Minced skin grafts for chronic wounds compared to conventional mesh grafts.

Background and Aims: Skin grafting is the single most effective method to close a chronic wound. The current standard of care is to use meshed split thickness skin grafts. This entails the use of surgical instruments that need to be autoclaved and to have a power source, which usually requires an OR facility. The minced skin technique uses single use, presterilized instruments and the procedure can be done under local anesthesia, by a wound care practitioner, in a wound clinic, a physician's office or even at the bedside. The current study was designed to determine if the results from micrografting were non inferior to conventional mesh grafting. Methods: In a prospective non inferiority study, 26 chronic ulcers were treated with micrografting (MSG) and 24 with conventional mesh grafts 1:3 (control group-CG) in a total of 21 patients, 10 male and 11 female. The donor site areas in the MSG group were predetermined to 2.5 × 5 cm and the mesh grafts expansion was set at 1:3. Results: In the first weeks postoperatively, micrograft healing initially lagged behind the conventional mesh grafts but at 60 days after grafting, all MSG wounds were healed. The MSG wounds had better pigmentation, less itching, and less scarring. The micrografting procedure was easy to learn and expeditious to perform. The MSG mean expansion was 9.1 compared to three times (CG). Conclusion: The MSG procedure is not inferior to conventional mesh grafting, requires smaller donor sites, and can be done with single use instruments, under local anesthesia, with early discharge.

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.

Bone Marrow Cells Transplant in Septic Mice Modulates Systemic Inflammatory Response via Cell-Cell Contact.

Sepsis is a dynamic disease, displaying an inflammatory profile that varies over time and for each organ. Controlling the inflammatory response based in targeting a single molecule has been proved useless. We hypothesized that treatment with bone marrow-derived mononuclear cells (BMDMCs) may be more efficient to modulate the systemic inflammatory response to infection. Adult male Balb/c mice were subjected to cecal ligation and puncture (CLP) or endotoxemia model of experimental sepsis. BMDMCs were separated under Ficoll gradient and injected intravenously 1 h after the procedures. Cytokines concentration was quantified in plasma, lungs, heart, and gut. Spleens, lymph nodes, and thymus were used for lymphocytes isolation and cell death assessment. All measurements were performed 2 h after BMDMCs injection. RAW264.7 macrophages and BMDMCs were cocultivated in vitro to investigate the mechanisms involved. Our data showed that an early single intravenous injection of BMDMCs in animals submitted to the murine model of endotoxemia led to the improvement of survival rate; BMDMCs persistency in lung, liver, and spleen after 24 h; decreased necrosis and apoptosis of mononuclear cells; lower TNF-α, but increased IL-10 concentration in plasma; and tissue-specific cytokine profile. In vitro experiments demonstrated that IL-6, IL-10, and nitric oxide production depends on direct contact of BMDMCs to macrophages and that TNF-α production is negatively regulated by PGE2. BMDMCs are efficient in protecting animals from endotoxemia and sepsis, reducing systemic inflammation as well as specifically modulating tissue inflammation, producing the necessary immune regulation to re-equilibrate the inflammatory response.

A mathematical model relates intracellular TLR4 oscillations to sepsis progression.

OBJECTIVE: Oscillations of physiological parameters describe many biological processes and their modulation is determinant for various pathologies. In sepsis, toll-like receptor 4 (TLR4) is a key sensor for signaling the presence of Gram-negative bacteria. Its intracellular trafficking rates shift the equilibrium between the pro- and anti-inflammatory downstream signaling cascades, leading to either the physiological resolution of the bacterial stimulation or to sepsis. This study aimed to evaluate the effects of TLR4 increased expression and intracellular trafficking on the course and outcome of sepsis. RESULTS: Using a set of three differential equations, we defined the TLR4 fluxes between relevant cell organelles. We obtained three different regions in the phase space: (1) a limit-cycle describing unstimulated physiological oscillations, (2) a fixed-point attractor resulting from moderate LPS stimulation that is resolved and (3) a double-attractor resulting from sustained LPS stimulation that leads to sepsis. We used this model to describe available hospital data of sepsis patients and we correctly characterize the clinical outcome of these patients.

Hydrogen sulfide modulates chromatin remodeling and inflammatory mediator production in response to endotoxin, but does not play a role in the development of endotoxin tolerance.

BACKGROUND: Pretreatment with low doses of LPS (lipopolysaccharide, bacterial endotoxin) reduces the pro-inflammatory response to a subsequent higher LPS dose, a phenomenon known as endotoxin tolerance. Moreover, hydrogen sulfide (H2S), an endogenous gaseous mediator (gasotransmitter) can exert anti-inflammatory effects. Here we investigated the potential role of H2S in the development of LPS tolerance. THP1 differentiated macrophages were pretreated with the H2S donor NaHS (1 mM) or the H2S biosynthesis inhibitor aminooxyacetic acid (AOAA, 1 mM). METHODS: To induce tolerance, cells were treated with a low concentration of LPS (0.5 µg/ml) for 4 or 24 h, and then treated with a high concentration of LPS (1 µg/ml) for 4 h or 24 h. In in vivo studies, male wild-type and CSE(-/-) mice were randomized to the following groups: Control (vehicle); Endotoxemic saline for 3 days before the induction of endotoxemia with 10 mg/kg LPS) mg/kg; Tolerant (LPS at 1 mg/kg for 3 days, followed LPS at 10 mg/kg). Animals were sacrificed after 4 or 12 h; plasma IL-6 and TNF-α levels were measured. Changes in histone H3 and H4 acetylation were analyzed by Western blotting. RESULTS: LPS tolerance decreased pro-inflammatory cytokine production. AOAA did not affect the effect of tolerance on reducing cytokine production. Treatment of the cells with the H2S donor reduced cytokine production. Induction of the tolerance increased the acetylation of H3; AOAA reduced histone acetylation. H2S donation increased histone acetylation. Tolerance did not affect the responses to H2S with respect to histone acetylation. CONCLUSIONS: In conclusion, both LPS tolerance and H2S donation decrease LPS-induced cytokine production in vitro and modulate histone acetylation. However, endogenous, CSE-derived H2S does not appear to play a significant role in the development of LPS tolerance.

Spontaneous hypothermia in human sepsis is a transient, self-limiting, and nonterminal response.

Hypothermia in sepsis is generally perceived as something dysregulated and progressive although there has been no assessment on the natural course of this phenomenon in humans. This was the first study on the dynamics of hypothermia in septic patients not subjected to active rewarming, and the results were surprising. A sample of 50 subjects presenting with spontaneous hypothermia during sepsis was drawn from the 2005-2012 database of an academic hospital. Hypothermia was defined as body temperature below 36.0°C for longer than 2 h, with at least one reading of 35.5°C or less. The patients presented with 138 episodes of hypothermia, 21 at the time of the sepsis diagnosis and 117 with a later onset. However, hypothermia was uncommon in the final 12 h of life of the patients that succumbed. The majority (97.1%) of the hypothermic episodes were transient and self-limited; the median recovery time was 6 h; body temperature rarely fell below 34.0°C. Bidirectional oscillations in body temperature were evident in the course of hypothermia. Nearly half of the hypothermic episodes had onset in the absence of shock or respiratory distress, and the incidence of hypothermia was not increased during either of these conditions. Usage of antipyretic drugs, sedatives, neuroleptics, or other medications did not predict the onset of hypothermia. In conclusion, hypothermia appears to be a predominantly transient, self-limiting, and nonterminal phenomenon that is inherent to human sepsis. These characteristics resemble those of the regulated hypothermia shown to replace fever in animal models of severe systemic inflammation.

Hydrogen sulfide attenuates cytokine production through the modulation of chromatin remodeling.

Hydrogen sulfide (H2S) is an endogenous gaseous biological mediator, which regulates, among others, the oxidative balance of cells under normal physiological conditions, as well as in various diseases. Several previous studies have reported that H2S attenuates inflammatory mediator production. In this study, we investigated the role of H2S in chromatin modulation in an in vitro model of lipopolysaccharide (LPS)-induced inflammation and evaluated its effects on inflammatory cytokine production. Tamm-Horsfall protein 1 (THP-1) differentiated macrophages were pre-treated with sodium hydrosulfide (NaHS) (an H2S donor) at 0.01, 0.1, 0.5 or 1 mM for 30 min. To stimulate cytokine production, the cells were challenged with bacterial LPS (1 µg/ml) for 1, 4, 8 or 24 h. Histone H3 acetylation was analyzed by chromatin immunoprecipitation (ChIP), cytokine production was measured by ELISA and histone deacetylase (HDAC) activity was analyzed using a standard biochemical assay. H2S inhibited the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in a concentration-dependent manner; it was most effective at the two highest concentrations used. This effect was associated with a decrease in histone H3 acetylation at the IL-6 and TNF-α promoters in the cells exposed to H2S or H2S + LPS. The findings of the present study suggest that H2S suppresses histone acetylation, which, in turn, inhibits chromatin openness, leading to a decrease in the gene transcription of various pro-inflammatory cytokines. Therefore, this mechanism may contribute to the previously demonstrated anti-inflammatory effects of H2S and various H2S donors.

Endotoxaemic myocardial dysfunction: the role of coronary driving pressure in subendocardial perfusion.

OBJECTIVE: To investigate the role of coronary driving pressure (CDP) in myocardial microcirculatory blood flow during sepsis. We hypothesised that in septic shock there is an impaired autoregulation of microcirculation, and blood flow is totally dependent on CDP. We analysed the effect of lipopolysaccharide (LPS)-induced shock on myocardial microcirculation, separating subendocardial and epicardial areas. We then studied the effect of CDP increases using noradrenaline (NOR) or metaraminol (Aramine [ARA]) on myocardial microcirculation and function, and we analysed the effect of volume infusion on CDP and myocardial function. DESIGN AND SETTING: Endotoxaemia was induced in male Wistar rats by an intraperitoneal injection of LPS 10 mg/kg. Animals were divided into a control (CT) group, an LPS-injected group, and an LPS-injected group treated with saline fluid, NOR or ARA. MAIN OUTCOME MEASURES: Ninety minutes later, a haemodynamic evaluation was performed. NOR or ARA were used to manage the mean arterial pressure (MAP) and CDP, and we inserted a catheter into the left ventricle to measure cardiac parameters. To measure blood flow in the myocardium and other organs, microspheres were introduced into the left ventricle using an infusion pump. RESULTS: After LPS treatment, left ventricular (LV) systolic function (dP/dt max) and diastolic function (dP/dt min) decreased by 34% and 15%, respectively, and load-independent indices (LV contractility in ejection phase and dP/dt max÷end-diastolic volume) were reduced. The CDP was also reduced (by 58%) in the endotoxaemic rats. Myocardial blood flow was reduced (by 80%) in animals with an MAP≤65 mmHg. NOR increased the CDP (LPS, 38 mmHg [SEM, 2 mmHg]; LPS+NOR, 59 mmHg [SEM, 3 mmHg]) and microcirculatory perfusion (LPS, 2 mL/min/g tissue [SEM, 0.6 mL/min/g]; LPS+NOR, 6.2 mL/min/g [SEM, 0.8 mL/min/g]). ARA was also effective in improve microcirculation but saline volume infusion was ineffective in improving CDP or myocardial function. CDP showed a significant correlation with subendocardial blood flow. CONCLUSIONS: Myocardial blood flow in the LV subendocardium and the right ventricle decreases in endotoxaemic rats. Increasing CDP improves myocardial blood flow and function. Thus, in endotoxaemia, microcirculatory blood flow is pressure dependent, suggesting that it may be beneficial to treat patients with sepsis using a higher CDP.

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.

Coadministration of branched-chain amino acids and lipopolysaccharide causes matrix metalloproteinase activation and blood-brain barrier breakdown.

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a severe deficiency in the activity of the branched-chain α-keto acid dehydrogenase complex, leading to accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine, and valine. Infections have a significant role in precipitating acute metabolic decompensation in patients with MSUD; however, the mechanisms underlying the neurotoxicity in this disorder are poorly understood. In this study, we subjected rats to the coadministration of lipopolysaccharide (LPS), which is a major component of gram-negative bacteria cell walls, and high concentrations of BCAA (H-BCAA) to determine their effects on the permeability of the blood-brain barrier (BBB) and on the levels of matrix metalloproteinases (MMP-2 and MMP-9). Our results demonstrated that the coadministration of H-BCAA and LPS causes breakdown of the BBB and increases the levels of MMP-2 and MMP-9 in the hippocampus of these rats. On the other hand, examination of the cerebral cortex of the 10- and 30-day-old rats revealed a significant difference in Evan's Blue content after coadministration of H-BCAA and LPS, as MMP-9 levels only increased in the cerebral cortex of the 10-day-old rats. In conclusion, these results suggest that the inflammatory process associated with high levels of BCAA causes BBB breakdown. Thus, we suggest that BBB breakdown is relevant to the perpetuation of brain inflammation and may be related to the brain dysfunction observed in MSUD patients.

Omega-6 polyunsaturated fatty acids prevent atherosclerosis development in LDLr-KO mice, in spite of displaying a pro-inflammatory profile similar to trans fatty acids.

The development of atherosclerosis and the inflammatory response were investigated in LDLr-KO mice on three high-fat diets (40% energy as fat) for 16 weeks: trans (TRANS), saturated (SAFA) or ω-6 polyunsaturated (PUFA) fats. The following parameters were measured: plasma lipids, aortic root total cholesterol (TC), lesion area (Oil Red-O), ABCA1 content and macrophage infiltration (immunohistochemistry), collagen content (Picrosirius-red) and co-localization of ABCA1 and macrophage (confocal microscopy) besides the plasma inflammatory markers (IL-6, TNF-α) and the macrophage inflammatory response to lipopolysaccharide from Escherichia coli (LPS). As expected, plasma TC and TG concentrations were lower on the PUFA diet than on TRANS or SAFA diets. Aortic intima macrophage infiltration, ABCA1 content, and lesion area on PUFA group were lower compared to TRANS and SAFA groups. Macrophages and ABCA1 markers did not co-localize in the atherosclerotic plaque, suggesting that different cell types were responsible for the ABCA1 expression in plaques. Compared to PUFA, TRANS and SAFA presented higher collagen content and necrotic cores in atherosclerotic plaques. In the artery wall, TC was lower on PUFA compared to TRANS group; free cholesterol was lower on PUFA compared to TRANS and SAFA; cholesteryl ester concentration did not vary amongst the groups. Plasma TNF-α concentration on PUFA and TRANS-fed mice was higher compared to SAFA. No difference was observed in IL-6 concentration amongst groups. Regarding the macrophage inflammatory response to LPS, TRANS and PUFA presented higher culture medium concentrations of IL-6 and TNF-α as compared to SAFA. The PUFA group showed the lowest amount of the anti-inflammatory marker IL-10 compared to TRANS and SAFA groups. In conclusion, PUFA intake prevented atherogenesis, even in a pro-inflammatory condition.

Dyslipidemia: a prospective controlled randomized trial of intensive glycemic control in sepsis.

PURPOSE: Metabolic disturbances are quite common in critically ill patients. Glycemic control appears to be an important adjuvant therapy in such patients. In addition, disorders of lipid metabolism are associated with worse prognoses. The purpose of this study was to investigate the effects that two different glycemic control protocols have on lipid profile and metabolism. METHODS: We evaluated 63 patients hospitalized for severe sepsis or septic shock, over the first 72 h of intensive care. Patients were randomly allocated to receive conservative glycemic control (target range 140­180 mg/dl) or intensive glycemic control (target range 80­110 mg/dl). Serum levels of lowdensity lipoprotein, high-density lipoprotein, triglycerides, total cholesterol, free fatty acids, and oxidized low-density lipoprotein were determined. RESULTS: In both groups, serum levels of low-density lipoprotein, high-density lipoprotein, and total cholesterol were below normal, whereas those of free fatty acids, triglycerides, and oxidized lowdensity lipoprotein were above normal. At 4 h after admission, free fatty acid levels were higher in the conservative group than in the intensive group, progressively decreasing in both groups until hour 48 and continuing to decrease until hour 72 only in the intensive group. Oxidized low-density lipoprotein levels were elevated in both groups throughout the study period. CONCLUSIONS: Free fatty acids respond to intensive glycemic control and, because of their high toxicity, can be a therapeutic target in patients with sepsis.

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.

Endotoxin tolerance: selective alterations in gene expression and protection against lymphocyte death.

Extensive lymphocyte apoptosis may be an important cause of immune suppression in sepsis. Here we investigated the effect of LPS tolerance on lymphocyte apoptosis in an experimental model of polymicrobial infection. Tolerance was induced by the injection of lipopolysaccharide (1.0mg/kg/subcutaneously) once a day for 5 days. Macroarray analysis of mRNA isolated from T-(CD4) lymphocytes was used to identify genes that are differentially expressed during LPS tolerance. In addition, assessment of the expression of apoptosis-associated lymphocyte gene products and apoptotic events was performed on the 8th day; 6h after the terminal challenge with polymicrobial infection or high-dose LPS administration. Survival studies with polymicrobial infection were also conducted. LPS tolerance induced a broad reprogramming of cell death pathways, including a suppression of receptor-mediated and mitochondrial apoptotic pathways, inflammatory caspases, alternate apoptotic pathways, as well as reduced expression of genes involved in necrosis. These alterations led to a marked resistance of lymphocytes against cell death during the subsequent period of sepsis. In addition, LPS tolerance produced an increased differentiation of T-lymphocytes to T(H)1 and T(H)2, with a T(H)1 differentiation predominance. Thus, in the current study we provide an evidence for a marked reprogramming of gene expression of multiple cell death pathways during LPS tolerance. These alterations may play a significant role in the observed protection of the animals from a subsequent lethal polymicrobial sepsis challenge.

Hypertonic saline reduces metalloproteinase expression in liver during pancreatitis.

1. We recently demonstrated that hypertonic saline reduces inflammation and mortality in acute pancreatitis. The present study investigated the effects of hypertonic saline in metalloproteinase (MMP) regulation and pancreatitis-associated hepatic injury. 2. Wistar rats were divided into four groups: (i) control, not subjected to insult or treatment; (ii) no treatment (NT), induction of pancreatitis (retrograde infusion of 2.5% sodium taurocholate (1.0 mL/kg)), but no further treatment; (iii) normal saline (NS), induction of pancreatitis and treatment with normal saline (0.9% NaCl, 34 mL/kg, i.v. bolus, 1 h after the induction of pancreatitis); and (iv) hypertonic saline (HS), induction of pancreatitis and treatment with hypertonic saline (7.5% NaCl, 4 mL/kg administered over a period of 5 min, 1 h after the induction of pancreatitis). In all four groups, 4, 12 and 24 h after the induction of pancreatitis, liver tissue samples were assayed to determine levels of MMP-2, MMP-9, 47 kDa heat shock protein (HSP47) and collagen (Type I and III). 3. Compared with the control group, MMP-9 expression and activity was increased twofold in the NS and NT groups 4 and 12 h after the induction of pancreatitis, but remained at basal levels in the HS group. In contrast, MMP-2 expression was increased twofold 12 h after the induction of pancreatitis only in the NS group, whereas the expression of HSP47 was increased 4 h after the induction of pancreatitis in the NS and NT groups. Greater extracellular matrix remodelling occurred in the NS and NT groups compared with the HS group, probably as a result of the hepatic wound-healing response to repeated injury. However, the collagen content in hepatic tissue remained at basal levels in the HS group. 4. In conclusion, the results of the present study indicate that hypertonic saline is hepatoprotective and reduces hepatic remodelling, maintaining the integrity of the hepatic extracellular matrix during pancreatitis. Hypertonic saline-mediated regulation of MMP expression may have clinical relevance in pancreatitis-associated liver injury.

Metabolic acidosis in patients with severe sepsis and septic shock: a longitudinal quantitative study.

OBJECTIVE: To describe the composition of metabolic acidosis in patients with severe sepsis and septic shock at intensive care unit admission and throughout the first 5 days of intensive care unit stay. DESIGN: Prospective, observational study. SETTING: Twelve-bed intensive care unit. PATIENTS: Sixty patients with either severe sepsis or septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Data were collected until 5 days after intensive care unit admission. We studied the contribution of inorganic ion difference, lactate, albumin, phosphate, and strong ion gap to metabolic acidosis. At admission, standard base excess was -6.69 +/- 4.19 mEq/L in survivors vs. -11.63 +/- 4.87 mEq/L in nonsurvivors (p < .05); inorganic ion difference (mainly resulting from hyperchloremia) was responsible for a decrease in standard base excess by 5.64 +/- 4.96 mEq/L in survivors vs. 8.94 +/- 7.06 mEq/L in nonsurvivors (p < .05); strong ion gap was responsible for a decrease in standard base excess by 4.07 +/- 3.57 mEq/L in survivors vs. 4.92 +/- 5.55 mEq/L in nonsurvivors with a nonsignificant probability value; and lactate was responsible for a decrease in standard base excess to 1.34 +/- 2.07 mEq/L in survivors vs. 1.61 +/- 2.25 mEq/L in nonsurvivors with a nonsignificant probability value. Albumin had an important alkalinizing effect in both groups; phosphate had a minimal acid-base effect. Acidosis in survivors was corrected during the study period as a result of a decrease in lactate and strong ion gap levels, whereas nonsurvivors did not correct their metabolic acidosis. In addition to Acute Physiology and Chronic Health Evaluation II score and serum creatinine level,inorganic ion difference acidosis magnitude at intensive care unit admission was independently associated with a worse outcome. CONCLUSIONS: Patients with severe sepsis and septic shock exhibit a complex metabolic acidosis at intensive care unit admission, caused predominantly by hyperchloremic acidosis,which was more pronounced in nonsurvivors. Acidosis resolution in survivors was attributable to a decrease in strong ion gap and lactate levels.

Effects of dobutamine on gut mucosal nitric oxide production during endotoxic shock in rabbits.

BACKGROUND: Dobutamine is the agent of choice for increasing cardiac output during myocardial depression in humans with septic shock. Studies have shown that beta-adrenoceptor agonists influence nitric oxide generation, probably by modulating cyclic adenosine monophosphate. We investigated the effects of dobutamine on the systemic and luminal gut release of nitric oxide during endotoxic shock in rabbits. MATERIAL/METHODS: Twenty anesthetized and ventilated New Zealand rabbits received placebo or intravenous lipopolysaccharide with or without dobutamine (5 micro g/kg/min). Ultrasonic flow probes placed around the superior mesenteric artery and the abdominal aorta continuously estimated the flow. A segment from the ileum was isolated and perfused, and serum nitrate/nitrite levels were measured in the perfusate solution and the serum every hour. RESULTS: The mean arterial pressure decreased with statistical significance in the lipopolysaccharide group but not in the lipopolysaccharide/dobutamine group. The abdominal aortic flow decreased statistically significantly after lipopolysaccharide administration in both groups but recovered to baseline in the lipopolysaccharide/dobutamine group. The flow in the superior mesenteric artery was statistically significantly higher in the lipopolysaccharide/dobutamine group than in the lipopolysaccharide group at 2 hours. The serum nitrate/nitrite levels were higher in the lipopolysaccharide group and lower in the lipopolysaccharide/dobutamine group than those in the control group. The gut luminal perfusate serum nitrate/nitrite level was higher in the lipopolysaccharide group than in the lipopolysaccharide/dobutamine group. CONCLUSIONS: Dobutamine can decrease total and intestinal nitric oxide production in vivo. Those effects seem to be inversely proportional to the changes in blood flow.

Prevention and reversion of nonalcoholic steatohepatitis in OB/OB mice by S-nitroso-N-acetylcysteine treatment.

OBJECTIVE: To evaluate the role oral administration of S-nitroso-N-acetylcysteine (SNAC), a NO donor drug, in the prevention and reversion of NASH in two different animal models. METHODS: NASH was induced in male ob/ob mice by methionine-choline deficient (MCD) and high-fat (H) diets. Two animal groups received or not SNAC orally for four weeks since the beginning of the treatment. Two other groups were submitted to MCD and H diets for 60 days receiving SNAC only from the 31(st) to the 60(th) day. RESULTS: SNAC administration inhibited the development of NASH in all groups, leading to a marked decrease in macro and microvacuolar steatosis and in hepatic lipid peroxidation in the MCD group. SNAC treatment reversed the development of NASH in animals treated for 60 days with MCD or H diets, which received SNAC only from the 31(st) to the 60(th) day. CONCLUSIONS: Oral administration of SNAC markedly inhibited and reversed NASH induced by MCD and H diets in ob/ob mice.