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.

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.

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.

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.

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.

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.

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.

[Relationship of TLR2 and TLR4 expressions on the surface of peripheral blood mononuclear cells to small intestinal bacteria overgrowth in patients with hepatocellular carcinoma].

Objective: To investigate TLR2 and TLR4 expressional situation on the surface of peripheral blood mononuclear cells (PBMC) in patients with hepatocellular carcinoma (HCC) and their relationship with small intestinal bacterial overgrowth (SIBO). Methods: Flow cytometry was used to detect TLR2 and TLR4 expressional situation on the surface of PBMC in 78 cases with HCC, 56 cases with cirrhosis and 33 healthy controls. Furthermore, lactose hydrogen breath test (LHBT) was used to detect small intestinal bacterial overgrowth. Results: Of the 78 cases with HCC, 56 cases (71.8%) were SIBO-positive, 23 cases (41.1%) were SIBO- positive in 56 cases with cirrhosis, and 1 (3.0%) was SIBO-positive in 33 healthy controls. The incidence of SIBO in HCC patients was higher than cirrhosis patients (χ(2) = 12.72, P < 0.05) and healthy controls (χ(2) = 41.18, P < 0.05). The expression levels of TLR2 and TLR4 in HCC patients (100.55 ± 24.22, 42.76 ± 15.96) were significantly higher than cirrhosis (67.42 ± 18.36, 24.38 ± 8.68)and healthy control group (33.06 ± 11.72, 12.52 ± 4.46) (P < 0.05). Furthermore, the expression levels of TLR2 and TLR4 in SIBO-positive patients (108.75 ± 20.40, 48.1 ± 14.98) were higher than SIBO-negative patients (79.67 ± 20.60, 28.62 ± 7.36) (P < 0.05). Conclusion: The expression of TLR2 and TLR4 and the incidence of SIBO in HCC patients are significantly higher than cirrhosis and healthy control group. Moreover, the high expressions of TLR2 and TLR4 in SIBO-positive HCC patients may promote the development of HCC.

Reduction of Articular and Systemic Inflammation by Kava-241 in a Porphyromonas gingivalis-Induced Arthritis Murine Model.

Rheumatoid arthritis (RA) is an inflammatory disease that has been linked to several risk factors, including periodontitis. Identification of new anti-inflammatory compounds to treat arthritis is needed. We had previously demonstrated the beneficial effect of Kava-241, a kavain-derived compound, in the management of Porphyromonas gingivalis-induced periodontitis. The present study evaluated systemic and articular effects of Kava-241 in an infective arthritis murine model triggered by P. gingivalis bacterial inoculation and primed with a collagen antibody cocktail (CIA) to induce joint inflammation and tissular destruction. Clinical inflammation score and radiological analyses of the paws were performed continuously, while histological assessment was obtained at sacrifice. Mice exposed to P. gingivalis and a CIA cocktail and treated concomitantly with Kava-241 exhibited a reduced clinical inflammatory score and a decreased number of inflammatory cells and osteoclasts within joint. Kava-241 treatment also decreased significantly tumor necrosis factor alpha (TNF-α) in serum from mice injected with a Toll-like receptor 2 or 4 (TLR-2/4) ligand, P. gingivalis-lipopolysaccharide (LPS). Finally, bone marrow-derived macrophages infected with P. gingivalis and exposed to Kava-241 displayed reduced TLR-2/4, reduced mitogen-activated protein kinase (MAPK)-related signal elements, and reduced LPS-induced TNF-α factor (LITAF), all explaining the observed reduction of TNF-α secretion. Taken together, these results emphasized the novel properties of Kava-241 in the management of inflammatory conditions, especially TNF-α-related diseases such as infective RA.

TLR-2, IL-10 and IL-17-mediated immunity in experimental chemotherapy murine model of systemic candidiasis; cyclophosphamides' impact and roles.

The majority of immune components such as Toll-like receptor (TLR)-2, interleukin (IL)-17, neutrophils, and IL-10 play pivotal roles in immunity to Candida albicans (C. albicans) through identifying and launching inflammatory and regulatory responses. Chemotherapy is one of the most potent risk factors for systemic candidiasis through inducing immunosuppression (mostly cyclophosphamide induced immunosuppression) and there is a sensible lack of study around the immunity to C. albicans in such a situation. In this study, following the establishment of infection and immunosuppression in Balb/c mice model, the mRNA/protein levels of TLR-2, IL-10, IL-17, and Myeloperoxidase (MPO) in serum/kidney were measured using Real-time PCR and ELISA respectively. The survival of mice was checked daily and organ fungal burden was calculated and the histology samples were prepared. Results indicated that the mRNA and protein levels of IL-10, IL-17 and MPO were significantly elevated in immunosuppressed-infected mice (P < 0.05). Conversely, the mRNA level of TLR-2 in this mice were significantly decreased (P < 0.05). We conclude that, I. cyclophosphamide could induce only a minor state of immunosuppression through depletion of serum neutrophils. II. TLR-2 does not have important roles in developing immune responses in immunosuppressed mice model of systemic candidiasis. Our findings can be applicable for further experimental investigations on patients in clinics for deep understanding of pathogenesis of systemic candidiasis, which could be useful to further broaden our insights for targeted therapy, especially targeting TLR-2 and IL-17, based on siRNA, miRNA or monoclonal antibodies.

Toll-Like Receptors 2 and 4 Predict New-Onset Atrial Fibrillation in Acute Myocardial Infarction Patients.

Myocardial infarction (MI) can cause new-onset atrial fibrillation (AF) due to cardiac remodeling. As a recent study has shown, inflammatory factors are closely tied to cell death and survival in myocardial ischemia injury. Toll-like receptors (TLRs) have been shown to participate in the process of myocardial infarction as innate immune factors.The subjects were divided into 3 groups: healthy controls (n = 82), MI patients (n = 84), and AFMI (new-onset atrial fibrillation after myocardial infarction) patients (n = 85). Peripheral blood mononuclear cell (PBMC) TLR mRNA expression was detected by rt-PCR. Western blot was used to analyze PBMC TLRs and their downstream signal protein expression. PBMCs were presented as TLR2 expression or TLR4 expression using flow cytometry.From mRNA to protein detection, PBMC TLR2 and TLR4 were significantly higher in the AFMI group than in the control group and MI group. A similar tendency was also observed in the expression of downstream signaling proteins. When further analyzed with TLR2 and TLR4 antibodies by flow cytometry, PBMC levels also appeared to be higher in AFMI patients than those in MI patients and the healthy control group.In our study, PBMC TLRs and their downstream signaling proteins were significantly higher in the acute myocardial infarction patients with new-onset atrial fibrillation compared with healthy people and acute myocardial infarction patients without new-onset atrial fibrillation. They have the potential to be novel biomarkers for new-onset atrial fibrillation after acute myocardial infarction.

New possible biomarkers for diagnosis of infections and diagnostic distinction between bacterial and viral infections in children.

Detailed information about patients with infections is required to ensure appropriate choice of treatment. Although white blood cell (WBC) counts, and C-reactive protein (CRP) levels are useful diagnostic indicators of infections, more rapid and easily assayed indicator(s) could improve diagnosis. Moreover, it is of pivotal importance to distinguish bacteria or viruses as causative pathogens. Overall, TLR2 and TLR4 expression levels in neutrophils derived from individuals (n = 118) with bacterial (n = 37) and viral (n = 34) infections were higher than those in control samples (n = 47). Significant higher levels of TNF-α in patients with both types of the infection were observed, and those of IL-4, IL-8, IL-10, and IL-12 also were observed in the present study. Levels of IL-2, IL-8, and IL-10 on day 1 post-viral infection were significantly higher than those on day 1 post-bacterial infection. Therefore, there is a possibility that IL-4, IL-8, IL-10, IL-12 and TNF-α might be biomarkers for infections, in addition to WBC counts and CRP levels, and that IL-2, IL-8 or IL-10 are potentially able to distinguish between bacterial and viral infections.

ANTIPHOSPHOLIPID SYNDROME AND MONOCYTES: NEW ASPECTS.

After discovery of antiphospholipid syndrome (APS) our understanding of molecular mechanisms of living matter has become more sophisticated and on this way monocytes has become crucial player, particularly in pathogenesis of APS. Thrombotic and non-thrombotic complications of APS could be explained by monocytes' activation too. But mechanisms underlying their activation are poorly investigated. So we aimed to determine transcriptional activity of monocytes after exposing them to low concentrations of lipopolysaccharide (LPS) and LPS+ATP using comparative of RT-PCR. Our study included eleven women suffering from recurrent miscarriages and APS (mean age 30±5,6 years). Nine healthy women (mean age of 29±8,5 years) without a positive family history of APS, autoimmune diseases and thrombosis were chosen as a control group. The results showed increasing levels of TLR2, IL-23, CCL2, CXCL10, IL-1ß and IL-6 in APS cells, while in healthy cells LPS resulted in IL-6 and STAT3 elevated mRNAs. Double stimulation of APS cells resulted in decreased mRNA levels of CCL-2, IL-1ß, and mRNA NLRP3 in healthy cells. At the same time TLR2 mRNAs were elevated in both groups after double stimulation. Thus increased sensitivity of APS cells to LPS may contribute to thrombus formation. Low concentration of ATP diminishes LPS-induced inflammatory state of APS monocytes, which might be one of potential regulatory mechanisms.

Acute serum amyloid A is an endogenous TLR2 ligand that mediates inflammatory and angiogenic mechanisms.

INTRODUCTION: Acute-phase serum amyloid A (A-SAA) has cytokine-like properties and is expressed at sites of inflammation. We examined whether A-SAA-induced pro-inflammatory mechanisms are mediated through Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA). METHODS: The effect of A-SAA on human embryonic kidney (HEK), TLR2 or TLR4 cells was quantified by nuclear factor (NF)-κB luciferase reporter assays. A-SAA-induced RASFC and dHMVEC function were performed in the presence of a specific neutralising anti-TLR2 mAb (OPN301) (1 µg/mL) and matched IgG isotype control Ab (1 µg/mL). Cell surface expression of intracellular adhesion molecule (ICAM)-1, chemokine expression, cell migration, invasion and angiogenesis were assessed by flow cytometry, ELISA, Matrigel invasion chambers and tube formation assays. MyD88 expression was assessed by real-time PCR and western blot. RESULTS: A-SAA induced TLR2 activation through induction of NF-κB (p<0.05), but failed to induce NF-κB in HEK-TLR4 cells, confirming specificity for TLR2. A-SAA-induced proliferation, invasion and migration were significantly inhibited in the presence of anti-TLR2 (all p<0.05), with no significant effect observed for tumour necrosis factor-α-induced events. Additionally, A-SAA-induced ICAM-1, interleukin-8, monocyte chemoattractant protein-1, RANTES and GRO-α expression were significantly reduced in the presence of anti-TLR2 (all p<0.05), as was A-SAA induced angiogenesis (p<0.05). Finally, A-SAA induced MyD88 signalling in RASFC and dHMVEC (p<0.05). CONCLUSIONS: A-SAA is an endogenous ligand for TLR2, inducing pro-inflammatory effects in RA. Blocking the A-SAA/TLR2 interaction may be a potential therapeutic intervention in RA.

Expression of toll-like receptors 2 and 4 in subjects with asthma by total serum IgE level.

BACKGROUND: Emerging data suggest that innate immunity may play a role in asthma, particularly the toll-like receptors (TLRs). Some studies pointed to an involvement of TLRs 2 and 4 in the pathogenesis of allergic asthma, and other studies related TLRs to IgE. However, there are not any studies that have comprehensively evaluated the expression of TLRs 2 and 4 in inflammatory cells, in peripheral blood and induced sputum specimens from asthmatic patients, according to their total serum IgE. METHODS: We studied 44 asthmatic patients (15 with high total serum IgE and 29 with normal total serum IgE). On a single visit, all patients underwent: induced sputum, pulmonary function tests, determination of exhaled nitric oxide fraction, venipuncture for blood analysis and skin prick allergy tests. The induced sputum cellularity was analyzed by flow cytometry, where expression of TLRs 2 and 4 was studied using fluorochrome-conjugated monoclonal antibodies. RESULTS: Asthmatic patients with high total serum IgE showed, a higher percentage of macrophages expressing TLR4 (42.99 % ± 22.49) versus asthmatic patients with normal total serum IgE (28.84 % ± 15.16) (P = 0.048). Furthermore, we observed a correlation (but weak) between the percentage of macrophages expressing TLR4 in induced sputum and the total serum IgE level (R = 0.314; P = 0.040). CONCLUSION: Asthmatic subjects with high total serum IgE show increased macrophage expression of TLR4 in induced sputum. This outcome may result from a link between innate immunity and IgE-mediated, adaptive immune responses in asthma, and point to TLR4 as a potential therapeutic target.

ß2-1 Fructan supplementation alters host immune responses in a manner consistent with increased exposure to microbial components: results from a double-blinded, randomised, cross-over study in healthy adults.

ß2-1 Fructans are purported to improve health by stimulating growth of colonic bifidobacteria, increasing host resistance to pathogens and stimulating the immune system. However, in healthy adults, the benefits of supplementation remain undefined. Adults (thirteen men, seventeen women) participated in a double-blinded, placebo-controlled, randomised, cross-over study consisting of two 28-d treatments separated by a 14-d washout period. Subjects' regular diets were supplemented with ß2-1 fructan or placebo (maltodextrin) at 3×5 g/d. Fasting blood and 1-d faecal collections were obtained at the beginning and at the end of each phase. Blood was analysed for clinical, biochemical and immunological variables. Determinations of well-being and general health, gastrointestinal (GI) symptoms, regularity, faecal SCFA content, residual faecal ß2-1 fructans and faecal bifidobacteria content were undertaken. ß2-1 Fructan supplementation had no effect on blood lipid or cholesterol concentrations or on circulating lymphocyte and macrophage numbers, but significantly increased serum lipopolysaccharide, faecal SCFA, faecal bifidobacteria and indigestion. With respect to immune function, ß2-1 fructan supplementation increased serum IL-4, circulating percentages of CD282+/TLR2+ myeloid dendritic cells and ex vivo responsiveness to a toll-like receptor 2 agonist. ß2-1 Fructans also decreased serum IL-10, but did not affect C-reactive protein or serum/faecal Ig concentrations. No differences in host well-being were associated with either treatment, although the self-reported incidence of GI symptoms and headaches increased during the ß2-1 fructan phase. Although ß2-1 fructan supplementation increased faecal bifidobacteria, this change was not directly related to any of the determined host parameters.

Inflammasome and toll-like receptor signaling in human monocytes after successful cardiopulmonary resuscitation.

BACKGROUND: Whole body ischemia-reperfusion injury (IRI) after cardiopulmonary resuscitation (CPR) induces a generalized inflammatory response which contributes to the development of post-cardiac arrest syndrome (PCAS). Recently, pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and inflammasomes, have been shown to mediate the inflammatory response in IRI. In this study we investigated monocyte PRR signaling and function in PCAS. METHODS: Blood samples were drawn in the first 12 hours, and at 24 and 48 hours following return of spontaneous circulation in 51 survivors after cardiac arrest. Monocyte mRNA levels of TLR2, TLR4, interleukin-1 receptor-associated kinase (IRAK)3, IRAK4, NLR family pyrin domain containing (NLRP)1, NLRP3, AIM2, PYCARD, CASP1, and IL1B were determined by real-time quantitative PCR. Ex vivo cytokine production in response to stimulation with TLR ligands Pam3CSK4 and lipopolysaccharide (LPS) was assessed in both whole blood and monocyte culture assays. Ex vivo cytokine production of peripheral blood mononuclear cells (PBMCs) from a healthy volunteer in response to stimulation with patients' sera with or without LPS was assessed. The results were compared to 19 hemodynamically stable patients with coronary artery disease. RESULTS: Monocyte TLR2, TLR4, IRAK3, IRAK4, NLRP3, PYCARD and IL1B were initially upregulated in patients following cardiac arrest. The NLRP1 and AIM2 inflammasomes were downregulated in resuscitated patients. There was a significant positive correlation between TLR2, TLR4, IRAK3 and IRAK4 expression and the degree of ischemia as assessed by serum lactate levels and the time until return of spontaneous circulation. Nonsurvivors at 30 days had significantly lower mRNA levels of TLR2, IRAK3, IRAK4, NLRP3 and CASP1 in the late phase following cardiac arrest. We observed reduced proinflammatory cytokine release in response to both TLR2 and TLR4 activation in whole blood and monocyte culture assays in patients after CPR. Sera from resuscitated patients attenuated the inflammatory response in cultured PBMCs after co-stimulation with LPS. CONCLUSIONS: Successful resuscitation from cardiac arrest results in changes in monocyte pattern recognition receptor signaling pathways, which may contribute to the post-cardiac arrest syndrome. TRIAL REGISTRATION: The trial was registered in the German Clinical Trials Register ( DRKS00009684 ) on 27/11/2015.

Reduced dietary intake of pro-inflammatory Toll-like receptor stimulants favourably modifies markers of cardiometabolic risk in healthy men.

BACKGROUND AND AIMS: Because pro-inflammatory stimulants of Toll-like receptor-2 and TLR4 (pathogen-associated molecular patterns, PAMPs), are abundant in some processed foods, we explored the effects of diets enriched or depleted in these molecules on markers of cardiometabolic risk in man. METHODS AND RESULTS: Adherence to a low PAMP diet for 7 days reduced LDL-cholesterol (-0.69 mM, P = 0.024) and abdominal circumference (-1.6 cm, P = 0.001) in 11 habitual consumers of high PAMP foodstuffs, and these markers, together with leukocyte counts (+14%, P = 0.017) increased significantly after 4 days consuming predominantly high PAMP foods. Change in LDL-cholesterol and leukocyte counts correlated well with change in frequency of intake of high PAMP foodstuffs per individual (r = 0.540, P = 0.0095 and r = 0.6551, P = 0.0009, respectively). In an independent group of 13 healthy men, leukocyte counts and expression of the activation marker CD11b on granulocytes and monocytes were significantly reduced after a fresh onion meal (P < 0.05), but these effects were reversed by a high PAMP equivalent meal. CONCLUSIONS: A low PAMP diet is associated with reduced levels of several cardiometabolic risk factors, while a high PAMP diet reverses these effects. These findings suggest a novel potential mechanistic explanation for the observed association between processed food consumption and risk of cardiometabolic diseases. The study is registered at clinicaltrials.org (reference NCT02430064).

Changes in blood monocyte Toll-like receptor and serum surfactant protein A reveal a pathophysiological mechanism for community-acquired pneumonia in patients with type 2 diabetes.

BACKGROUND: The lung is one of the target organs of microangiopathy in diabetes mellitus (DM); patients with type 2 diabetes mellitus (T2DM) are vulnerable to pneumonia, and a variety of pathophysiological mechanisms has been described. AIM: This study aimed to determine the pathophysiological mechanism of community-acquired pneumonia (CAP) in T2DM patients. METHODS: A total of 90 individuals was included in this study comprised of three groups (n = 30): healthy control, T2DM and T2DM+ CAP groups. Toll-like receptor (TLR)2 and 4 protein and messenger RNA expression in peripheral blood monocytes(PBMC) was assessed by western blot and reverse transcription-polymerase chain reaction, respectively, and surfactant protein A (SP-A) levels were examined in serum samples by enzyme-linked immunosorbent assay. RESULTS: In T2DM and T2DM+CAP groups, levels of both TLR2/4 protein and mRNA in PBMC were decreased compared with controls (P <0.05), with lower levels observed in the T2DM+CAP group in comparison with T2DM patients (P <0.05). The serum SP-A levels in T2DM+CAP individuals were significantly higher than the values obtained for T2DM patients (P <0.05). It also showed apparent increases when compared with that in controls although no statistical significance was detected. CONCLUSION: In T2DM patients with pneumonia, TLR2/4 levels in PBMC and serum SP-A were altered, maybe playing an important role in the susceptibility to pneumonia in T2DM patients.

Quercetin ameliorates LPS-induced inflammation in human peripheral blood mononuclear cells by inhibition of the TLR2-NF-κB pathway.

Quercetin, a dietary flavonoid abundant in fruits, vegetables, and herbs, presents various pharmacological effects. This study aimed to investigate the anti-inflammatory effect and the underlying mechanism of quercetin in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs). Cell viability was measured by the Cell Counting Kit-8 assay. The mRNA expression of Toll-like receptor 2 (TLR2) was assessed by quantitative real-time polymerase chain reaction. Inflammatory cytokine secretions and nuclear factor (NF)-kB levels were analyzed by enzyme-linked immunosorbent assay. Our findings showed that quercetin significantly reduced LPS-induced cytotoxicity in human PBMCs. Quercetin suppressed the secretion of tumor necrosis factor-a, interleukin (IL)-1b, and IL-6 in LPS-stimulated human PBMCs. Moreover, quercetin reduced the LPS-induced increase in the expression of TLR2 mRNA and decreased the NF-kB concentration in LPS-stimulated human PBMCs. The data indicates that quercetin plays an important role in LPS-induced inflammation in human PBMCs via suppression of the TLR2-NF-kB pathway.