas that of interferon-gamma was dependent on interleukin-2 and membrane-associated tumor necrosis factor; c) these cytokines up regulated MAM-induced nitric oxide production. Unraveling the mechanisms of cell activation induced by MAM might be helpful to design strategies to prevent immune system activation by superantigens and therefore in seeking amelioration of associated immunopathologies. (C) 2008 Elsevier Masson SAS. All rights reserved.

TICK INNATE IMMUNITY

Ticks are blood feeding parasites transmitting a wide variety of pathogens to their vertebrate hosts. The vector competence of ticks is tightly linked with their immune system. Despite its importance, our knowledge of tick innate immunity is still inadequate and the limited number of sufficiently characterized immune molecules and cellular reactions are dispersed across numerous tick species. The phagocytosis of microbes by tick hemocytes seems to be coupled with a primitive complement-like system, which possibly involves self/nonself recognition by fibrinogen-related lectins and the action of thioester-containing proteins. Ticks do not seem to possess a pro-phenoloxidase system leading to melanization and also coagulation of tick hemolymph has not been experimentally proven. They are capable of defending themselves against microbial infection with a variety of antimicrobial peptides comprising lysozymes, defensins and molecules not found in other invertebrates. Virtually nothing is known about the signaling cascades involved in the regulation of tick antimicrobial immune responses. Midgut immunity is apparently the decisive factor of tick vector competence. The gut content is a hostile environment for ingested microbes, which is mainly due to the antimicrobial activity of hemoglobin fragments generated by the digestion of the host blood as well as other antimicrobial peptides. Reactive oxygen species possibly also play an important role in the tick-pathogen interaction. The recent release of the Ixodes scapularis genome and the feasibility of RNA interference in ticks promise imminent and substantial progress in tick innate immunity research.

Innate immunity to Paracoccidioides brasiliensis infection

Innate immunity is based in pre-existing elements of the immune system that directly interact with all types of microbes leading to their destruction or growth inhibition. Several elements of this early defense mechanism act in concert to control initial pathogen growth and have profound effect on the adaptative immune response that further develops. Although most studies in paracoccidioidomycosis have been dedicated to understand cellular and humoral immune responses, innate immunity remains poorly defined. Hence, the main purpose of this review is to present and discuss some mechanisms of innate immunity developed by resistant and susceptible mice to Paracoccidioides brasiliensis infection, trying to understand how this initial host-pathogen interface interferes with the protective or deleterious adaptative immune response that will dictate disease outcome. An analysis of some mechanisms and mediators of innate immunity such as the activation of complement proteins, the microbicidal activity of natural killer cells and phagocytes, the production of inflammatory eicosanoids, cytokines, and chemokines among others, is presented trying to show the important role played by innate immunity in the host response to P. brasiliensis infection.

Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunised with human serum albumin

Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund`s adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated. (C) 2011 Elsevier Ltd. All rights reserved.

EFFECT OF EXERCISE ON GLUTAMINE SYNTHESIS AND TRANSPORT IN SKELETAL MUSCLE FROM RATS

P>Reductions in plasma glutamine are observed after prolonged exercise. Three hypotheses can explain such a decrease: (i) high demand by the liver and kidney; (ii) impaired release from muscles; and (iii) decreased synthesis in skeletal muscle. The present study investigated the effects of exercise on glutamine synthesis and transport in rat skeletal muscle. Rats were divided into three groups: (i) sedentary (SED; n = 12); (ii) rats killed 1 h after the last exercise bout (EX-1; n = 15); and (iii) rats killed 24 h after the last exercise bout (EX-24; n = 15). Rats in the trained groups swam 1 h/day, 5 days/week for 6 weeks with a load equivalent to 5.5% of their bodyweight. Plasma glutamine and insulin were lower and corticosterone was higher in EX-1 compared with SED rats (P < 0.05 and P < 0.01, respectively). Twenty-four hours after exercise (EX-24), plasma glutamine was restored to levels seen in SED rats, whereas insulin levels were higher (P < 0.001) and costicosterone levels were lower (P < 0.01) than in EX-1. In the soleus, ammonia levels were lower in EX-1 than in SED rats (P < 0.001). After 24 h, glutamine, glutamate and ammonia levels were lower in EX-24 than in SED and EX-1 rats (P < 0.001). Soleus glutamine synthetase (GS) activity was increased in EX-1 and was decreased in EX-24 compared with SED rats (both P < 0.001). The decrease in plasma glutamine concentration in EX-1 is not mediated by GS or glutamine transport in skeletal muscle. However, 24 h after exercise, lower GS may contribute to the decrease in glutamine concentration in muscle.

Expansion of CD4(+) CD25(+) Foxp3(+) T cells by bone marrow-derived dendritic cells

Dendritic cells (DCs) are the most important antigen-presenting cells of the immune system and have a crucial role in T-lymphocyte activation and adaptive immunity initiation. However, DCs have also been implicated in maintaining immunological tolerance. In this study, we evaluated changes in the CD4(+) CD25(+) Foxp3(+) T-cell population after co-culture of lymph node cells from BALB/c mice with syngeneic bone marrow-derived DCs. Our results showed an increase in CD4(+) CD25(+) Foxp3(+) T cells after co-culture which occurred regardless of the activation state of DCs and the presence of allogeneic apoptotic cells; however, it was greater when DCs were immature and were pulsed with the alloantigen. Interestingly, syngeneic apoptotic thymocytes were not as efficient as allogeneic apoptotic cells in expanding the CD4(+) CD25(+) Foxp3(+) T-cell population. In all experimental settings, DCs produced high amounts of transforming growth factor (TGF)-beta. The presence of allogeneic apoptotic cells induced interleukin (IL)-2 production in immature and mature DC cultures. This cytokine was also detected in the supernatants under all experimental conditions and enhanced when immature DCs were pulsed with the alloantigen. CD4(+) CD25(+) Foxp3(+) T-cell expansion during co-culture of lymph node cells with DCs strongly suggested that the presence of alloantigen enhanced the number of regulatory T cells (Tregs) in vitro. Our data also suggest a role for both TGF-beta and IL-2 in the augmentation of the CD4(+) CD25(+) Foxp3(+) population.