Curbing Inflammation in hemorrhagic trauma: a review

Trauma is one of the world's leading causes of death within the first 40 years of life and thus a significant health problem. Trauma accounts for nearly a third of the lost years of productive life before 65 years of age and is associated with infection, hemorrhagic shock, reperfusion syndrome, and inflammation. The control of hemorrhage, coagulopathy, optimal use of blood products, balancing hypo and hyperperfusion, and hemostatic resuscitation improve survival in cases of trauma with massive hemorrhage. This review discusses inflammation in the context of trauma-associated hemorrhagic shock. When one considers the known immunomodulatory effects of traumatic injury, allogeneic blood transfusion, and the overlap between patient populations, it is surprising that so few studies have assessed their combined effects on immune function. We also discuss the relative benefits of curbing inflammation rather than attempting to prevent it.

The role of the vagus nerve in hypertonic resuscitation of hemorrhagic shocked dogs

Previous studies have suggested a critical role for the vagi during the hypertonic resuscitation of hemorrhagic shocked dogs. Vagal blockade prevented the full hemodynamic and metabolic recovery and increased mortality. This interpretation, however, was challenged on the grounds that the blockade also abolished critical compensatory mechanisms and therefore the animals would die regardless of treatment. To test this hypothesis, 29 dogs were bled (46.0 ± 6.2 ml/kg, enough to reduce the mean arterial pressure to 40 mmHg) and held hypotensive for 45 min. After 40 min, vagal activity was blocked in a reversible manner (0ºC/15 min) and animals were resuscitated with 7.5% NaCl (4 ml/kg), 0.9% NaCl (32 ml/kg), or the total volume of shed blood. In the vagal blocked isotonic saline group, 9 of 9 dogs, and in the vagal blocked replaced blood group, 11 of 11 dogs survived, with full hemodynamic and metabolic recovery. However, in the hypertonic vagal blocked group, 8 of 9 dogs died within 96 h. Survival of shocked dogs which received hypertonic saline solution was dependent on vagal integrity, while animals which received isotonic solution or blood did not need this neural component. Therefore, we conclude that hypertonic resuscitation is dependent on a neural component and not only on the transient plasma volume expansion or direct effects of hyperosmolarity on vascular reactivity or changes in myocardial contraction observed immediately after the beginning of infusion.

High mobility group box 1 as a mediator of endotoxin administration after hemorrhagic shock-primed lung injury

High mobility group box 1 (HMGB1) was discovered as a novel late-acting cytokine that contributes to acute lung injury (ALI). However, the contribution of HMGB1 to two-hit-induced ALI has not been investigated. To examine the participation of HMGB1 in the pathogenesis of ALI caused by the two-hit hypothesis, endotoxin was injected intratracheally in a hemorrhagic shock-primed ALI mouse model. Concentrations of HMGB1 in the lung of the shock group were markedly increased at 16 h (1.63 ± 0.05, compared to the control group: 1.02 ± 0.03; P < 0.05), with the highest concentration being observed at 24 h. In the sham/lipopolysaccharide group, lung HMGB1 concentrations were found to be markedly increased at 24 h (1.98 ± 0.08, compared to the control group: 1.07 ± 0.03; P < 0.05). Administration of lipopolysaccharide to the hemorrhagic shock group resulted in a notable HMGB1 increase by 4 h, with a further increase by 16 h. Intratracheal lipopolysaccharide injection after hemorrhagic shock resulted in the highest lung leak at 16 h (2.68 ± 0.08, compared to the control group: 1.05 ± 0.04; P < 0.05). Compared to the hemorrhagic shock/lipopolysaccharide mice, blockade of HMGB1 at the same time as lipopolysaccharide injection prevented significantly pulmonary tumor necrosis factor-alpha, interleukin-1beta and myeloperoxidase. Lung leak was also markedly reduced at 16 h; blockade of HMGB1 24 h after lipopolysaccharide injection failed to alter lung leak or myeloperoxidase at 48 h. Our observations suggest that HMGB1 plays a key role as a late mediator when lipopolysaccharide is injected after hemorrhagic shock-primed ALI and the kinetics of its release differs from that of one-hit ALI. The therapeutic window to suppress HMGB1 activity should not be delayed to 24 h after the disease onset.

Treatment of hemorrhagic shock with hypertonic saline solution modulates the inflammatory response to live bacteria in lungs

Shock and resuscitation render patients more susceptible to acute lung injury due to an exacerbated immune response to subsequent inflammatory stimuli. To study the role of innate immunity in this situation, we investigated acute lung injury in an experimental model of ischemia-reperfusion (I-R) followed by an early challenge with live bacteria. Conscious rats (N = 8 in each group) were submitted to controlled hemorrhage and resuscitated with isotonic saline (SS, 0.9% NaCl) or hypertonic saline (HS, 7.5% NaCl) solution, followed by intratracheal or intraperitoneal inoculation of Escherichia coli. After infection, toll-like receptor (TLR) 2 and 4 mRNA expression was monitored by RT-PCR in infected tissues. Plasma levels of tumor necrosis factor α and interleukins 6 and 10 were determined by ELISA. All animals showed similar hemodynamic variables, with mean arterial pressure decreasing to nearly 40 mmHg after bleeding. HS or SS used as resuscitation fluid yielded equal hemodynamic results. Intratracheal E. coli inoculation per se induced a marked neutrophil infiltration in septa and inside the alveoli, while intraperitoneal inoculation-associated neutrophils and edema were restricted to the interseptal space. Previous I-R enhanced lung neutrophil infiltration upon bacterial challenge when SS was used as reperfusion fluid, whereas neutrophil influx was unchanged in HS-treated animals. No difference in TLR expression or cytokine secretion was detected between groups receiving HS or SS. We conclude that HS is effective in reducing the early inflammatory response to infection after I-R, and that this phenomenon is achieved by modulation of factors other than expression of innate immunity components.

Rabbit model of uncontrolled hemorrhagic shock and hypotensive resuscitation

Clinically relevant animal models capable of simulating traumatic hemorrhagic shock are needed. We developed a hemorrhagic shock model with male New Zealand rabbits (2200-2800 g, 60-70 days old) that simulates the pre-hospital and acute care of a penetrating trauma victim in an urban scenario using current resuscitation strategies. A laparotomy was performed to reproduce tissue trauma and an aortic injury was created using a standardized single puncture to the left side of the infrarenal aorta to induce hemorrhagic shock similar to a penetrating mechanism. A 15-min interval was used to simulate the arrival of pre-hospital care. Fluid resuscitation was then applied using two regimens: normotensive resuscitation to achieve baseline mean arterial blood pressure (MAP, 10 animals) and hypotensive resuscitation at 60% of baseline MAP (10 animals). Another 10 animals were sham operated. The total time of the experiment was 85 min, reproducing scene, transport and emergency room times. Intra-abdominal blood loss was significantly greater in animals that underwent normotensive resuscitation compared to hypotensive resuscitation (17.1 ± 2.0 vs 8.0 ± 1.5 mL/kg). Antithrombin levels decreased significantly in normotensive resuscitated animals compared to baseline (102 ± 2.0 vs 59 ± 4.1%), sham (95 ± 2.8 vs 59 ± 4.1%), and hypotensive resuscitated animals (98 ± 7.8 vs 59 ± 4.1%). Evidence of re-bleeding was also noted in the normotensive resuscitation group. A hypotensive resuscitation regimen resulted in decreased blood loss in a clinically relevant small animal model capable of reproducing hemorrhagic shock caused by a penetrating mechanism.

Myosin light chain kinase is necessary for post-shock mesenteric lymph drainage enhancement of vascular reactivity and calcium sensitivity in hemorrhagic-shocked rats

Vascular hyporeactivity is an important factor in irreversible shock, and post-shock mesenteric lymph (PSML) blockade improves vascular reactivity after hemorrhagic shock. This study explored the possible involvement of myosin light chain kinase (MLCK) in PSML-mediated vascular hyporeactivity and calcium desensitization. Rats were divided into sham (n=12), shock (n=18), and shock+drainage (n=18) groups. A hemorrhagic shock model (40±2 mmHg, 3 h) was established in the shock and shock+drainage groups. PSML drainage was performed from 1 to 3 h from start of hypotension in shock+drainage rats. Levels of phospho-MLCK (p-MLCK) were determined in superior mesenteric artery (SMA) tissue, and the vascular reactivity to norepinephrine (NE) and sensitivity to Ca2+ were observed in SMA rings in an isolated organ perfusion system. p-MLCK was significantly decreased in the shock group compared with the sham group, but increased in the shock+drainage group compared with the shock group. Substance P (1 nM), an agonist of MLCK, significantly elevated the decreased contractile response of SMA rings to both NE and Ca2+ at various concentrations. Maximum contractility (Emax) in the shock group increased with NE (from 0.179±0.038 to 0.440±0.177 g/mg, P<0.05) and Ca2+ (from 0.515±0.043 to 0.646±0.096 g/mg, P<0.05). ML-7 (0.1 nM), an inhibitor of MLCK, reduced the increased vascular response to NE and Ca2+ at various concentrations in the shock+drainage group (from 0.744±0.187 to 0.570±0.143 g/mg in Emax for NE and from 0.729±0.037 to 0.645±0.056 g/mg in Emax for Ca2+, P<0.05). We conclude that MLCK is an important contributor to PSML drainage, enhancing vascular reactivity and calcium sensitivity in rats with hemorrhagic shock.

Epidemia de dengue em Ribeirão Preto, SP, Brasil: nota prévia

Apresenta-se Nota preliminar de uma epidemia de dengue, ocorrida em Ribeirão Preto, São Paulo, Brasil, no período de novembro de 1990 a março de 1991. Descreve-se a ocorrência de um caso de "Dengue Hemorrhagic Fever/Dengue Shock Syndrome" Grau II (DHF/DSS) e de dois óbitos associados ao dengue devido a síndrome de choque. Alerta clínicos e epidemiologistas quanto à possibilidade de ocorrência de casos isolados de DHF/DSS durante epidemias de dengue clássico em áreas antes indenes à doença.

Thrombocytopenia and leptospirosis

The present study has intended to contribute to the elucidation of the pathogenic mechanisms, involved in the thrombocytopenia and in the bleeding diathesis seen in the course of Leptospirosis. The group of cases included in the present prospective study consisted of 30 patients with Leptospirosis, admitted to the Infectious and Parasitic Diseases Ward, Hospital das Clínicas, Faculty of Medicine, University of São Paulo. The following possible mechanisms of thrombocytopenia have been considered and therefore investigated: platelet consumption, due to disseminated intravascular coagulation; immune-mediated platelet destruction, due to platelet-associated antibodies and an inhibited platelet production in the bone marrow. Thrombocytopenia occurred in 86.6% of 30 patients and did not seem to be immune-mediated by platelet-associated antibodies. Furthermore it did not seem to be due to a disseminated intravascular coagulation consumption. Although there was a statistically-significant correlation between bone marrow platelet production and platelet counts we think that the static microscopic examination of a bone marrow aspirate cannot accurately depict the dynamic mechanisms of platelet production when these cells are being consumed in peripheral blood. Vasculitis should be considered as the most important factor for the pathogenesis of the bleeding disturbances in Leptospirosis. However, we believe that thrombocytopenia, uremia and coagulation disorders, individually or as a group, should be included among the contributing factors that lead to and worsen bleeding episodes, which represent the leading cause of death in this disease.

Laboratory acquired infection by the virus SP H 114202 (Arenavirus: Arenaviridae): clinical and laboratory findings

Here in is described the clinical and laboratorial findings of a laboratory-acquired infection caused by the virus SP H 114202 (Arenavirus, family Arenaviridae) a recently discovered agent responsible for a viral hemorrhagic fever. The patient was sick for 13 days. The disease had an abrupt onset characterized by high fever (39ºC.), headache, chills and myalgias for 8 days. In addition, on the 3rd day, the patient developed nauseas and vomiting, and in the 10th, epigastralgia, diarrheia and gengivorrhagia. Leucopenia was seen within the 1 st week of onset, with counts as low as 2,500 white cells per mm³. Counts performed after the 23th day of the onset were within normal limits. With the exception of moderate lymphocitosis, no changes were observed in differential counts. An increase in the liter of antibodies by complement fixation, neutralization and ELISA (IgM) was detected. Suckling mice and baby hamsters were inoculated intracerebrally with 0.02 ml of blood samples collected in the 2nd and 7th days of disease. Attempts to isolate the virus were also made in Vero cells. No virus was isolated. This virus was isolated before in a single occasion in São Paulo State, in 1990, from the blood of a patient with hemorrhagic fever with a fatal outcome. The manipulation of the virus under study, must be done carefully, since the transmission can occur through aerosols.

Involvement of the central nervous system in dengue fever: three serologically confirmed cases from Fortaleza, Ceará, Brazil

Three cases of dengue fever involving the central nervous system (CNS) are reported. All occurred in 1994 during a dengue (DEN) epidemic caused by serotypes DEN-1 and DEN-2. The first case examined was a 17-year-old girl who complained of fever, nuchal rigidity and genital bleeding. Three blood samples were positive by anti-dengue IgM ELISA and showed hemagglutination-inhibition (HI) test titers ³ 1,280. The second case concerned a 86-year-old woman with fever, muscle and joint pains, altered consciousness, syncope, nuchal rigidity and meningismus. Her blood sample showed an HI titer of 1:320 for flaviviruses, and an IgM ELISA positive for dengue. The third case was a 67-year-old woman with fever, abnormal behaviour, seizures, tremor of extremities, thrombocytopenia, increased hematocrit and leukopenia. The patient suffered a typical case of dengue hemorrhagic fever with ensuing shock and a fatal outcome. A single blood sample showed HI antibodies of ³ 1,280 and an IgM ELISA positive for dengue. No virus could be isolated from any patient by inoculation of blood into C6/36 cells and suckling mice. No other agent of disease was encountered in the patient.

Heparin-antivenom association: differential neutralization effectiveness in Bothrops atrox and Bothrops erythromelas envenoming

Heparin, in some regions of Brazil has been used in the treatment of bothropic accidents, but the data found in the literature are inconclusive about its effectiveness. The venoms of Bothrops atrox and of B. erythromelas were characterized according to their biological activities. The capacity of heparin in neutralizing these activities was tested with doses of 3 and 6 IU in isolated form and associated to Antibothropic Serum (ABS). It was verified that heparin, in doses of 3 and 6 IU, was not effective in neutralizing the desfibrinating and edema-forming activities of B. atrox venom and the hemorrhagic and coagulant actions of both venoms. Heparin diminished the effectiveness of the ABS in the neutralization of the hemorrhagic and edema-forming activities of the B. atrox venom. However, heparin in the 6 IU dose was capable of neutralize the edema-forming of the B. erythromelas and increase the effectiveness of the ABS. Heparin also neutralized the phospholipasic A2 activity of B. atrox (14.3%) and B. erythromelas (28.0%) venoms. For B. erythromelas venom, the associated treatment, heparin and ABS, was more effective in the neutralization of its lethal activity.

Evaluation of hemostasis disorders and anticardiolipin antibody in patients with severe leptospirosis

A prospective study was designed to evaluate disorders of hemostasis and levels of anticardiolipin antibodies (ACL) in 30 patients with severe leptospirosis and acute renal failure (ARF) (ARF was defined as serum creatinine > or = 1.5 mg/dL). The patients had been admitted to the Walter Cantídio University Hospital, São José Infectious Diseases Hospital and General Hospital of Fortaleza, Ceará, from August 1999 to July 2001. They all were male, with a mean age of 32 ± 14 years and with clinical and laboratory diagnoses of ARF leptospirosis. The time elapsed between onset of symptoms and the first hemorrhagic manifestation was 9 ± 4 days. Bleeding was observed in 86% of the patients. Laboratory tests showed significantly high levels of urea (181 ±95 mg/dl), fibrinogen, (515 ± 220 mg/dl), prothrombin time (13.3 ± 0.9 seconds) and low platelet counts (69 ± 65x10³/mm³) on admission. There was no elevation in activated partial thromboplastin time or thrombin time. Levels of IgM and IgG ACL concentrations were significantly increased (p < 0.05) in leptospirosis patients when compared to control patients (28.5 ± 32.4 vs. 11.5 ± 7.9MPL U/ml and 36.7 ± 36.1 vs. 6.5 ± 2.5 GPL U/ml), respectively. Vasculitis, thrombocytopenia and uremia should be considered important factors for the pathogenesis of hemorrhagic disturbances and the main cause of death in severe leptospirosis.

Immunohistochemical changes in kidney glomerular and tubular proteins caused by rattlesnake (Crotalus vegrandis) venom

Renal damage is an important cause of death in patients who have survived the early effects of severe crotalid envenomation. Extracellular matrix of renal tissue is altered by Crotalus toxin activities. The aim of this study was to describe how cytoskeletal proteins and basal membrane components undergo substantial alterations under the action of Crotalus vegrandis crude venom and its hemorrhagic fraction (Uracoina-1) in mice. To detect the proteins in question, the immunoperoxidase method with monoclonal and polyclonal antibodies was used. Cell types within renal lesions were characterized by phenotypic identification, by means of immunohistologic analysis of marker proteins using different primary antibodies against mesangial cells, endothelial cells, cytoskeletal proteins (intermediate filament), extracellular matrix and basal membranes. Samples for morphological study by standard procedures (biotin-streptavidin-peroxidase technique) using light microscopy were processed. Positive and negative controls for each antigen tested in the staining assay were included. After crude venom and hemorrhagic fraction inoculation of mice, the disappearance of cytoskeletal vimentin and desmin and collagen proteins in the kidney was observed. In extracellular matrix and basal membranes, collagen type IV from envenomed animals tends to disappear from 24 h to 120 h after venom injection.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.

Cytokine and nitric oxide production by mouse macrophages infected with brazilian flaviviruses

The Flaviviridae family, Flavivirus genus includes viruses that are transmitted to vertebrates by infected mosquitoes or ticks. The genus Flavivirus includes a variety of viruses that cause diseases such as acute febrile illness, encephalitis, and hemorrhagic fever. Flaviviruses primarily infect blood monocytes and tissue macrophages, which have been shown to be permissive, supporting viral replication and serving as virus reservoirs. On the other hand, these cells may have an important antiviral activity related to modulation by cytokine production and by the capacity of these cells to synthesize reactive free radicals such as nitric oxide (NO) which can have a microbicidal effect. The present study was performed in order to determine the production of cytokines interleukin-1beta (IL-1β), tumor necrosis factor -alpha (TNF-α), transforming growth factor- beta (TGF-β) and interferon -alpha (IFN-α) and NO by macrophages infected with one of four Brazilian flaviviruses, Bussuquara virus (BUSV), Yellow Fever virus (YFV), Rocio virus (ROCV) and Encephalitis Saint Louis virus (SLEV), and to verify the possible antiviral effect of NO during macrophage infection with ROCV. Moreover, we asked if the different viruses were able to regulate bacterial lipopolysaccharide (LPS) induced cytokine production. Our results showed that YFV and SLEV reduced the production of IL-1β and TGF-β by LPS-stimulated macrophages, while ROCV only diminished LPS-stimulated TGF-β synthesis. On the other hand, BUSV more likely favored an enhancement of the LPS-induced production of IL-1β by macrophages. Additionally, while most of the viruses stimulated the production of IFN-α, none of them altered the production of TNF-α by murine macrophages. Interestingly, all viruses induced synthesis of NO that was not correlated with antiviral activity for ROCV.