Melatonin mediates intestinal barrier dysfunction and systemic inflammation in moderate-severe OSA patients.

BACKGROUND: Intestinal barrier dysfunction and systemic inflammation are common in obstructive sleep apnoea (OSA). We aimed to investigate the role of melatonin, an anti-inflammatory mediator, in mediating the relationships between OSA, intestinal barrier dysfunction and systemic inflammation. METHODS: Two hundred and thirty-five male participants who complained with sleep problems and underwent whole night polysomnography at our sleep centre between 2017 and 2018 were enrolled. Polysomnographic data, anthropometric measurements and biochemical indicators were collected. Serum melatonin, intestinal barrier function biomarker zonula occludens-1 (ZO-1) and inflammatory biomarkers C-reactive protein (CRP) with lipopolysaccharide (LPS) were detected. Spearman's correlation analysis assessed the correlations between sleep parameters, melatonin and biomarkers (ZO-1, LPS and CRP). Mediation analysis explored the effect of OSA on intestinal barrier dysfunction and systemic inflammation in moderate-severe OSA patients. RESULTS: As OSA severity increased, serum melatonin decreased, whereas ZO-1, LPS and CRP increased. Spearman's correlation analysis showed that serum melatonin was significantly negatively correlated with ZO-1 (r = -0.19, p < .05) and LPS (r = -0.20, p < .05) in the moderate-OSA group; serum melatonin was significantly negatively correlated with ZO-1 (r = -0.46, p < .01), LPS (r = -0.35, p < .01) and CPR (r = -0.30, p < .05) in the severe-OSA group. Mediation analyses showed melatonin explain 36.12% and 35.38% of the effect of apnoea-hypopnea index (AHI) on ZO-1 and LPS in moderate to severe OSA patients. CONCLUSIONS: Our study revealed that melatonin may be involved in mediating intestinal barrier dysfunction and systemic inflammation in moderate-to-severe OSA patients.

ZO-1 Serum Levels as a Potential Biomarker for Psychotic Disorder.

OBJECTIVE: There are limited studies in the literature on the relationship between intestinal and blood-brain barrier permeability and the etiology of schizophrenia. We hypothesized that the difference in serum ZO-1 levels in patients with schizophrenia may affect the severity of the disease. The aim of this study was to investigate the role of changes in serum ZO-1 concentrations in the etiopathogenesis of patients with schizophrenia. METHODS: A total of 46 patients, 34 with schizophrenia, 12 with a first psychotic attack, and 37 healthy controls, were included in the study. Symptom severity was determined by applying the Positive and Negative Syndrome Scale and the Clinical Global Impression-Severity Scale. Serum ZO-1 levels were measured from venous blood samples. RESULTS: Serum ZO-1 levels were higher in patients with psychotic disorder compared to healthy controls. There was no statistically significant difference between the groups in the first psychotic attack group and the schizophrenia patients. There was a statistically significant positive correlation between serum ZO-1 levels and Positive and Negative Syndrome Scale positive symptom score. CONCLUSIONS: These findings regarding ZO-1 levels suggest that dysregulation of the blood-brain barrier in psychotic disorder may play a role in the etiology of the disorder.

Circulating tight-junction proteins are potential biomarkers for blood-brain barrier function in a model of neonatal hypoxic/ischemic brain injury.

BACKGROUND: Neonatal encephalopathy often leads to lifelong disabilities with limited treatments currently available. The brain vasculature is an important factor in many neonatal neurological disorders but there is a lack of diagnostic tools to evaluate the brain vascular dysfunction of neonates in the clinical setting. Measurement of blood-brain barrier tight-junction (TJ) proteins have shown promise as biomarkers for brain injury in the adult. Here we tested the biomarker potential of tight-junctions in the context of neonatal brain injury. METHODS: The levels of TJ-proteins (occluding, claudin-5, and zonula occludens protein 1) in both blood plasma and cerebrospinal fluid (CSF) as well as blood-brain barrier function via 14C-sucrose (342 Da) and Evans blue extravasation were measured in a hypoxia/ischemia brain-injury model in neonatal rats. RESULTS: Time-dependent changes of occludin and claudin-5 levels could be measured in blood and CSF after hypoxia/ischemia with males generally having higher levels than females. The levels of claudin-5 in CSF correlated with the severity of the brain injury at 24 h post- hypoxia/ischemia. Simultaneously, we detected early increase in blood-brain barrier-permeability at 6 and 24 h after hypoxia/ischemia. CONCLUSIONS: Levels of circulating claudin-5 and occludin are increased after hypoxic/ischemic brain injuries and blood-brain barrier-impairment and have promise as early biomarkers for cerebral vascular dysfunction and as a tool for risk assessment of neonatal brain injuries.

Probiotic Bacillus Spores Protect Against Acetaminophen Induced Acute Liver Injury in Rats.

Acetaminophen (APAP) is one of the most used analgesics and antipyretic agents in the world. Intoxication with APAP is the main cause of acute liver toxicity in both the US and Europe. Spore-forming probiotic bacteria have the ability to resist harsh gastric and intestinal conditions. The aim of this study was to investigate the possible protective effect of Bacillus (B) species (sp) spores (B. licheniformis, B. indicus, B. subtilis, B. clausii, B. coagulans) against hepatotoxicity induced by APAP in rats. A total of 35 rats were randomly divided into seven groups: group I served as control; group II received silymarin; group III received MegaSporeBioticTM (MSB); group IV received APAP and served as the model of hepatotoxicity; group V received APAP and silymarin; group VI received APAP and MSB; group VII received APAP, silymarin and MSB. The livers for histopathological examination and blood samples were collected on the last day of the experiment. We determined aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total antioxidant capacity (TAC) levels and zonula occludens (ZO-1), tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) expression. APAP overdose increased AST and ALT. It slowly decreased TAC compared to the control group, but pretreatment with silymarin and MSB increased TAC levels. Elevated plasma concentrations were identified for ZO-1 in groups treated with APAP overdose compared with those without APAP or receiving APAP in combination with silymarin, MSB or both. The changes were positively correlated with the levels of other proinflammatory cytokines (TNF-α, IL-1ß). In addition, histopathological hepatic injury was improved by preadministration of MSB or silymarin versus the disease model group. Bacillus sp spores had a protective effect on acute hepatic injury induced by APAP. Pretreatment with MSB resulted in a significant reduction in serum AST, ALT, TNF-α, IL-1ß, ZO-1, TAC and also hepatocyte necrosis, similar to the well-known hepatoprotective agent-silymarin.

The Prognostic Value of Endotoxemia and Intestinal Barrier Biomarker ZO-1 in Bacteremic Sepsis.

BACKGROUND: Intestinal barrier dysfunction exerts a pivotal pathophysiological role in the development of multiple organ dysfunction in sepsis. The present study was undertaken to investigate the potential role of serum intestinal fatty acid-binding protein (I-FABP) and zonula occludens-1 (ZO-1) levels as biomarkers of intestinal barrier dysfunction in bacteremic sepsis. METHODS: Seventy-five patients with bacteremic sepsis of abdominal origin (n = 34) or nonabdominal origin (n = 41) and 12 healthy controls were retrospectively studied. Blood samples collected upon sepsis diagnosis were analyzed for serum ZO-1, I-FABP and endotoxin levels. Prognostic scores Sequential Organ Failure Assessment (SOFA), quickSOFA and Acute Physiology and Chronic Health Evaluation (APACHE-II) were determined over the first 24 hours after sepsis diagnosis and patients' outcome in terms of 28-day mortality was recorded. RESULTS: Serum ZO-1 levels were significantly higher in bacteremic septic patients as compared to controls with no difference between patients with abdominal or extra-abdominal source of infection. Serum I-FABP levels were significantly lower in septic patients as compared to control and this reduction was more evident in patients with bacteremic abdominal sepsis. Serum ZO-1 and endotoxin concentrations were found significantly higher in patients who did not survive from sepsis. In receiver operating characteristic curve analysis, both endotoxin and ZO-1 predicted 28-day mortality. In addition, ZO-1 and endotoxin were correlated with the prognostic scores of qSOFA, SOFA and APACHE II. CONCLUSIONS: The results of this study indicate that serum ZO-1 might be a reliable biomarker of gut barrier dysfunction in sepsis, not affected by the abdominal or extra-abdominal site of infection. ZO-1, measured early at sepsis diagnosis, might represent a valuable additional prognostic tool for patients' outcome.

Effects of lipocalin-2 on brain endothelial adhesion and permeability.

Lipocalin-2 (LCN2) is a stress protein, and can be hyper-produced by many kinds of cells after exposure to injury or disease conditions. In this study, we asked whether LCN2 may play a protective role in cerebral endothelium. After focal cerebral ischemia in rats, plasma levels of LCN2 were significantly elevated at 6, 12, and 24 hrs, and persisted until 3 days post-stroke. To assess the vascular mechanisms of LCN2, we used brain endothelial cell cultures to investigate its effects on neutrophil adhesion and endothelial barrier integrity. LCN2 did not affect neutrophil adhesion to endothelial cells either under normal conditions or after TNFα stimulation. TNFα significantly increased endothelial permeability, and LCN2 rescued endothelial permeability. Concomitantly, LCN2 restored the membrane distribution of the tight junction protein ZO-1 and the adherens junction protein VE-cadherin. Our findings suggest that elevated LCN2 in the blood after ischemic stroke might affect endothelial function, in part by reducing damage to endothelial junctional proteins and maintain blood-brain barrier integrity.

Increased systemic zonula occludens 1 associated with inflammation and independent biomarker in patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a frequent type of primary liver cancer, and its prevalence is increasing worldwide. Indeed, the underlying molecular mechanism is not well understood. Previous studies have shown evidence that tight junction (TJ) components were correlated with carcinogenesis and tumor development. Our aims were to determine the serum levels of tight junction protein Zonula Occludens (ZO)-1 and an inflammatory marker such as high-sensitive C-reactive protein (hs-CRP) in HCC patients compared to healthy volunteers and also to identify the association between ZO-1 and inflammation in HCC. METHODS: Thirty HCC patients and 30 healthy volunteers were recruited in the current study. Clinical data regarding child class, BCLC staging, the number of lesions, tumor size, absence or presence of metastasis, cirrhosis and hepatitis infection were also collected in HCC patients. Plasma ZO-1 and serum hsCRP were analyzed by EIA and ELISA respectively and biochemical parameters by autoanalyser (AU680 Beckman Coulter, USA). Furthermore, hepatic ZO-1 protein expression and tissue localization were examined. RESULTS: Compared to healthy individuals, the serum levels of bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP) were elevated significantly (P < 0.0001) whilst serum albumin level was significantly (P < 0.0001) decreased in HCC patients. Furthermore, tight junction protein ZO-1 concentration was significantly elevated in HCC patients compared to control subjects (648 ± 183.8 vs. 396.4 ± 135.8 pg/ml, respectively; P < 0.0001). Serum hsCRP level was also significantly increased in HCC patients compared to control subjects (17.25 ± 3.57 vs. 5.54 ± 2.62 mg/L, respectively; P < 0.0001). Moreover, decreased protein expression of ZO-1 was found in liver tissue obtained from HCC patients. CONCLUSION: Our findings show for the first time that the systemic concentration of ZO-1 was significantly elevated in HCC patients and is positively correlated with inflammatory markers. Thus, the current study showing evidence that inflammation promotes plasma ZO-1 concentration and raises the possibility that it could be used as a potential diagnostic biomarker for HCC progression.

Elevated systemic zonula occludens 1 is positively correlated with inflammation in cirrhosis.

BACKGROUND/AIM: It has been well established that disruption of the intestinal barrier function and increased intestinal permeability contribute to endotoxemia and associated liver injury in patients with cirrhosis. However, the relationship between systemic inflammation and tight junction protein in cirrhosis remain unidentified. The aim of this study was to assess and compare the blood concentrations of ZO-1 with systemic hsCRP in patients with cirrhosis and healthy individuals. METHODS: 30 cirrhotic patients and 30 healthy individuals were enrolled in the study. Blood ZO-1 and hsCRP were measured by ELISA and biochemical parameters by AU680 Beckman Coulter (USA) autoanalyser. RESULTS: The serum ALT, AST, bilirubin, gamma GT, ALP and ammonia were significantly (P < 0.0001) elevated whilst serum albumin concentration was decreased in cirrhotic patients when compared to healthy individuals. Systemic tight junction protein ZO-1 concentration [590.0 ±â€¯32.79 vs. 349.9 ±â€¯18.76 pg/ml, respectively; P < 0.0001] and hsCRP [10.50 ±â€¯1.05 vs 5.31 ±â€¯0.65 mg/L, respectively; P < 0.001] were significantly elevated in patients with cirrhosis compared to controls. Significant positive correlation was found between increased ZO-1 and hsCRP (r = 0.2680 P < 0.01). CONCLUSION: Our results suggested that increased systemic ZO-1 concentration was associated with inflammation in cirrhosis. Thus, elevated blood ZO-1 levels could be a prognostic marker of cirrhotic patients with intestinal TJ disruption on the background of inflammation.

Sodium Tanshinone IIA Sulfonate Enhances Effectiveness Rt-PA Treatment in Acute Ischemic Stroke Patients Associated with Ameliorating Blood-Brain Barrier Damage.

Treatment with sodium tanshinone IIA sulfonate (STS) may ameliorate blood-brain barrier (BBB) damage in acute ischemic stroke patients receiving recombinant tissue plasminogen activator (rt-PA) thrombolysis and improve stroke patients' outcome. This randomized, single-center, placebo-controlled clinical trial investigated the potential effects and underlying mechanisms of STS. Forty-two acute ischemic stroke patients receiving intravenous rt-PA thrombolysis were randomized to intravenous administration either with STS (60 mg/day) (n = 21) or with equivalent volume of saline as a placebo (n = 21) after randomization for 10 days. Clinical outcomes, computer tomography perfusion (CTP) imaging with permeability-surface area product (PS) maps and serum levels of BBB damage biomarkers, were compared between the two groups. The percentage of patients with excellent functional outcome indicated by a 90-day mRS ≤1 was significantly higher in the STS group than in the placebo group (p = 0.028). For patients with CTP imaging (n = 30), PS in the ipsilateral lesion (p = 0.034) and relative PS (p = 0.013) were significantly lower in the STS group than that in placebo. STS-treated patients also had lower levels of matrix metalloproteinase (MMP)-9 (p = 0.036) and claudin-5 (p = 0.026), but higher levels of tissue inhibitor of metalloproteinase (TIMP)-1 (p = 0.040) than those in the placebo group. Post-stroke STS treatment could improve neurologic functional outcomes for acute ischemic stroke patients following rt-PA treatment by reducing BBB leakage and damage, which might be mechanistically associated with MMP-9 inhibition.

Circulating tight junction proteins mirror blood-brain barrier integrity in leukaemia central nervous system metastasis.

The aim of this study was to evaluate the clinical significance of circulating tight junction (TJ) proteins as biomarkers reflecting of leukaemia central nervous system (CNS) metastasis. TJs [claudin5 (CLDN5), occludin (OCLN) and ZO-1] concentrations were measured in serum and cerebrospinal fluid (CSF) samples obtained from 45 leukaemia patients. Serum ZO-1 was significantly higher (p < 0.05), but CSF ZO-1 levels were not significantly higher in the CNS leukaemia (CNSL) compared to the non-CNSL. The CNSL patients also had a lower CLDN5/ZO1 ratio in both serum and CSF than in non-CNSL patients (p < 0.05). The TJ index was negatively associated with WBCCSF , ALBCSF and BBB values in leukaemia patients. Among all of the parameters studied, CLDN5CSF had the highest specificity in discriminating between CNSL and non-CNSL patients. Therefore, analysing serum and CSF levels of CLDN5, OCLN and the CLDN5/ZO1 ratio is valuable in evaluating the potential of leukaemia CNS metastasis. Copyright © 2016 John Wiley & Sons, Ltd.

PROGNOSTIC VALUE OF PLASMA TIGHT-JUNCTION PROTEINS FOR SEPSIS IN EMERGENCY DEPARTMENT: AN OBSERVATIONAL STUDY.

BACKGROUND: Studies have illustrated that the breakdown of tight junction (TJ) contributed to an increase in vascular permeability in response to stimulation of inflammatory cytokines. Additionally, the release of TJ-associated proteins into the circulation was observed in many diseases. The present study was designed to investigate whether plasma levels of TJ-associated proteins could serve as predictors of severity and clinical outcome of sepsis. METHODS: In total, 51 septic patients were enrolled. The peripheral blood samples were collected for each patient on emergency department arrival. Plasma levels of occludin (OCLN), claudins (CLDN)-5, and zonula occludens (ZO)-1 and serum levels of procalcitonin (PCT) and lactate were measured. In addition, APACHE II score as well as SOFA score was calculated. The prognostic values of OCLN, CLDN-5, and ZO-1 were compared with the first 24-h maximum APACHE II score and SOFA score. RESULTS: The median levels of OCLN and ZO-1 were elevated with sepsis severity. The levels of plasma OCLN and ZO-1 were positively correlated with APACHE II score, SOFA score as well as lactate levels of the patients. The levels of ZO-1 revealed valuable diagnostic capacity to diagnose MODS, and the areas under the receiver-operating characteristic (AUC) curves of ZO-1 were similar to those of lactate levels, but better than those of PCT levels. The prognostic value for in-hospital mortality of ZO-1 was comparable to that of lactate levels, APACHE II score, and SOFA score, and superior to OCLN or PCT. CONCULSIONS: OCLN and ZO1 levels appear to be early prognostic markers in patients suffering from sepsis.

Platelet activating factor contributes to vascular leak in acute dengue infection.

BACKGROUND: Although plasma leakage is the hallmark of severe dengue infections, the factors that cause increased vascular permeability have not been identified. As platelet activating factor (PAF) is associated with an increase in vascular permeability in other diseases, we set out to investigate its role in acute dengue infection. MATERIALS AND METHODS: PAF levels were initially assessed in 25 patients with acute dengue infection to determine if they were increased in acute dengue. For investigation of the kinetics of PAF, serial PAF values were assessed in 36 patients. The effect of dengue serum on tight junction protein ZO-1 was determined by using human endothelial cell lines (HUVECs). The effect of dengue serum on and trans-endothelial resistance (TEER) was also measured on HUVECs. RESULTS: PAF levels were significantly higher in patients with acute dengue (n = 25; p = 0.001) when compared to healthy individuals (n = 12). In further investigation of the kinetics of PAF in serial blood samples of patients (n = 36), PAF levels rose just before the onset of the critical phase. PAF levels were significantly higher in patients with evidence of vascular leak throughout the course of the illness when compared to those with milder disease. Serum from patients with dengue significantly down-regulated expression of tight junction protein, ZO-1 (p = 0.004), HUVECs. This was significantly inhibited (p = 0.004) by use of a PAF receptor (PAFR) blocker. Serum from dengue patients also significantly reduced TEER and this reduction was also significantly (p = 0.02) inhibited by prior incubation with the PAFR blocker. CONCLUSION: Our results suggest the PAF is likely to be playing a significant role in inducing vascular leak in acute dengue infection which offers a potential target for therapeutic intervention.

Increased circulatory levels of lipopolysaccharide (LPS) and zonulin signify novel biomarkers of proinflammation in patients with type 2 diabetes.

Emerging data indicate that gut-derived endotoxin (metabolic endotoxemia) may contribute to low-grade systemic inflammation in insulin-resistant states. Specific gut bacteria seem to serve as lipopolysaccharide (LPS) sources and several reports claim a role for increased intestinal permeability in the genesis of metabolic disorders. Therefore, we investigated the serum levels of LPS and zonulin (ZO-1, a marker of gut permeability) along with systemic levels of tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) in patients with type 2 diabetes mellitus (T2DM) compared to control subjects. Study subjects were recruited from the Chennai Urban Rural Epidemiology Study [CURES], Chennai, India. Study group (n = 45 each) comprised of a) subjects with normal glucose tolerance (NGT) and (b) patients with T2DM. LPS, ZO-1, TNF-α, and IL-6 levels were measured by ELISA. Serum levels of LPS [p < 0.05], LPS activity [p < 0.001], ZO-1 [p < 0.001], TNFα [p < 0.001], and IL-6 [p < 0.001] were significantly increased in patients with T2DM compared to control subjects. Pearson correlation analysis revealed that LPS activity was significantly and positively correlated with ZO-1, fasting plasma glucose, 2 h post glucose, HbA1c, serum triglycerides, TNF-α, IL-6, and negatively correlated with HDL cholesterol. Regression analysis showed that increased LPS levels were significantly associated with type 2 diabetes [odds ratio (OR) 13.43, 95 % CI 1.998-18.9; p = 0.003]. In Asian Indians who are considered highly insulin resistant, the circulatory LPS levels, LPS activity, and ZO-1 were significantly increased in patients with type 2 diabetes and showed positive correlation with inflammatory markers and poor glycemic/lipid control.

Serum tight-junction proteins predict hemorrhagic transformation in ischemic stroke patients.

OBJECTIVE: To evaluate the significance of circulating tight-junction (TJ) proteins as predictors of hemorrhagic transformation (HT) in ischemic stroke patients. METHODS: We examined 458 consecutive ischemic stroke patients, 7.2% of whom had clinically evident HT. None of the patients was treated with thrombolytic drugs. Serum levels of standard markers of blood-brain barrier (BBB) breakdown (S100B, neuron-specific enolase), TJ proteins (occludin [OCLN], claudin 5 [CLDN5], zonula occludens 1 [ZO1]), and molecules involved in BBB disintegration (matrix metalloproteinase 9 and vascular endothelial growth factor [VEGF]) were assessed upon admission to the emergency department. A clinical deterioration caused by HT (cdHT) was defined as an increase of ≥4 points in the NIH Stroke Scale score in combination with a visible HT on a CT scan performed immediately after the onset of new neurologic symptoms. RESULTS: Patients with cdHT had higher concentrations of OCLN, S100B, and the CLDN5/ZO1 ratio, and a lower level of VEGF than those without cdHT. CLDN5 levels also correlated with cdHT occurrence when estimated within 3 hours of stroke onset. We also demonstrated correlations between the levels of circulating TJ molecules and the level of S100B, which is a previously established marker of BBB disruption. CONCLUSIONS: Analyzing serum levels of TJ proteins, like CLDN5, OCLN, and CLDN5/ZO1 ratio, as well as S100B and VEGF, is an effective way to screen for clinical deterioration caused by HT in ischemic stroke patients, both within and after the IV thrombolysis time window.