Sex Bias in Experimental Immune-Mediated,Drug-Induced Liver Injury in BALB/c Mice: Suggested Roles for Tregs,Estrogen, and IL-6

Background and Aims

Immune-mediated, drug-induced liver injury (DILI) triggered by drug haptens is more prevalent in women than in men. However, mechanisms responsible for this sex bias are not clear. Immune regulation by CD4+CD25+FoxP3+ regulatory T-cells (Tregs) and 17β-estradiol is crucial in the pathogenesis of sex bias in cancer and autoimmunity. Therefore, we investigated their role in a mouse model of immune-mediated DILI.

Methods

To model DILI, we immunized BALB/c, BALB/cBy, IL-6–deficient, and castrated BALB/c mice with trifluoroacetyl chloride-haptenated liver proteins. We then measured degree of hepatitis, cytokines, antibodies, and Treg and splenocyte function.

Results

BALB/c females developed more severe hepatitis (p<0.01) and produced more pro-inflammatory hepatic cytokines and antibodies (p<0.05) than did males. Castrated males developed more severe hepatitis than did intact males (p<0.001) and females (p<0.05). Splenocytes cultured from female mice exhibited fewer Tregs (p<0.01) and higher IL-1β (p<0.01) and IL-6 (p<0.05) than did those from males. However, Treg function did not differ by sex, as evidenced by absence of sex bias in programmed death receptor-1 and responses to IL-6, anti-IL-10, anti-CD3, and anti-CD28. Diminished hepatitis in IL-6-deficient, anti-IL-6 receptor α-treated, ovariectomized, or male mice; undetectable IL-6 levels in splenocyte supernatants from ovariectomized and male mice; elevated splenic IL-6 and serum estrogen levels in castrated male mice, and IL-6 induction by 17β-estradiol in splenocytes from naïve female mice (p<0.05) suggested that 17β-estradiol may enhance sex bias through IL-6 induction, which subsequently discourages Treg survival. Treg transfer from naïve female mice to those with DILI reduced hepatitis severity and hepatic IL-6.

Conclusions

17β-estradiol and IL-6 may act synergistically to promote sex bias in experimental DILI by reducing Tregs. Modulating Treg numbers may provide a therapeutic approach to DILI.     

Establishment of a Rat Model of Portal Vein Ligation Combined with In Situ Splitting

Background

Portal vein ligation (PVL) combined with in situ splitting (ISS) has been shown to induce remarkable liver regeneration in patients. The purpose of this study was to establish a model of PVL+ISS in rats for exploring the possible mechanisms of liver regeneration using these techniques.

Materials and Methods

Rats were randomly assigned to three experimental groups: selective PVL, selective PVL+ISS and sham operation. The hepatic regeneration rate (HRR), Ki-67, liver biochemical determinations and histopathology were assessed at 24, 48, and 72 h and 7 days after the operation. The microcirculation of the median lobes before and after ISS was examined by laser speckle contrast imaging. Meanwhile, cytokines such as TNF-α, IL-6, HGF and HSP70 in regenerating liver lobes at 24 h was investigated by RT-PCR and ELISA.

Results

The HRR of PVL+ISS was much higher than that of the PVL at 72 h and 7 days after surgery (p<0.01). The expression of Ki-67 in hepatocytes in the regenerating liver lobe was stronger in the PVL+ISS group than in the PVL group at 48 and 72 h (p<0.01). There was a significant reduction in microcirculation blood perfusion of the left median lobe before and after ISS. Liver biochemical determinations and histopathology demonstrated more severe hepatocyte injury in the PVL+ISS group. Both the mRNA levels of TNF-α and IL-6 and the protein levels of TNF-α, IL-6 and HGF in regenerating liver lobes were higher in the PVL+ISS than the PVL alone.

Conclusions

The higher HRR in the PVL+ISS compared with the PVL confirmed that we had successfully established a PVL+ISS model in rats. The possible mechanisms included the reduced microcirculation blood perfusion of the left median lobe and up-regulation of cytokines in the regenerating lobes after ISS.     

Inhibition of UII/UTR System Relieves Acute Inflammation of Liver through Preventing Activation of NF-κB Pathway in ALF Mice

Urotensin II (UII) is implicated in immune inflammatory diseases through its specific high-affinity UT receptor (UTR). Enhanced expression of UII/UTR was recently demonstrated in the liver with acute liver failure (ALF). Here, we analysed the relationship between UII/UTR expression and ALF in lipopolysaccharide (LPS)/D-galactosamine (GalN)-challenged mice. Thereafter, we investigated the effects produced by the inhibition of UII/UTR system using urantide, a special antagonist of UTR, and the potential molecular mechanisms involved in ALF. Urantide was administered to mice treated with LPS/GalN. Expression of UII/UTR, releases of proinflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interferon-γ (IFN-γ), and activation of nuclear factor κB (NF-κB) signaling pathway were assessed in the lethal ALF with or without urantide pretreatment. We found that LPS/GalN-challenged mice showed high mortality and marked hepatic inflammatory infiltration and cell apoptosis as well as a significant increase of UII/UTR expression. Urantide pretreatment protected against the injury in liver following downregulation of UII/UTR expression. A close relationship between the acutely flamed hepatic injury and UII/UTR expression was observed. In addition, urantide prevented the increases of proinflammatory cytokines such as TNF-α, IL-1β and IFN-γ, and activation of NF-κB signaling pathway induced by LPS/GalN in mice. Thus, we conclude that UII/UTR system plays a role in LPS/GalN-induced ALF. Urantide has a protective effect on the acutely inflamed injury of liver in part through preventing releases of proinflammatory cytokines and activation of NF-κB pathway.     

Hepatotoxicity in Mice of a Novel Anti-parasite Drug Candidate Hydroxymethylnitrofurazone: A Comparison with Benznidazole

Background

Treatment of Chagas disease, caused by Trypanosoma cruzi, relies on nifurtimox and benznidazole (BZL), which present side effects in adult patients, and natural resistance in some parasite strains. Hydroxymethylnitrofurazone (NFOH) is a new drug candidate with demonstrated trypanocidal activity; however, its safety is not known.

Methods

HepG2 cells dose response to NFOH and BZL (5–100 µM) was assessed by measurement of ROS, DNA damage and survival. Swiss mice were treated with NFOH or BZL for short-term (ST, 21 d) or long-term (LT, 60 d) periods. Sera levels of cellular injury markers, liver inflammatory and oxidative stress, and fibrotic remodeling were monitored.

Results

HepG2 cells exhibited mild stress, evidenced by increased ROS and DNA damage, in response to NFOH, while BZL at 100 µM concentration induced >33% cell death in 24 h. In mice, NFOH ST treatment resulted in mild-to-no increase in the liver injury biomarkers (GOT, GPT), and liver levels of inflammatory (myeloperoxidase, TNF-α), oxidative (lipid peroxides) and nitrosative (3-nitrotyrosine) stress. These stress responses in NFOH LT treated mice were normalized to control levels. BZL-treated mice exhibited a >5-fold increase in GOT, GPT and TNF-α (LT) and a 20–40% increase in liver levels of MPO activity (ST and LT) in comparison with NFOH-treated mice. The liver inflammatory infiltrate was noted in the order of BZL>vehicle≥NFOH and BZL>NFOH≥vehicle, respectively, after ST and LT treatments. Liver fibrotic remodeling, identified after ST treatment, was in the order of BZL>vehicle>NFOH; lipid deposits, indicative of mitochondrial dysfunction and in the order of NFOH>vehicle>BZL were evidenced after LT treatment.

Conclusions

NFOH induces mild ST hepatotoxicity that is normalized during LT treatment in mice. Our results suggest that additional studies to determine the efficacy and toxicity of NFOH are warranted.     

Bone Marrow and Nonbone Marrow Toll Like Receptor 4 Regulate Acute Hepatic Injury Induced by Endotoxemia

Background

Toll-like receptors (TLRs) are expressed in immune cells and hepatocytes. We examined whether hepatic Toll-like receptor 4 (TLR4) is involved in the acute hepatic injury caused by the administration of lipopolysaccharide (LPS) (septic shock model).

Methods

Wild type (WT), TLR4-deficient and chimera mice underwent myeloablative bone marrow transplantation to dissociate between TLR4 expression in the liver or in the immune-hematopoietic system. Mice were injected with LPS and sacrificed 4 hours later.

Results

Compared to TLR4 deficient mice, WT mice challenged with LPS displayed increased serum liver enzymes and hepatic cellular inflammatory infiltrate together with increased serum and hepatic levels of interleukin 1β (IL-1β), tumor necrosis factor α (TNFα) ,Up-regulation of hepatic mRNA encoding TLR4, IκB and c-jun expressions. TLR4 mutant mice transplanted with WT bone marrow were more protected than WT chimeric mice bearing TLR4 mutant hemopoietic cells from LPS, as seen by IL-1β and TNFα levels. We then used hepatocytes (Huh7) and macrophages from monocytic cell lines to detect TLR mRNA expression. Macrophages expressed a significantly higher level of TLR4 mRNA and TLR2 (more than 3000- and 8000-fold respectively) compared with the hepatocyte cell line. LPS administration induced TLR4 activation in a hepatocyte cell line in a dose dependent manner while TLR2 mRNA hardly changed.

Conclusions

These results suggest that TLR4 activation of hepatocytes participate in the immediate response to LPS induced hepatic injury. However, in this response, the contribution of TLR4 on bone marrow derived cells is more significant than those of the hepatocytes. The absence of the TLR4 gene plays a pivotal role in reducing hepatic LPS induced injury.     

Serum γ-glutamyl Transferase Levels,Insulin Resistance and Liver Fibrosis in Patients with Chronic Liver Diseases

Background and Aims

Serum levels of γ-glutamyl-transpeptidase(γ-GT) were associated with liver disease severity and metabolic alterations, which in turn are able to affect hepatic damage. In patients with nonalcoholic fatty liver disease (NAFLD), genotype 1 chronic hepatitis C (G1CHC) and chronic hepatitis B (CHB), we assessed the link between liver fibrosis and γ-GT serum levels, and we evaluated if normal or high γ-GT serum levels affect the association between insulin resistance (IR) and severity of liver fibrosis.

Methods

843 consecutive patients with chronic liver disease (CLD)(193 NAFLD, 481 G1CHC, 169 CHB) were evaluated by liver biopsy (Kleiner and Scheuer scores) and clinical and metabolic measurements. IR was diagnosed if HOMA>3. A serum γ-GT concentration of >36 IU/L in females and >61 IU/L in males was considered the threshold value for identifying high levels of γ-GT.

Results

By multivariate logistic regression analysis, abnormal γ-GT serum levels were independently linked to severe liver fibrosis in patients with NAFLD (OR2.711,CI1.120–6.564,p = 0.02), G1CHC (OR3.461,CI2.138–5.603,p<0.001) and CHB (OR2.778,CI1.042–7.414,p = 0.04), together with IR and liver necroinflammation, and with a negative predictive value>80%. Interestingly, among patients with high or normal γ-GT values, even if IR prevalence was significantly higher in patients with severe fibrosis compared to those without, IR remained significantly associated with severe fibrosis in patients with abnormal γ-GT values only (OR4.150,CI1.079–15.970,p = 0.03 for NAFLD; OR2.250,CI1.211–4.181,p = 0.01 for G1CHC; OR3.096,CI2.050–34.220,p = 0.01 for CHB).

Conclusions

In patients with CLD, IR is independently linked to liver fibrosis only in patients with abnormal γ-GT values, without differences according to liver disease etiology, and suggesting a role of γ-GT as a marker of metabolic-induced liver damage. These data could be useful for the clinical and pharmacologic management of patients with CLD.     

Circulating Annexin A5 Levels after Atrial Switch for Transposition of the Great Arteries: Relationship with Ventricular Deformation and Geometry

Background

Inflammatory cytokines, cardiomyocyte apoptosis, and altered collagen turnover may contribute to unfavourable ventricular remodeling. This unfavourable ventricular remodelling is well documented in patients after atrial switch operation for complete transposition of the great arteries. We therefore tested if levels of circulating markers of inflammation, apoptosis, collagen synthesis, and extracellular matrix degradation are altered in patients after atrial switch operation for transposition of the great arteries.

Methods and Results

Circulating tumour necrosis factor (TNF)-α, annexin A5 (AnxA5), carboxy-terminal propeptide of type I procollagen (PICP), amino-terminal propeptide of type III procollagen (PIIINP), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels were determined in 27 patients aged 25.2±3.1 years and 20 controls. Ventricular myocardial deformation and left ventricular eccentricity index (EI) were determined by speckle tracking and two-dimensional echocardiography, respectively. Compared with controls, patients had significantly higher circulating AnxA5 (p<0.001) and TNF-α (p = 0.018) levels, but similar PICP, PIIINP, MMP-1 and TIMP-1 levels. For the whole cohort, plasma AnxA5 correlated with serum TNF-α (p = 0.002), systemic ventricular global longitudinal strain (GLS) and systolic and early diastolic strain rate (all p<0.001), and subpulmonary ventricular GLS and early diastolic strain rate (both p<0.001). In patients, plasma AnxA5 level correlated positively with subpulmonary ventricular EI (p = 0.027). Multiple linear regression analysis identified systemic ventricular GLS (β = −0.50, p<0.001) and serum TNF-α (β = 0.29, p = 0.022) as significant correlates of plasma AnxA5.

Conclusions

Elevated plasma AnxA5 level in patients after atrial switch operation is associated with impaired systemic myocardial deformation, increased subpulmonary ventricular eccentricity, and increased serum TNF-α level.     

Neutrophil-Derived Myeloperoxidase Aggravates Non-Alcoholic Steatohepatitis in Low-Density Lipoprotein Receptor-Deficient Mice

Background

Chronic inflammation and oxidative stress play fundamental roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). Previously, we reported that myeloperoxidase (MPO), an aggressive oxidant-generating neutrophil enzyme, is associated with NASH severity in man. We now investigated the hypothesis that MPO contributes to the development and progression of NASH.

Methodology

Low-density lipoprotein receptor-deficient mice with an MPO-deficient hematopoietic system (LDLR^−/−/MPO^−/−tp mice) were generated and compared with LDLR^−/−/MPO^+/+tp mice after induction of NASH by high-fat feeding.

Results

High-fat feeding caused a ∼4-fold induction of liver MPO in LDLR^−/−/MPO^+/+ mice which was associated with hepatic sequestration of MPO-positive neutrophils and high levels of nitrotyrosine, a marker of MPO activity. Importantly, LDLR^−/−/MPO^−/−tp mice displayed markedly reduced hepatic neutrophil and T-lymphocyte infiltration (p<0.05), and strong down regulation of pro-inflammatory genes such as TNF-α and IL-6 (p<0.05, p<0.01) in comparison with LDLR^−/−/MPO^+/+tp mice. Next to the generalized reduction of inflammation, liver cholesterol accumulation was significantly diminished in LDLR^−/−/MPO^−/−tp mice (p = 0.01). Moreover, MPO deficiency appeared to attenuate the development of hepatic fibrosis as evident from reduced hydroxyproline levels (p<0.01). Interestingly, visceral adipose tissue inflammation was markedly reduced in LDLR^−/−/MPO^−/−tp mice, with a complete lack of macrophage crown-like structures. In conclusion, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in response to a high fat diet, supporting an important role for neutrophils in the pathogenesis of metabolic disease.     

Sputum IgE and Cytokines in Asthma: Relationship with Sputum Cellular Profile

Background

Local IgE production may play a role in asthma pathogenesis. The aim of the study was to assess sputum total IgE and cytokines in asthmatics according to sputum cellular phenotype.

Methods

We studied 122 subjects including 22 non atopic healthy subjects, 41 eosinophilic (sputum eosinophils ≥3%), 16 neutrophilic (sputum neutrophils >76%) and 43 pauci-granulocytic asthmatics (sputum eosinophils <3% and sputum neutrophils ≤76%) recruited from the asthma clinic at CHU Liege.Sputum supernatant total IgE (tIgE) was measured by ImmunoCAP and sputum supernatant cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ and TNF-α) were measured with the Luminex xMAP Technology by using commercially available Fluorokine MAP kits.

Results

After concentrating sputum samples, total IgE was detectable in the majority of subjects. Sputum IgE was raised in asthmatics when compared to healthy subjects. Overall, asthmatics did not significantly differ from healthy subjects with respect to cytokine levels. The eosinophilic asthma phenotype, however, was characterised by raised sputum tIgE, IL-5 and IL-13 compared to healthy subjects (p<0.001, p<0.001 and p<0.05 respectively) and pauci-granulocytic asthma (p<0.01, p<0.001 and p<0.05 respectively) and raised IL-5 compared to neutrophilic asthma (p<0.01). When patients were classified according to sputum IgE levels, it appeared that IL-5, IL-6, IL-17 and TNF-α sputum supernatant levels were raised in the “IgE high” asthmatics (IgE ≥0.1 kU/l) when compared to “IgE low” asthmatics (IgE<0.1 kU/l).

Conclusion

The eosinophilic asthma phenotype was associated with raised sputum IgE and a Th2 cytokine profile. Raised sputum IgE was associated with a heterogeneous cytokine overproduction.     

Exercise training is more effective than resveratrol supplementation on alleviation of inflammation in peritoneal macrophages of high fat diet mice

[Purpose]

This study investigated the effect of exercise training and resveratrol supplementation with low fat diet on proinflammatory profiles by Lipopolysaccharide (LPS)-stimulation in peritoneal macrophage of high fat diet mice.

[Methods]

To accomplish the purpose of this study, C57BL/6 male mice were fed high fat diet (45% fat diet) for 8 weeks. Then these mice were divided into 3 groups; HLC (high fat diet and low fat diet for 8 weeks as the control, n=10), HLR (high fat diet and low fat diet for 8 weeks with resveratrol supplementation, n=10). HLE (high fat diet and low fat diet for 8 weeks with moderate exercise training, n=10). Resveratrol (10 mg/kg) was administrated once a day, 5 days/week for 8 weeks. Exercise training was performed for 8 weeks on a treadmill running for 30-60 min/day at 10-22 m/min, 0% grade, 5 days/week. After exercise training, all the peritoneal macrophage was collected and LPS (0, 0.5, 1.0 μg/ml) were used to stimulate the cells. Then peritoneal macrophage TNF-α, IL-6, MCP-1, IL12p70, IFN-γ, IL-10 were measured by BD cytometric bead array mouse inflammation kit.

[Results]

As a result, body weight and total cholesterol were significantly reduced in HLE compared with HLC (p<.05). Also, TNF-α and MCP-1 were decreased in HLE compared with HLC (p<.05) by LPS-stimulation (0, 0.5, 1.0 μg/ml) and IL-6, IL-12p70 and IFN-r were decreased in HLE compared with HLC (p<.05) by LPS-stimulation (1.0 μg/ml). But resveratrol supplementation did not affect the result.

[Conclusion]

These findings suggest that exercise training has beneficial effects on body weight, total cholesterol, peritoneal macrophage and proinflammatory cytokine in high fat diet mice.     

Secreted Protein Acidic and Rich in Cysteine (SPARC) Exacerbates Colonic Inflammatory Symptoms in Dextran Sodium Sulphate-Induced Murine Colitis

Background

Secreted Protein Acidic and Rich in Cysteine (SPARC) is expressed during tissue repair and regulates cellular proliferation, migration and cytokine expression. The aim was to determine if SPARC modifies intestinal inflammation.

Methods

Wild-type (WT) and SPARC-null (KO) mice received 3% dextran sodium sulphate (DSS) for 7 days. Inflammation was assessed endoscopically, clinically and histologically. IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12/IL23p40, TNF-α, IFN-γ, RANTES, MCP-1, MIP-1α, MIP-1β, MIG and TGF-β1 levels were measured by ELISA and cytometric bead array. Inflammatory cells were characterised by CD68, Ly6G, F4/80 and CD11b immunofluorescence staining and regulatory T cells from spleen and mesenteric lymph nodes were assessed by flow cytometry.

Results

KO mice had less weight loss and diarrhoea with less endoscopic and histological inflammation than WT animals. By day 35, all (n = 13) KO animals completely resolved the inflammation compared to 7 of 14 WT mice (p<0.01). Compared to WTs, KO animals at day 7 had less IL1β (p = 0.025) and MIG (p = 0.031) with higher TGFβ1 (p = 0.017) expression and a greater percentage of FoxP3+ regulatory T cells in the spleen and draining lymph nodes of KO animals (p<0.01). KO mice also had fewer CD68+ and F4/80+ macrophages, Ly6G+ neutrophils and CD11b+ cells infiltrating the inflamed colon.

Conclusions

Compared to WT, SPARC KO mice had less inflammation with fewer inflammatory cells and more regulatory T cells. Together, with increased TGF-β1 levels, this could aid in the more rapid resolution of inflammation and restoration of the intestinal mucosa suggesting that the presence of SPARC increases intestinal inflammation.     

Separation of Hepatic Iron and Fat by Dual-Source Dual-Energy Computed Tomography Based on Material Decomposition: An Animal Study

Objective

To explore the feasibility of dual-source dual-energy computed tomography (DSDECT) for hepatic iron and fat separation in vivo.

Materials and Methods

All of the procedures in this study were approved by the Research Animal Resource Center of Shanghai Ruijin Hospital. Sixty rats that underwent DECT scanning were divided into the normal group, fatty liver group, liver iron group, and coexisting liver iron and fat group, according to Prussian blue and HE staining. The data for each group were reconstructed and post-processed by an iron-specific, three-material decomposition algorithm. The iron enhancement value and the virtual non-iron contrast value, which indicated overloaded liver iron and residual liver tissue, respectively, were measured. Spearman''s correlation and one-way analysis of variance (ANOVA) were performed, respectively, to analyze statistically the correlations with the histopathological results and differences among groups.

Results

The iron enhancement values were positively correlated with the iron pathology grading (r = 0.729, p<0.001). Virtual non-iron contrast (VNC) values were negatively correlated with the fat pathology grading (r = −0.642,p<0.0001). Different groups showed significantly different iron enhancement values and VNC values (F = 25.308,p<0.001; F = 10.911, p<0.001, respectively). Among the groups, significant differences in iron enhancement values were only observed between the iron-present and iron-absent groups, and differences in VNC values were only observed between the fat-present and fat-absent groups.

Conclusion

Separation of hepatic iron and fat by dual energy material decomposition in vivo was feasible, even when they coexisted.     

Inflammatory Kidney and Liver Tissue Response to Different Hydroxyethylstarch (HES) Preparations in a Rat Model of Early Sepsis

Background

Tissue hypoperfusion and inflammation in sepsis can lead to organ failure including kidney and liver. In sepsis, mortality of acute kidney injury increases by more than 50%. Which type of volume replacement should be used is still an ongoing debate. We investigated the effect of different volume strategies on inflammatory mediators in kidney and liver in an early sepsis model.

Material and Methods

Adult male Wistar rats were subjected to sepsis by cecal ligation and puncture (CLP) and assigned to three fluid replenishment groups. Animals received 30mL/kg of Ringer’s lactate (RL) for 2h, thereafter RL (75mL/kg), hydroxyethyl starch (HES) balanced (25mL/kg), containing malate and acetate, or HES saline (25mL/kg) for another 2h. Kidney and liver tissue was assessed for inflammation. In vitro rat endothelial cells were exposed to RL, HES balanced or HES saline for 2h, followed by stimulation with tumor necrosis factor-α (TNF-α) for another 4h. Alternatively, cells were exposed to malate, acetate or a mixture of malate and acetate, reflecting the according concentration of these substances in HES balanced. Pro-inflammatory cytokines were determined in cell supernatants.

Results

Cytokine mRNA in kidney and liver was increased in CLP animals treated with HES balanced compared to RL, but not after application of HES saline. MCP-1 was 3.5fold (95% CI: 1.3, 5.6) (p<0.01) and TNF-α 2.3fold (95% CI: 1.2, 3.3) (p<0.001) upregulated in the kidney. Corresponding results were seen in liver tissue. TNF-α-stimulated endothelial cells co-exposed to RL expressed 3529±1040pg/mL MCP-1 and 59±23pg/mL CINC-1 protein. These cytokines increased by 2358pg/mL (95% CI: 1511, 3204) (p<0.001) and 29pg/ml (95% CI: 14, 45) (p<0.01) respectively when exposed to HES balanced instead. However, no further upregulation was observed with HES saline. PBS supplemented with acetate increased MCP-1 by 1325pg/mL (95% CI: 741, 1909) (p<0.001) and CINC-1 by 24pg/mL (95% CI: 9, 38) (p<0.01) compared to RL. Malate as well as HES saline did not affect cytokine expression.

Conclusion

We identified HES balanced and specifically its component acetate as pro-inflammatory factor. How important this additional inflammatory burden on kidney and liver function is contributing to the sepsis-associated inflammatory burden in early sepsis needs further evaluation.     

Tumor Necrosis Factor - Alpha Is Essential for Angiotensin II-Induced Ventricular Remodeling: Role for Oxidative Stress

The functional crosstalk between angiotensin II (Ang II) and tumor necrosis factor (TNF)-α has been shown to cause adverse left ventricular remodeling and hypertrophy in hypertension. Previous studies from our lab showed that mice lacking TNF-α (TNF-α^-/-) have attenuated hypertensive response to Ang II; however, the signaling mechanisms involved are not known. In this study, we investigated the signaling pathways involved in the Ang II and TNF-α interaction. Chronic Ang II infusion (1μg/kg/min, 14 days) significantly increased cardiac collagen I, collagen III, CTGF and TGF-β mRNA and protein expression in wild-type (WT) mice, whereas these changes were decreased in TNF-α^-/- mice. TNF-α^-/- mice with Ang II infusion showed reduced myocardial perivascular and interstitial fibrosis compared to WT mice with Ang II infusion. In WT mice, Ang II infusion increased reactive oxygen species formation and the expression of NADPH oxidase subunits, indicating increased oxidative stress, but not in TNF-α^-/- mice. In addition, treatment with etanercept (8 mg/kg, every 3 days) for two weeks blunted the Ang II-induced hypertension (133±4 vs 154±3 mmHg, p<0.05) and cardiac hypertrophy (heart weight to body weight ratio, 4.8±0.2 vs 5.6±0.3, p<0.05) in WT mice. Furthermore, Ang II-induced activation of NF-κB, p38 MAPK, and JNK were reduced in both TNF-α^-/- mice and mice treated with etanercept. Together, these findings indicate that TNF-α contributes to Ang II-induced hypertension and adverse cardiac remodeling, and that these effects are associated with changes in the oxidative stress dependent MAPK/TGF-β/NF-κB pathway. These results may provide new insight into the mechanisms of Ang II and TNF-α interaction.     

Exercises in Hot and Humid Environment Caused Liver Injury in a Rat Model

Objective

To investigate injury pattern during intense exercises in hot and humid environment particularly on liver in a rat exertional heat stroke model.

Methods

We randomly divided 30 rats into a control group (CG), a normal temperature (25±2°C, 60%±5% humidity) exercise group (NTEG) and a high temperature and high humidity (35±2°C, 80%±10% humidity) exercising group (HTEG), each comprising 10 animals. The NTEG and HTEG rats were forced to run in a treadmill for 1 hour maximum at 20 rpm. We analyzed liver cells of all three groups with JC-1 dye and flow cytometry for apoptosis rates in addition to liver tissue 8 - hydroxy deoxyguanosine (8 - OhdG) and blood serum IL–6, tumor necrosis factor alpha (TNF-α), alanine aminotransferase ALT, aspartate amino transferase (AST), serum creatinine (CREA), blood urea nitrogen (BUN), lactate dehydrogenase (LDH), creatine phosphate kinase (CK) concentrations.

Result

Compared with NTEG rats, beside reduced exercise tolerance (60±5 vs. 15±3 minutes) (p = 0.002) the 8-OhdG liver tissue concentrations were significantly higher (p = 0.040) in the HTEG rats. The HTEG developed more organ tissue damage and cellular fragmentations of liver cells. In both exercise groups TNF-α and IL-6 serum concentrations were enhanced significantly (p<0.001) being highest in the HTEG animals. Serum ALT, AST, LDH, CREA, BUN and CK concentrations were significantly enhance in both exercise groups.

Conclusion

In our exertional heat stroke rat model, we found tissue damage particularly in livers during exercises in hot and humid environment that was related to inflammation, oxidative stress and apoptosis.     

A Combined Supplementation of Omega-3 Fatty Acids and Micronutrients (Folic Acid,Vitamin B12) Reduces Oxidative Stress Markers in a Rat Model of Pregnancy Induced Hypertension

Objectives

Our earlier studies have highlighted that an altered one carbon metabolism (vitamin B₁₂, folic acid, and docosahexaenoic acid) is associated with preeclampsia. Preeclampsia is also known to be associated with oxidative stress and inflammation. The current study examines whether maternal folic acid, vitamin B₁₂ and omega-3 fatty acid supplementation given either individually or in combination can ameliorate the oxidative stress markers in a rat model of pregnancy induced hypertension (PIH).

Materials and Methods

Pregnant Wistar rats were assigned to control and five treatment groups: PIH; PIH + vitamin B₁₂; PIH + folic acid; PIH + Omega-3 fatty acids and PIH + combined micronutrient supplementation (vitamin B₁₂ + folic acid + omega-3 fatty acids). L-Nitroarginine methylester (L-NAME; 50 mg/kg body weight/day) was used to induce hypertension during pregnancy. Blood Pressure (BP) was recorded during pregnancy and dams were dissected at d20 of gestation.

Results

Animals from the PIH group demonstrated higher (p<0.01 for both) systolic and diastolic BP; lower (p<0.01) pup weight; higher dam plasma homocysteine (p<0.05) and dam and offspring malondialdehyde (MDA) (p<0.01), lower (p<0.05) placental and offspring liver DHA and higher (p<0.01) tumor necrosis factor–alpha (TNF–ά) levels as compared to control. Individual micronutrient supplementation did not offer much benefit. In contrast, combined supplementation lowered systolic BP, homocysteine, MDA and placental TNF-ά levels in dams and liver MDA and protein carbonyl in the offspring as compared to PIH group.

Conclusion

Key constituents of one carbon cycle (folic acid, vitamin B₁₂ and DHA) may play a role in reducing oxidative stress and inflammation in preeclampsia.     

Tumor Necrosis Factor Alpha Blocking Agents as Treatment for Ulcerative Colitis Intolerant or Refractory to Conventional Medical Therapy: A Meta-Analysis

Background

Efficacy of tumor necrosis factor alpha (TNF-α) blockers for treatment of ulcerative colitis that is unresponsive to conventional therapy is unclear due to recent studies yielding conflicting results.

Aim

To assess the efficacy and safety of anti-TNF-α agents for treatment of ulcerative colitis patients who were intolerant or refractory to conventional medical therapy.

Methods

Pubmed, Embase, and the Cochrane database were searched. Analysis was performed on randomized controlled trials that assessed anti-TNF-α therapy on ulcerative colitis patients that had previously failed therapy with corticosteroids and/or immunosuppressants. The primary outcome focused on was the frequency of patients that achieved clinical remission. Further trial outcomes of interest included rates of remission without patient use of corticosteroids during the trial, extent of mucosal healing, and the number of cases that resulted in colectomy and serious side effects.

Results

Eight trials from seven studies (n = 2122) met the inclusion criteria and were thus included during analysis. TNF-α blockers demonstrated clinical benefit as compared to placebo control as evidenced by an increased frequency of clinical remission (p<0.00001), steroid-free remission (p = 0.01), endoscopic remission (p<0.00001) and a decrease in frequency of colectomy (p = 0.03). No difference was found concerning serious side effects (p = 0.05). Three small trials (n = 57) comparing infliximab to corticosteroid treatment, showed no difference in frequency of clinical remission (p = 0.93), mucosal healing (p = 0.80), and requirement for a colectomy (p = 0.49). One trial compared infliximab to cyclosporine (n = 115), wherein no difference was found in terms of mucosal healing (p = 0.85), colectomy frequency (p = 0.60) and serious side effects (p = 0.23).

Conclusion

TNF-α blockers are effective and safe therapies for the induction and maintenance of long-term remission and prevention of treatment by colectomy for patients with refractory ulcerative colitis where conventional treatment was previously ineffective. Furthermore, infliximab and cyclosporine were found to be comparable for treating acute severe steroid-refractory ulcerative colitis.     

Hepatic Hemodynamics and Fetal Growth: A Relationship of Interest for Further Research

Background

It is well known that hepatic hemodynamics is an important physiologic mechanism in the regulation of cardiac output (CO). It has been reported that maternal cardiac output relates to neonatal weight at birth.

Aims

In this study, we assessed the correlation between maternal hepatic vein Doppler flow parameters, cardiac output and neonatal birth weight.

Methods

Healthy women with uncomplicated second or third trimester pregnancy attending the outpatient antenatal clinic of Ziekenhuis Oost-Limburg in Genk (Belgium), had a standardized combined electrocardiogram-Doppler ultrasound with Impedance Cardiography, for measurement of Hepatic Vein Impedance Index (HVI  =  [maximum velocity – minimum velocity]/maximum velocity), venous pulse transit time (VPTT  =  time interval between corresponding ECG and Doppler wave characteristics) and cardiac output (heart rate x stroke volume). After delivery, a population-specific birth weight chart, established from a cohort of 27000 neonates born in the index hospital, was used to define customized birth weight percentiles (BW%). Correlations between HVI, VPTT, CO and BW% were calculated using Spearman''s ρ, linear regression analysis and R² goodness of fit in SPSS 22.0.

Results

A total of 73 women were included. There was a negative correlation between HVI and VPTT (ρ = −0.719, p<0.001). Both HVI and VPTT correlated with CO (ρ = −0.403, p<0.001 and ρ = 0.332, p<0.004 resp.) and with BW% (ρ = −0.341, p<0.003 and ρ = 0.296, p<0.011 resp.)

Conclusion

Our data illustrate that the known contribution of hepatic hemodynamics in the regulation of cardiac output is also true for women with uncomplicated pregnancies. Our study is the first to illustrate a potential link between maternal hepatic hemodynamics and neonatal birth weight. Whether this link is purely associative or whether hepatic vascular physiology has a direct impact on fetal growth is to be evaluated in more extensive clinical and experimental research.     

Red Blood Cell Distribution Width during the First Week Is Associated with Severity and Mortality in Septic Patients

Objective

Higher values of red blood cell distribution width (RDW) have been found in non-surviving than in surviving septic patients. However, it is unknown whether RDW during the first week of sepsis evolution is associated with sepsis severity and early mortality, oxidative stress and inflammation states, and these were the aims of the study.

Methods

We performed a prospective, observational, multicenter study in six Spanish Intensive Care Units with 297 severe septic patients. We measured RDW, serum levels of malondialdehyde (MDA) to assess oxidative stress, and tumour necrosis factor (TNF)-α to assess inflammation at days 1, 4, and 8. The end-point was 30-day mortality.

Results

We found higher RDW in non-surviving (n = 104) than in surviving (n = 193) septic patients at day 1 (p = 0.001), day 4 (p = 0.001), and day 8 (p = 0.002) of ICU admission. Cox regression analyses showed that RDW at day 1 (p<0.001), 4 (p = 0.005) and 8 (p = 0.03) were associated with 30-day mortality. Receiver operating characteristic curves showed that RDW at day 1 (p<0.001), 4 (p<0.001), and 8 (p<0.001) could be used to predict 30-day mortality. RDW showed a positive correlation with serum MDA levels at day 1 and day 4, with serum TNF-α levels at days 4 and 8, and with SOFA score at days 1, 4 and 8.

Conclusions

The major findings of our study were that non-surviving septic patients showed persistently higher RDW during the first week of ICU stay than survivors, that RDW during the first week was associated with sepsis severity and mortality, that RDW during the first week could be used as biomarker of outcome in septic patients, and that there was an association between RDW, serum MDA levels, and serum TNF-α levels during the first week.