Pathological pulmonary vascular remodeling is induced by type V collagen in a model of scleroderma.

OBJECTIVE: The pulmonary vascular remodeling in systemic sclerosis (SSc) is poorly understood and animal models are lacking. Type V collagen (COLV) is elevated in SSc and is implicated in the pathogenesis, and immunization with human COLV induces SSc-like skin and lung changes in rabbits and mice. Here we tested the hypothesis that COLV immunization will induce pathological and functional changes that phenocopy SSc-associated pulmonary vascular disease. METHODS: Pulmonary vascular changes in rabbits immunized with human COLV were extensively characterized by a combination of histology, electron microscopy and immunohistochemistry. Physiologic changes induced by COLV in explanted pulmonary artery rings were evaluated. The pattern of histopathologic alterations and gene expression induced in immunized rabbits were compared to those in SSc patients. RESULTS: COLV immunization was accompanied by striking pulmonary vascular abnormalities, characterized by reduced capillary density, perivascular inflammation, endothelial cell injury and collagen accumulation, that closely phenocopy changes seen in SSc patients. Moreover, pulmonary arteries from immunized rabbits showed impaired ex vivo vascular relaxation. Expression of COL5A2 was significantly increased in the lungs from immunized rabbits (p = 0.02), as well as in patients with SSc (P = 0.02). CONCLUSION: COLV immunity in rabbits is associated with marked vascular remodeling in the lung that phenocopies early-stage human SSc-associated pulmonary vascular disease. COLV immunization therefore represents a novel approach to model SSc pulmonary vascular pathology. Moreover, our findings suggest that COLV might represent a novel pathogenic autoantigen in SSc and future studies with the present model should be developed for possible association with PAH.

Endotoxin tolerance modulates TREG and TH17 lymphocytes protecting septic mice.

Background: Tolerance induces a regulated immune response to infection. We hypothesized that tolerance induction modulated profile of T regulatory cell (Treg) and T lymphocyte 17 (Th17) cells and is related cytokine released in septic animals. Methods: Male black C57/6 mice received subcutaneous (s.c.) injections of lipopolysaccharide (LPS) (1 mg/kg) for 5 days, on day 8th was made cecal ligation and puncture (CLP). Blood and spleen tissue were collected for cell analysis and cytokines measurements. Results: Cytokines (interleukin 2 (IL-2), interleukin (IL-6), transforming growth factor ß (TGF-ß) and interferon γ (INF-γ)) related to Treg and Th17 stimulation were elevated in the spleen of tolerant animals compared to sham. Treg and Th17 lymphocytes showed an increased amount in blood (Treg: 920 ± 84 cells vs. 1946 ± 65 cells, sham vs. tolerant; Th17:38321± 1954 cells vs. 43526 ± 7623 cells, sham vs. tolerant) and spleen (Treg: 5947 ± 273 cells vs. 16521 ± 486 cells, sham vs. tolerant; Th17: 26543 ± 2944 cells vs. 64567 ± 5523 cells, sham vs. tolerant). Treg (135±23 cells) and Th17 (1590 ± 256 cells) cells were reduced in blood of septic animals compared to sham, while CLP tolerant animals presented an increasing number of these cells. Lymphocyte Th17IL6+ were elevated in tolerant and CLP tolerant animals in the blood compared to sham. Conclusion: LPS tolerance was associated with increasing population of Treg and Th17. LPS tolerance reduces the hyper inflammatory response with immunoregulation exerted by Treg and Th17 cells protecting from septic damage.

Influence of Body Mass Index on Inflammatory Profile at Admission in Critically Ill Septic Patients.

Introduction. Inflammation is ubiquitous during sepsis and may be influenced by body mass index (BMI). We sought to evaluate if BMI was associated with serum levels of several cytokines measured at intensive care unit admission due to sepsis. Methods. 33 septic patients were included. An array of thirty-two cytokines and chemokines was measured using Milliplex technology. We assessed the association between cytokine levels and BMI by generalized additive model that also included illness severity (measured by SAPS 3 score); one model was built for each cytokine measured. Results. We found that levels of epidermal growth factor, vascular endothelial growth factor, and interleukins 4, 5, and 13 were associated with BMI in a complex, nonlinear way, independently of illness severity. Higher BMI was associated with higher levels of anti-inflammatory interleukins. Conclusion. BMI may influence host response to infection during critical illness. Larger studies should confirm these findings.

Pro-atherosclerotic markers and cardiovascular risk factors one year after liver transplantation.

AIM: To investigate pro-atherosclerotic markers (endothelial dysfunction and inflammation) in patients one year after liver transplantation. METHODS: Forty-four consecutive liver transplant (LT) outpatients who were admitted between August 2009 and July 2010, were followed-up by for 1 year, exhibited no evidences of infection or rejection, all of them underwent tacrolimus-based immunosuppressive regimens were consecutively enrolled. Inflammatory cytokines (TNFα, IFNγ, IL-8, and IL-10), endothelial biomarkers (sVCAM-1, sICAM-1, MPO, adiponectin, PAI-1, SAP, SAA, E-selectin, and MMP-9), high sensitive C-reactive protein, and Framingham risk score (FRS) were assessed. The anthropometric data, aminotransferases, metabolic syndrome features, glucose and lipid profiles, and insulin resistance data were also collected. The LT recipients were compared to 22 biopsy-proven non-alcoholic steatohepatitis (NASH) patients and 20 healthy controls (non-obese, non-diabetics, and non-dyslipidemic). RESULTS: The LT recipients had significantly younger ages and lower body mass indices, aminotransferases, fasting glucose and insulin levels, glucose homeostasis model and metabolic syndrome features than the NASH patients. Classic cardiovascular risk markers, such as Hs-CRP and FRS [2.0 (1.0-8.75)], were lower in the LT patients compared to those observed in the NASH patients (P = 0.009). In contrast, the LT recipients and NASH patients had similar inflammatory and endothelial serum markers compared to the controls (pg/mL): lower IL-10 levels (32.3 and 32.3 vs 62.5, respectively, P = 0.019) and higher IFNγ (626.1 and 411.9 vs 67.9, respectively, P < 0.001), E-selectin (48.5 and 90.03 vs 35.7, respectively, P < 0.001), sVCAM-1 (1820.6 and 1692.4 vs 1167.2, respectively, P < 0.001), and sICAM-1 (230.3 and 259.7 vs 152.9, respectively, P = 0.015) levels. CONCLUSION: Non-obese LT recipients have similar pro-atherosclerotic serum profiles after a short 1-year follow-up period compared to NASH patients, suggesting a high risk of atherosclerosis in this population.

Relationship between acid-base status and inflammation in the critically ill.

INTRODUCTION: There is a complex interplay between changes in acid-base components and inflammation. This manuscript aims to explore associations between plasma cytokines and chemokines and acid-base status on admission to intensive care. METHODS: We conducted a prospective cohort study in a 13-bed ICU in a tertiary-care center in Brazil. 87 unselected patients admitted to the ICU during a 2-year period were included. We measured multiple inflammatory mediators in plasma using multiplex assays and evaluated the association between mediator concentrations and acid-base variables using a variety of statistical modeling approaches, including generalized linear models, multiadaptive regression splines and principal component analysis. RESULTS: We found a positive association between strong ion gap (SIG) and plasma concentrations of interleukin (IL)6, 8, 10 and tumor necrosis factor (TNF); whereas albumin was negatively associated with IL6, IL7, IL8, IL10, TNF and interferon (IFN)α. Apparent strong ion difference (SIDa) was negatively associated with IL10 and IL17. A principal component analysis including SAPS 3 indicated that the association between acid-base components and inflammatory status was largely independent of illness severity, with both increased SIG and decreased SIDa (both drivers of acidosis) associated with increased inflammation. CONCLUSION: Acid-base variables (especially increased SIG, decreased albumin and decreased SIDa) on admission to ICU are associated with immunological activation. These findings should encourage new research into the effects of acid-base status on inflammation.

Small interfering RNA targeting focal adhesion kinase prevents cardiac dysfunction in endotoxemia.

Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.

Pro- and anti-inflammatory cytokines in steatosis and steatohepatitis.

BACKGROUND: Fatty liver disease is a problem in both bariatric patients and in patients with moderate obesity. Tumor necrosis factor (TNF)-alpha has been frequently measured in nonalcoholic steatohepatitis (NASH) with or without diabetes, but less is known about interleukin (IL)-6 and IL-10. METHODS: Moderately obese patients (n = 80) with histologically proven steatosis (n = 29) and NASH (n = 51) were recruited. Serum levels of cytokines were documented along with clinical information. The aim was to identify the correlates of such biomolecules in a stable population. RESULTS: Diabetes tended to be more associated with NASH (52.5% instead of 41.4%, P = 0.015), with no difference of age, gender, or body mass index regarding steatosis. For the entire population, cytokine changes were not significant, including TNF-alpha and IL-6. In diabetics only, all markers tended to diminish with NASH, especially IL-10 (P = 0.000). IL-10 correlated with homeostatic model assessment index (P = 0.000) and other variables of glucose homeostasis in diabetes, thus representing a major marker of the disease. CONCLUSIONS: (1) Generally inconsistent changes in pro- and anti-inflammatory cytokines occurred when NASH was globally compared to steatosis. (2) In contrast, downregulation of IL-6 and IL-10 was perceived in diabetics with NASH. (3) Arterial hypertension did not play a role in these circumstances. (4) IL-10 maintained strong correlations with glucose metabolism indices. (5) TNF-alpha could not be incriminated for progressive liver damage, as values failed to increase in NASH. (6) Investigations of IL-10 and other counterregulatory cytokines are lacking in this context and deserve further studies.

Increased gastric cytokine production after Roux-en-Y gastric bypass for morbid obesity.

HYPOTHESIS: Mucosal cytokines may be involved in the process of gastric bacterial contamination that may occur after Roux-en-Y bypass for morbid obesity in both gastric chambers, with inflammation and gastritis mostly in the excluded stomach. DESIGN: A prospective observational study in a homogeneous population with nonspecific complaints. SETTING: Outpatient clinic of a large, public, academic hospital. PATIENTS: Subjects (n = 37; 26 [70.3%] female; mean +/- SD age, 42.4 +/- 9.9 years) seen a mean +/- SD of 7.3 +/- 1.4 years after Roux-en-Y gastric bypass and nonoperated on morbidly obese control subjects (n = 10; 7 [70%] female; mean +/- SD age, 44.0 +/- 8.9 years). INTERVENTION: Enteroscopy was performed to collect samples for cytokine assays and bacteriologic studies. MAIN OUTCOME MEASURES: Concentrations of tumor necrosis factor alpha and transforming growth factor beta in the gastric mucosa of both chambers in patients undergoing Roux-en-Y gastric bypass and correlation with bacterial overgrowth and Helicobacter pylori infection. RESULTS: High microbial counts (>10(5) colony-forming units per milliliter) were detected in 22 (59.5%) and 7 (18.9%) of the 37 samples from the functional pouch and excluded reservoir, respectively; and H pylori investigation was positive in 6 of 37 samples (16.2%). The tumor necrosis factor alpha concentration (mean +/- SD, 2.1 +/- 1.9 pg/g of protein) and the transforming growth factor beta concentration (mean +/- SD, 24.2 +/- 12.8 pg/g of protein) in the excluded stomach, but not in the proximal pouch, were elevated with regard to the corpus or antrum of controls, and correlation with bacterial overgrowth and with H pylori infection was demonstrated. CONCLUSION: Overexpression of tumor necrosis factor alpha and transforming growth factor beta occurred in the distal stomach, positive cytokine correlation with microbial invasion by H pylori and nonspecific germs was seen, and further studies addressing phenotypic and genotypic changes of gastric mucosa are recommended.

Microbial flora of the stomach after gastric bypass for morbid obesity.

BACKGROUND: The normal stomach is virtually sterile but the effect of Roux-en-Y gastric bypass (RYGBP) on bacterial flora in the used (very small proximal pouch) and unused (large bypassed) gastric chambers is not known. In a prospective study, this variable was documented. METHODS: Bariatric subjects (n=37) were submitted to endoscopic examination of both gastric reservoirs via FUJINON enteroscope model EN-450P5, 7.3 +/- 1.4 years after RYGBP. Age was 42.4 +/- 9.9 years (70.2% females), preoperative BMI was 53.5 +/- 10.6, and current BMI was 32.6 +/- 7.8 kg/m2. Methods included quantitative culture of gastric secretion along with gastric pH and lactulose/hydrogen breath test. RESULTS: None of the subjects displayed diarrhea, malabsorption or other complaints suggestive of GI bacterial overgrowth. Elevated counts of bacteria and fungi were identified in both chambers, with predominance of aerobes and anaerobes, but not molds and yeasts, in the proximal stomach. Gram-positive cocci, bacilli and coccobacilli represented the majority of the isolates. Gastric pH was neutral (pH 7.0 +/- 0.2) in the proximal pouch, whereas the distal chamber mostly but not always conserved the expected acidity (pH 3.3 +/- 2.2, P<0.001). The breath test for bacterial overgrowth was positive in 40.5% of the population. CONCLUSIONS: 1) Frequent colonization of both gastric chambers was detected; 2) Aerobes, anaerobes and fungi were represented in both situations; 3) Gastric pH as well as bacterial count was higher in the functioning proximal stomach; 4) Breath test was positive in 40.5% of the subjects; 5) Clinical manifestation such as diarrhea, malabsorption or pneumonia were not demonstrated; 6) Further histologic and microbiologic studies of both the stomach and the small bowel are recommended.

Systemic inflammation in morbidly obese subjects: response to oral supplementation with alpha-linolenic acid.

BACKGROUND: Morbidly obese patients frequently display asymptomatic chronic activation of acute phase response, with potentially adverse metabolic and cardiovascular consequences. Nutritional preparations to improve this phenomenon have rarely been administered. Aiming to investigate the supplementation of flaxseed flour, a source of omega-3 fatty acids, a prospective randomized double-blind cross-over study was designed. METHODS: Outpatient obese subjects (n=41) were clinically and biochemically screened, and results for 24 randomized subjects are shown. Age was 40.8 +/- 11.6 years (83.3% females) and body mass index (BMI) was 47.1 +/- 7.2 kg/m2. Flaxseed flour (Farinha de Linhaca Dourada LinoLive, Cisbra, Brazil) in the amount of 30 g/day (5 g of alpha-linolenic acid - omega-3) and an equal mass of placebo (manioc flour) were administered for 2 weeks each. Variables included general biochemical investigation, white blood cell count (WBC), C-reactive protein (CRP), serum amyloid A (SAA) and fibronectin. RESULTS: No intolerance was registered. Body weight and general biochemical indices remained stable. Initial CRP and SAA were elevated (13.7 +/- 9.9 and 17.4 +/- 8.0 ). WBC (8100 +/- 2100/mm3) and fibronectin (463.2 +/- 61.3 mg/dL) were acceptable but in the upper normal range. Corresponding findings after supplementation of flaxseed were 10.6 +/- 6.2 mg/L, 14.3 +/- 9.2 mg/L, 7300 +/- 1800/mm3 and 412.8 +/- 38.6 respectively (P<0.05). No change during the control period regarding baseline occurred when placebo was randomized to be given first; however, when it followed omega-3 supplementation, CRP and SAA recovered, whereas WBC and fibronection remained depressed during those 2 weeks (7500 +/- 2100/mm3 and 393.2 +/- 75.8 mg/dL, P<0.05). CONCLUSIONS: 1) Various inflammatory markers were elevated in the studied population, although not necessarily exceeding the normal range; 2) Significant reduction could be demonstrated; 3) Some persistent effects of flaxseed supplement 2 weeks after discontinuation were observed.

Nitrated lipids decompose to nitric oxide and lipid radicals and cause vasorelaxation.

Nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite have been receiving increasing attention as mediators of nitric oxide toxicity. Indeed, nitrated and nitrosated compounds have been detected in biological fluids and tissues of healthy subjects and in higher yields in patients under inflammatory or infectious conditions as a consequence of nitric oxide overproduction. Among them, nitrated lipids have been detected in vivo. Here, we confirmed and extended previous studies by demonstrating that nitrolinoleate, chlolesteryl nitrolinoleate, and nitrohydroxylinoleate induce vasorelaxation in a concentration-dependent manner while releasing nitric oxide that was characterized by chemiluminescence-and EPR-based methodologies. As we first show here, diffusible nitric oxide production is likely to occur by isomerization of the nitrated lipids to the corresponding nitrite derivatives that decay through homolysis and/or metal ion/ascorbate-assisted reduction. The homolytic mechanism was supported by EPR spin-trapping studies with 3,5-dibromo-4-nitrosobenzenesulfonic acid that trapped a lipid-derived radical during nitrolinoleate decomposition. In addition to provide a mechanism to explain nitric oxide production from nitrated lipids, the results support their role as endogenous sources of nitric oxide that may play a role in endothelium-independent vasorelaxation.