Cleavage and reduced CD36 ectodomain density on heart and spleen macrophages in the spontaneously hypertensive rat.

Metabolic disease is accompanied by a range of cellular defects ("comorbidities") whose origin is uncertain. To investigate this pathophysiological phenomenon we used the Spontaneously Hypertensive Rat (SHR), which besides an elevated arterial blood pressure also has many other comorbidities, including a defective glucose and lipid metabolism. We have shown that this model of metabolic disease has elevated plasma matrix metalloproteinase (MMP) activity, which cleaves the extracellular domain of membrane receptors. We hypothesize here that the increased MMP activity also leads to abnormal cleavage of the scavenger receptor and fatty acid transporter CD36. To test this idea, chronic pharmaceutical MMP inhibition (CGS27023A) of the SHR and its normotensive control, the Wistar Kyoto Rat (WKY), was used to determine if inhibition of MMP activity serves to maintain CD36 receptor density and function. Surface density of CD36 on macrophages from the heart, spleen, and liver was determined in WKY, SHR, CGS-treated WKY (CGS WKY), and CGS-treated SHR (CGS SHR) by immunohistochemistry with an antibody against the CD36 ectodomain. The extracellular CD36 density was lower in SHR heart and spleen macrophages compared to that in the WKY. MMP inhibition by CGS served to restore the reduced CD36 density on SHR cardiac and splanchnic macrophages to levels of the WKY. To examine CD36 function, culture assays with murine macrophages (RAW 264.7) after incubation in fresh WKY or SHR plasma were used to test for adhesion of light-weight donor red blood cell (RBC) by CD36. This form of RBC adhesion to macrophages was reduced after incubation in SHR compared WKY plasma. Analysis of the supernatant macrophage media by Western blot shows a higher level of CD36 extracellular protein fragments following exposure to SHR plasma compared to WKY. MMP inhibition in the SHR plasma compared to untreated plasma, served to increase the RBC adhesion to macrophages and decrease the number of receptor fragments in the macrophage media. In conclusion, these studies bring to light that plasma in the SHR model of metabolic disease has an unchecked MMP degrading activity which causes cleavage of a variety of membrane receptors, including CD36, which attenuates several cellular functions typical for the metabolic disease, including RBC adhesion to the scavenger receptor CD36. In addition to other cell dysfunctions chronic MMP inhibition restores CD36 in the SHR.

Enteral Tranexamic Acid Decreases Proteolytic Activity in the Heart in Acute Experimental Hemorrhagic Shock.

The mechanisms for cardiac injury after hemorrhagic shock (HS) are unresolved. We hypothesize that remote organ damage can be caused by uncontrolled pancreatic proteolytic activity, as enteral protease inhibition improves outcomes in experimental HS. Uncontrolled proteolysis in the heart may disrupt cardiac metabolism and adrenergic control with subsequent deleterious outcomes. To test this hypothesis, the heart rate-pressure product (RPP) as an index of myocardial oxygen consumption and the levels of fatty acid transporter proteins CD36 and FATP6 as surrogates for metabolic activity in the heart were measured in rats subjected to experimental HS (n = 6/group) with and without the enteral protease inhibitor tranexamic acid (TXA). Plasma troponin I and heart fatty acid-binding protein (HFABP) concentrations were measured as indices of myocardial damage. Expression of the adrenergic receptors ß1, α1D, and ß2 was also measured in the heart to determine the possible effects of shock with and without enteral TXA on the adrenergic control of heart function. Hemorrhagic shock was induced by reduction in mean arterial blood pressure to 35 mm Hg for 2 hours before reperfusion of shed blood. The RPP was maintained in shocked animals treated enterally with TXA but not in those subjected to HS alone; this group also demonstrated decreased HFABP and plasma troponin I levels. Serine protease (trypsin, chymotrypsin, and elastase) and matrix metalloproteinase (MMP)-2 and MMP-9 activity was elevated in cardiac tissue and plasma after HS and abrogated by enteral TXA. Levels of CD36, FATP6, ß1, α1D, and ß2 were also increased after HS in cardiac tissue, and the increases were mitigated by TXA treatment. These results suggest that increased proteolytic activity may contribute to cardiac injury after HS. Enteral TXA prevents these changes, indicating a potential therapeutic option in the management of shock with resultant cardiac injury.

Enteral tranexamic acid attenuates vasopressor resistance and changes in α1-adrenergic receptor expression in hemorrhagic shock.

BACKGROUND: Irreversible hemorrhagic shock is characterized by hyporesponsiveness to vasopressor and fluid therapy. Little is known, however, about the mechanisms that contribute to this phenomenon. Previous studies have shown that decreased intestinal perfusion in hemorrhagic shock leads to proteolytically mediated increases in gut permeability, with subsequent egress of vasoactive substances systemically. Maintenance of blood pressure is achieved in part by α1 receptor modulation, which may be affected by vasoactive factors; we thus hypothesized that decreases in hemodynamic stability and vasopressor response in shock can be prevented by enteral protease inhibition. METHODS: Rats were exposed to experimental hemorrhagic shock (35 mm Hg mean arterial blood pressure for 2 hours, followed by reperfusion for 2 hours) and challenged with phenylephrine (2 µg/kg) at discrete intervals to measure vasopressor responsiveness. A second group of animals received enteral injections with the protease inhibitor tranexamic acid (TXA) (127 mM) along the small intestine and cecum 1 hour after induction of hemorrhagic shock. RESULTS: Blood pressure response (duration and amplitude) to phenylephrine after reperfusion was significantly attenuated in animals subjected to hemorrhagic shock compared with baseline and control nonshocked animals and was restored to near baseline by enteral TXA. Arteries from shocked animals also displayed decreased α1 receptor density with restoration to baseline after enteral TXA treatment. In vitro, rat shock plasma decreased α1 receptor density in smooth muscle cells, which was also abrogated by enteral TXA treatment. CONCLUSION: Results from this study demonstrate that experimental hemorrhagic shock leads to decreased response to the α1-selective agonist phenylephrine and decreased α1 receptor density via circulating shock factors. These changes are mitigated by enteral TXA with correspondingly improved hemodynamics. Proteolytic inhibition in the lumen of the small intestine improves hemodynamics in hemorrhagic shock, possibly by restoring α1 adrenergic functionality necessary to maintain systemic blood pressure and perfusion.

Peptidomic Analysis of Rat Plasma: Proteolysis in Hemorrhagic Shock.

It has been previously shown that intestinal proteases translocate into the circulation during hemorrhagic shock and contribute to proteolysis in distal organs. However, consequences of this phenomenon have not previously been investigated using high-throughput approaches. Here, a shotgun label-free quantitative proteomic approach was utilized to compare the peptidome of plasma samples from healthy and hemorrhagic shock rats to verify the possible role of uncontrolled proteolytic activity in shock. Plasma was collected from rats after hemorrhagic shock (HS) consisting of 2-h hypovolemia followed by 2-h reperfusion, and from healthy control (CTRL) rats. A new two-step enrichment method was applied to selectively extract peptides and low molecular weight proteins from plasma, and directly analyze these samples by tandem mass spectrometry. One hundred twenty-six circulating peptides were identified in CTRL and 295 in HS animals. Ninety-six peptides were present in both conditions; of these, 57 increased and 30 decreased in shock. In total, 256 peptides were increased or present only in HS confirming a general increase in proteolytic activity in shock. Analysis of the proteases that potentially generated the identified peptides suggests that the larger relative contribution to the proteolytic activity in shock is due to chymotryptic-like proteases. These results provide quantitative confirmation that extensive, system-wide proteolysis is part of the complex pathologic phenomena occurring in hemorrhagic shock.

Set up of a protocol for rat plasma peptidomics in hemorrhagic shock model in presence of heparin.

A preliminary mass spectrometry based shotgun protocol was set up to compare the peptidome of plasma samples from healthy and hemorrhagic shock rats with the aim of verifying the possible role of uncontrolled proteolytic activity in circulatory shock. Since the hemorrhagic shock model requires heparin as anticoagulant, a preliminary experiment using plasma sample obtained in the presence/absence of heparin from healthy rats was performed to determine whether its presence is fully compatible with the peptidomic protocol proposed. The entire protocol was tested in a pilot experiment to compare the peptidome of healthy or heparin-anticoagulated rats subjected to hemorrhagic shock.

Factores de riesgo a enfermedad crítica en niños con influenza AH1N1 en Hospitales de EsSalud 2009-2010. Lima-Perú / Risk factors for critical illness in children with influenza AH1N1 in hospitals of EsSalud 2009-2010. Lima-Peru

Introducción: En 2009, la pandemia por Influenza A H1N1 causó una morbilidad significativa y mortalidad en todo el mundo. Objetivos. Determinar los factores de riesgo a enfermedad crítica en niños con Influenza AH1N1 en Hospitales de EsSalud. Población y métodos: Se Revisaron todas las Historias Clínicas de casos confirmados por Influenza AH1N1 de niños de 0-14 años internados en 03 centros desde el 1/6/09 al 31/7/10 en un estudio de cohorte retrospectivo. El diagnóstico viral se confirmó por método RT-PCR. Se realizó un análisis descriptivo de las variables continuas o discretas, también un análisis bivariado entre los casos de Enfermedad crítica y los que no tenían Enfermedad crítica con Influenza AH1N1, mediante el t de students o chi al cuadrado. Resultados: Número total de casos: 51; 59 por ciento Mujeres, contacto previo con personas con sintomatología respiratoria 68.63 por ciento, síntomas más frecuentes: Fiebre 100 por ciento, Tos 92 por ciento. 19.6 por ciento se internaron en la UCIP. La letalidad encontrada en el estudio fue de 9.8 por ciento. Rango de 2-5 y 5-14 años con RR 0.11, RR 0.04 y p=0.019, p=0.02 respectivamente en relación menores de 2 años. Enfermedad Neurológica crónica RR 5.12 (p=0.046). Desnutrición Crónica RR 22.28 (p=0.009). Días de tratamiento con Oseltamivir y Días de Estancia Hospitalaria tuvieron RR de 1.79 y 1.20, así como p=0.041 y p=0.002 respectivamente. Conclusiones: La Enfermedad Neurológica crónica, Desnutrición Crónica y menores de 2 años son los factores de riesgo para desarrollar Enfermedad Critica por Influenza AH1N1.

Introduction: In 2009, the pandemic influenza A H1N1 caused significant morbidity and mortality worldwide. Objectives: Identify risk factors critical illness in children with influenza AHIN1 EsSalud Hospital. Population and methods: We identified all medical records of confirmed cases of Influenza AH1N1 hospitalized in 03 centers and included all confirmed cases of 0-14 years from 1/6/09 to 31/7/10 in a retrospective cohort study. The viral diagnosis was confirmed by RT-PCR method. We performed a descriptive analysis of continuous or discrete variables, also a bivariate analysis between the cases of critical illness and those without Critical illness with Influenza AH1N1, by Student t test or chi2. Results: Total cases: 51, 59 per cent female, previous contact with persons with respiratory symptoms 68.63 per cent, most common symptoms: fever 100 per cent, cough 92 per cent. 19.6 per cent were admitted to the PICU. The mortality found in the study was 9.8 per cent. Range of 2-5 and 5-14 years RR 0.11, RR 0.04 and p=0.019, p=0.02 respectively on children under 2 years. Chronic neurological disease RR 5.12 (p=0.046). Chronic malnutrition RR 22.28 (p=0.009). Days oftreatment with oseltamivir and hospital stay were 1.79 and 1.20 RR and p=0.041 and p=0.002 respectively. Conclusions: Chronic Neurological Disease, Chronic Malnutrition and children under 2 years are risk factors for developing Critical Illness Influenza AH1N1.