José Renan Cunha-Melo

Serum levels of cytokines in patients envenomed by Tityus serrulatus scorpion sting.

Seventeen patients stung by Tityus serrulatus scorpion were classified as mild (pain at the site of the sting, n = 6), moderate (local pain and one of the following manifestations: vomiting, psychomotor agitation, prostration, sweating, tachypnea, tachycardia and mild arterial hypertension, n = 10) and severe cases (equal moderate cases plus cardiac failure, pulmonary edema and shock, n = 1). Venous blood was sampled for biochemical and hematological analysis and for IL-1alpha, IL-6, IL-10, TNF-alpha, IFN-gamma and GM-CSF ELISAs at the time of hospital admission, 6 h (moderate and severe cases), and 12, 18, 36 and 72 h (severe case) later. Ten age-matched healthy volunteers were used as control. Increased serum levels of IL-1alpha was noticed in all patients, high levels of IL-6, IFN-gamma and GM-CSF were observed only in a patient with severe envenomation. Our data suggest that a systemic inflammatory response-like syndrome is triggered during severe envenomation caused by T. serrulatus sting and that release of cytokines may be involved in this response.

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‐β‐activated kinase 1, which was then transmitted downstream through JNK, p38 MAP kinase, and AP‐1. Stimulation of the Ca2+/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.

The abdominal compartment syndrome as a second insult during systemic neutrophil priming provokes multiple organ injury.

In our recent clinical study of damage control laparotomy, the abdominal compartment syndrome (ACS) emerged as an independent risk factor for postinjury multiple organ failure (MOF). We and others have shown previously that the ACS promotes the systemic production of proinflammatory cytokines. Our study objective was to develop a clinically relevant two-event animal model of postinjury MOF using the ACS as a second insult during systemic neutrophil priming to provoke organ dysfunction. Male adult rats underwent hemorrhagic shock (30 mmHg × 45 min) and were resuscitated with crystalloids and shed blood. The timing of postshock systemic neutrophil (PMN) priming was determined by the surface expression of CD11b via flow cytometry. Finding maximal PMN priming at 8 h, but no priming at 2 h (early) and 18 h (late), the ACS (25 mmHg × 60 min) was introduced at these time points. At 24 h postshock, lung injury was assessed by lung elastase concentration and Evans blue dye extravasation in bronchoalveolar lavage. Liver and renal injuries were determined by serum alanine aminotransferase, serum creatinine, and blood urea nitrogen. The ACS during the time of maximal systemic PMN priming (8 h) provoked lung and liver injury, but did not if introduced at 2 or 18 h postshock when there was no evidence of systemic PMN priming. The 24-h mortality of this two-event model was 33%. These findings corroborate the potential for the ACS to promote multiple organ injury when occurring at the time of systemic PMN priming. This clinically relevant two-event animal model of PMN organ injury may be useful in elucidating therapy strategies to prevent postinjury MOF.

Induction of Telomerase Activity During Development of Human Mast Cells from Peripheral Blood CD34+ Cells: Comparisons with Tumor Mast-Cell Lines

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+/CD117+/CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+/CD117+/CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3′-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.

Cardiovascular and respiratory effects induced by a purified scorpion toxin (tityustoxin) in unanesthetized rats.

Intravenous injection of a purified scorpion toxin (tityustoxin) into unanesthetized rats induced acute pulmonary edema, which was directly related to the dose and the time of intoxication. To study the cardiovascular and respiratory effects evoked by an edematogenic dose of the toxin (1 mg/kg), the following parameters were recorded in unanesthetized rats: systolic, diastolic and mean arterial pressure; central venous pressure; electrocardiogram; respiratory movements. The toxin induced acute systolic and diastolic hypertension, bradycardia and bradypnea. During a 1 hr period, the systolic, diastolic and mean arterial pressure fell progressively to control values, whereas the central venous pressure did not change significantly. The cardiac and respiratory rates remained lower than the control values during a 1 hr period. Several changes in the respiratory pattern were recorded, such as gasping, prolonged and labored expiration, ataxic rhythm and noisy inspiration with the mouth open. These respiratory changes were explained, in part, by the presence of edema in the lungs and froth in the trachea. From a group of 24 rats, 6 died 18-30 min after tityustoxin injection. The cause of death was apnea. The female rats were more susceptible to pulmonary edema and death than the male rats.

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.

Clinical Forms of Human Schistosoma mansoni Infection Are Associated with Differential Activation of T-Cell Subsets and Costimulatory Molecules

The current study has compared the activationstatus and the expression of the CD28 molecule oncirculating CD4+ and CD8+lymphocytes from patients with different clinical formsof schistosomiasis. The data show that patients with acuteschistosomiasis have an increase on the mean percentageof CD4+HLA-DR+ cells, whereaschronic asymptomatic patients exhibit an increased meanpercentage of CD8+HLA-DR+ cells. Patients with the hepatosplenic diseaseshowed an increase in bothCD4+HLA-DR+ andCD8+HLA-DR+ cells. Despite thehigh levels of CD8+HLA-DR+ cellsin hepatosplenic patients, they presented a decreased ratio ofCD8+CD28+/CD8+ cells.These findings of a different percentage of circulatingCD8+CD28+ cells might explain thedifferent in vitro cellular reactivity of asymptomaticand hepatosplenic patients and the defects in the cytokine secretionpatterns reported in individuals with hepatosplenicschistosomiasis.

Effects of purified scorpion toxin (tityustoxin) on gastric secretion in the rat

An i.v. bolus injection of a purified scorpion toxin (tityustoxin, TsTX) in urethane anesthetized rats induced a dramatic increase in volume, acid and pepsin output of gastric juice and a significant decrease in its pH. The maximal stimulatory effects of TsTX on gastric secretion were obtained with a dose of 0.25 mg/kg acting for 60 min. Hexamethonium did not prevent the gastric secretion evoked by TsTX, whereas atropine or cimetidine abolished partially or totally the toxin effects. Acute bilateral cervical or abdominal vagotomy did not prevent the effects of TsTX on gastric secretion, but chronic abdominal vagotomy abolished the toxin effects. Chronic antrectomy diminished the effect of TsTX on gastric secretion. In the pylorus-ligated group of rats, the gastric secretion evoked by TsTX was not different from that observed in the pylorus-intact group. It is concluded that the changes in gastric volume, acid output, pH and pepsin output induced by TsTX in the rat are due to the release of chemical mediators from postganglionic autonomic nerve fibers which would act through muscarinic and H2-receptors stimulation.

Acute gastric mucosal injury induced by toxins from Tityus serrulatus scorpion venom: a novel experimental model in the rat

The effect of a partially purified fraction (T1) and toxin gamma purified from Tityus serrulatus scorpion venom, on gastric mucosa were investigated in anesthetized rats. The animals were injected i.v. with the T1 fraction (37.5 micrograms/100 g) or with saline and 60 min later were sacrificed and the stomachs resected. The gastric juice was measured and stereoscopic examination of the stomachs made. In animals injected with the T1 fraction there was an increase in volume, acidity and pepsin output of rat stomach. The T1 fraction also induced acute gastric injuries in the glandular mucosa, consisting of circular or linear ulcers, and punctiform lesions. Intravenous injection of 20 micrograms/100 g of a pure toxin obtained from Tityus serrulatus scorpion venom (toxin gamma) also induced similar lesions in the rat stomach. Our data indicate that the injection of T1 fraction or toxin gamma are good models to induce acute gastric ulcers in a short period of time in anesthetized rats.

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

IntroductionThe 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.MethodsTwenty 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.ResultsIntra-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.ConclusionsHypotensive 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.