Important GABAergic mechanism within the NTS and the control of sympathetic baroreflex in SHR

Inhibitory neurotransmission has an important role in the processing of sensory afferent signals in the nucleus of the solitary tract (NTS), particularly in spontaneously hypertensive rats (SHR). In the present study, we tested the hypothesis that gamma-aminobutyric acid (GABA) mediated neurotransmission within the NTS produces an inhibition of the baroreflex response of splanchnic sympathetic nerve discharge (sSND). In urethane-anesthetized, artificially ventilated and vagotomized male SHR and Wistar Kyoto (WKY) rats we compared baroreflex-response curves evoked after bilateral injections into the NTS of the GABA-A antagonist bicuculline (25 pmol/50 nl) or the GABA-B antagonist CGP 35348 (5 nmol/50 nl). Baseline MAP in SHR was higher than the WKY rats (SHR: 153+/-5, vs. WKY: 112+/-6 mm Hg, p<0.05). Bilateral injection of bicuculline or CGP 35348 into the NTS induced a transient (5 min) reduction in MAP (Delta = -26+/-4 and -41+/-6 mm Hg, respectively vs. saline Delta = +4+/-3 mm Hg, p<0.05) and sSND (Delta = -21+/-13 and -78+/-7%, respectively vs. saline: Delta = +6+/-4% p<0.05). Analysis of the baroreceptor curve revealed a decrease in the lower plateau (43+/-11 and 15+/-5%, respectively vs. saline: 78+/-6%, p<0.05) and an increase in the sympathetic gain of baroreflex (6.3+/-0.3, 7.2+/-0.8% respectively vs. saline: 4.2+/-0.4%, p<0.05). Bicuculline or CGP35348 into the NTS in WKY rats did not change MAP, sSND and sympathetic baroreflex gain. These data indicate that GABAergic mechanisms within the NTS act tonically reducing sympathetic baroreflex gain in SHR. Crown Copyright (C) 2010 Published by Elsevier By. All rights reserved.

The periaqueductal gray and its potential role in maternal behavior inhibition in response to predatory threats

Animals faced with conflicting cues, such as predatory threat and a given rewarding stimulus, must make rapid decisions to engage in defensive versus other appetitive behaviors. The brain mechanisms mediating such responses are poorly understood. However, the periaqueductal gray (PAG) seems particularly suitable for accomplishing this task. The PAG is thought to have, at least, two distinct general roles on the organization of motivated responses, i.e., one on the execution of defensive and reproductive behaviors, and the other on the motivational drive underlying adaptive responses. We have presently examined how the PAG would be involved in mediating the behavioral choice between mutually incompatible behaviors, such as reproduction or defense, when dams are exposed to pups and cat odor. First, we established the behavioral protocol and observed that lactating rats, simultaneously exposed to pups and cat odor, inhibited maternal behavior and expressed clear defensive responses. We have further revealed that cat odor exposure up-regulated Fos expression in the dorsal PAG, and that NMDA cytotoxic lesions therein were able to restore maternal responses, and, at the same time, block defensive responsiveness to cat odor. Potential paths mediating the dorsal PAG influences on the inhibition of appetitive (i.e., retrieving behavior) and consummatory (i.e., nursing) maternal responses are discussed. Overall, we were able to confirm the dual role of the PAG, where, in the present case, the dorsal PAG, apart from organizing defensive responses, also appears to account for the behavioral inhibition of non-defensive responses. (C) 2010 Elsevier B.V. All rights reserved.

THE ROLE OF THE SUPERIOR COLLICULUS IN PREDATORY HUNTING

Combining the results of behavioral, neuronal immediate early gene activation, lesion and neuroanatomical experiments, we have presently investigated the role of the superior colliculus (SC) in predatory hunting. First, we have shown that insect hunting is associated with a characteristic large increase in Fos expression in the lateral part of the intermediate gray layer of the SC (Wig). Next, we have shown that animals with bilateral NMDA lesions of the lateral parts of the SC presented a significant delay in starting to chase the prey and longer periods engaged in other activities than predatory hunting. They also showed a clear deficit to orient themselves toward the moving prey and lost the stereotyped sequence of actions seen for capturing, holding and killing the prey. Our Phaseolus vulgaris-leucoagglutinin analysis revealed that the lateral SCig, besides providing the well-documented descending crossed pathway to premotor sites in brainstem and spinal cord, projects to a number of midbrain and diencephalic sites likely to influence key functions in the context of the predatory behavior, such as general levels of arousal, motivational level to hunt or forage, behavioral planning, appropriate selection of the basal ganglia motor plan to hunt, and motor output of the primary motor cortex. In contrast to the lateral SC lesions, medial SC lesions produced a small deficit in predatory hunting, and compared to what we have seen for the lateral SCig, the medial SCig has a very limited set of projections to thalamic sites related to the control of motor planning or motor output, and provides conspicuous inputs to brainstem sites involved in organizing a wide range of anti-predatory defensive responses. Overall, the present results served to clarify how the different functional domains in the SC may mediate the decision to pursue and hunt a prey or escape from a predator. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

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.

Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat

Background and purpose: Recent findings suggest that the noxious gas H(2)S is produced endogenously, and that physiological concentrations of H(2)S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H(2)S to modulate carrageenan-induced synovitis in the rat knee. Experimental approach: Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H(2)S formation (dl-propargylglycine) or an H(2)S donor [Lawesson`s reagent (LR)]. Key results: Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters. Conclusions and implications: Whereas exogenous H(2)S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, locally produced H(2)S exerts little immunomodulatory effect. These data further support the development and use of H(2)S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms.

p38 alpha MAP Kinase Controls IL-17 Synthesis in Vogt-Koyanagi-Harada Syndrome and Experimental Autoimmune Uveitis

PURPOSE. Interleukin (IL)-17, which is responsible for the initial influx of leukocytes into the target tissue, was recently described as the main cytokine involved in autoimmune diseases. Vogt-Koyanagi-Harada (VKH) syndrome is a significant cause of noninfectious blindness in the world. Herein the authors aimed at unraveling the involvement of IL-17 in VKH and in experimental autoimmune uveitis, focusing on the signaling pathways involved in IL-17 synthesis. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin. Draining lymph node cells, harvested 21 days after immunization, were cultured in the presence or absence of p38 alpha mitogen-activated protein kinase (MAPK) inhibitor (SB203580) and assayed for cytokine production and quantification of CD4(+)IL-17(+) cells. Mice received intraocular injections of SB203580, and disease severity was evaluated by histologic examination of the enucleated eyes at day 21. CD4(+) lymphocytes from MSK-1/2-deficient mice, human CD4(+) cells silenced with MSK1 siRNA, or peripheral blood mononuclear cells (PBMCs) from VKH patients were cultured in the presence or absence of p38 alpha MAPK inhibitor and then assayed for IL-17, IFN-gamma, and IL-4 production. RESULTS. The inhibition of p38 alpha MAPK fully blocked the synthesis of IL-17 by PBMCs from VKH patients and lymphocytes from EAU mice. The absence of the msk1/2 gene resulted in failure to produce IL-17 by murine and human lymphocytes. Interestingly, intraocular injections of SB203580 in EAU mice did not suppress development of the disease. CONCLUSIONS. These data show that p38 alpha MAPK-MSK1/2 is involved in the control of IL-17 synthesis by CD4(+) T cells and that inhibition of p38 alpha MAPK in vitro suppresses IL-17 synthesis but that inhibition of this kinase in vivo did not protect from EAU. (Invest Ophthalmol Vis Sci. 2010;51:3567-3574) DOI: 10.1167/iovs.09-4393

Evaluation of Experimental Autoimmune Uveitis in Mice Treated with FTY720

PURPOSE. FTY720 (fingolimod) is an immunomodulatory drug capable of preventing T-cell migration to inflammatory sites by binding to and subsequently downregulating the expression of sphingosine-1 phosphate receptor 1 (S1P(1)) leading in turn to T-cell retention in lymphoid organs. Additional effects of FTY720 by increasing functional activity of regulatory T cells have recently been demonstrated, raising the conversion of conventional T cells into regulatory T cells and affecting the sequestration of regulatory T cells in normal mice. In this study, the action of FTY720 in the ocular autoimmune model in mice was investigated. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin and were treated with 1 mg/kg/d FTY720 by gavage (7-21 days postimmunization [dpi]) or left untreated. Spleen cells, harvested 21 dpi, were cultured and assayed for cytokine production. Draining lymph node, spleen, and eye cells 21 dpi were assayed for quantification of T-cell populations. Disease severity was evaluated by histologic examination of the enucleated eyes at 21 and 49 dpi. In addition, anti-IRBP antibodies were analyzed by ELISA. RESULTS. FTY720 was effective in suppressing the experimental autoimmune uveitis score. Although there was a reduction in the number of eye-infiltrating cells, FTY did not prevent Treg accumulation at this site. FTY720 leads to a significant increase of CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cell percentages in lymph nodes, suggesting that this site could be the source of Treg cells found in the eye. CONCLUSIONS. The data showed that treatment in vivo with FTY720 was able to suppress EAU in mice. These results are indicative of the possible therapeutic use of FTY720 in ocular autoimmune processes. (Invest Ophthalmol Vis Sci. 2010;51:2568-2574) DOI:10.1167/iovs.09-4769

Cohabitation with a B16F10 melanoma-bearer cage mate influences behavior and dendritic cell phenotype in mice

This study evaluated the effects of cohabitation with a B16F10 melanoma-bearer cage mate on behavior and immune functions in mice. Five different experiments were conducted. In each of them, the female mice were divided into two groups: control and experimental. One mouse of each control pair was kept undisturbed and called ""companion of health partner"" (CHP). One mouse of each experimental pair was inoculated with B16FI0 cells and the other, the subject of this study, was called ""companion sick partner"" (CSP). On Day 20 of cohabitation, behavior and immune parameters from CHP and CSP mice were analyzed. In comparison to the CHP, the CSP mice: (1) presented an increased general locomotion in the open field and a decreased exploration time and number of entries in the plus-maze open arms; (2) had an enhanced expression of the CD80 costimulatory molecule on Iab(+)CD11c(+) spleen cells, but no differences were found on lymph nodes cells; (3) presented an altered differentiation of bone marrow cells in the presence of GM-CSF, IL-4, and LPS in vitro, resulting in a lower percentage of Iab(+)CD80(+) cells; (4) had a deficit in the establishment of a Delayed Type of Hypersensitivity to ovalbumin, which was associated to an in vitro proliferation of an IL-10-producing lymphocyte subpopulation after ovalbumin stimulation. Corticosterone levels detected on Day 20 of cohabitation were similar in CHP and CSP mice. It is shown here that DCs phenotype in mice is affected by conditions associated with behavioral alterations indicative of an anxiety-like state induced by the cohabitation with a tumor-bearer conspecific. This phenomenon occurred probably through a nondependent corticosterone mechanism. (C) 2009 Elsevier Inc. All rights reserved.

A role for regulatory T cells in renal acute kidney injury

Ischemia reperfusion injury (IRI) is a potential contributor for the development of chronic allograft nephropathy. T cells are important mediators of injury, even in the absence of alloantigens. We performed a depletion of TCD4(+)CTLA4(+)Foxp3(+) cells with anti-CD25(PC61), a treatment with anti-GITR (DTA-1) and rat-IgG, followed by 45 min of ischemia and 24/72 h of reperfusion, and then analyzed blood urea, kidney histopathology and gene expression in kidneys by QReal Time PCR. After 24 h of reperfusion, depletion of TCD4(+)CTLA4(+)Foxp3(+) cells reached 30.3%(spleen) and 67.8%(lymph nodes). 72 h after reperfusion depletion reached 43.1%(spleen) and 90.22%(lymph nodes) and depleted animals presented with significantly poorer renal function, while DTA-1 (anti-GITR)-treated ones showed a significant protection, all compared to serum urea from control group (IgG: 150.10 +/- 50.04; PC61: 187.23 +/- 31.38; DTA-1: 64.53 +/- 25.65, mg/dL, p<0.05). These data were corroborated by histopathology. We observed an increase of HO-1 expression in animals treated with DTA-1 at 72 h of reperfusion with significant differences. Thus, our results suggest that PC61 (anti-CD25) mAb treatment is deleterious, while DTA-1 (anti-GITR) mAb treatment presents a protective role in the renal IRI, indicating that some regulatory populations of T cells might have a role in IRI. (C) 2009 Elsevier B.V. All rights reserved.

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-kappa B ACTIVATION IN LPS-INDUCED ACUTE LUNG INJURY IN RATS

Diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment. The goal of this study was to investigate whether insulin regulates transcription of cytokines and intercellular adhesion molecule 1 (ICAM-1) via nuclear factor-kappa B (NF-kappa B) signaling pathway in Escherichia coli LIPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, iv., 10 days) and controls were instilled intratracheally with saline containing LPS (750 mu g/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LIPS. Analyses performed 6 h after LPS included: (a) lung and mesenteric lymph node IL-1 beta, TNF-alpha, IL-10, and ICAM-1 messenger RNA (mRNA) were quantified by real-time reverse transcriptase-polymerase chain reaction; (b) number of neutrophils in the bronchoalveolar lavage (BAL) fluid, and concentrations of IL-1 beta, TNF-alpha, and IL-10 in the BAL were determined by the enzyme-linked immunosorbent assay; and (c) activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were quantified by Western blot analysis. Relative to controls, diabetic rats exhibited a reduction in lung and mesenteric lymph node IL-1 beta (40%), TNF-alpha (similar to 30%), and IL-10 (similar to 40%) mRNA levels and reduced concentrations of IL-1 beta (52%), TNF-alpha (62%), IL-10 (43%), and neutrophil counts (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were almost suppressed in diabetic rats. Treatment of diabetic rats with insulin completely restored mRNA and protein levels of these cytokines and potentiated lung ICAM-1 mRNA levels (30%) and number of neutrophils (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were partially restored by insulin treatment. In conclusion, data presented suggest that insulin regulates transcription of proinflammatory (IL-1 beta, TNF-alpha) and anti-inflammatory (IL-10) cytokines, and expression of ICAM-1 via the NF-kappa B signaling pathway.

Functional interferences in host inflammatory immune response by airway allergic inflammation restrain experimental periodontitis development in mice

Aims Periodontal disease (PD) and airway allergic inflammation (AL) present opposing inflammatory immunological features and clinically present an inverse correlation. However, the putative mechanisms underlying such opposite association are unknown. Material and Methods Balb/C mice were submitted to the co-induction of experimental PD (induced by Actinobacillus actinomycetemcomitans oral inoculation) and AL [induced by sensitization with ovalbumin (OVA) and the subsequent OVA challenges], and evaluated regarding PD and AL severity, immune response [cytokine production at periodontal tissues, and T-helper transcription factors in submandibular lymph nodes (LNs)] and infection parameters. Results PD/AL co-induction decreased PD alveolar bone loss and periodontal inflammation while experimental AL parameters were unaltered. An active functional interference was verified, because independent OVA sensitization and challenge not modulate PD outcome. PD+AL group presented decreased tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, -gamma, IL-17A, receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells ligand and matrix metalloproteinase (MMP)-13 levels in periodontal tissues, while IL-4 and IL-10 levels were unaltered by AL co-induction. AL co-induction also resulted in upregulated T-bet and related orphan receptor gamma and downregulated GATA3 levels expression in submandibular LNs when compared with PD group. Conclusion Our results demonstrate that the interaction between experimental periodontitis and allergy involves functional immunological interferences, which restrains experimental periodontitis development by means of a skewed immune response.

Infection by Trypanosoma vivax in goats and sheep in the Brazilian semiarid region: From acute disease outbreak to chronic cryptic infection

A study was undertaken to investigate the role of Trypanosoma vivax in sheep and goat mortality and abortions in the Brazilian semiarid region, where outbreaks Had been previously reported in bovines. For this purpose, 177 goats and 248 sheep (20% of herds) were randomly sampled on four farms in the State of Paraiba in May and October 2008. The animals were screened for trypanosomes by the buffy coat technique (BCT) and PCR. Infected animals, similar to 25% in both surveys, manifested apathy, pale mucous membranes, enlarged lymph nodes, weakness, weight loss, opacity of the cornea, blindness and abortion. However, the animals with acute and severe disease showing the highest levels of parasitemia and fever, which many times resulted in death, were only detected in the first survey. These severely diseased animals exhibited progressive weight loss and had the smallest packed cell volume (PCV) values. During survey 2, done in October 2008 on the same farms, only animals with low parasitemia and normal temperatures, PCV values and body weights were detected. Therefore, animals that spontaneously recovered from acute infection developed chronic and asymptomatic disease. This finding demonstrated for the first time that sheep and goats, which are the most important livestock in the semiarid region of Brazil, may be severely injured by T. vivax infection and also play a role as asymptomatic carriers and important sources of T. vivax to ruminants in general. Published by Elsevier B.V.

Retinoic acid inhibits dendritic cell differentiation driven by interleukin-4

All-trans-retinoic acid (atRA) appears to affect Th1-Th2 differentiation and its effects on immune responses might also be mediated by dendritic cell (DC). Nonetheless, studies have been showing contradictory results since was observed either induction or inhibition of DC differentiation. Our aim was to investigate atRA action on human monocyte derived DC differentiation. For this purpose we tested pharmacological and physiological doses of atRA with or without cytokines. Cell phenotypes were analyzed by flow cytometry and function was investigated by phagocytosis and respiratory burst. DC, positive control group, was differentiated with GM-CSF and IL-4 and maturated with TNF-alpha. We demonstrated that atRA effects depend on the dose used as pharmacological doses inhibited expression of all phenotypic markers tested while a physiological dose caused cell differentiation. However, atRA combined or not with cytokines did not promote DC differentiation. In fact, atRA was detrimental on IL-4 property as a DC inductor. (C) 2009 Elsevier Inc. All rights reserved.

Maximal inflammatory response benefits syngeneic skin graft acceptance

Objective: We investigated the influence of acute inflammation in skin isograft acceptance. Methods: Two mouse lines selected for maximal (AIR(MAX)) or minimal inflammatory response (AIR(MIN)) were transplanted with syngeneic skin. Cellular infiltrates and cytokine production were measured 1, 3, 7 or 14 days post-transplantation. The percentage of CD4(+) CD25(+) Foxp3(+) cells in the lymph nodes was also evaluated. Results: Grafts were totally accepted in 100% of AIR(MAX) and in 26% of AIR(MIN) mice. In the latter, partial acceptance was observed in 74% of the animals. Emigrated cells were basically PMN and were enhanced in AIR(MAX) transplants. IL-10 production by graft infiltrating cells showed no interline differences. IFN-gamma was increased in AIR(MIN) grafts at day 14 and lower percentages of CD4(+)CD25(+)Foxp3(+) cells in the lymph nodes were observed in these mice. Conclusions: Our data suggest that differences in graft acceptance might be due to a lack of appropriate regulation of the inflammatory response in AIR(MIN) mice compromising the self/non-self recognition.

Allogeneic apoptotic thymocyte-stimulated dendritic cells expand functional regulatory T cells

P>Dendritic cells (DCs) play an important role in the clearance of apoptotic cells. The removal of apoptotic cells leads to peripheral tolerance, although their role is still not clear. We show that the uptake of apoptotic thymocytes by DCs converts these cells into tolerogenic DCs resistant to maturation by lipopolysaccharide, modulating the production of interleukin-12 and up-regulating the expression of transforming growth factor-beta(1) latency associated peptide. We also observed that DCs pulsed with apoptotic cells in the allogeneic context were more efficient in the expansion of regulatory T cells (Tregs), and that this expansion requires contact between DCs and the T cell. The Tregs sorted from in vitro culture suppressed the proliferation of splenocytes in vitro in a specific and non-specific manner. In the in vivo model, the transfer of CD4+ CD25- cells to Nude mice induced autoimmunity, with cell infiltrate found in the stomach, colon, liver and kidneys. The co-transfer of CD4+ CD25- and CD4+ CD25+ prevented the presence of cell infiltrates in several organs and increased the total cell count in lymph nodes. Our data indicate that apoptotic cells have an important role in peripheral tolerance via induction of tolerogenic DCs and CD4+ CD25+ Foxp3+ cells that present regulatory functions.