Serum TLR2 and TLR9 in Prostate Cancer Patients in Relation to EBV Status.

The relationship between Toll-like receptors (TLRs) and prostate cancer (PCa) is complex due to the presence of the Epstein-Barr virus (EBV) infection, which has been identified as a predisposing factor for some cancers, including PCa. The present study aims to investigate these complex links by examining the levels of selected TLRs and the potential impact of EBV infection on PCa. Therefore, we examined the serum of patients with PCa. The study compared EBV(+) patients to risk groups, the Gleason score (GS), and the T-trait. Additionally, the correlation between TLR and antibody levels was examined. The results indicated that higher levels of TLR-2 and TLR-9 were observed in more advanced PCa. The findings of this study may contribute to a deeper understanding of the role of viral infections in PCa and provide information on future strategies for the diagnosis, prevention, and treatment of these malignancies.

The association between TLR2/4 and clinical outcome in intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Toll-like receptors (TLRs) are involved in innate immunity and inflammatory responses in various diseases. Our study aimed to investigate the association between the levels of soluble TLR4 (sTLR4) and soluble TLR2 (sTLR2) and clinical outcomes following intracerebral hemorrhage (ICH). METHODS: Patients admitted to department of Neurology with acute ICH were included. Plasma levels of sTLR4 and sTLR2 after ICH were measured by enzyme-linked immunosorbent assay. Poor clinical outcome was defined as a modified Rankin score (mRS) of 3-6 at 3-month and 12-month after onset. RESULTS: All 207 patients with ICH and 100 non-stroke controls were included in our analysis. The mean sTLR4 level was 4.53±1.51 ng/ml and mean sTLR2 level was 3.65±0.72 ng/ml. There was significant trend towards worse clinical outcomes with increasing sTLR4 and sTLR2 terciles at 3 and 12 months. According to receiver operating curve (ROC), the sTLR4 was reliable predictor for poor clinical outcome at 3 months (ROC=0.75) and 12 months (ROC=0.74). The sTLR2 was less reliable predictor for poor clinical outcome at 3 months (ROC=0.64) and 12 months (ROC=0.65). The level of sTLR4 was an independent predictor of poor clinical outcome at 12-month (OR 1.24, 95 % CI 1.16-1.80; P=0.019). CONCLUSIONS: The sTLR4 quantification may provide accurate prognostic information after ICH.

Correlation of HMGB1, TLR2 and TLR4 with left ventricular diastolic dysfunction in sepsis patients.

Left ventricular diastolic dysfunction (LVDD) is a common consequence of sepsis due to dysregulated inflammatory responses. Here we aim to investigate high mobility group box 1 (HMGB1), toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) as serum biomarkers to assess LVDD risk of patients with sepsis. We recruited 120 patients with sepsis, among which 52 had ultrasonically confirmed LVDD and 68 were without LVDD. Blood samples were collected, and enzyme-linked immunosorbent assay (ELISA) was used to analyse levels of HMGB1, TLR2 and TLR4 in serum. Multivariate analysis was performed to assess the odds ratio of the serum biomarkers. Spearman's correlation analysis was conducted to evaluate the correlation between the serum biomarkers to B-type natriuretic peptide (BNP) and cardiac troponin I (cTnl) levels and the ratios of early diastolic mitral inflow velocity to early diastolic mitral annulus velocity (E/e' ratios) in ultrasound. Receiver operating curve was used to measure the sensitivity and specificity of HMGB1, TLR2 and TLR4 individually and in combination as diagnostic markers. Elevated HMGB1, TLR2 and TLR4 had significant values in predicting LVDD suggested by high odds ratio (all P < .05). A significant correlation was found between these values and cTnl, the current gold standard for LVDD analysis. HMGB1, TLR2 and TLR4 also showed a high diagnostic sensitivity and specificity in ROC analysis. HMGB1, TLR2 and TLR4 are potentially valuable in predicting LVDD risk among patients with sepsis, providing additional tools with the capability of potentially assisting the clinical management of patients with sepsis.

The Effects of 16 Weeks of Exercise Training on Neutrophil Functions in Breast Cancer Survivors.

Following therapy, breast cancer survivors (BCS) have an increased risk of infections because of age and cancer dysregulation of inflammation and neutrophil functions. Neutrophil functions may be improved by exercise training, although limited data exist on exercise and neutrophil functions in BCS.Sixteen BCS [mean age: 56 (SD 11) years old] completed 16 weeks of community-based exercise training and a 45-minute acute bout of cycling before (Base) and after (Final) the exercise training program. Exercise training consisted of 3 x 40 - 60 minute mixed mode aerobic exercises, comprising 10 - 30 minutes aerobic and 30 minutes resistance training. At Base and Final, we took BCS blood samples before (PRE), immediately after (POST), and 1 hour after (1Hr) acute exercise to determine neutrophil counts, phenotype, bacterial killing, IL-6, and IL-8 levels. Eleven healthy, age- and physical activity levels-matched women (Control) completed the acute bout of exercise once as a healthy response reference. Resting Responses. BCS and Controls had similar Base PRE absolute neutrophil counts [mean (SD): 3.3 (1.9) v 3.1 (1.2) x 109/L, p=0.801], but BCS had lower bacterial phagocytosis [3991 (1233) v 4881 (417) MFI, p=0.035] and higher oxidative killing [6254 (1434) v 4709 (1220) MFI, p=0.005], lower CD16 [4159 (1785) v 7018 (1240) MFI, p<0.001], lower CXCR2 [4878 (1796) v 6330 (1299) MFI, p=0.032] and higher TLR2 [98 (32) v 72 (17) MFI, p=0.022] expression, while IL-6 [7.4 (5.4) v 4.0 (2.7) pg/mL, p=0.079] levels were marginally higher and IL-8 [6.0 (4.7) v 7.9 (5.0) pg/mL, p=0.316] levels similar. After 16 weeks of training, compared to Controls, BCS Final PRE phagocytosis [4510 (738) v 4881 (417) MFI, p=0.146] and TLR2 expression [114 (92) v 72 (17) MFI, p=0.148] were no longer different. Acute Exercise Responses. As compared to Controls, at Base, BCS phagocytic Pre-Post response was lower [mean difference, % (SD): 12% (26%), p=0.042], CD16 Pre-Post response was lower [12% (21%), p=0.016] while CD16 Pre-1Hr response was higher [13% (25%), p=0.022], TLR2 Pre-Post response was higher [15% (4%) p=0.002], while IL-8 Pre-Post response was higher [99% (48%), p=0.049]. As compared to Controls, following 16 weeks of training BCS phagocytic Pre-Post response [5% (5%), p=0.418], CD16 Pre-1Hr response [7% (7%), p=0.294], TLR2 Pre-Post response [6% (4%), p=0.092], and IL-8 Pre-Post response [1% (9%), p=0.087] were no longer different. Following cancer therapy, BCS may have impaired neutrophil functions in response to an acute bout of exercise that are partially restored by 16 weeks of exercise training. The improved phagocytosis of bacteria in BCS may represent an exercise-induced intrinsic improvement in neutrophil functions consistent with a reduced risk of infectious disease. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03760536.

Toll-like Receptor 2 as a Marker Molecule of Advanced Ovarian Cancer.

Ovarian cancer is a global problem that affects women of all ages. Due to the lack of effective screening tests and the usually asymptomatic course of the disease in the early stages, the diagnosis is too late, with the result that less than half of the patients diagnosed with ovarian cancer (OC) survive more than five years after their diagnosis. In this study, we examined the expression of TLR2 in the peripheral blood of 50 previously untreated patients with newly diagnosed OC at various stages of the disease using flow cytometry. The studies aimed at demonstrating the usefulness of TLR2 as a biomarker in the advanced stage of ovarian cancer. In this study, we showed that TLR2 expression levels were significantly higher in women with more advanced OC than in women in the control group. Our research sheds light on the prognostic potential of TLR2 in developing new diagnostic approaches and thus in increasing survival in patients with confirmed ovarian cancer.

Toll-like receptor 10 controls TLR2-induced cytokine production in monocytes from patients with Parkinson's disease.

Peripheral inflammation, particularly mediated by monocytes, can cause neuroinflammation in Parkinson's disease (PD). We investigated the mechanism of TLR2-induced cytokine impairment in peripheral monocytes from PD patients and the association between the presence of CD14+ TLR10+ monocytes and PD severity. Peripheral blood mononuclear cells from PD patients and healthy individuals were evaluated for TLR expression on monocyte subsets (CD14 and CD16 expression) using flow cytometry. Moreover, cytokines were evaluated using flow cytometry after stimulation with Pam3 Cys (TLR2/TLR1 agonist) in the absence or presence of neutralizing antibodies to TLR10. The severity of PD was assessed using the unified PD rating scale (UPDRS) and motor activity, anxiety (BAI), depression (BDI), and fatigue (PD Fatigue Scale-16) scales. The frequency of CD14+ TLR10+ monocytes and expression intensity of TLR2 and TLR10 were higher in patients with PD than healthy individuals. The frequency of intermediate monocytes (CD14++ CD16+ ) was not significantly increased in patients with PD, but was the main monocyte subset expressing TLR10. The TLR2/TLR1-impaired cytokine production (IL-6, TNFα, IL-8, and IL-10) in PD patients was reversed by neutralizing TLR10. The high frequency of total CD14+ TLR10+ monocytes was associated with a reduction in the severity of PD according to the evaluation of motor and nonmotor symptoms. Peripheral monocytes from patients with PD showed phenotypic and functional alterations. The expression of TLR10 on monocytes can protect against PD by controlling TLR2-induced cytokine production. Furthermore, data suggested that a low frequency of CD14+ TLR10+ monocytes indicates the severity of PD. The results identified new opportunities for the development of novel PD neuroprotective therapies.

Lipidomic analysis of local inflammation models shows a specific systemic acute phase response to lipopolysaccharides.

Toll-like receptors (TLR) are crucial for recognizing bacterial, viral or fungal pathogens and to orchestrate the appropriate immune response. The widely expressed TLR2 and TLR4 differentially recognize various pathogens to initiate partly overlapping immune cascades. To better understand the physiological consequences of both immune responses, we performed comparative lipidomic analyses of local paw inflammation in mice induced by the TLR2 and TLR4 agonists, zymosan and lipopolysaccharide (LPS), respectively, which are commonly used in models for inflammation and inflammatory pain. Doses for both agonists were chosen to cause mechanical hypersensitivity with identical strength and duration. Lipidomic analysis showed 5 h after LPS or zymosan injection in both models an increase of ether-phosphatidylcholines (PC O) and their corresponding lyso species with additional lipids being increased only in response to LPS. However, zymosan induced stronger immune cell recruitment and edema formation as compared to LPS. Importantly, only in LPS-induced inflammation the lipid profile in the contralateral paw was altered. Fittingly, the plasma level of various cytokines and chemokines, including IL-1ß and IL-6, were significantly increased only in LPS-treated mice. Accordingly LPS induced distinct changes in the lipid profiles of ipsilateral and contralateral paws. Here, oxydized fatty acids, phosphatidylcholines and phosphatidylethanolamines were uniquely upregulated on the contralateral side. Thus, both models cause increased levels of PC O and lyso-PC O lipids at the site of inflammation pointing at a common role in inflammation. Also, LPS initiates systemic changes, which can be detected by changes in the lipid profiles.

Cytomegalovirus Infection Downregulates Vitamin D Receptor in Patients Undergoing Hematopoietic Stem Cell Transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative option for patients with hematologic diseases but is associated with high mortality and morbidity. Cytomegalovirus (CMV) infection is common in HSCT patients and modulates vitamin D metabolism in vitro. We aimed at validating CMV-associated vitamin D metabolism in vivo in HSCT. METHODS: Patients treated for significant CMV viremia after HSCT were evaluated for CMV load before, during, and after antiviral treatment. RNA was isolated from whole-blood samples to test for regulation of key components of the vitamin D receptor (VDR) pathway during different phases of CMV viremia. RESULTS: CMV viremia developed a mean time of 102 (±34) d post-HSCT. Maximum levels of CMV-DNA reached a mean of 5668 (±7257) copies/mL. VDR expression was downregulated to a mean of 64.3% (±42.5%) relative to the VDR expression pre-CMV viremia (P = 0.035) and lagged in recovery following antiviral treatment. Toll-like receptor (TLR) 2 mRNA was upregulated to 225.4% during CMV viremia relative to the expression pre-CMV viremia (P = 0.012) but not TLR6/7/8 and the TLR-adaptor protein MyD88. Levels of 25-OH vitamin D were reduced in all viremic patients (48.0 ± 4.8 versus 25.1 ± 3.7 ng/mL) and were even lower after periods of CMV viremia compared with the control group (48.3 ± 3.5 versus 17.8 ± 1.8 ng/mL; P = 0.008). CONCLUSIONS: CMV viremia is associated with significant dysregulation of vitamin D metabolism in HSCT patients.

Association of a Serum Protein Signature With Rheumatoid Arthritis Development.

OBJECTIVE: The pathophysiologic events that precede the onset of rheumatoid arthritis (RA) remain incompletely understood. This study was undertaken to identify changes in the serum proteome that precede the onset of RA, with the aim of providing new insights into the pathogenic mechanisms that lead to its development. METHODS: In a cohort of first-degree relatives of Indigenous North American RA patients, the SomaScan proteomics platform was used to determine the levels of 1,307 proteins in multiple longitudinal serum samples from 17 individuals who were followed up prospectively to the time of disease onset. Proteomic signatures from this group of individuals (designated the progressor group) were compared to those in a group of individuals who were considered at risk of developing RA, stratified as either positive (n = 63) or negative (n = 47) for anti-citrullinated protein antibodies (ACPAs) (designated the at-risk group). Machine learning was used to identify a protein signature that could accurately classify those individuals at highest risk of future RA development. RESULTS: A preclinical proteomic signature that differentiated RA progressors from at-risk individuals, irrespective of ACPA status, was identified (area under the curve 0.913, accuracy 91.2%). Importantly, the predictive preclinical proteomic signature was present not only in serum samples obtained close to the onset of RA, but also in serum samples obtained a median of 30.9 months prior to onset. Network analysis implicated the activation of Toll-like receptor 2 and production of tumor necrosis factor and interleukin-1 as key events that precede RA progression. CONCLUSION: Alterations in the serum proteome in the preclinical phase of RA can emerge years prior to the onset of disease. Our findings suggest that the serum proteome provides a rich source of proteins serving both to classify at-risk individuals and to identify molecular pathways involved in the development of clinically detectable RA.

Physical activity prevents acute inflammation in a gout model by downregulation of TLR2 on circulating neutrophils as well as inhibition of serum CXCL1 and is associated with decreased pain and inflammation in gout patients.

OBJECTIVES: Gout is the most prevalent inflammatory arthritis. To study the effects of regular physical activity and exercise intensity on inflammation and clinical outcome, we examined inflammatory pathogenesis in an acute model of murine gout and analyzed human gout patient clinical data as a function of physical activity. METHODS: NF-κB-luciferase reporter mice were organized into four groups and exercised at 0 m/min (non-exercise), 8 m/min (low-intensity), 11 m/min (moderate-intensity), and 15 m/min (high-intensity) for two weeks. Mice subsequently received intra-articular monosodium urate (MSU) crystal injections (0.5mg) and the inflammatory response was analyzed 15 hours later. Ankle swelling, NF-κB activity, histopathology, and tissue infiltration by macrophages and neutrophils were measured. Toll-like receptor (TLR)2 was quantified on peripheral monocytes/neutrophils by flow cytometry and both cytokines and chemokines were measured in serum or synovial aspirates. Clinical data and questionnaires accessing overall physical activity levels were collected from gout patients. RESULTS: Injection of MSU crystals produced a robust inflammatory response with increased ankle swelling, NF-κB activity, and synovial infiltration by macrophages and neutrophils. These effects were partially mitigated by low and moderate-intensity exercise. Furthermore, IL-1ß was decreased at the site of MSU crystal injection, TLR2 expression on peripheral neutrophils was downregulated, and expression of CXCL1 in serum was suppressed with low and moderate-intensity exercise. Conversely, the high-intensity exercise group closely resembled the non-exercised control group by nearly all metrics of inflammation measured in this study. Physically active gout patients had significantly less flares/yr, decreased C-reactive protein (CRP) levels, and lower pain scores relative to physically inactive patients. CONCLUSIONS: Regular, moderate physical activity can produce a quantifiable anti-inflammatory effect capable of partially mitigating the pathologic response induced by intra-articular MSU crystals by downregulating TLR2 expression on circulating neutrophils and suppressing systemic CXCL1. Low and moderate-intensity exercise produces this anti-inflammatory effect to varying degrees, while high-intensity exercise provides no significant difference in inflammation compared to non-exercising controls. Consistent with the animal model, gout patients with higher levels of physical activity have more favorable prognostic data. Collectively, these data suggest the need for further research and may be the foundation to a future paradigm-shift in conventional exercise recommendations provided by Rheumatologists to gout patients.

Trimetazidine Improves the Outcome of EECP Therapy in Patients with Refractory Angina Pectoris.

INTRODUCTION: Cardiovascular disease (CAD) associated with death and disability remains a serious medical problem. In some patients the initial clinical coronary artery disease presentation is stable angina pectoris. AIM: The aim of the study was to evaluate the effect of EECP therapy with or without trimetazidine (TMZ) in patients with refractory angina via modulating peripheral monocyte expression of Toll like receptor2 (TLR2) and its downstream signaling. METHODS: This is a double-blind randomized prospective study in which 88 stable refractory angina patients allocated into two groups, Enhanced External Counter Pulsation (EECP) group: included 44 patients with stable refractory angina, and were treated with EECP-Therapy. TMZ-EECP group: included 44 patients with stable refractory angina, we gave TMZ 35 mg twice daily in addition to EECP-Therapy. RESULTS: TLR2 expression in peripheral monocyte investigated by flow cytometry and 8-iso-prostaglandin F2ß (8-iso-PGF2 ß), interleukin1ß (IL-1ß), heat shock protein 60 (HSP60) and monocytes chemoattractant protein-1(MCP-1) were also measured before the EECP-therapy and before giving TMZ to patients, and after 35 hours of EECP treatment (7 consecutive weeks). Inhibition in TLR2 expression in peripheral monocyte was observed among the EECP group (P<0.05). Inflammatory cytokine MCP-1 was remarkably decreased in both study groups but (heat shock protein 60 (HSP60), MCP-1 and interleukin-1ß (IL-1ß)) significantly decreased levels were observed among the TMZ-EECP group (P<0.05). Also, the oxidative stress biomarker 8-iso-prostaglandin F2ß (8-iso-PGF2ß) was decreased in both study groups but significantly decreased levels were observed among the TMZ-EECP group (P<0.05). TMZ and EECP therapy in patients with stable refractory angina remarkably decreased the inflammatory markers HSP60, MCP-1 and IL-1ß in serum levels also the decreased levels were found in serum levels of oxidative stress marker 8-iso-PGF2ß serum level. CONCLUSION: EECP-therapy decreased the expression of TLR2 on peripheral monocytes in patients with chronic stable refractory angina which yield improvement in the quality of patients' life by decreasing the frequency of angina episodes, decreasing the Short-acting nitrate use and change the exercise tolerance and distance.

Toll-like receptor-2 mediates systemic inflammation in gentamicin-induced rat nephrotoxicity.

Gentamicin is an aminoglycoside antibiotic commonly administrated to patients with Gram-negative infections. Gentamicin induced nephrotoxicity by functional and structural impairment. Toll-like receptors (TLRs) as key mediators in the innate and adaptive immune system response involved in gentamicin-induced nephrotoxicity. The present study aimed to investigate the gene expression of TLR2 and pro-inflammatory cytokines in the renal tissues and buffy coat of the whole blood in gentamicin-treated rats. Twenty adult male Sprague Dawley rats weighing 180-200 were randomly divided into gentamicin (100 mg/kg, i.p) and control groups (n = 10). After 10 days, the serum creatinine (Cr) levels and blood urea nitrogen (BUN) were measured. The mRNA levels of TLR2, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and monocyte chemoattractant peptide (MCP)-1 were investigated in the renal tissue and buffy coat by qRT-PCR. Kidney histological analysis performed by hematoxylin-eosin (H&E) staining. Functional disturbance is characterized by a significant increase in the serum levels of Cr and BUN in the gentamicin group. Renal tissue slides of the gentamicin group indicated severe glomerular and tubular damage including lobulation of the glomerular tuft, Bowman's space enlargement, acute tubular necrosis, and proximal tubular destruction. The mRNA levels of IL-1ß, TNF-α, MCP-1, and TLR2 increased in the buffy coat, but all of them except TLR2 decreased in the renal tissues in the gentamicin group compared with controls. Gentamicin administration induced relative systemic inflammation, which may be related to an increase in the mRNA levels of TLR2 results in gene expression of pro-inflammatory chemokines and cytokines including IL-1ß, TNF-α, and MCP-1 in immune cells.

Ischemic postconditioning attenuates the inflammatory response in ischemia/reperfusion myocardium by upregulating miR­499 and inhibiting TLR2 activation.

Toll-like receptor 2 (TLR2)-mediated myocardial inflammation serves an important role in promoting myocardial ischemic/reperfusion (I/R) injury. Previous studies have shown that miR­499 is critical for cardioprotection after ischemic postconditioning (IPostC). Therefore, the present study evaluated the protective effect of IPostC on the myocardium by inhibiting TLR2, and also assessed the involvement of microRNA (miR)­499. Rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. The IPostC was 3 cycles of 30 sec of reperfusion and 30 sec of re­occlusion prior to reperfusion. In total, 90 rats were randomly divided into six groups (n=15 per group): Sham; I/R; IPostC; miR­499 negative control adeno­associated virus (AAV) vectors + IPostC; miR­499 inhibitor AAV vectors + IPostC; and miR­499 mimic AAV vectors + IPostC. It was identified that IPostC significantly decreased the I/R­induced cardiomyocyte apoptotic index (29.4±2.03% in IPostC vs. 42.64±2.27% in I/R; P<0.05) and myocardial infarct size (48.53±2.49% in IPostC vs. 66.52±3.1% in I/R; P<0.05). Moreover, these beneficial effects were accompanied by increased miR­499 expression levels (as demonstrated by reverse transcription­quantitative PCR) in the myocardial tissue and decreased TLR2, protein kinase C (PKC), interleukin (IL)­1ß and IL­6 expression levels (as demonstrated by western blotting and ELISA) in the myocardium and serum. The results indicated that IPostC + miR­499 mimics significantly inhibited inflammation and the PKC signaling pathway and enhanced the anti­inflammatory and anti­apoptotic effects of IPostC. However, IPostC + miR­499 inhibitors had the opposite effect. Therefore, it was speculated that IPostC may have a miR­499­dependent cardioprotective effect. The present results suggested that miR­499 may be involved in IPostC­mediated ischemic cardioprotection, which may occur via local and systemic TLR2 inhibition, subsequent inhibition of the PKC signaling pathway and a decrease in inflammatory cytokine release, including IL­1ß and IL­6. Moreover, these effects will ultimately lead to a decrease in the myocardial apoptotic index and myocardial infarct size via the induction of the anti­apoptotic protein Bcl­2, and inhibition of the pro­apoptotic protein Bax in myocardium.

ASSOCIATIONS BETWEEN EFFICACY OF THE THERAPY AND CIRCADIAN FLUCTUATIONS OF ENDOTHELIAL NITRIC OXIDE SYNTHASE WITH TOLL-LIKE RECEPTORS 2 EXPRESSION, AND NOS3 POLYMORPHISM IN FEMALES WITH RHEUMATOID ARTHRITIS.

Despite significant progress in treatment of rheumatoid arthritis (RA), a considerable part of patients remains resistant to the current therapy, apparently for the reasons of undefined mechanisms of its pathogenesis. Recently, the disturbances of circadian regulation of inflammatory processes in RA have been highlighted as important ones. Endothelial nitric oxide synthase (NOS3) and soluble toll-like receptors 2 (sTLR2) take part in the regulation of angiogenesis, osteoclastogenesis, immune responses but their circadian rhythms and predictive significance in RA patients are still unknown. Aim - to estimate the associations between efficacy of treatment and the circadian rhythms of NOS3 and sTLR2, and NOS3 polymorphism in females with rheumatoid arthritis, Ukraine. 97 RA patients (100% female) aged 46.3±8.89 years with disease duration 8.44±6.52 years were examined. All patients as a disease-modifying therapy received methotrexate (MTX) orally in a dose ≤15 mg/week, folic acid 5 mg/week, NSAIDs and corticosteroids (CS) ≤10 mg/day by prednisone. Doses of MTX, NSAIDs and CS were stable 4 weeks prior to the enrolment and during the whole period of study. The efficacy end points included DAS28, RAID and American College of Rheumatology response criteria (ACR20/50/70). Serum levels of NOS3 and sTLR2 were determined at 08:00 and 20:00 using Cloud-Clone Corp kits (USA). NOS3 T-786С polymorphism was determined by Real-Time PCR. The SPSS22 software package was used for statistical processing of the results. The study was performed in accordance to the bioethical standards. After 12-week treatment among RA patients were revealed 52.6% ACR 20 responders and 47.4% non-responders. Opposite diurnal variation of NOS3 and sTLR2 serum levels were found in RA patients. There were significant differences in NOS3/sTLR2 ratio at 08:00 accordingly to NOS3 T786C genotype. The disturbances in daily variability of NOS3 or sTLR2 serum levels were more significant in non-responders compare to responders. Decrease of NOS3/sTLR2 ratio was a predictor of non-response to treatment in RA patients (ß=0.366, р=0.000). In RA patients the disturbances of circadian rhythms of endothelial nitric oxide synthase or toll-like receptors 2 expression are associated with an increase of resistance to disease-modifying therapy with methotrexate.

Relationship between Small Intestinal Bacterial Overgrowth and Peripheral Blood ET, TLR2 and TLR4 in Ulcerative Colitis.

OBJECTIVE: To investigate the relationship between small intestinal bacterial overgrowth (SIBO) and Endotoxin (ET) concentration in peripheral blood, and levels of toll-like receptors (TLR) 2 and TLR4 expression on surface of peripheral blood mononuclear cells (PBMCs) in patients with ulcerative colitis. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: The First Hospital of Hebei Medical University, from July 2018 to October 2019. METHODOLOGY: The 130 patients with ulcerative colitis were included in case group. Another 72 healthy cases were selected as control group. SIBO, ET, TLR2, and TLR4, were determined, and compared. RESULTS: Positive rate of SIBO in case group was higher than that in control group (p <0.001). Lactulose hydrogen breath test (LHBT) intestine set value, peripheral blood ET concentration, and TLR2 and TLR4 expression levels on surface of PBMCs in case group were higher than those in control group (all p <0.001); the above indexes in SIBO-positive patients in case group were higher than those in SIBO-negative patients in case group (all p <0.001). Pearson's correlation analysis showed that LHBT intestine set value of SIBO-positive patients in case group was positively correlated with ET concentration, and TLR2 and TLR4 expression levels on surface of PBMCs (r= 0.910, p <0.001; r = 0.970, p <0.001; and r = 0.965, p <0.001 respectively). ET concentration of SIBO-positive patients in case group was positively correlated with expression levels of TLR2 and TLR4 on surface of PBMCs (r=0.962, p <0.001; and r = 0.829 p <0.001 respectively). CONCLUSION: Patients with ulcerative colitis are easy to occur SIBO, and SIBO increases blood endotoxin, TLR2 and TLR4 levels. Synergistic effects of endotoxin and endotoxin receptors TLR2 and TLR4 overexpression mediate body inflammation and may be involved in progression of ulcerative colitis. Patients with ulcerative colitis with excessive growth of small intestinal bacteria are more likely to have hypertoxemia.

Toll-like receptor 2 and 9 expression on circulating neutrophils is associated with increased mortality in critically ill patients.

BACKGROUND: Toll-like receptors (TLRs) play an important role in inflammatory processes in critically ill patients by binding to pathogen-associated molecular patterns and danger-associated molecular patterns (DAMPs). Whether neutrophil or monocyte TLR expression patterns are associated with outcome in critical illness is unknown. OBJECTIVES: To answer this question, we conducted a prospective, observational study including 215 consecutive patients admitted to a medical ICU at a tertiary care center. METHODS: Blood was drawn at admission and expression of TLR-2, TLR-4, and TLR-9 on neutrophils and monocytes were analyzed by flow cytometry. RESULTS: Median Acute Physiology and Chronic Health Evaluation II (APACHE II) score was 19, and 30-day mortality was 26%. TLR-2 expression on neutrophils was associated with APACHE II, Simplified Acute Physiology Score II, and Sepsis-related Organ Failure Assessment score. TLR-2 (P < 0.001) and TLR-9 (P < 0.05) expression on neutrophils was significantly higher in nonsurvivors. In contrast, neutrophil TLR-4 expression and monocyte TLR expression were not associated with survival. Neutrophil TLR-2 (odds ratio 3.8; 95% confidence interval 1.4-10.2; P < 0.05) and TLR-9 (odds ratio 4.0; 95% confidence interval 2.0-8.1; P < 0.001) expression in the third tertile predicted mortality independent from APACHE II, serum lactate, serum creatinine, and procalcitonin, respectively. CONCLUSION: We provide evidence for prognostic properties of neutrophil TLR-2 and TLR-9 expression regarding 30-day mortality in unselected critically ill patients, independent from baseline clinical characteristics, and laboratory values. These findings suggest that specific TLR-dependent activation of the innate immune system via neutrophils possibly caused by cell damage and release of otherwise intracellular components may play a significant role in the pathophysiology of critical illness.

Higher Levels of Serum TLR2 and TLR4 in Patients with Hashimoto's Thyroiditis.

OBJECTIVE: Hashimoto's thyroiditis (HT) is an autoimmune disorder caused by the interaction between genes and environmental triggers. HT is the most common endocrine disorder, as well as the most common cause of hypothyroidism. Autoimmunity plays a crucial role in the pathogenesis of HT and recent studies suggest that Toll-like receptor (TLR) signals lead to increased inflammatory response. The aim of our study is to investigate whether TLR-2 and TLR-4 levels and gene polymorphisms contribute to the damaged immune response leading to HT. METHODS: Using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, single-nucleotide polymorphisms (SNPs) of TLR2 gene Arg677Trp, Arg753Gln, 196-174 del and TLR4 gene Asp299Gly, Thr399Ile were studied in 100 patients with HT and 100 healthy controls. Also, we investigated serum levels of TLR-2 and TLR-4 in the immunopathogenesis of HT. TLR-2 and TLR-4 serum levels were found to be significantly higher in HT patients than the control group. However, no statistical significance was found between patient and control groups in terms of genotype frequencies and allele frequency distribution of TLR2 gene Arg677Trp, Arg753Gln, 196-174 del and TLR4 gene Asp299Gly, Thr399Ile polymorphisms. RESULT: TLR2 gene Arg677Trp, Arg753Gln, 196-174 del and TLR4 gene Asp299Gly, Thr399Ile polymorphism do not appear to have a role in the development of HT disease. However, in our study, serum levels of TLR-2 and TLR-4 were found to be higher in HT patients than control groups. CONCLUSION: These findings suggest that TLR-2 and TLR-4 play an important role in the immunopathologic mechanism of disease by causing an increase in proinflammatory response.

Mulberry leaf reduces inflammation and insulin resistance in type 2 diabetic mice by TLRs and insulin Signalling pathway.

BACKGROUND: It has been testified that Diabetes mellitus (DM) has a close association with chronic inflammation and Toll-like Receptors (TLRs), and DM could be prevented by mulberry leaf. Therefore, a hypothesis came into being that mulberry leaf could ameliorate proinflammation and insulin resistance (IR) through TLRs and insulin signalling pathways. METHODS: Water extracts of mulberry leaf (WEM) was given to diabetic mice by gavage for 10 weeks, and the diabetic mice was injected with low-dose streptozocin, fed with high-fat and high-sugar diet. Oral glucose tolerance tests (OGTTs) were conducted. At the same time, homeostasis model assessment of insulin (HOMA-IR) and the level of the inflammatory factor, tumour necrosis factor-α (TNF-α) was measured. The expressions of critical nodes of TLRs and insulin signalling pathway were also examined. RESULTS: WEM contributed to a significant decrease in fasting blood glucose, AUC from the investigation of OGTTs and HOMA-IR. The levels of the inflammatory factor, tumour necrosis factor-α (TNF-α) also declined. Moreover, WEM suppressed the expression of TLR2, myeloid differentiation primary-response protein 88 (MyD88), tumour-necrosis-factor receptor-associated factor 6 (TRAF6), nuclear factor kappa B (NF-κB) in the skeletal muscle. WEM could up-regulate the expression of insulin receptor (InsR) and insulin receptor substrate 1 (IRS1), and down-regulate the phosphorylation of IRS1 in adipose tissue. CONCLUSION: Through this study, a conclusion could be made that WEM mitigates hyperglycemia, IR, and inflammation through the interactions among TLR2 signalling pathway, insulin signalling pathway and TNF-α.

Assessment of TLR-2 concentration in tick-borne encephalitis and neuroborreliosis.

The aim of the study was to check whether measurement of TLR-2 in serum or cerebrospinal fluid (CSF) can help differentiate between neuroborreliosis (NB) and tick-borne encephalitis (TBE). Eighty patients with meningitis and meningoencephalitis were divided into two groups: Group I - patients with NB (n = 40) and Group II - patients with TBE (n = 40). Diagnosis was based on the clinical picture, CSF examination and presence of specific antibodies in serum and CSF. The control group (CG) consisted of healthy blood donors (n = 25) and patients in whom inflammatory process in central nervous system was excluded (n = 25). Concentration of TLR-2 was measured using a commercial kit [TLR-2 Elisa Kit (EIAab, China)]. The serum and CSF TLR-2 concentration of NB patients was significantly higher than in CG. The serum and CSF TLR-2 concentration in TBE patients was significantly higher than in the CG. Receiver operating characteristic analysis of the serum TLR-2 concentration showed significant differences between the group of patients with NB and a group of patients with TBE. TLR-2 is involved in the development of inflammatory process in the CNS caused by both tick-borne pathogens: viral and bacterial as TLR-2 concentration in both CSF and serum differentiates these groups from healthy patients. Although TLR-2 cannot be used as a sole and reliable biomarker differentiating NB from TBE, results of our study are a step forward toward discovering such biomarker in the future.

Serum TLR2 and TLR4 levels in colorectal cancer and their association with systemic inflammatory markers, tumor characteristics, and disease outcome.

Toll-like receptors (TLRs) are involved in colorectal cancer (CRC) pathogenesis. However, the significance of serum TLR concentrations in CRC is unknown. We analyzed serum TLR2 and TLR4 concentrations with ELISA in preoperative samples from 118 patients with CRC and 88 matched controls. We also assessed tissue TLR expression with immunohistochemistry and by detecting serum determinants of systemic inflammation. Most participants (>70%) had undetectable serum TLR2. The mean serum TLR4 levels were lower in patients than in controls (1.1 vs 1.8 ng/mL; p = 0.015). Undetectable TLR4 was more common in stage I (39%) than in stages II-IV (11%, p < 0.001). TLR2 or TLR4 expression in tumor cells did not correlate with serum levels, but abundant TLR2 expression in normal colon epithelium was associated with detectable serum TLR2 (p = 0.034). Undetectable serum TLR2 was linked to high modified Glasgow prognostic scores (p = 0.010), high CRP levels (p = 0.013), blood vessel invasion (p = 0.013), and tended to be associated with worse 5-year survival (p = 0.052). In conclusion, serum TLR2 levels were inversely associated with systemic inflammation in patients with CRC. Moreover, serum TLR2 levels might depend more on normal colorectal mucosa contributions than on tumor tissue contributions. Further studies are required to assess the prognostic value of serum TLR2.