Mast cell-T cell axis alters development of colitis-dependent and colitis-independent colorectal tumours: potential for therapeutically targeting via mast cell inhibition.

BACKGROUND: Colorectal cancer (CRC) has a high mortality rate and can develop in either colitis-dependent (colitis-associated (CA)-CRC) or colitis-independent (sporadic (s)CRC) manner. There has been a significant debate about whether mast cells (MCs) promote or inhibit the development of CRC. Herein we investigated MC activity throughout the multistepped development of CRC in both human patients and animal models. METHODS: We analyzed human patient matched samples of healthy colon vs CRC tissue alongside conducting a The Cancer Genome Atlas-based immunogenomic analysis and multiple experiments employing genetically engineered mouse (GEM) models. RESULTS: Analyzing human CRC samples revealed that MCs can be active or inactive in this disease. An activated MC population decreased the number of tumor-residing CD8 T cells. In mice, MC deficiency decreased the development of CA-CRC lesions, while it increased the density of tumor-based CD8 infiltration. Furthermore, co-culture experiments revealed that tumor-primed MCs promote apoptosis in CRC cells. In MC-deficient mice, we found that MCs inhibited the development of sCRC lesions. Further exploration of this with several GEM models confirmed that different immune responses alter and are altered by MC activity, which directly alters colon tumorigenesis. Since rescuing MC activity with bone marrow transplantation in MC-deficient mice or pharmacologically inhibiting MC effects impacts the development of sCRC lesions, we explored its therapeutic potential against CRC. MC activity promoted CRC cell engraftment by inhibiting CD8+ cell infiltration in tumors, pharmacologically blocking it inhibits the ability of allograft tumors to develop. This therapeutic strategy potentiated the cytotoxic activity of fluorouracil chemotherapy. CONCLUSION: Therefore, we suggest that MCs have a dual role throughout CRC development and are potential druggable targets against this disease.

Mast Cells and Serotonin Synthesis Modulate Chagas Disease in the Colon: Clinical and Experimental Evidence.

BACKGROUND: Trypanosoma cruzi (T. cruzi) infects millions of Latin Americans each year and can induce chagasic megacolon. Little is known about how serotonin (5-HT) modulates this condition. Aim We investigated whether 5-HT synthesis alters T. cruzi infection in the colon. MATERIALS AND METHODS: Forty-eight paraffin-embedded samples from normal colon and chagasic megacolon were histopathologically analyzed (173/2009). Tryptophan hydroxylase 1 (Tph1) knockout (KO) mice and c-KitW-sh mice underwent T. cruzi infection together with their wild-type counterparts. Also, mice underwent different drug treatments (16.1.1064.60.3). RESULTS: In both humans and experimental mouse models, the serotonergic system was activated by T. cruzi infection (p < 0.05). While treating Tph1KO mice with 5-HT did not significantly increase parasitemia in the colon (p > 0.05), rescuing its synthesis promoted trypanosomiasis (p < 0.01). T. cruzi-related 5-HT release (p < 0.05) seemed not only to increase inflammatory signaling, but also to enlarge the pericryptal macrophage and mast cell populations (p < 0.01). Knocking out mast cells reduced trypanosomiasis (p < 0.01), although it did not further alter the neuroendocrine cell number and Tph1 expression (p > 0.05). Further experimentation revealed that pharmacologically inhibiting mast cell activity reduced colonic infection (p < 0.01). A similar finding was achieved when 5-HT synthesis was blocked in c-KitW-sh mice (p > 0.05). However, inhibiting mast cell activity in Tph1KO mice increased colonic trypanosomiasis (p < 0.01). CONCLUSION: We show that mast cells may modulate the T. cruzi-related increase of 5-HT synthesis in the intestinal colon.

Mast Cell Coupling to the Kallikrein-Kinin System Fuels Intracardiac Parasitism and Worsens Heart Pathology in Experimental Chagas Disease.

During the course of Chagas disease, infectious forms of Trypanosoma cruzi are occasionally liberated from parasitized heart cells. Studies performed with tissue culture trypomastigotes (TCTs, Dm28c strain) demonstrated that these parasites evoke neutrophil/CXCR2-dependent microvascular leakage by activating innate sentinel cells via toll-like receptor 2 (TLR2). Upon plasma extravasation, proteolytically derived kinins and C5a stimulate immunoprotective Th1 responses via cross-talk between bradykinin B2 receptors (B2Rs) and C5aR. Awareness that TCTs invade cardiovascular cells in vitro via interdependent activation of B2R and endothelin receptors [endothelin A receptor (ETAR)/endothelin B receptor (ETBR)] led us to hypothesize that T. cruzi might reciprocally benefit from the formation of infection-associated edema via activation of kallikrein-kinin system (KKS). Using intravital microscopy, here we first examined the functional interplay between mast cells (MCs) and the KKS by topically exposing the hamster cheek pouch (HCP) tissues to dextran sulfate (DXS), a potent "contact" activator of the KKS. Surprisingly, although DXS was inert for at least 30 min, a subtle MC-driven leakage resulted in factor XII (FXII)-dependent activation of the KKS, which then amplified inflammation via generation of bradykinin (BK). Guided by this mechanistic insight, we next exposed TCTs to "leaky" HCP-forged by low dose histamine application-and found that the proinflammatory phenotype of TCTs was boosted by BK generated via the MC/KKS pathway. Measurements of footpad edema in MC-deficient mice linked TCT-evoked inflammation to MC degranulation (upstream) and FXII-mediated generation of BK (downstream). We then inoculated TCTs intracardiacally in mice and found a striking decrease of parasite DNA (quantitative polymerase chain reaction; 3 d.p.i.) in the heart of MC-deficient mutant mice. Moreover, the intracardiac parasite load was significantly reduced in WT mice pretreated with (i) cromoglycate (MC stabilizer) (ii) infestin-4, a specific inhibitor of FXIIa (iii) HOE-140 (specific antagonist of B2R), and (iv) bosentan, a non-selective antagonist of ETAR/ETBR. Notably, histopathology of heart tissues from mice pretreated with these G protein-coupled receptors blockers revealed that myocarditis and heart fibrosis (30 d.p.i.) was markedly and redundantly attenuated. Collectively, our study suggests that inflammatory edema propagated via activation of the MC/KKS pathway fuels intracardiac parasitism by generating infection-stimulatory peptides (BK and endothelins) in the edematous heart tissues.

Mast cells control insulitis and increase Treg cells to confer protection against STZ-induced type 1 diabetes in mice.

Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low-dose streptozotocin (MLD-STZ) treatments, using two strains of mast cell-deficient mice (W/W(v) or Wsh/Wsh) and the adoptive transfer of mast cells. Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL-10, TGF-ß, and IL-6 expression in the pancreatic tissue. Interestingly, IL-6-deficient mice are more susceptible to T1D associated with reduced Treg-cell numbers in the PLNs, but mast cell transfer from wild-type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD-STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL-17-producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ-induced T1D via an immunological tolerance mechanism mediated by Treg cells.

Fresh frozen plasma: red blood cells (1:2) coagulation effect is equivalent to 1:1 and whole blood.

BACKGROUND: Preemptive treatment of trauma-associated coagulopathy involves transfusion of fresh frozen plasma (FFP) at 1:1 ratio with red blood cells (RBCs), but the optimal ratio remains controversial. In combat theaters, fresh whole blood (FWB) is also an option. The objective of this study was to determine the effect of FFP:RBC ratios 1:1, 1:2, 1:3 and FWB on coagulation during resuscitation. MATERIALS AND METHODS: Thirty-six rats were randomized in the following six groups: Group 1: sham; Group 2: hemorrhage followed by sole lactated Ringer (LR) infusion; Group 3: FFP:RBC (1:1); Group 4: FFP:RBC (1:2); Group 5: FFP:RBC (1:3); Group 6: FWB transfusion. Another 25 animals were used for blood harvesting. Hemorrhage was induced by withdrawing 40% of total blood volume, mean arterial pressure (MAP) decreased to 45% of baseline, and laparotomy. Animals underwent LR infusion followed by blood product transfusion preset for each group. Blood samples were obtained at baseline and in the 105th minute for thromboelastometry and lactate. RESULTS: Hemorrhage caused a significant decrease in MAP and increase in lactate (P < 0.05). MAP was persistently low in group 2 despite fluid infusion (P < 0.05), but not in the other groups after 20 min of resuscitation. Mean clot formation time, alpha angle, and maximum clot firmness decreased significantly (P < 0.05) in group 2 (LR) and group 5 (1:3) compared with other groups. CONCLUSIONS: FFP:RBC in a 1:2 ratio optimally harnessed hemostatic resuscitation and prudent use of blood products compared with 1:1 and 1:3 ratios and to FWB transfusion.

IL-33 induces a hyporesponsive phenotype in human and mouse mast cells.

IL-33 is elevated in afflicted tissues of patients with mast cell (MC)-dependent chronic allergic diseases. Based on its acute effects on mouse MCs, IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of prolonged IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. In this study, we found that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to Ag. This reduction required >72 h exposure to IL-33 for onset and 1-2 wk for reversion following IL-33 removal. This hyporesponsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation, including Ag-mediated calcium mobilization and cytoskeletal reorganization, potentially as a consequence of downregulation of the expression of phospholipase Cγ(1) and Hck. These findings suggest that IL-33 may play a protective, rather than a causative, role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to downregulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease.

Heart failure survival score in patients with Chagas disease: correlation with functional variables.

INTRODUCTION AND OBJECTIVES: Chagas disease is a prevalent cause of heart failure in Latin America, and its prognosis is worse than other etiologies. The Heart Failure Survival Score has been used to assess prognosis in patients with heart failure; however, this score has not yet been studied in patients with Chagas cardiopathy. METHODS: The Heart Failure Survival Score was calculated in 55 patients with severe left ventricular systolic dysfunction due to Chagas disease. Correlations were assessed between the Heart Failure Survival Score and variables obtained from, cardiopulmonary exercise tests, quality-of-life measures, and 6-minute walking tests. RESULTS: Patients were distributed among New York Heart Association classes II-IV; 89% were taking angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, 62% were taking beta-blockers, 86% were taking diuretics, and 74% were taking aldosterone receptor blockers. The mean Heart Failure Survival Score was 8.75 (0.80). The score correlated well with cardiopulmonary test variables such as peak oxygen uptake (0.662; P<.01), oxygen uptake at the anaerobic threshold (0.644; P<.01), ventilation carbon dioxide efficiency slope (-0.417; P<.01), oxygen pulse (0.375; P<.01), oxygen uptake efficiency slope (0.626; P<.01), 6-minute walking test (0.370; P<.01), left ventricle ejection fraction (0.650; P=.01), and left atrium diameter (-0.377; P<.01). There was also a borderline significant correlation between the Heart Failure Survival Score and quality of life (-0.283; P<.05). CONCLUSIONS: In heart failure patients with Chagas disease, the Heart Failure Survival Score correlated well with the main prognostic functional test variables.

Permissive hypotension does not reduce regional organ perfusion compared to normotensive resuscitation: animal study with fluorescent microspheres.

INTRODUCTION: The objective of this study was to investigate regional organ perfusion acutely following uncontrolled hemorrhage in an animal model that simulates a penetrating vascular injury and accounts for prehospital times in urban trauma. We set forth to determine if hypotensive resuscitation (permissive hypotension) would result in equivalent organ perfusion compared to normotensive resuscitation. METHODS: Twenty four (n=24) male rats randomized to 4 groups: Sham, No Fluid (NF), Permissive Hypotension (PH) (60% of baseline mean arterial pressure - MAP), Normotensive Resuscitation (NBP). Uncontrolled hemorrhage caused by a standardised injury to the abdominal aorta; MAP was monitored continuously and lactated Ringer's was infused. Fluorimeter readings of regional blood flow of the brain, heart, lung, kidney, liver, and bowel were obtained at baseline and 85 minutes after hemorrhage, as well as, cardiac output, lactic acid, and laboratory tests; intra-abdominal blood loss was assessed. Analysis of variance was used for comparison. RESULTS: Intra-abdominal blood loss was higher in NBP group, as well as, lower hematocrit and hemoglobin levels. No statistical differences in perfusion of any organ between PH and NBP groups. No statistical difference in cardiac output between PH and NBP groups, as well as, in lactic acid levels between PH and NBP. NF group had significantly higher lactic acidosis and had significantly lower organ perfusion. CONCLUSIONS: Hypotensive resuscitation causes less intra-abdominal bleeding than normotensive resuscitation and concurrently maintains equivalent organ perfusion. No fluid resuscitation reduces intra-abdominal bleeding but also significantly reduces organ perfusion.

Evaluation of serum levels of chemokines during interferon-ß treatment in multiple sclerosis patients: a 1-year, observational cohort study.

BACKGROUND: The molecules that provide access to activated T cells in the CNS, including chemokines, have been considered to be a crucial step in the pathogenesis of multiple sclerosis (MS). AIMS: In this study, we investigated serial serum chemokine levels in patients with relapsing-remitting MS over 1 year and the association of these chemokine levels with treatment regimens, lesions on MRI and patients' characteristics. METHODS: Serum CXCL9, CXCL10, CCL2, CCL4 and CCL5 levels were evaluated using ELISA every 2 months for a year in 28 healthy controls and 28 MS patients during their treatment with interferon (IFN)-ß. Patients underwent MRI and were evaluated using the Expanded Disability Status Scale (EDSS) at the first and final evaluations. RESULTS: CXCL10 serum levels were higher in MS patients compared with controls, were positively correlated with T2 lesions on MRI and were slightly increased during relapses. Treatment with IFNß-1a or IFNß-1b was associated with increased CXCL10 levels when evaluated more than 36 hours after subcutaneous injection. The CXCL9 levels were higher after MS relapse. There was significant variability in CCL4 and CCL5 levels in the serial evaluations, associated with gender and treatment. CCL2 levels were higher in treated MS patients than healthy controls, particularly among those patients with a stable form of the disease. CONCLUSION: Serum is a feasible resource for searching for an immunological marker in MS. Peripheral chemokine levels correlated in different ways with IFNß therapy and with disease and patient characteristics. CLINICAL TRIAL REGISTRATION NUMBER: ISRCTN45526724.

Amplification of cytokine production through synergistic activation of NFAT and AP-1 following stimulation of mast cells with antigen and IL-33.

IL-33 is associated with atopic and autoimmune diseases and, as reported here, it interacts synergistically with Ag to markedly enhance production of inflammatory cytokines in rodent mast cells even in the absence of degranulation. Investigation of the underlying mechanisms revealed that synergy in signaling occurred at the level of TGF-ß-activated kinase 1, which was then transmitted downstream through JNK, p38 MAP kinase, and AP-1. Stimulation of the Ca(2+) /calcineurin/NFAT pathway by Ag, which IL-33 did not, was critical for the synergy between Ag and IL-33. For example, selective stimulation of the NFAT pathway by thapsigargin also markedly enhanced responses to IL-33 in a calcineurin-dependent manner. As indicated by luciferase-reporter assays, IL-33 failed to stimulate the transcriptional activities of NFAT and AP-1 but augmented the activation of these transcription factors by Ag or thapsigargin. Robust stimulation of NF-κB transcriptional activity by IL-33 was also essential for the synergy. These and pharmacologic data suggested that the enhanced production of cytokines resulted in part from amplification of the activation of AP-1 and NFAT as well as co-operative interactions among transcription factors. IL-33 may retune mast cell responses to Ag toward enhanced cytokine production and thus determine the symptoms and severity of Ag-dependent allergic and autoimmune diseases.

Endotoxin tolerance and cross-tolerance in mast cells involves TLR4, TLR2 and FcepsilonR1 interactions and SOCS expression: perspectives on immunomodulation in infectious and allergic diseases.

BACKGROUND: The study of the endotoxin tolerance phenomenon in light of the recently defined roles of mast cells and toll-like receptors as essential components of the innate immune response and as orchestrators of acquired immunity may reveal potentially useful mechanisms of immunomodulation of infectious and allergic inflammatory responses, such as sepsis or asthma. Here we evaluated the phenomenon of direct tolerance of endotoxins, as well as the induction of cross-tolerance and synergism by stimulation with toll-like receptor-2 (TLR2) and FcepsilonR1 agonists, in murine mast cells prestimulated with lipopolysaccharide (LPS). Additionally, we evaluated some stimulatory and inhibitory signaling molecules potentially involved in these phenomena. METHODS: MC/9 cells and primary bone marrow-derived mast cells obtained from C57BL/6 and TLR4-/- knock-out mice were sensitized to DNP-HSA (antigen) by incubation with DNP-IgE and were prestimulated with LPS for 18 hr prior to stimulation. Cultures were stimulated with LPS or Pam3Cys-Ser-(Lys)4 3HCl (P3C), a TLR2 agonist, individually or in combination with antigen. The production of IL-6 and TNFalpha, the phosphorylation of NFkappaB and p38 MAPK, and the expression of TLR4 and SOCS-1 and -3 were analyzed. RESULTS: We found that production of TNFalpha and IL-6 in murine mast cells that have been pretreated with LPS and challenged with TLR4 (LPS) or -2 (P3C) agonists was reduced, phenomena described as endotoxin tolerance (LPS) and cross-tolerance (P3C), respectively. The expression of TLR4 was not affected by LPS pretreatment. Our results show that the FcepsilonR1 agonist DNP-HSA (antigen) interacts synergistically with LPS or P3C to markedly enhance production of cytokines (TNFalpha and IL-6). This synergistic effect with LPS and P3C was also attenuated by LPS pretreatment and was mediated by TLR4. These results may be attributed to the reduction in phosphorylation of the mitogen-activated protein kinase (MAPK), p38, and the transcription factor NFkappaB, as well as to an increase in the expression of the suppressors of cytokine signaling (SOCS)-1 and -3 proteins in LPS-pretreated mast cells. CONCLUSIONS: These findings can be explored with respect to the modulation of inflammatory responses associated with infectious and allergic processes in future studies.

Permissive hypotension and desmopressin enhance clot formation.

BACKGROUND: Experimental studies of uncontrolled hemorrhage demonstrated that permissive hypotension (PH) reduces blood loss, but its effect on clot formation remains unexplored. Desmopressin (DDAVP) enhances platelet adhesion promoting stronger clots. We hypothesized PH and DDAVP have additive effects and reduce bleeding in uncontrolled hemorrhage. METHODS: Rabbits (n = 42) randomized as follows: sham; normal blood pressure (NBP) resuscitation; PH resuscitation-60% baseline mean arterial pressure; NBP plus DDAVP 1 hour before (DDAVP NBP) or 15 minutes after beginning of shock (DDAVP T1 NBP); and PH plus DDAVP 1 hour before (DDAVP PH) or 15 minutes after beginning of shock (DDAVP T1 PH). Fluid resuscitation started 15 minutes after aortic injury and ended at 85 minutes. Intraabdominal blood loss was calculated, aortic clot sent for electron microscopy. Activated partial thromboplastin time, platelet count, thromboelastometry, arterial blood gases, and complete blood count were performed at baseline and 85 minutes. Analysis of variance was used for comparison. RESULTS: NBP received more fluid volume and had greater intraabdominal blood loss. DDAVP, when administered preshock, significantly reduced blood loss in NBP and fluid requirement when given postshock. Platelets, arterial blood gas, complete blood count, and activated partial thromboplastin time were similar at 85 minutes. NBP delayed clot formation and worsened thrombodynamic potential on thromboelastometry, whereas PH and DDAVP improved. Electron microscopy showed lack of fibrin on NBP clots, whereas DDAVP and PH clots displayed exuberant fibrin/platelet aggregates. DDAVP NBP presented intermediate clots. CONCLUSION: PH reduced bleeding and improved hemostasis compared with normotensive resuscitation. DDAVP given preshock exerted similar effects with normotensive resuscitation.

Invasive and noninvasive correlations of B-type natriuretic peptide in patients with heart failure due to Chagas cardiomyopathy.

Heart failure due to Chagas cardiomyopathy (HFCC) differs from failure with other etiologies because of the occurrence of intense inflammatory infiltrate and right ventricle compromise. This article investigates correlations of B-type natriuretic peptide (BNP) levels with parameters of severity in HFCC. Twenty-eight patients and 8 normal controls underwent heart catheterization and clinical and laboratory analyses. BNP levels were higher in patients with HFCC (P<.0001) and correlated with New York Heart Association (NYHA) class; right atrial pressure; wedge pressure; cardiac output; levels of serum sodium, hemoglobin, urea, and tumor necrosis factor-alpha; and ejection fraction. Interferon-gamma and transforming growth factor-beta did not correlate with BNP level. The authors conclude that BNP levels are elevated in patients experiencing HFCC, irrespective of NYHA class, and that the occurrence of HFCC correlates with severity of disease.

Cobertura temporária da cavidade abdominal com curativo a vácuo / Vacuum pack technique for temporary abdominal wound closure

OBJETIVO: Descrever técnica de curativo para cobertura temporária da cavidade abdominal que utiliza sistema de vácuo. MÉTODO: A técnica foi aplicada em 12 pacientes. Inicialmente coloca-se sobre a laparostomia a bolsa plástica fenestrada, em seguida a primeira camada de compressas. Sobre esta, coloca-se o tubo de látex. Este é recoberto por outra camada de compressas as quais são fixadas sobre o curativo com o campo cirúrgico auto-aderente. O tubo de látex é conectado ao sistema de vácuo com pressão de -10 a -50 mmHg. Trocam-se os curativos a cada 12 horas. Material utilizado bolsa plástica de solução salina, compressas cirúrgicas, tubo de látex, campo cirúrgico auto-aderente de 50cm x 30cm e vácuo do sistema de gases hospitalares. RESULTADOS:A peritonite grave foi a indicação mais freqüente para laparostomia, seguida da síndrome de compartimento abdominal. Fechamento definitivo da cavidade abdominal foi possível em oito pacientes (67 por cento) em média após 11 dias (9 a 21 dias) da laparostomia. Não houve complicações associadas ao método. O custo diário aproximado do curativo foi de R$ 50,00. CONCLUSÃO: O curativo a vácuo proporcionou boa contenção das vísceras abdominais, controlou o extravasamento de secreções e o edema. Permitiu o fechamento definitivo da cavidade abdominal na maioria dos casos e foi de baixo custo.

BACKGROUND: We describe a vacuum pack technique for a temporary abdominal wound closure. METHODS: The vacuum pack materials were a plastic sheet, laparotomy pads, latex tube, 50cm x 30cm adhesive-backed plastic, and a vacuum source. Twelve patients underwent the procedure as follows: we cut several slits in the plastic sheet, which is applied directly over the abdominal contents. We put laparotomy pads over the plastic sheet, and then a latex tube is placed over the laparotomy pads. Another layer of laparotomy pads is placed over the latex tube, which is stuck by an adhesive-backed plastic. We connect the tube to a vacuum source with negative pressure between -10 to -50 mmHg. The pack is changed every 12 hours. RESULTS: Severe peritonitis was the most common indication followed by the abdominal compartment syndrome. Definitive abdominal closure was performed in eight patients (67 percent) after an average of 11 days (9 to 21 days) from the first day of laparostomy. There were no complications directly associated with the method. Daily cost was approximately R$ 50.00. CONCLUSION: The vacuum pack kept the underlying abdominal viscera secure beneath the dressing; in addition, it controlled fluid leakage and edema. Definitive abdominal wound closure was possible in the majority of the patients. This was achieved at a reasonable financial cost.

Toll-like receptors, new horizons in sepsis.

Sepsis and septic shock, its more severe form, have shown alarming increases in incidence and a persistently high mortality rate, despite technological advancement allowing adequate support of vital functions in intensive care units. Progress in understanding of physiopathology has directed the therapeutic approach, until recently limited to sustaining failing organ systems and combating infectious agents, towards the alterations provoked by an unbalanced systemic inflammatory response and its deleterious effects on cellular function. Less than 10 years ago, the discovery of Toll-Like Receptor proteins, which allow the detection of pathogen molecular patterns, initiate and modulate the immune response, opened up new and exciting possibilities in approaches to sepsis. The elucidation of the transduction pathways triggered by Toll-Like Receptors activation signals exposes promising therapeutic targets. Currently, mechanisms associated within the context of Toll-Like Receptor signalization are identified in the tolerance phenomena described in the past. The description of genetic polymorphisms associated with Toll-Like Receptors, and the different patterns of response to infectious insults have defined high-risk subgroups of imbalanced immune response with greater specificity. A better understanding of the molecular structures involved in the process and the negative-regulation of some of them have opened up possibilities in antagonizing and modulating the response to the inflammatory activation mediated by Toll-Like Receptors. Having understood how the immune system recognizes pathogens and organizes the inflammatory response upon the discovery of Toll-Like Receptors and their signaling pathways, we gained an insight into the possibilities of specific treatment instead of supportive measures for sepsis.

Scorpion venom increases mRNA expression of lung cytokines.

Previous studies have demonstrated that scorpion toxins increase the serum levels of IL-1, IL-6, INF-gamma, and GM-CSF in patients with severe shock and pulmonary edema. Moreover, it has been shown that experimental models of scorpion envenomation presented an increase in serum levels of IL-1, IL-6, IFN-gamma and nitric oxide. Thus, it is possible that the cytokine release may contribute to the onset and maintenance of the pulmonary edema induced by scorpion venom. This study was designed to investigate whether inflammatory and non-inflammatory cytokines, contribute to the pulmonary injury induced by infusion of Tityus serrulatus scorpion toxin in rats. We show that scorpion venom not only increases the expression of mRNA pulmonary inflammatory cytokines but also non-inflammatory cytokines as well. Moreover, the expression of IL-1alpha, IL-1beta and IL-6 mRNA was shown to be higher among the remaining detectable cytokines. The findings of this study provide additional insight towards the understanding of the pathophysiology of the pulmonary edema induced by scorpion venom. The increased level of pulmonary cytokines observed during the pulmonary edema may be responsible for the exacerbation and maintenance of the inflammatory response to scorpion venom in the lungs.

FcepsilonR1 and toll-like receptors mediate synergistic signals to markedly augment production of inflammatory cytokines in murine mast cells.

Mast cells mediate both IgE-dependent allergic reactions and protective responses against acute infections, possibly through the activation of Toll-like receptors (TLRs). We find that antigen interacts synergistically with TLR2 and TLR4 ligands to markedly enhance production of cytokines in murine mast cell lines. However, the TLR ligands neither stimulated degranulation and release of arachidonic acid nor influenced such responses to antigen, probably because these ligands failed to generate a necessary calcium signal. The enhanced cytokine production could be attributed to synergistic activation of mitogen-activated protein kinases in addition to the engagement of a more effective repertoire of transcription factors for cytokine gene transcription. The synergistic interactions of TLR ligands and antigen might have relevance to the exacerbation of IgE-mediated allergic diseases by infectious agents.

Lung compliance, plasma electrolyte levels and acid-base balance are affected by scorpion envenomation in anesthetized rats under mechanical ventilation.

To determine the effects of Tityus serrulatus scorpion toxin on lung compliance and resistance, ionic equilibrium and acid-base balance over time in anesthetized and mechanically ventilated rats, we measured air flow, tracheal and esophageal pressure. Lung volume was obtained by electronic integration of airflow signal. Arterial blood samples were collected through a catheter at baseline (before) and 5, 15, 30 and 60 min after scorpion toxin injection for arterial blood gases, bicarbonate, and alkali reserve levels as well as for, sodium, potassium, magnesium, glucose, lactate, hematocrit, and osmolality analysis. Injection of the gamma fraction of the T. serrulatus scorpion venom in rats under mechanical ventilatory support leads to a continuous decrease in lung compliance secondary to pulmonary edema, but no change in airway resistance. The changes in arterial blood gases characterizing metabolic acidosis were accompanied by an increase in arterial lactate and glucose values, suggesting a scorpion toxin-induced lactic acidosis, in association with poor tissue perfusion (hypotension and low cardiac output). Moreover, scorpion toxin injection resulted in hyperosmolality, hyperkalemia, hypermagnesemia and an increase in hematocrit. The experiments have shown a clinically relevant animal model to study severe scorpion envenoming and may help to better understand the scorpion envenoming syndrome.

Kit and FcepsilonRI mediate unique and convergent signals for release of inflammatory mediators from human mast cells.

In human mast cells, derived from CD34(+) peripheral blood cells, we observed that Kit ligand (KL) failed to induce degranulation but acted in synergy with antigen to markedly enhance degranulation, levels of cytokine gene transcripts, and production of cytokines. Further examination revealed that antigen and KL activated common and unique signaling pathways to account for these varied responses. KL, unlike antigen, failed to activate protein kinase C but activated phospholipase Cgamma and calcium mobilization and augmented these signals as well as degranulation when added together with antigen. Both KL and antigen induced signals that are associated with cytokine production, namely phosphorylation of the mitogen-activated protein kinases, phosphatidylinositol 3-kinase-dependent phosphorylation of protein kinase B (also known as Akt), and phosphorylation of nuclear factor kappaB (NFkappaB). However, only KL stimulated phosphorylation of signal transducer and activator of transcription 5 (STAT5) and STAT6, whereas antigen weakly stimulated the protein kinase C-dependent induction and phosphorylation of c-Jun and associated activating protein-1 (AP-1) components, an action that was markedly potentiated by costimulation with KL. Interestingly, most signals were down-regulated on continuous exposure to KL but were reactivated along with cytokine gene transcription on addition of antigen. The findings, in total, indicated that a combination of FcepsilonRI and Kit-mediated signals and transcriptional processes were required for optimal physiologic responses of human mast cells to antigen.

Dexamethasone suppresses antigen-induced activation of phosphatidylinositol 3-kinase and downstream responses in mast cells.

Dexamethasone and other glucocorticoids suppress FcepsilonRI-mediated release of inflammatory mediators from mast cells. Suppression of cytokine production is attributed to repression of cytokine gene transcription but no mechanism has been described for the suppression of degranulation. We show that therapeutic concentrations of dexamethasone inhibit intermediate signaling events, in particular the activation of phosphatidylinositol (PI)3-kinase and downstream signaling events that lead to degranulation in rat basophilic leukemia 2H3 cells. This inhibitory action is mediated via the glucocorticoid receptor and is not apparent when cells are stimulated via Kit in a mouse bone marrow-derived mast cell line. The primary perturbation appears to be the failure of the regulatory p85 subunit of PI3-kinase to engage with the adaptor protein Grb2-associated binder 2 leading to suppression of phosphorylation of phospholipase Cgamma2, the calcium signal, and degranulation. Suppression of PI3-kinase activation by dexamethasone may also contribute to reduced cytokine production because the PI3-kinase inhibitor LY294002, like dexamethasone, inhibits Ag-induced transcription of cytokine genes as well as degranulation.