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Helicobacter pylori has been of interest to scientists and clinicians for many years, often causing diagnostic difficulties, especially in the youngest age group, in children. The presence of this bacterium in the population depends on the geographic region. However, it is assumed that even half of the world's population may be infected with H. pylori. Children infected with H. pylori—the study group (Hp(+)) and control group (Hp(−)), were chosen for further examination. The aim of the study was to analyze the concentrations of selected inflammatory markers in saliva (TNF-α, IL-8) and other markers (neutrophil defensin-1, sICAM-1, calprotectin, metalloproteinase-9, metalloproteinase-2, lactotransferrin, TLR-2) using ELISA technique. We confirmed the increased concentrations of IL-8, ND-1, and TLR-2 in the group of children infected with Helicobacter pylori. Moreover, there was also a positive, significant correlation between the concentration of ND-1 and MMP-2, sICAM-1, and calprotectin as well as MMP-9 and MMP-2 in the group of infected children. The study created new possibilities of insight into the pathogenetic mechanisms of developing inflammation in the mouth. This type of comprehensive research is also used to monitor the current disease process and create new opportunities for better in-depth diagnostics of children infected with H. pylori. [ABSTRACT FROM AUTHOR]

The present study aimed to identify in patients with severe COVID-19 and acute respiratory distress syndrome (ARDS) the association between rs3804099 and rs3804100 (TLR2) and evaluate the expression of TLR-2 on the cell surface of innate and adaptive cells of patients' carriers of C allele in at least one genetic variant. We genotyped 1018 patients with COVID-19 and ARDS. According to genotype, a subgroup of 12 patients was selected to stimulate peripheral blood mononuclear cells (PBMCs) with spike and LPS + spike. We evaluated soluble molecules in cell culture supernatants. The C allele in TLR2 (rs3804099, rs3804100) is not associated with a risk of severe COVID-19; however, the presence of the C allele (rs3804099 or rs3804100) affects the TLR-2 ability to respond to a spike of SARS-CoV-2 correctly. The reference group (genotype TT) downregulated the frequency of non-switched TLR-2+ B cells in response to spike stimulus; however, the allele's C carriers group is unable to induce this regulation, but they produce high levels of IL-10, IL-6, and TNF-α by an independent pathway of TLR-2. Findings showed that TT genotypes (rs3804099 and rs3804100) affect the non-switched TLR-2+ B cell distribution. Genotype TT (rs3804099 and rs3804100) affects the TLR-2's ability to respond to a spike of SARS-CoV-2. However, the C allele had increased IL-10, IL-6, and TNF-α by stimulation with spike and LPS. [ABSTRACT FROM AUTHOR]

The aim of this study was to verify milk productivity associated with biochemical parameters and expression of stress marker genes (HSP70, HSP90?, HSP90?) and immunity (TLR-2 and TLR-4) in dairy buffaloes. For this purpose, 12 buffaloes with an average production of 5.93 liters/day (highest production group, n=6) and 3.46 liters/day (lowest production group, n=6) were evaluated. As a result, the groups did not differ (P>0.05) regarding the biochemical analysis of proteins (4.16% versus 4.61%, p=0.13), lipids (5.80% versus 4.87%, p=0.69), lactose (4.74% versus 4.43%, p=0.83) and cortisol quantification (0.67 versus 0.79, p=0.12). The somatic cell count (106.4 versus 139, p=0.36) was within the normal range, indicating the absence of an inflammatory process in the breasts. Relative quantification was higher in the lower yield group compared to the higher yield group (P

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. [ABSTRACT FROM AUTHOR]

Background: Defective intestinal epithelial tight junction (TJ), characterized by an increase in intestinal TJ permeability, has been shown to play a critical role in the pathogenesis of inflammatory bowel disease (IBD). Tumor necrosis factor-a (TNF-α) is a key pro-inflammatory cytokine involved in the immunopathology of IBD and has been shown to cause an increase in intestinal epithelial TJ permeability. Although TNF-α antibodies and other biologics have been advanced for use in IBD treatment, these therapies are associated with severe side effects and have limited efficacy, and there is an urgent need for therapieswith benign profiles and high therapeutic efficacy. Probiotic bacteria have beneficial effects and are generally safe and represent an important class of potential therapeutic agents in IBD. Lactobacillus acidophilus (LA) is one of the most used probiotics for wide-ranging health benefits, including in gastrointestinal, metabolic, and inflammatory disorders. A specific strain of LA, LA1, was recently demonstrated to have protective and therapeutic effects on the intestinal epithelial TJ barrier. However, the mechanisms of actions of LA1 remain largely unknown. Methods: The primary aim of this study was to investigate microbial-epithelial interactions and novel signaling pathways that regulate the effect of LA1 on TNFa- induced increase in intestinal epithelial TJ permeability, using cell culture and animal model systems. Results and Conclusion: Pre-treatment of filter-grown Caco-2 monolayers with LA1 prevented the TNF-α-induced increase in intestinal epithelial TJ permeability by inhibiting TNF-α-induced activation of NF-kB p50/p65 and myosin light chain kinase (MLCK) gene and kinase activity in a TLR-2-dependent manner. LA1 produced a TLR-2- and MyD88-dependent activation of NF-kB p50/p65 in immune cells; however, LA1, in intestinal cells, inhibited the NF-kB p50/p65 activation in a TLR-2-dependent but MyD88-independent manner. In addition, LA1 inhibition of NF-kB p50/p65 and MLCK gene was mediated by TLR-2 pathway activation of phosphatidylinositol 3-kinase (PI3K) and IKK-a phosphorylation. Our results demonstrated novel intracellular signaling pathways by which LA1/TLR-2 suppresses the TNF-α pathway activation of NF-kB p50/p65 in intestinal epithelial cells and protects against the TNF-α-induced increase in intestinal epithelial TJ permeability. [ABSTRACT FROM AUTHOR]

BackgroundDefective intestinal epithelial tight junction (TJ), characterized by an increase in intestinal TJ permeability, has been shown to play a critical role in the pathogenesis of inflammatory bowel disease (IBD). Tumor necrosis factor-α (TNF-α) is a key pro-inflammatory cytokine involved in the immunopathology of IBD and has been shown to cause an increase in intestinal epithelial TJ permeability. Although TNF-α antibodies and other biologics have been advanced for use in IBD treatment, these therapies are associated with severe side effects and have limited efficacy, and there is an urgent need for therapies with benign profiles and high therapeutic efficacy. Probiotic bacteria have beneficial effects and are generally safe and represent an important class of potential therapeutic agents in IBD. Lactobacillus acidophilus (LA) is one of the most used probiotics for wide-ranging health benefits, including in gastrointestinal, metabolic, and inflammatory disorders. A specific strain of LA, LA1, was recently demonstrated to have protective and therapeutic effects on the intestinal epithelial TJ barrier. However, the mechanisms of actions of LA1 remain largely unknown.MethodsThe primary aim of this study was to investigate microbial-epithelial interactions and novel signaling pathways that regulate the effect of LA1 on TNF-α-induced increase in intestinal epithelial TJ permeability, using cell culture and animal model systems.Results and ConclusionPre-treatment of filter-grown Caco-2 monolayers with LA1 prevented the TNF-α-induced increase in intestinal epithelial TJ permeability by inhibiting TNF-α-induced activation of NF-κB p50/p65 and myosin light chain kinase (MLCK) gene and kinase activity in a TLR-2-dependent manner. LA1 produced a TLR-2- and MyD88-dependent activation of NF-κB p50/p65 in immune cells; however, LA1, in intestinal cells, inhibited the NF-κB p50/p65 activation in a TLR-2-dependent but MyD88-independent manner. In addition, LA1 inhibition of NF-κB p50/p65 and MLCK gene was mediated by TLR-2 pathway activation of phosphatidylinositol 3-kinase (PI3K) and IKK-α phosphorylation. Our results demonstrated novel intracellular signaling pathways by which LA1/TLR-2 suppresses the TNF-α pathway activation of NF-κB p50/p65 in intestinal epithelial cells and protects against the TNF-α-induced increase in intestinal epithelial TJ permeability.

Abstract Background Tuberculosis spondylitis accounts for approximately 50% of all cases of skeletal tuberculosis. Vitamin D plays a role in the immune system. Vitamin D helps in the activation of TLR-2 and TLR-4, which play a role in the process of tuberculosis infection. The objective of this study was to investigate the effect of oral supplementation with vitamin D on TLR-2 and TLR-4 levels in tuberculosis spondylitis patients. Methods The true Experiment Design Pretest–Posttest with Control Group (Pretest–Posttest with Control Group) was used for this research. TLR-2 and TLR-4 were measured by ELISA. Repeated ANOVA, ANOVA tests, and Kolmogorov–Smirnov normality tests on the SPSS program were used to statistically analyze the results. Result In the dose groups of 10,000 IU and 5000 IU, significant increases in the levels of vitamin D, TLR-2, and TLR-4 were observed at weeks 4 and 8 (p 50 ng/dl to optimally act as an immunomodulator.

Background: Tuberculosis spondylitis accounts for approximately 50% of all cases of skeletal tuberculosis. Vitamin D plays a role in the immune system. Vitamin D helps in the activation of TLR-2 and TLR-4, which play a role in the process of tuberculosis infection. The objective of this study was to investigate the effect of oral supplementation with vitamin D on TLR-2 and TLR-4 levels in tuberculosis spondylitis patients. Methods: The true Experiment Design Pretest–Posttest with Control Group (Pretest–Posttest with Control Group) was used for this research. TLR-2 and TLR-4 were measured by ELISA. Repeated ANOVA, ANOVA tests, and Kolmogorov–Smirnov normality tests on the SPSS program were used to statistically analyze the results. Result: In the dose groups of 10,000 IU and 5000 IU, significant increases in the levels of vitamin D, TLR-2, and TLR-4 were observed at weeks 4 and 8 (p < 0.05). In the control group, there was no significant increase. Conclusions: Vitamin D supplements can significantly increase TLR-2 and TLR-4 levels. Supplementation with vitamin D 10,000 IU/day for 8 weeks can increase vitamin D levels > 50 ng/dl to optimally act as an immunomodulator. [ABSTRACT FROM AUTHOR]

The present study investigated the effect of linseed supplementation on serum profiling and differential expression levels of IL-2 and TLR 2 genes involved in the immune system of lactating goats. The study was conducted on 15 lactating Beetal goats for 3 months, which were divided into three groups. Treatment group T1 was kept as a control group with no linseed supplementation, whereas treatment group T2 received ground linseed at the rate 10% of concentrate per day per animal, and treatment group T3 received 20% ground linseed. The concentration of triglycerides, cholesterol and LDL concentration were found to be significantly lower (P < 0.05) in both 10% and 20% linseed supplemented group than the control group. Whereas, HDL concentration in T2 and T3 group were found to be significantly higher than the control group. The differential expression of targeted genes comparatively revealed highest relative expression in the group that received 20% linseed supplementation. Although the study did not show a significant effect of linseed supplementation on the expression of TLR-2 and IL-2 genes in goats, it highlights the potential benefits of omega-3 PUFA supplementation on immune system modulation in animals. [ABSTRACT FROM AUTHOR]

The role of microRNAs (miRNAs) in Mycobacterium fortuitum pathogenesis is not well illustrated. Using zebrafish kidney macrophages (ZFKM) we observed that M. fortuitum triggers miR-155 expression, and the TLR-2/NF-κB axis plays a key role in initiating the process. We report that mir-155 activates the pro-inflammatory Stat1/TBX21 pathway in M. fortuitum-infected ZFKM. Our results further reveal the role of miR-155 in M1-macrophage polarisation during M. fortuitum infection. We observed that miR-155 inhibits socs1 expression augmenting the expression of tnf-α, il-12 and ifn-γ in infected ZFKM. Additionally, attenuating miR-155 inhibited ZFKM apoptosis and increased the intracellular bacterial load implicating its pro-apoptotic and bactericidal role in M. fortuitum pathogenesis. This is the first report on the role of miRNA in regulating innate immunity to mycobacteria in fish. We propose that the TLR-2/NF-κB axis triggers miR-155 expression, which in turn represses socs1 and promotes the development of M1-macrophages. Thus, the functional miR-155/Stat1/TBX21pathway induces a pro-inflammatory milieu favouring ZFKM apoptosis and M. fortuitum clearance. Therefore, our study unveils the role of miR-155 in the hierarchy of events leading to M. fortuitum-induced apoptosis and bacterial clearance in fish that is still not explored in detail.

Abstract Background Toll like receptors (TLR) 2 and 4 present on innate immune cells of the dental pulp detect cariogenic bacteria. Along with bacteria, C. albicans may also be present in dental caries. The presence of C. albicans can be detected by Dectin-1 a C type Lectin receptor. Expression of Dectin-1 in human pulpits has not been reported. Similarly, cytokines are released as a consequence of dental pulp inflammation caused by cariogenic bacteria. The T helper (Th) 1 inflammatory response leads to exacerbation of inflammation and its relationship with Osteopontin (OPN) is not known in pulp inflammation. Objective The aim of this study was to observe the expression of Dectin-1, TLR-2, OPN and pro-inflammatory cytokines in irreversibly inflamed human dental pulp and to observe relationship between Dectin-1/TLR-2 and OPN/Pro-inflammatory cytokines in the presence of appropriate controls. Methods A total of 28 subjects diagnosed with irreversible pulpitis were included in this ex-vivo study. Fifteen samples were subjected to standard hematoxylin and Eosin (H&E) and immunohistochemistry staining. Whereas, gene expression analysis was performed on 13 samples to observe mRNA expression of pro-inflammatory cytokines; tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 beta (ß), IL-6 Dectin-1, OPN, TLR-2 and TLR-4. SPSS version 21 was used for statistical analysis. One way analysis of variance (ANOVA), Pearson correlation and Chi-square test were used at p ≤ 0.05. Results Gene expressions of Dectin-1, TLR-2 and TLR-4 were observed in all samples. Dectin-1 and TLR-2 expressions were significantly correlated (r = 0.5587, p = 0.0002). Similarly, OPN and TNF-α expression showed a significant correlation (r = 0.5860, p = 0001). The agreement between histologic and clinical diagnosis was 69.2% in the cases of irreversible pulpitis. Conclusion Dectin-1 was expressed by inflamed human dental pulp. Dectin-1 and TLR-2 expression pattern was suggestive of a collaborative receptor response in inflamed pulp environment. OPN and TNF-α expressions showed a positive correlation indicating a possible relationship.

The common goldfish is the most widespread teleosts in the world. Due to its peculiar characteristics, such as the high resistance, easy availability and stabulation, and for its evolutionary characteristics, this fish lends itself to be one of the most used experimental models. This study aimed to characterize the mast cells in the intestine of Carassius auratus using anti‐TLR‐2, anti‐S100, anti‐VIP, anti‐serotonin (5‐HT) and anti‐Piscidin antibodies. The intestine of goldfish, like that of all vertebrates, plays an important role in the immunology of the animal. The gut‐associated lymphoid tissue GALT is an immune component containing several specific cells such as lymphocytes, macrophages and mast cells. In addition, the presence of goblet cells in the intestinal epithelium strengthens the defence system, secreting many cytokines and chemokines and displaying antibacterial properties. Our results show mast cells labelled with antibodies that are highly conserved between fish and mammals, demonstrating an active role of these cells in the immune response. [ABSTRACT FROM AUTHOR]

Damage-associated molecular patterns (DAMPs) and their receptors (TLR-2 and -4) may play important roles in renal fibrosis, of which the pathogenesis is complicated. We used rat renal lesions induced by a single intraperitoneal injection of cisplatin at 6 mg/kg body weight; consisting of tissue damage of renal tubules on days 1 and 3, further damage and regeneration with inflammation mainly on days 5 and 7, and interstitial fibrosis on days 9, 12, 15, and 20. Microarray analyses on days 5 (the commencement of inflammation) and 9 (the commencement of interstitial fibrosis) showed that DAMPs increased by more than two-fold relative to control included common extra-cellular matrix (ECM) components such as laminin (Lamc2) and fibronectin, and heat shock protein family, as well as fibrinogen, although it was limited analysis; Lamc2, an element of basement membrane, may be regarded as an indicator for damaged renal tubules. In the real-time RT-PCR analyses, TLR-2 significantly increased transiently on day 1, whereas TLR-4 significantly increased on days 9 and 15, almost in agreement with the increased biglycan (a small leucine-rich proteoglycan as ubiquitous ECM component). As M1/M2 macrophages participated in renal lesions, such as inflammation and fibrosis, presumably, TLR-4, which may be expressed in immune cells, could play crucial roles in the formation of renal lesions in association with biglycan. [ABSTRACT FROM AUTHOR]

The role of microRNAs (miRNAs) in Mycobacterium fortuitum pathogenesis is not well illustrated. Using zebrafish kidney macrophages (ZFKM) we observed that M. fortuitum triggers miR-155 expression, and the TLR-2/NF-κB axis plays a key role in initiating the process. We report that mir-155 activates the pro-inflammatory Stat1/TBX21 pathway in M. fortuitum-infected ZFKM. Our results further reveal the role of miR-155 in M1-macrophage polarisation during M. fortuitum infection. We observed that miR-155 inhibits socs1 expression augmenting the expression of tnf-α, il-12 and ifn-γ in infected ZFKM. Additionally, attenuating miR-155 inhibited ZFKM apoptosis and increased the intracellular bacterial load implicating its pro-apoptotic and bactericidal role in M. fortuitum pathogenesis. This is the first report on the role of miRNA in regulating innate immunity to mycobacteria in fish. We propose that the TLR-2/NF-κB axis triggers miR-155 expression, which in turn represses socs1 and promotes the development of M1-macrophages. Thus, the functional miR-155/Stat1/TBX21pathway induces a pro-inflammatory milieu favouring ZFKM apoptosis and M. fortuitum clearance. Therefore, our study unveils the role of miR-155 in the hierarchy of events leading to M. fortuitum-induced apoptosis and bacterial clearance in fish that is still not explored in detail. [ABSTRACT FROM AUTHOR]

In patients with mild osteoarthritis (OA), two to four monthly injections are required for 6 months due to the degradation of hyaluronic acid (HA) by peroxidative cleavage and hyaluronidase. However, frequent injections may lead to local infection and also cause inconvenience to patients during the COVID‐19 pandemic. Herein, we developed a novel HA granular hydrogel (n‐HA) with improved degradation resistance. The chemical structure, injectable capability, morphology, rheological properties, biodegradability, and cytocompatibility of the n‐HA were investigated. In addition, the effects of the n‐HA on the senescence‐associated inflammatory responses were studied via flow cytometry, cytochemical staining, Real time quantitative polymerase chain reaction (RT‐qPCR), and western blot analysis. Importantly, the treatment outcome of the n‐HA with one single injection relative to the commercial HA product with four consecutive injections within one treatment course in an OA mouse model underwent anterior cruciate ligament transection (ACLT) was systematically evaluated. Our developed n‐HA exhibited a perfect unification of high crosslink density, good injectability, excellent resistance to enzymatic hydrolysis, satisfactory biocompatibility, and anti‐inflammatory responses through a series of in vitro studies. Compared to the commercial HA product with four consecutive injections, a single injection of n‐HA contributed to equivalent treatment outcomes in an OA mouse model in terms of histological analysis, radiographic, immunohistological, and molecular analysis results. Furthermore, the amelioration effect of the n‐HA on OA development was partially ascribed to the attenuation of chondrocyte senescence, thereby leading to inhibition of TLR‐2 expression and then blockade of NF‐κB activation. Collectively, the n‐HA may be a promising therapeutic alternative to current commercial HA products for OA treatment. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B (CHB) has a wide range of outcomes depending on host immune responses mainly Toll-like receptors (TLRs) signaling and released cytokines. Toll-like receptor 2 (TLR2) single nucleotide polymorphisms (SNPs) and interleukin 6 (IL-6) may influence the course of CHB. We aimed to elucidate the relation between TLR-2 polymorphism, IL-6 profile, and CHB progression. We analyzed TLR-2 polymorphism (SNP; rs3804099) in 185 CHB patients and 60 controls using TaqMan allelic discrimination assay. Serum IL-6 levels were assessed by ELISA. IL-6 levels were considerably higher in active CHB and cirrhotic patients compared with inactive carriers and controls (P < 0.001). IL-6 showed positive correlation with ALT and advanced fibrosis in active CHB patients (r = 0.31, P = 0.02). A significant positive correlation was noticed between IL-6 and HBV DNA PCR in all CHB groups. TT genotype of rs3804099/TLR-2 was significantly more prevalent in inactive carriers compared to active hepatitis patients (P = 0.04, OR = 0.39 and 95% CI: 0.16–0.95). Both heterozygous CT and mutant TT genotypes were significantly more frequent among inactive carriers compared to cirrhotic patients (P = 0.01, OR = 0.33, 95% CI: 0.13–0.81 and P = 0.009, OR = 0.32, 95% CI: 0.13–0.77). TT genotype was significantly related to lower IL-6 levels in active hepatitis and cirrhotic groups (P = 0.005 and P = 0.001, respectively) showing that TLR mutations would be associated with milder hepatitis activity and lower possibility for disease progression. There may be a positive association between TLR2 rs3804099 polymorphism and hepatitis B activity. IL-6 is a good indicator of CHB disease progression. [ABSTRACT FROM AUTHOR]

Background: UTIs (urinary tract infections) are the most frequent bacterial disorder requiring medical management with a reported incidence of 8 million where nearly 20% need an emergency visit. Aim: The present study was done to study the systemic immunity and bacteriological profile of subjects having urinary tract infections in Indian subjects. The study focused on isolating the uropathogenic bacteria, assessing their antibiotic sensitivity, and evaluating the serum TLR2 levels in subjects with UTIs and healthy controls. Methods: In 200 blood and mid-stream urine study samples and 100 healthy samples of urine and blood as controls were subjected to TLR-2 detection. The samples from subjects with urinary tract infections were assessed for the presence of Uropathogenic bacteria (both grampositive and gram-negative). Results: The most common isolated bacteria were E. coli seen in 80% (n=160) study subjects followed by enterococcus faecalis in 50% (n=100) study subjects, Klebsiella pneumonia, enterococcus facium, and pseudomonas aeruginosa in 40% (n=80) subjects each, staphylococcus aureus in 36% (n=72) subjects. The mean TLR 2 level was significantly higher for cases subjects with urinary tract infections with a mean value of 3.875±1.495 compared to the control healthy subjects without urinary tract infections where the mean serum TLR2 value was 3.316±4.731. This difference was statistically significant with p=0.04 Conclusion: The present study concludes that subjects with active urinary tract infection commonly show more gram-negative bacteria than gram-positive bacteria with a predominance of E. coli, Klebsiella pneumonia, and staphylococcus spp. Also, toll-like receptor 2 is significantly higher in subjects with urinary tract infections compared to their control subjects. [ABSTRACT FROM AUTHOR]

Background: Toll like receptors (TLR) 2 and 4 present on innate immune cells of the dental pulp detect cariogenic bacteria. Along with bacteria, C. albicans may also be present in dental caries. The presence of C. albicans can be detected by Dectin-1 a C type Lectin receptor. Expression of Dectin-1 in human pulpits has not been reported. Similarly, cytokines are released as a consequence of dental pulp inflammation caused by cariogenic bacteria. The T helper (Th) 1 inflammatory response leads to exacerbation of inflammation and its relationship with Osteopontin (OPN) is not known in pulp inflammation. Objective: The aim of this study was to observe the expression of Dectin-1, TLR-2, OPN and pro-inflammatory cytokines in irreversibly inflamed human dental pulp and to observe relationship between Dectin-1/TLR-2 and OPN/Pro-inflammatory cytokines in the presence of appropriate controls. Methods: A total of 28 subjects diagnosed with irreversible pulpitis were included in this ex-vivo study. Fifteen samples were subjected to standard hematoxylin and Eosin (H&E) and immunohistochemistry staining. Whereas, gene expression analysis was performed on 13 samples to observe mRNA expression of pro-inflammatory cytokines; tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 beta (ß), IL-6 Dectin-1, OPN, TLR-2 and TLR-4. SPSS version 21 was used for statistical analysis. One way analysis of variance (ANOVA), Pearson correlation and Chi-square test were used at p ≤ 0.05. Results: Gene expressions of Dectin-1, TLR-2 and TLR-4 were observed in all samples. Dectin-1 and TLR-2 expressions were significantly correlated (r = 0.5587, p = 0.0002). Similarly, OPN and TNF-α expression showed a significant correlation (r = 0.5860, p = 0001). The agreement between histologic and clinical diagnosis was 69.2% in the cases of irreversible pulpitis. Conclusion: Dectin-1 was expressed by inflamed human dental pulp. Dectin-1 and TLR-2 expression pattern was suggestive of a collaborative receptor response in inflamed pulp environment. OPN and TNF-α expressions showed a positive correlation indicating a possible relationship. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae causes a wide variety of infectious diseases. Lipopolysaccharide (LPS) is a large heatstable polymer that is gram-negative bacteria's major outer membrane component, accounting for roughly 75% of the surface area and 5-10% of the total dry weight. Therefore the current in vivo study was carried out to investigate the immunomodulatory effect of purified lipopolysaccharide produced from local clinical Klebsiella pneumoniae isolates compared with ZnO-NPs and LPS-ZnO NPs. To do the experimental evaluations 35, Balb/c mouse was injected intramuscularly (i.m.) with different concentrations of the purified LPS, ZnoNPs and LPS-ZnoNPs for 12 days and immunized with 10% SRBCs (i.p) on day 4 and 8 of the schedule, while K. pneumoniae suspension and normal saline for positive and negative control groups. Focus on estimating body weight before and after treatment, Arthus and delayed-type hypersensitivity, and detecting serum level of cytokines (TLR-2, IL1Beta, IL4, and IL10) using sandwich ELISA. The data showed the highest value before and after treatment with LPS-ZnO NPs recorded in 2µg/mouse was 27. 92±1.48 and 31.50±0.4, respectively. In Arthurs reaction and Delayed type hypersensitivity, the highest results showed in the positive control group injected with K. pneumoniae 4.08±0.17 and 4.86±80.02, respectively. The results of TLR-2 showed the highest value in the positive control group, 242.17±3.98 pg/ml, followed by Group LPS at 135.51.58 pg/ml. The results of Interleukin-1Beta showed the highest value in the positive control group, 254.88±3.51 pg/ml, followed by Group LPS 174.3± 1.46 pg/ml. The concentration of IL-4 in serum of treated albino mice showed the highest value in the positive control group, 136.2±1.12 pg/ml, followed by Group LPS 86.12±1.49 pg/ml. While the highest value of IL-10 was recorded in the positive control group, 98.58± 4.09 pg/ml, followed by Group LPSZnoNP in concentration 4µg/ mouse was 86.018±0.69 pg/ml. The results of the statistical analysis showed a significant difference (P=0.05) between LPS, ZnoNPs, and LPs-ZnoNPs treated groups and control groups (positive & negative). In the present study, we can conclude that LPS-ZnO NPs had a positive immunomodulatory effect on immune response in immunized mice. As shown in the results of the level of IL-1 beta, IL-4, IL-10, and TLRs-2, Abs titer, and Arthus and DTH reactions. [ABSTRACT FROM AUTHOR]

• Uterine inflammatory conditions have socioeconomic impacts. • Uncontrolled inflammation and oxidative stress contribute to these conditions. • Berberine has antioxidant, anti-inflammatory, and antiapoptotic properties. • Berberine shows potential as a therapeutic agent against uterine inflammation induced by an iodine mixture. • Berberine treatment restored normal uterine histopathology and reduced oxidative stress markers. • Berberine modulated inflammatory and apoptotic genes and the TLR-2/p-PI3K/p-AKT axis. Uterine inflammation affects 8% of women in the United States and 32% in developing nations, often caused by uncontrolled inflammation and oxidative stress. This condition significantly impacts women's health, productivity, and quality of life, and increases the risk of related morbidities leading to higher healthcare costs. Research now focuses on natural antioxidants and anti-inflammatory, particularly berberine (BBR), an isoquinoline alkaloid known for its antioxidant, anti-inflammatory, and antiapoptotic activities. The present study sought to examine the potential therapeutic efficacy of BBR against uterine inflammation induced by the intrauterine infusion of an iodine (I 2) mixture in an experimental setting. Female Sprague Dawley rats (n = 6) were divided into five groups, control, sham, I 2 , I 2 and BBR 10 mg/kg, and I 2 and BBR 25 mg/kg-treated groups. Compared to I 2 infusion, BBR treatment effectively restored normal uterine histopathology and reduced inflammatory markers such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), nuclear factor- kappa B (NF-κB), monocyte chemoattractant protein 1 (MCP1), and myeloperoxidase (MPO). It lowered oxidative markers like malondialdehyde (MDA), and increased antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). It balanced apoptotic genes by upregulating B-cell lymphoma 2 (Bcl-2) and downregulating Bcl-2-associated X protein (Bax). Furthermore, BBR reduced the expression of Toll-like receptor 2 (TLR-2), phosphorylated phosphatidylinositol 3‑kinase (p-PI3K), and phosphorylated protein kinase B (p-AKT) in the rats treated with intrauterine I 2. Ultimately, the therapeutic benefits of BBR can be attributed, to some extent, to its antioxidant, anti-inflammatory, and antiapoptotic properties, in addition to its ability to modulate the TLR-2/p-PI3K/p-AKT axis. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic autoimmune joint disease that causes cartilage and bone damage or even disability, seriously endangering human health. Chronic synovial inflammation has been shown to play a vital role in disease sustainability. Therefore, downregulation of synovial inflammation is considered to be an effective discipline for RA therapy. Polyene phosphatidylcholine (PPC) is a hepatoprotective agent, which was observed to inhibit inflammation in macrophages and prevent collagen-induced arthritis (CIA) of rats in our previous study. However, the underlying mechanism remains unclear. The present study further reported that PPC can inhibit synovial inflammation. In lipopolysaccharide (LPS)-stimulated primary synovial fibroblasts (SFs) of mice, PPC significantly decreased pro-inflammatory cytokines production while increasing anti-inflammatory cytokines level. In this process, PPC downregulated the expression of TLR-2 and their downstream signaling molecules such as MyD88, p-ERK1/2, p-JNK1/2, and p-P38 in MAPK pathway and p-IκBα and NF-κB-p65 in NF-κB pathway. Moreover, the inhibitory effect of PPC on the above molecules and cytokines was weakened after pre-treatment with TLR-2 agonist Pam3CSK4. In addition, PPC lost its anti-inflammatory effect and its suppressing capability on MAPK and NF-κB pathways in TLR-2−/− primary SFs after exposure to LPS. Collectively, this study demonstrated that PPC can alleviate synovial inflammation through TLR-2-mediated MAPK and NF-κB pathways, which can be proposed to be a potential drug candidate for RA prevention. [ABSTRACT FROM AUTHOR]

Microglial inflammatory responses play a central role in the pathogenesis of S. aureus induced brain infections. Upon activation, microglia produces free radicals (ROS/RNS) and disrupts the cellular antioxidant defense to combat invading microorganisms. Despite conventional antibiotic or steroid therapy, microglial over-activation could not be controlled. So, an attempt had been taken by using a natural antioxidant ascorbic acid along with ciprofloxacin to regulate microglial over-activation by involving TLR-2 and glucocorticoid receptor (GR) in an in-vitro cell culture-based study. Combinatorial treatment during TLR-2 neutralization effectively reduced the bacterial burden at 60 min compared to the GR blocking condition (p < 0.05). Moreover, the infection-induced H2O2, O2.−, and NO release in microglial cell culture was diminished possibly by enhancing SOD and catalase activities in the same condition (p < 0.05). The arginase activity was markedly increased after TLR-2 blocking in the combinatorial group compared to single treatments (p < 0.05). Experimental results indicated that combinatorial treatment may act through up-regulating GR expression by augmenting endogenous corticosterone levels. However, better bacterial clearance could further suppress the TLR-2 mediated pro-inflammatory NF-κB signaling. From Western blot analysis, it was concluded that ciprofloxacin-ascorbic acid combination in presence of anti-TLR-2 antibody exhibited 81.25% inhibition of TLR-2 expression while the inhibition for GR was 3.57% with respect to the infected group. Therefore, during TLR-2 blockade ascorbic acid combination might be responsible for the restoration of redox balance in microglia via modulating TLR-2/GR interaction. The combination treatment could play a major role in the neuroendocrine-immune regulation of S. aureus induced microglial activation. [ABSTRACT FROM AUTHOR]

Cutibacterium acnes (C. acnes) has been implicated in inflammatory acne where highly mutated Christie–Atkins–Munch–Petersen factor (CAMP)1 displays strong toll like receptor (TLR)-2 binding activity. Using specific antibodies, we showed that CAMP1 production was independent of C. acnes phylotype and involved in the induction of inflammation. We confirmed that TLR-2 bound both mutated and non-mutated recombinant CAMP1, and peptide array analysis showed that seven peptides (A14, A15, B1, B2, B3, C1 and C3) were involved in TLR-2 binding, located on the same side of the three-dimensional structure of CAMP1. Both mutated and non-mutated recombinant CAMP1 proteins induced the production of C-X-C motif chemokine ligand interleukin (CXCL)8/(IL)-8 in vitro in keratinocytes and that of granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, IL-1β and IL-10 in ex vivo human skin explants. Only A14, B1 and B2 inhibited the production of CXCL8/IL-8 by keratinocytes and that of (GM-CSF), TNF-α, IL-1β and IL-10 in human skin explants stimulated with rCAMP1 and C. acnes. Following pretreatment with B2, RNA sequencing on skin explants identified the 10 genes displaying the strongest differential expression as IL6, TNF, CXCL1, CXCL2, CXCL3, CXCL8, IL-1β, chemokine ligand (CCL)2, CCL4 and colony stimulating factor (CSF)2. We, thus, identified a new CAMP1-derived peptide as a TLR-2 modulator likely to be a good candidate for clinical evaluation. [ABSTRACT FROM AUTHOR]

Irg1 is an enzyme that generates itaconate, a metabolite that plays a key role in the regulation of inflammatory responses. Previous studies have implicated Irg1 as an important mediator in preventing excessive inflammation and tissue damage in Mycobacterium tuberculosis (Mtb) infection. Here, we investigated the pattern recognition receptors and signaling pathways by which Mtb triggers Irg1 gene expression by comparing the responses of control and genetically deficient BMDMs. Using this approach, we demonstrated partial roles for TLR-2 (but not TLR-4 or -9), MyD88 and NFκB signaling in Irg1 induction by Mtb bacilli. In addition, drug inhibition studies revealed major requirements for phagocytosis and endosomal acidification in Irg1 expression triggered by Mtb but not LPS or PAM3CSK4. Importantly, the Mtb -induced Irg1 response was highly dependent on the presence of the bacterial ESX-1 secretion system, as well as host STING and Type I IFN receptor (IFNAR) signaling with Type II IFN (IFN-γ) signaling playing only a minimal role. Based on these findings we hypothesize that Mtb induces Irg1 expression in macrophages via the combination of two independent triggers both dependent on bacterial phagocytosis: 1) a major signal stimulated by phagocytized Mtb products released by an ESX-1-dependent mechanism into the cytosol where they activate the STING pathway leading to Type I-IFN production, and 2) a secondary TLR-2, MyD88 and NFκB dependent signal that enhances Irg1 production independently of Type I IFN induction. [ABSTRACT FROM AUTHOR]

In the course of infection and intense endotoxemia processes, induction of a catabolic state leading to weight loss is observed in mice and humans. However, the late effects of acute inflammation on energy homeostasis, regulation of body weight and glucose metabolism are yet to be elucidated. Here, we addressed whether serial intense endotoxemia, characterized by an acute phase response and weight loss, could be an aggravating or predisposing factor to weight gain and associated metabolic complications. Male Swiss Webster mice were submitted to 8 consecutive doses of lipopolysaccharide (10 mg/kg LPS), followed by 10 weeks on a high-fat diet (HFD). LPS-treated mice did not show changes in weight when fed standard chow. However, when challenged by a high-fat diet, LPS-treated mice showed greater weight gain, with larger fat depot areas, increased serum leptin and insulin levels and impaired insulin sensitivity when compared to mice on HFD only. Acute endotoxemia caused a long-lasting increase in mRNA expression of inflammatory markers such as TLR-4, CD14 and serum amyloid A (SAA) in the adipose tissue, which may represent the key factors connecting inflammation to increased susceptibility to weight gain and impaired glucose homeostasis. In an independent experimental model, and using publicly available microarray data from adipose tissue from mice infected with Gram-negative bacteria, we performed gene set enrichment analysis and confirmed upregulation of a set of genes responsible for cell proliferation and inflammation, including TLR-4 and SAA. Together, we showed that conditions leading to intense and recurring endotoxemia, such as common childhood bacterial infections, may resound for a long time and aggravate the effects of a western diet. If confirmed in humans, infections should be considered an additional factor contributing to obesity and type 2 diabetes epidemics. [ABSTRACT FROM AUTHOR]

Irg1 is an enzyme that generates itaconate, a metabolite that plays a key role in the regulation of inflammatory responses. Previous studies have implicated Irg1 as an important mediator in preventing excessive inflammation and tissue damage in Mycobacterium tuberculosis (Mtb) infection. Here, we investigated the pattern recognition receptors and signaling pathways by which Mtb triggers Irg1 gene expression by comparing the responses of control and genetically deficient BMDMs. Using this approach, we demonstrated partial roles for TLR-2 (but not TLR-4 or -9), MyD88 and NFκB signaling in Irg1 induction by Mtb bacilli. In addition, drug inhibition studies revealed major requirements for phagocytosis and endosomal acidification in Irg1 expression triggered by Mtb but not LPS or PAM3CSK4. Importantly, the Mtb-induced Irg1 response was highly dependent on the presence of the bacterial ESX-1 secretion system, as well as host STING and Type I IFN receptor (IFNAR) signaling with Type II IFN (IFN-γ) signaling playing only a minimal role. Based on these findings we hypothesize that Mtb induces Irg1 expression in macrophages via the combination of two independent triggers both dependent on bacterial phagocytosis: 1) a major signal stimulated by phagocytized Mtb products released by an ESX-1-dependent mechanism into the cytosol where they activate the STING pathway leading to Type I-IFN production, and 2) a secondary TLR-2, MyD88 and NFκB dependent signal that enhances Irg1 production independently of Type I IFN induction.

The mechanisms underlying Mycobacterium fortuitum -induced mycobacteriosis remain unexplored. Using head kidney macrophages (HKM) from catfish (Clarias gariepinus), we report that Ca2+ surge across mitochondrial-Ca2+ uniporter (MICU), and consequent mitochondrial ROS (mtROS) production, is imperative for mycobactericidal activity. Inhibition of mtROS alleviated HKM apoptosis and enhanced bacterial survival. Based on RNA interference (RNAi) and inhibitor studies, we demonstrate that the Toll-like receptor (TLR)-2–endoplasmic reticulum (ER) stress–store-operated calcium entry (SOCE) axis is instrumental for activating the mt-Ca2+/mtROS cascade in M. fortuitum -infected HKM. Additionally, pharmacological inhibition of mtROS attenuated the expression of CHOP , STIM1 , and Orai1 , which suggests a positive feedback loop between ER-stress-induced SOCE and mtROS production. Elevated tumor necrosis factor alpha (TNF-α) levels and caspase-8 activity were observed in HKM consequent to M. fortuitum infection, and our results implicate that mtROS is crucial in activating the TNF-mediated caspase-8 activation. Our results for the first time demonstrate mitochondria as an innate immune signaling center regulating mycobacteriosis in fish. We conclude that M. fortuitum- induced persistent SOCE signaling leads to mtROS production, which in turn activates the TNF-α/caspase-8 axis culminating in HKM apoptosis and bacterial clearance. [ABSTRACT FROM AUTHOR]

The mechanisms underlying Mycobacterium fortuitum-induced mycobacteriosis remain unexplored. Using head kidney macrophages (HKM) from catfish (Clarias gariepinus), we report that Ca2+ surge across mitochondrial-Ca2+ uniporter (MICU), and consequent mitochondrial ROS (mtROS) production, is imperative for mycobactericidal activity. Inhibition of mtROS alleviated HKM apoptosis and enhanced bacterial survival. Based on RNA interference (RNAi) and inhibitor studies, we demonstrate that the Toll-like receptor (TLR)-2–endoplasmic reticulum (ER) stress–store-operated calcium entry (SOCE) axis is instrumental for activating the mt-Ca2+/mtROS cascade in M. fortuitum-infected HKM. Additionally, pharmacological inhibition of mtROS attenuated the expression of CHOP, STIM1, and Orai1, which suggests a positive feedback loop between ER-stress-induced SOCE and mtROS production. Elevated tumor necrosis factor alpha (TNF-α) levels and caspase-8 activity were observed in HKM consequent to M. fortuitum infection, and our results implicate that mtROS is crucial in activating the TNF-mediated caspase-8 activation. Our results for the first time demonstrate mitochondria as an innate immune signaling center regulating mycobacteriosis in fish. We conclude that M. fortuitum-induced persistent SOCE signaling leads to mtROS production, which in turn activates the TNF-α/caspase-8 axis culminating in HKM apoptosis and bacterial clearance.

Background: The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, SARS-CoV-2 initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. Methods: SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 106 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E). Findings: We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs (p=

Objective: To determine the effects of toll-like receptor 2 (TLR-2) agonist, Pam3CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum. Methods: Six groups of mice (n=6 per group) received intraperitoneal phosphate buffered saline T80 (PBS-T80), BCG or rBCG in the presence or absence of Pam3CSK4. Enzyme- linked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2a, and IgG2b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN- γ via enzyme-linked immunosorbent assay. Results: The production of total IgG and the isotype IgG1, IgG2a and IgG2b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3CSK4 further enhanced their productions. A similar pattern was also observed in IFN- γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3CSK4. Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2a, IgG2b, as well as IFN- γ in response to rBCG. However, the presence or absence of Pam3CSK4 had no effect on IL-4 production.

Background: Immunosuppressed conditions due to long-term corticosteroid and tetracycline consumption are susceptible to fungal invasion, especially by Candida albicans (C. albicans), that requires treatment of oral candidiasis. Toll like receptor-2 (TLR-2) plays a role in candida recognition. Nystatin is regularly employed for oral candidiasis, but produces certain side-effects. Chloroform extract of Acanthus ilicifolius (A. ilicifolius) leaves represents both a potent inhibitor of C. albicans growth and an antioxidant. Purpose: This study aimed to compare the effect of A. ilicifolius leaf chloroform extract and nystatin treatment on TLR-2 expression in oral candidiasis immunosupressed models. Methods: This study constitutes a true experimental investigation incorporating a post test-only control group design. 20 healthy male Rattus novergicus (Wistar), aged 12 weeks and with an average weight of 250g, were immunosuppressed through oral administration of dexamethasoneand tetracycline for a period of 21 days before being induced with C. albicans (ATCC-10231) 6 x 108 for two weeks. The subjects were divided into five groups (n=4/group): healthy (H), no-treatment(P), nystatin treatment(N), A. Ilicifollius (8%) treatment (AI-1) and A. ilicifollius (16%) treatment (AI-2). The subjects were treated for 14 days, with their tongue being subsequently biopsied. TLR-2 expression was subjected to immunohistochemical examination, observed under a microscope (400x magnification) and statistically analyzed (one-way Anova, LSD-test, p

Abstract Background The clinical trials conducted at Chingleput India suggest that BCG fails to protect against tuberculosis (TB) in TB-endemic population. Recent studies advocate that non-tuberculous mycobacteria and latent Mycobacterium tuberculosis (Mtb) infection interferes in the antigen processing and presentation of BCG in inducing protective immunity against Mtb. Thereby, indicating that any vaccine that require extensive antigen processing may not be efficacious in TB-endemic zones. Recently, we have demonstrated that the vaccine candidate L91, which is composed of lipidated promiscuous MHC-II binder epitope, derived from latency associated Acr1 antigen of Mtb is immunogenic in the murine and Guinea pig models of TB and conferred better protection than BCG against Mtb. Methods In this study, we have used a multi-stage based bi-epitope vaccine, namely L4.8, comprising of MHC-I and MHC-II binding peptides of active (TB10.4) and latent (Acr1) stages of Mtb antigens, respectively. These peptides were conjugated to the TLR-2 agonist Pam2Cys. Results L4.8 significantly elicited both CD8 T cells and CD4 T cells immunity, as evidenced by increase in the enduring polyfunctional CD8 T cells and CD4 T cells. L4.8 efficiently declined Mtb-burden and protected animals better than BCG and L91, even at the late stage of Mtb infection. Conclusions The BCG-L4.8 prime boost strategy imparts a better protection against TB than the BCG alone. This study emphatically denotes that L4.8 can be a promising future vaccine candidate for controlling active and latent TB.

Currently, there are single data on the relationship between TLR-2 polymorphisms (Arg753Gln) and TLR-4 (Asp299Gly) polymorphisms and susceptibility to brucellosis. Therefore, the aim of the study was to determine the frequency of TLR-2 (Arg753Gln) and TLR-4 (Asp299Gly) polymorphisms in patients with acute brucellosis with cardiovascular lesions in the Republic of Azerbaijan. 178 patients with a brucellosis clinic were examined. According to the criteria for inclusion in the study, only 120 people fully met all the criteria, which made up the main group. The control group consisted of 30 healthy individuals. TLR-2 (Arg753Gln) and TLR-4 (Asp299Gly) polymorphisms were also determined for all patients in both groups. In order to assess the state of the cardiovascular system, an electrocardiogram was recorded, blood pressure was measured, and an ultrasound scan of the heart was performed for all patients. It was found that 93 patients (77.50 ± 3.13%) with acute brucellosis had some or other signs of impairment in the work of the cardiovascular system, identified clinically or as a result of functional examination. Among carriers of the Asp/Gly genotype of the TLR-4 gene, an increased risk of brucellosis with CVS diseases was determined (χ 2 = 30.19; p

Abstract Background Excretory-secretory products released by Echinococcus granulosus protoscoleces (EgPSC-ESPs) are well-known to regulate T cell responses. However, their direct influence on the differentiation of B cell subsets remains largely elusive. This study investigated the effects of EgPSC-ESPs on the differentiation of IL-10-producing B cells (B10), and explored the possible role of Toll-like receptor 2 (TLR-2) signaling in this process. Results In comparison to phosphate buffered saline (PBS), B cells exposed to the excretory–secretory products (ESPs) generated higher percentages of B10 cells, with higher expression of IL-10 mRNA, and larger amount of IL-10 production, which were in a dose dependent way. The mRNA and protein expression of TLR-2 in the ESPs-stimulated B cells were significantly higher than those in PBS, which was consistent to the results in B cells isolated from EgPSC infected mice. Moreover, TLR-2−/− B cells in response to ESPs stimulation expressed lower levels of IL-10 mRNA and produced undetectable IL-10 in comparison to those in normal B cells. In addition, Phosphatase and tensin homolog deleted on chromosome ten/AKT/Phosphatidylinositol-3 kinase (PTEN/AKT/PI3K) pathway was activated in ESPs-treated B cells, which was also dependent on TLR-2 signaling. Pam3CSK4, the agonist of TLR-2, could mock the effects of ESPs on the expression of PTEN, AKT and PI3K. Conclusion Overall, this study revealed that TLR-2 signaling was required for B10 induction mediated by EgPSC-ESPs, which might be an immunomodulatory target against the parasite infection.

Abstract Background Melanoma is one of the most common types of skin malignancies. Since current therapies are suboptimal, considerable interest has focused on novel natural-based treatments. Toll-like receptors (TLRs) play an important role in evoking innate immunity against cancer cells. Zymosan, a known TLR-2 agonist, is a glucan derived from yeast cell walls with promising immunomodulatory effects. The aim of this study was to evaluate whether Saccharomyces cerevisiae-derived zymosan-modulated skin melanoma progression by regulation of TLR-2 and TLR-4 expression in peritoneal macrophages and serum TNF-α level. Methods Male C57BL/6 mice were divided into four groups: i) zymosan-treated (Z), ii) Melanoma-bearing mice (M), iii) Melanoma-bearing mice treated with zymosan (ZM) and iv) a healthy control group (negative control). 15 days after melanoma induction, mice were injected i.p. with zymosan (10 μg) daily for 4 consecutive days. Mice were CO2-euthanized and serum TNF-α level, TLR-2 and TLR-4 expression in peritoneal macrophages and tumor growth measured. Splenocytes were treated ex-vivo with zymosan to determine viability and proliferation. Results Tumor weight significantly decreased following therapeutic dosing with zymosan (P

• TLR2 and NLRP3 were responsible for PGD 2 synthesis in S. aureus infected or Pam2CSK4-simulated macrophages. • COX-2 and HPGDS was responsible for the increased PGD 2 secretion in S. aureus -infected or Pam2CSK4-simulated macrophages. • PGD 2 participated in the regulation of inflammatory mediators secretion and macrophage phagocytosis. • The exogenous PGD 2 pretreatment affected the secretion of inflammatory mediators, while had no significant effect on macrophage phagocytosis. • The exogenous PGD 2 enhanced NLRP3 expression, while decreased TLR2 expression in macrophages infected by S. aureus. Staphylococcus aureus (S. aureus) is one of the most infamous and widespread bacterial pathogens, causing a hard-to-estimate number of uncomplicated skin infections and probably hundreds of thousands to millions of more severe, invasive infections globally per year. S. aureus may also be acquired from animals, especially in the livestock industry. The interaction mechanism of host and S. aureus has significance for finding ways to against S. aureus infection and control inflammatory response of host, while the molecular biological activities after S. aureus infection, particular in inflammatory and immune cells are not fully clear. The present study aimed to explore whether pattern recognition receptors (PRRs) mediate prostaglandin D 2 (PGD 2) synthesis and PGD 2 participates in the regulation of inflammatory response in macrophages during S. aureus infection or synthetic bacterial lipopeptide (Pam2CSK4) stimulation. PGD 2 secretion level was enhanced by mice peritoneal macrophages infected with the S. aureus. The results indicated that PGD 2 secretion was impaired in S. aureus infected-macrophages from toll-like receptors 2 (TLR2)-deficient and NLR pyrin domain-containing 3 (NLRP3)-deficient mice. PGD 2 synthetase (hematopoietic PGD synthase, HPGDS) inhibitors could reduce the activation of macrophage mitogen-activated protein kinase (MAPK)/nuclear factor-κ-gene binding (NF-κB) signaling pathways. HPGDS inhibition impaired cytokines (TNF-α, IL-1β, IL-10 and RANTES) secretion and macrophage phagocytosis during S. aureus infection. In addition, inhibition of endogenous PGD 2 synthesis was unable to affect the TLR2 and NLRP3 expression in S. aureus -infected macrophages. Taken together, macrophage PGD 2 secretion after S. aureus infection depended on receptors TLR2 and NLRP3, and the induced PGD 2 participated in the regulation of inflammatory response in S. aureus -infected macrophages. Interestingly, it was found that exogenous PGD 2 down-regulated the cytokines secretion and had no effect on phagocytosis in the S. aureus -infected macrophages. [ABSTRACT FROM AUTHOR]

Calcined Jade (CJ) is a metasilicate frequently used in traditional system of medicine as tonic to vital organs with several other pharmacological activities. X-ray powder diffraction (XRPD), inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectroscopy (AAS) and CHNS analyzer techniques were used to characterize CJ sample. CJ was administered orally to Swiss albino mice at a dose of 50, 75, 100 and 200 μg/kg body weight for 10 days and modulation of the macrophage mediated innate immune responses was studied. Flow cytometric analysis of TLR-2/4 on peritoneal macrophage revealed elevated expression of TLR-2 as compared to control. Significant increase in phagocytic activity was observed in peritoneal macrophage. The lymphoid organs weight and other toxicity parameters did not exhibit any harmful effect. To evaluate the presence of nanoparticles, CJ was dissolved in milli Q water, filtered and lyophilized. Transmission electron microscopic (TEM) analysis revealed the presence of spherical nanoparticles in CJ [14.7–142.0 nm dimension with average particle size of 64.6 nm]. In conclusion, we report stimulation of innate immune responses by CJ may partly be due to the formation of nanoparticles. Further experiments using isolated nanoparticles may further validate the role of nanoparticles.

Abstract Objective We have previously demonstrated that unencapsulated Enterococcus faecalis cps2 inhibits biofilm formation of Candida albicans, a fungus commonly found with E. faecalis in periapical lesion. In this study, we compared encapsulated and unencapsulated E. faecalis cps2 strains relationship with osteoblastic (MG-63) cells, whereas E. faecalis ATCC 29212 were used as a reference strain. Results The binding capacity of E. faecalis to MG-63 cells as shown by each tested strain was comparable, but the unencapsulated strain was less invasive compared to the encapsulated and the reference strains. Moreover, quantitative real time-PCR (qPCR) results showed that infecting unencapsulated E. faecalis cps2 is a stronger stimulator for toll like receptor 2 (TLR2) and interleukin-1β (IL-1β) mRNAs, but not for inducible nitric oxide synthase (iNOS) mRNA in osteoblastic cells. In conclusion, the performance of unencapsulated E. faecalis cps2 when the bacterium interacts with osteoblastic cells is quite different from that of encapsulated E. faecalis cps2 and reference strains. It appears that the unencapsulated strain might contribute to the persistence of the periapical inflammatory response, depending on down-regulation of iNOS mRNA expression.

This study aimed to investigate whether the classic hepatoprotective drug polyene phosphatidylcholine (PPC) regulates macrophage polarization and explores the potential role of TLR-2 in this process. In RAW264.7 macrophages and murine bone marrow-derived macrophages (BMDMs) stimulated by lipopolysaccharide (LPS), PPC significantly inhibited the production of IL-6, TNF-α, and the mRNA expression of M1-type macrophage markers. Consistently, PPC reduced the mRNA expression of several key enzymes in the pathways of glycolysis and lipid synthesis while increasing the expression of key enzymes associated with lipid oxidation. Moreover, blocking the glycolytic pathway using 2-deoxy-d-glucose (2-DG) significantly enhanced the anti-inflammatory effect of PPC. However, inhibition of lipid oxidation using GW9662 (an inhibitor of PPAR-γ) and GW6471 (an inhibitor of PPAR-α) abolished the anti-inflammatory effect of PPC. Interestingly, TLR-2 expression in macrophages was significantly downregulated after exposure to PPC. Moreover, pre-activation of TLR-2 hampered the anti-inflammatory effect of PPC. In addition, PPC did not inhibit the secretion of IL-6 and TNF-α in TLR-2−/− BMDMs that were activated by LPS. This was consistent with the increased expression of M1 markers and glycolytic and lipid synthesis enzymes but decreased lipid oxidation-related enzymes. These results showed that PPC inhibits the differentiation of M1-type macrophages, which was most likely related to TLR-2-mediated metabolic reprogramming. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective: To determine the effects of toll-like receptor 2 (TLR-2) agonist, Pam3CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum. Methods: Six groups of mice (n=6 per group) received intraperitoneal phosphate buffered saline T80 (PBS-T80), BCG or rBCG in the presence or absence of Pam3CSK4. Enzyme- linked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2a, and IgG2b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN- γ via enzyme-linked immunosorbent assay. Results: The production of total IgG and the isotype IgG1, IgG2a and IgG2b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3CSK4 further enhanced their productions. A similar pattern was also observed in IFN- γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3CSK4. Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2a, IgG2b, as well as IFN- γ in response to rBCG. However, the presence or absence of Pam3CSK4 had no effect on IL-4 production. [ABSTRACT FROM AUTHOR]

Abstract The progressive degeneration of cartilage marks the advancement of osteoarthritis (OA), which requires specific targeted treatment for effective cartilage repair. However, there is still no efficient cartilage delivery system or novel magnetic resonance (MR) contrast agent (CA). Herein, we report the synthesis of a novel class of MR CA, Gd 2 (CO 3) 3 -based nanoparticles (NPs), from a simpler and "greener" approach than previous ones. After the coating of polydopamine (PDA) onto the Gd 2 (CO 3) 3 core, we further anchored a cartilage-targeting peptide and loaded hesperetin (Hes) into NPs (Hes-Gd 2 (CO 3) 3 @PDA-PEG-DWpeptide, HGdPDW), showing excellent cartilage affinity and MR suitability. Additionally, the synthesized HGdPDW exerted significant protective effects against IL-1β stimulation, as shown by the decreased apoptosis and inflammation and increased maturation of chondrocytes in vitro. More importantly, RNA-seq analyses showed the significant reduction of TLR-2 in IL-1β-treated chondrocytes, and this reduction was followed by the inactivation of NF-κB/Akt signaling, leading to the protective effect of HGdPDW. By the establishment of anterior cruciate ligament transection (ACLT) OA mice, the bimodal MRI/IVIS imaging demonstrated the effective cartilage-binding ability of HGdPDW in OA knees with low cytotoxicity, which alleviated the gradual degeneration of articular cartilage in vivo by inhibiting TLR-2 in chondrocytes. Taken together, these results suggest that HGdPDW could target cartilage effectively, thereby protecting chondrocytes from apoptosis and inflammation via TLR-2/NF-κB/Akt signaling. We hope this new class of MRI CA could be applied in not only other fields using MRI technology but also the treatment of general cartilage-related diseases; this application will undoubtedly extend the treatment of OA clinically. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Ovine ruminal epithelial cells (ORECs) not only have a physical barrier function but also can secrete host defence peptides (HDPs), such as sheep β-defensin-1 (SBD-1). As a feed additive, Saccharomyces cerevisiae can enhance the host's innate immunity. β-glucan, a cell wall component of Saccharomyces cerevisiae, can stimulate innate immune responses and trigger the up-regulation of SBD-1 in ORECs. The signaling mechanisms involved in β-glucan-induced SBD-1 expression are not completely understood. The aim of this study was to identify the receptors and intracellular pathways involved in the up-regulation of SBD-1 induced by β-glucan. ORECs were cultured, and the regulatory mechanisms of β-glucan-induced up-regulation of SBD-1 were detected using quantitative real-time PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and western blotting. TLR-2 and MyD88 knockdown or inhibition attenuated β-glucan-induced SBD-1 expression. We also showed that inhibition of MAPK and NF-κB pathways significantly reduced β-glucan-induced SBD-1 expression. These results demonstrate that β-glucan-induced SBD-1 expression is TLR-2-MyD88-dependent and may be regulated by both MAPK and NF-κB pathways. Since NF-κB inhibition had a greater effect on the down-regulation of β-glucan-induced SBD-1 expression, the NF-κB pathway may be the dominant signaling pathway involved in the regulation of defensin expression. Our studies demonstrate that β-glucan-induced SBD-1 expression is mediated through the TLR-2-MyD88-NF-κB/MAPK pathway. Our results would contribute to the understanding of immunological modulations in the gastrointestinal tract triggered by probiotic yeast cell wall components. [ABSTRACT FROM AUTHOR]

Periprosthetic Joint Infection (PJI) represents one of the leading causes of revision prosthetic surgery, accounting for 25% of failed Total Knee Replacement (TKR) and 15% of failed Total Hip Replacement (THR). The search for a biomarker that, together with clinical and radiological findings, could improve the management of such a kind of patients is currently a big challenge for orthopaedic surgeons. This review aims (1) to assess the accuracy and the limitations of the traditional (Serum Erythrocytes Sedimentation Rate, C-reactive Protein, Procalcitonin, Interleukin 6, Tumor Necrosis Factor alpha), (2) and to analyse the emerging serum biomarkers (Presepsin, Tolllike Receptor 2, soluble urokinase-type Plasminogen Activator Receptor, Chemokine Ligand 2 and Osteopontin) in the diagnosis of PJI. A special attention will be given to the emerging serum biomarkers, that could play an important role as first-line investigations, in the screening of PJI in a close future. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Garcinia mangostana pericarp extract (GME) completely healed MRSA-infected wound. • GME reformed dermal-epidermal junction of the wound without mast cell accumulation. • GME restored the expressions of TNF-α, IL-6, and IL-1β via TLR-2 pathway. • GME possessed anti-bacterial, anti- inflammatory, and healing effects. • GME showed superior anti-inflammatory activity to α-mangostin with healing benefit. Abstract Garcinia mangostana (mangosteen) pericarp has antibacterial effects; however, information regarding its anti-inflammatory activity in vivo is limited. The anti-inflammatory effect of G. mangostana pericarp extract against methicillin-resistant Staphylococcus aureus (MRSA)-induced superficial skin infection was investigated in mice using a tape stripping model. G. mangostana pericarp ethanolic extract (GME) and its constituent, α-mangostin, were topically administered to mice with MRSA-induced superficial skin infection. MRSA-infected wounds treated with GME were completely healed on the 10th day of the study and the number of MRSA-colonies decreased from the first day of the study, whereas α-mangostin-treated wounds never completely healed with higher numbers of MRSA colonies. The epidermis of GME-treated wounds had nearly completely regenerated, with no inflammatory cell infiltration. In contrast, α-mangostin-treated wounds exhibited neutrophil infiltration and accumulation of mast cells. MRSA-infected wounds without treatment showed high expression of TNF-α, IL-6, IL-1β, and TLR-2 genes. In contrast, GME decreased mRNA levels, restoring expression of those genes to normal levels. Notably, α-mangostin did not down-regulate the expression of pro-inflammatory cytokines to the same extent as GME. Hence, GME is a promising alternative MRSA treatment because of its antibacterial, anti-inflammatory, and wound healing effects. [ABSTRACT FROM AUTHOR]

RA associated with oxidative stress and chronic inflammation has been a major health problem among the population worldwide. In this study protective effect of methanolic extract of Adhatoda vasica leaf (AVE) was evaluated on Collagen-induced arthritis in male Swiss albino mice. Post oral administration of AVE at 50, 100 and 200 mg/kg body weight doses decreased the arthritic index and footpad swelling. AVE administration diminished pro-inflammatory cytokines in serum and synovial tissues. Reduced chemokines and neutrophil infiltration in synovial tissues after AVE administration dictated its protective effect against RA. Decreased LPO content and SOD activity along with concomitant rise in GSH and CAT activities from liver, spleen and synovial tissues indicated regulation of oxidative stress by AVE. In addition decreased CRP in serum along with suppressed TLR-2 expression in CIA mice after AVE treatment was also observed. Protective effect of AVE in RA is further supported from histopathological studies which showed improvement during bone damage. In conclusion this study demonstrated A. vasica is capable of regulating oxidative stress during CIA and therefore down regulated local and systemic release of pro-inflammatory mediators, which might be linked to mechanism of decreasing synovial TLR-2 expression via downregulating release of its regular endogenous ligands like CRP.

A probiotic strain Lactobacillus paracasei L9,which was isolated from human intestine, was investigated for its immunomodulatory activity in vivo. Results showed that L9 improved systemic immunity by enhancing the phagocytic activity of peritoneal macrophages, the proliferation ratio of splenocytes, the IgG level in the serum and the level of IgA in the mucosa. Further, L9induced theTh1-polarized immune response by elevating the IFN-γ/IL-4 ratio in the mucosa. This effect was confirmed by the enhanced IL-12-inducing activity of macrophages after in vitro stimulation of L9. Also detected was increased expression of TLR-2mRNA in the mucosa. We predict that L9 could enhance innate immunity by activating TLR-2 in the mucosa, and enhance acquired immunity by promoting Th1 polarization through induced production of IL-12 by macrophages.

Background: One option for the palliative treatment of recurrent malignant pleural effusion is powdered talc using thoracoscopy. This paper presents the results of selected systemic and local manifestations of the talc-induced inflammatory reaction using a videothoracoscope. Method: A total of 114 patients with repeated malignant pleural effusion were treated at the Cardiac Surgery Clinic in Hradec Kralove from January 2010 to December 2012. Those with a life expectancy more than ≥ 3 months were eligible for talcage surgery. The group was retrospectively divided according to treatment results into Group A (N1 = 98 - successful) and Group B (N2 = 16 - relapsing). The pleural effusion was quantified using ultrasound over 1 year at 3-month intervals. Systemic changes due to the inflammatory reaction (body temperature, serum leukocyte and CRP levels) were evaluated. Local indicators of inflammation included changes in the leukocyte cell population in the effusion and changes in the pleural CRP levels. The dynamics of local expression of membrane receptors TLR-2 and CD-64 on granulocyte and monocyte cell populations in the pleural effusion were also evaluated. Results: The reaction after talcage, included a significant increase in axillary temperature and leukocyte count, 12 h after the procedure. The dynamics were different in the two groups. The dynamics of local inflammatory changes were an early increase in the pleural CRP levels in both groups. The time interval of local inflammatory development and duration was related to the treatment efficacy and showed a significant rise 2 h after talcage in Group A. In Group B the local inflammatory reaction was slower and the rise was only observed 24 h after talc application. A decrease in lymphocyte count and an increase in granulocyte count 2 h after talcage were found. After an initial drop in monocyte level, a rise occurred within 24 h after talcage. Changes in the expression of TLR-2 and CD-64 receptors in relation to their cell carriers were observed depending on time after talcage. Conclusion: The differences in the serum and pleural effusion CRP levels suggest that the surgical stress manifests itself locally in the pleural space with a lower intensity and time delay. The TLR-2 and CD-64 receptors exhibit different behaviour depending on the type of cell membrane where they are found. The inverse relation between the granulocyte increase and TLR-2 receptor decrease in the membrane immediately after talcage is a new finding. The dynamics of TLR-2 expression on the monocytes demonstrates a direct proportion between the increasing expression of the TLR-2 receptor and increasing percent fraction of the cell carrier.

Recent studies show that CD4+CD25+Foxp3+ regulatory cells (Tregs) produce effector cytokines under inflammatory conditions. However, the direct role of microbial agents that serve as toll-like receptor (TLR) ligands in the induction of effector cytokines in Tregs is less clear. Here we show that CD4+Foxp3+Tregs produce the effector cytokine IL-17A during oropharyngeal candidiasis (OPC) and inflammatory bowel disease in a TLR-2/Myd88 signaling dependent manner. TLR-2 ligands promote proliferation in Tregs in the presence and absence of TCR signals and inflammatory cytokines in vitro. The proliferation is directly dependent on TLR-2 expression in Tregs. Consistent with this, Tlr2−/− mice harbor fewer thymically derived Tregs and peripheral Tregs under homeostatic conditions in vivo. However, under Th17 inducing conditions, IL-6 and TLR-2 signaling both in Tregs as well as antigen presenting cells (APC) are critical for maximal ROR-γt and IL-17A up-regulation in Foxp3+ Tregs. The minimal and transient loss of Foxp3 expression and suppressive properties are due to the presence of IL-6 in the milieu, but not the direct effect of TLR-2 signaling in Tregs. Taken together, our data reveal that TLR-2 signaling promotes not only proliferation, but also IL-17A in Tregs, depending on the cytokine milieu. These IL-17A producing Tregs may be relevant in mucosal infections and inflammation.

Abstract Both vaccine and therapeutic approaches to malaria are based on conventional paradigms; whole organism or single antigen epitope-based vaccines administered with or without an adjuvant, and chemotherapeutics (anti-malaria drugs) that are toxic to the parasite. Two major problems that limit the effectiveness of these approaches are i) high levels of antigenic variation within parasite populations rendering vaccination efficacy against all variants difficult, and ii) the capacity of the parasite to quickly evolve resistance to drugs. We describe a new approach to both protection from and treatment of malaria parasites that involves the direct stimulation of the host innate immune response through the administration of a Toll-Like Receptor-2 (TLR2) agonist. The activity of PEG-Pam 2 Cys against the hepatocytic stages, erythrocytic stages and gametocytes of the rodent malaria parasite Plasmodium yoelii was investigated in laboratory mice. We show that administration of PEG-Pam 2 Cys, a soluble form of the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine (Pam 2 Cys), significantly and dramatically reduces the numbers of malaria parasites that grow in the livers of mice following subsequent challenge with sporozoites. We also show that treatment can also clear parasites from the liver when administered subsequent to the establishment of infection. Finally, PEG-Pam 2 Cys can reduce the numbers of mosquitoes that are infected, and the intensity of their infection, following blood feeding on gametocytaemic mice. These results suggest that this compound could represent a novel liver stage anti-malarial that can be used both for the clearance of parasites following exposure and for the prevention of the establishment of infection. Graphical abstract Image 1 Highlights • TLR-2 agonist Pam 2 Cys reduces malaria parasite burden in the liver when administered prior to sporozoite challenge. • It also reduces malaria parasite burden in the liver when administered 24 h after sporozoite challenge. • It reduces the transmissibility of a malaria infection to mosquitoes. [ABSTRACT FROM AUTHOR]