Expression of sterile-α and armadillo motif containing protein (SARM) in rheumatoid arthritis monocytes correlates with TLR2-induced IL-1ß and disease activity.

OBJECTIVE: Cartilage and bone damage in RA are associated with elevated IL-1ß. The effects of IL-1ß can be reduced by biological therapies that target IL-1ß or TNF-α. However, the mechanisms responsible for increased IL-1ß and the effect of anti-TNF-α have not been fully elucidated. Recently, sterile-α and armadillo motif containing protein (SARM) was identified as a negative regulator of toll-like receptor (TLR) induced IL-1ß secretion through an interaction with the inflammasome. This study set out to investigate SARM during TLR-induced IL-1ß secretion in RA peripheral blood monocytes and in patients commencing anti-TNF-α treatment. METHODS: Monocytes were isolated from RA patients and healthy controls; disease activity was measured by DAS28. IL-1ß secretion was measured by ELISA following TLR1/2, TLR4 and TLR7/8 stimulation. The mRNA expression of SARM1, IL-1ß and the components of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome were measured by quantitative PCR. SARM protein expression was measured by western blotting. RESULTS: TLR1/2 activation induced elevated IL-1ß in RA monocytes compared with healthy controls (P = 0.0009), which negatively correlated with SARM1 expression (P = 0.0086). Lower SARM expression also correlated with higher disease activity (P = 0.0246). Additionally, patients responding to anti-TNF-α treatment demonstrated a rapid upregulation of SARM, which was not observed in non-responders. CONCLUSION: Together, these data highlight a potential contribution from SARM to RA pathophysiology where decreased SARM may lead to elevated IL-1ß associated with RA pathogenesis. Furthermore, the data additionally present a potential mechanism by which TNF-α blockade can modify IL-1ß secretion.

Thymic stromal lymphopoietin participates in the TLR2-and TLR4-dependent immune response triggered by Aspergillus fumigatus in human corneal cells.

Fungal keratitis constitutes a serious vision-threatening disease. Toll-like receptors (TLRs) comprise key mediators of innate immunity triggered by Aspergillus fumigatus (AF) in the cornea, but the messenger between innate and adaptive immunity remained unknown. Thymic stromal lymphopoietin (TSLP) represents a critical factor of adaptive immunity. Here we investigated the expression of TSLP in corneal epithelial and stromal cells challenged by AF and its relationship with TLRs. We stimulated corneal cells with TLR ligands zymosan or lipopolysaccharide (LPS), human recombinant TSLP, or AF hyphae for various periods, with or without prior TLR2, TLR4, or TSLP inhibition. TLR2, TLR4, TSLP, IL-8, and TNF-α release and expression were measured via enzyme-linked immunosorbent analysis, quantitative polymerase chain reaction, or western blot. Corneal cell stimulation with zymosan or LPS induced up-regulated TSLP expression. Enhanced TSLP expression was associated with AF treatment in human corneal cells; TLR2 or TLR4 inhibition impaired the AF-induced TSLP levels. Human recombinant TSLP augmented TLR2 and TLR4 expression; RNA interference of TSLP attenuated TLR, IL-8, and TNF-α expression stimulated by AF hyphae. These findings indicated that TSLP participates in the immune response of corneal cells triggered by AF, which is closely related to TLR function, and the innate immunity mediated by TLRs could be enhanced by TSLP. Innate immunity may therefore transmit inflammatory signals to adaptive immunity through activation of TSLP; in turn, adaptive immunity likely exerts certain regulatory effects on innate immunity via TSLP. That is, TSLP could interact with innate immunity mediated by TLR2 and TLR4 in human corneal cells challenged by AF and thus may serve as a messenger between the innate and adaptive immune responses in AF keratitis.

Revisiting bupropion anti-inflammatory action: involvement of the TLR2/TLR4 and JAK2/STAT3.

There are accumulating reports regarding poor response to common antidepressant therapy. Antidepressant resistance is often linked to inflammatory system activation and patients displaying inflammation prior to the treatment are less responsive to antidepressants. We hypothesized that the inefficacy of antidepressant therapy in some patients may be attributable to the drugs' inflammatory mode of action, which has been overlooked because of their substantial therapeutic benefit. Bupropion is a commonly prescribed antidepressant that is often used to treat seasonal affective disorders as well. Nevertheless, research suggests that bupropion causes inflammation and worsens depressive symptoms. Therefore, we investigated the impact of bupropion on cytokines of innate and adaptive immunity, as well as immune signaling pathways. We treated lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs) with different doses of bupropion. Pro-/anti-inflammatory cytokines [tumor necrosis factor alpha (TNFα), interleukin-1ß (IL-1ß), IL-17, and IL-10] were assessed at both transcriptional and translational levels as well as the involvement of JAK2 /STAT3, TLR2, and TLR4 signaling in this process. Bupropion reduced IL-17A, TNFα, and IL-1ß protein levels in the cultures. Nonetheless, bupropion increased IL-1ß (P < 0.0001), TNFα (P < 0.0001), and IL-17A (P < 0.05) mRNA levels. Treatment enhanced both IL-10 concentration (P < 0.0001) and gene expression (P < 0.0001). TLR2 (P < 0.0001), TLR4 (P < 0.0001), JAK2 (P < 0.0001), and STAT3 (P < 0.0001) gene expression also rose in response to bupropion. The findings imply that bupropion, particularly 50 µM and 100 µM, has pro-inflammatory effects and should be co-administered with anti-inflammatory medications, at least in patients with inflammatory conditions.

Insight into the expression of toll-like receptors 2 and 4 in patients with abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is a relatively common, life-threatening disease prevalent in persons over the age of 65. In recent years, an increasing number of studies have suggested that pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), may serve as important regulators in the development of AAAs. In this study, we evaluated the TLR2 and TLR4 expression in the aortic wall and blood of patients with AAA. The TLR2 and TLR4 mRNA expression were significantly higher in the blood of patients with AAA than in the blood of healthy volunteers (p = 0.009 and p = 0.010, respectively). The expression of TLR2 and TLR4 transcripts was also higher in the blood compared with the aortic wall tissue of AAA patients (p = 0.001 for both). Higher TLR2 protein expression was observed in the aortic wall of AAA patients compared with the blood (p = 0.026). A significantly higher concentration of TNF-α and IL-4 in patients with AAA than in healthy volunteers (p < 0.001 for both) was noticed. This study suggests that TLR2 may play a role in the inflammatory response in the aorta, both locally and systemically, in patients with AAA.

Gene Signatures of Early Response to Anti-TNF Drugs in Pediatric Inflammatory Bowel Disease.

Around a 20-30% of inflammatory bowel disease (IBD) patients are diagnosed before they are 18 years old. Anti-TNF drugs can induce and maintain remission in IBD, however, up to 30% of patients do not respond. The aim of the work was to identify markers that would predict an early response to anti-TNF drugs in pediatric patients with IBD. The study population included 43 patients aged <18 years with IBD who started treatment with infliximab or adalimumab. Patients were classified into primary responders (n = 27) and non-responders to anti-TNF therapy (n = 6). Response to treatment could not be analyzed in 10 patients. Response was defined as a decrease in over 15 points in the disease activity indexes from week 0 to week 10 of infliximab treatment or from week 0 to week 26 of adalimumab treatment. The expression profiles of nine genes in total RNA isolated from the whole-blood of pediatric IBD patients taken before biologic administration and after 2 weeks were analyzed using qPCR and the 2-∆∆Ct method. Before initiation and after 2 weeks of treatment the expression of SMAD7 was decreased in patients who were considered as non-responders (p value < 0.05). Changes in expression were also observed for TLR2 at T0 and T2, although that did not reach the level of statistical significance. In addition, the expression of DEFA5 decreased 1.75-fold during the first 2 weeks of anti-TNF treatment in responders, whereas no changes were observed in non-responders. Expression of the SMAD7 gene is a pharmacogenomic biomarker of early response to anti-TNF agents in pediatric IBD. TLR2 and DEFA5 need to be validated in larger studies.

Innate immune response in astrocytes infected with herpes simplex virus 1.

Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, infects epithelial surfaces and establishes latency in the central nervous system, where astrocytes are a major immune cell type. Here, we report changes that occur in the expression of pathogen recognition receptors, such as Toll-like receptors, DNA and RNA sensors, interferons, and interferon-stimulated genes, when astrocytes are infected with HSV-1 strain F. We observed upregulation of Toll-like receptors 2, 6 and 9, MDA5, and DAI along with an increase in the expression of type I interferons and interferon-stimulated genes such as IFIT1, IFIT3 and RNase L. These genes encode proteins that mediate the antiviral immune response.

Effects of guluronic acid (G2013) on gene expression of TLR2, TLR4, MyD88, TNF-α and CD52 in multiple sclerosis under in vitro conditions.

Context: Multiple sclerosis (MS) is an autoimmune and chronic inflammatory disease of CNS. The α-L-guluronic acid (G2013) as novel NSAID with immunomodulatory effects has shown its positive effects in various investigations.Objective: Present research aimed to study the potency of G2013 on gene expression of TLR2, TLR4, MyD88, TNF-α and CD52 in PBMCs of MS patients under in vitro conditions. Materials and methods: 24 blood samples from MS patients and healthy controls were considered for RT-PCR and flow cytometry techniques under two different doses of G2013.Results: Our research indicated that this drug could significantly decrease the gene expression of TLR2, TLR4 and TNF-α compared to untreated group. Conclusion: Data demonstrated that the guluronic acid is able to modify the expression levels of TLR2, TLR4 and TNF-α genes to less than the pathogenic boarder line level, which it might be recommended for reducing the pathological process in multiple sclerosis.

[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.

Platelet Toll-like receptor and its ligand HMGB-1 expression is increased in the left atrium of atrial fibrillation patients.

BACKGROUND: Atrial fibrillation(AF) is the most common sustained arrhythmia. Its most feared sequelae are stroke and peripheral thromboembolism due to atrial thrombi formation. Mechanisms underlying the relationship between platelet activation and left atrial thrombi have not been clearly elucidated yet. We aimed to investigate whether immune-mediated platelet activation occurred in AF patients in this cross-sectional study. METHODS: Persistent and paroxysmal AF patients who underwent cryoballoon-based AF ablation between March 2015 and July 2016 were included as the patient group. Patients without AF in whom transseptal puncture was performed at the same period for purposes other than AF ablation were included as the control group. Peripheral and left atrial blood samples were obtained for determination of platelet Toll-like receptor(TLR)-2, TLR-4 and high mobility group box-1(HMGB-1) expression levels. RESULTS: A total of 75 subjects (53 patients with AF and 22 control subjects) [mean: 60.33 (SD: 6.14) years, 57.33% male] were included. Left atrial and peripheral TLR-2, 4 and HMGB-1 expression levels were significantly higher in the patient group when compared to the controls. Left atrial platelet TLR-2 and TLR-4 expression and serum HMGB-1 levels were higher in persistent AF patients compared to paroxysmal AF patients. In the patient group, left atrial expression of TLR-2, 4 and HMGB-1 were significantly higher than the peripheral expression levels. CONCLUSION: Findings of our study suggest evidence for immune-mediated platelet activation in the left atria of AF patients.

Killing of S. aureus in murine peritoneal macrophages by Ascorbic acid along with antibiotics Chloramphenicol or Ofloxacin: Correlation with inflammation.

Alarming increase of death due to S. aureus sepsis demands newer treatment strategies. Enhancement of antibiotic resistant S. aureus strains caused increased mortality. Only antibiotic treatment for Staphylococcal sepsis has been found insufficient to improve outcomes. In the innate immune response, phagocytosis mediated killing of pathogen and further triggering of intracellular signaling cascades by the PRRs culminates in the release of a variety of pro inflammatory cytokines, which orchestrate together in the early host response to infection. Increased production of inflammatory cytokines not only delineate pathogen burden but also affects host cell by triggering inflammation. Therefore, combinational therapy of Ascorbic acid is used along with antibiotics Ofloxacin (OFX) or Chloramphenicol (CHL) to kill S. aureus by mouse peritoneal macrophages. For this ROS like H2O2, superoxide anion and NO production was accessed, TLR2 and COX2 expression was monitored. Pro-inflammatory cytokines along with antioxidant levels were also analyzed. Ascorbic acid along with antibiotics OFX or CHL promoted bacterial clearance at early infection by increasing H2O2 and O2-.NO production has been found to decrease, providing protection against harmful per-oxynitril ion. Increase in TLR-2 expression resulted in enhanced phagocytosis and subsequently more killing. Treatment with Ascorbic acid decreased proinflammatory cytokines and inflammatory markers like iNOS and COX2. This combination increased antioxidant enzymes like SOD, Catalase, GSH as well as decreased LPO, thus balancing ROS and antioxidant status inside the cell. Thus in-vitro augmentation of bacterial clearance along with regulated inflammation as found by decrease in proinflammatory cytokines like TNF-α IFN-γ,IL-6 and inflammatory markers like COX2 may be considered as a novel and important therapeutic strategy.

TLR2/MyD88/NF-κB signaling pathway regulates IL-1ß production in DF-1 cells exposed to Mycoplasma gallisepticum LAMPs.

Mycoplasma gallisepticum (M. gallisepticum) is one of the most important pathogens that cause chronic respiratory disease in chickens. M. gallisepticum-derived lipid-associated membrane proteins (LAMPs) are thought to be one of the major factors in mycoplasma pathogenesis and are potent inducers of the host innate immune response. However, the interaction of pathogenic M. gallisepticum-derived LAMPs with Toll-like receptors (TLRs) and the signaling pathways responsible for activating inflammation and NF-κB have not been fully elucidated. In this study, we found that IL-1ß expression was induced in DF-1 cells stimulated with M. gallisepticum LAMPs. Subcellular localization experiments using immunofluorescence assays (IFAs) showed p65 translocation from the cytoplasm to the nucleus in DF-1 cells following stimulation with M. gallisepticum LAMPs. Phosphorylation of p65 was detected in LAMP-stimulated DF-1 cells. Treatment with an NF-κB-specific inhibitor showed that NF-κB is required for M. gallisepticum LAMP-induced IL-1ß expression. In addition, the results indicated that TLR2 and myeloid differentiation primary-response protein 88 (MyD88)-dependent signaling pathways were involved in the activation of NF-κB by M. gallisepticum LAMPs. Together, these results provide evidence that M. gallisepticum LAMPs activate IL-1ß production through the NF-κB pathway via TLR2 and MyD88.

Leishmania amazonensis induces modulation of costimulatory and surface marker molecules in human macrophages.

Manipulation of costimulatory and surface molecules that shape the extent of immune responses by Leishmania is suggested as one of the mechanisms of evading the host's defences. The experiments reported here were designed to evaluate the expressions of CD11b, CD11c, CD14, CD18, CD54, CD80, CD86, CD206, MHC class II and TLR-2 (Toll-like receptor 2) in human macrophages infected with L. amazonensis. Phenotypic evaluation revealed a negative modulation in CD11b, CD11c, CD14, CD18, CD54 and MHC class II molecules, depending on the level of infection. The results showed that as early as 1 hour after infection no reduction in marker expression occurs, whereas after 24 hours, downregulation of these molecules was observed in macrophages. No significant changes were observed in the expressions of CD80, CD86, CD206 and TLR2. Evidence of the differential modulation of markers expression and that after parasite uptake no reduction in surface marker expression occurs indicates that parasite internalization is not involved in the phenomena of down-modulation.

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.

Toll-like receptor 2bright cells identify circulating monocytes in human and non-human primates.

Polychromatic flow cytometry is a useful tool for monitoring circulating whole blood monocytes, although gating strategies often vary depending on the study. Increased analyses of the myeloid system have revealed monocytes to be more plastic than previously understood and uncovered changes among surface markers previously considered to be stable. The myeloid system has also been found to have disparate surface markers between mouse, human, and non-human primate studies, which further complicates examination between species. This study has found bright Toll-like receptor 2 (TLR2) expression to be a consistent surface marker of circulating whole blood monocytes in humans and two species of macaques. Furthermore, within our pigtailed macaque model of HIV-associated CNS disease, where monocyte surface markers have previously been shown to reorganize during acute infection, TLR2 remains stably expressed on the surface of classical, intermediate, and non-classical monocytes. Our findings demonstrate that TLR2 is a useful surface marker for including all monocytes during other phenotypic changes that may alter the expression of common surface receptors. These results provide a practical tool for studying all types of monocytes during inflammation and infection within humans and macaques. © 2017 International Society for Advancement of Cytometry.

Effects of Lactobacillus plantarum Strain OLL2712 Culture Conditions on the Anti-inflammatory Activities for Murine Immune Cells and Obese and Type 2 Diabetic Mice.

Studies on the health-promoting effects of lactic acid bacteria (LAB) are numerous, but few provide examples of the relationship between LAB function and culture conditions. We verified the effect of differences in culture conditions on Lactobacillus plantarum OLL2712 functionality; this strain exhibits anti-inflammatory activity and preventive effects against metabolic disorders. We measured interleukin-10 (IL-10) and IL-12 production in murine immune cells treated with OLL2712 cells prepared under various culture conditions. The results showed that the IL-10-inducing activities of OLL2712 cells on murine immune cells differed dramatically between OLL2712 groups at different culture phases and using different culture medium components, temperatures, and neutralizing pHs. In particular, exponential-phase cells had much more IL-10-inducing activity than stationary-phase cells. We confirmed that the Toll-like receptor 2 (TLR2) stimulation activity of OLL2712 cells depended on culture conditions in conjunction with IL-10-inducing activity. We also demonstrated functional differences by culture phases in vivo; OLL2712 cells at exponential phase had more anti-inflammatory activity and anti-metabolic-disorder effects on obese and diabetic mice than those by their stationary-phase counterparts. These results suggest that culture conditions affect the functionality of anti-inflammatory LAB.IMPORTANCE While previous studies demonstrated that culture conditions affected the immunomodulatory properties of lactic acid bacteria (LAB), few have comprehensively investigated the relationship between culture conditions and LAB functionality. In this study, we demonstrated several culture conditions of Lactobacillus plantarum OLL2712 for higher anti-inflammatory activity. We also showed that culture conditions concretely influenced the health-promoting functions of OLL2712 in vivo, particularly against metabolic disorders. Further, we characterized a novel mechanism by which changing LAB culture conditions affected immunomodulatory properties. Our results suggest that culture condition optimization is important for the production of LAB with anti-inflammatory activity.

Association of mRNA expression of toll-like receptor 2 and 3 with hepatitis B viral load in chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

During Hepatitis B virus infection, the pathogen sensors Toll-like receptors (TLRs) play a role in innate immunity system. The study aimed to investigate mRNA expression levels of TLR2 and TLR3 in Hepatitis B virus (HBV) mediated chronic hepatitis B (CHB), cirrhosis (CIRR), and hepatocellular carcinoma (HCC), and to correlate viral load with severity of these diseases and expression of TLRs. A total of 180 HBV DNA positive samples were selected for the study. HVB-DNA was detected by multiplex PCR. Viral load estimation was done by using the Ampisure HBV Quantitative kit as per manufacture instructions. Expression levels of TLR2 and TLR3 were determined by real time PCR. The viral load was estimated to be 6.64log10 IU/ml in CHB, 4.88log10 IU/ml in CIRR, and 4.86log10 IU/ml in HCC. No significant association of viral load was found with increasing age. Upregulation of TLR2 expression in CHB when individually compared with CIRR and HCC was found to be statistically significant. Downregulation of TLR3 expressions in CIRR when compared to both CHB and HCC individually were found to be statistically significant. No significant effect of viral load on the expression of TLR2 and 3 were found. With severity of the disease from CHB to HCC, the HBV load decreases. The study suggests the possibility of HBV interacting with signalling of both analysed TLR receptors which partially explains the induction of immune tolerance pathways by Hepatitis B virus. J. Med. Virol. 89:1008-1014, 2017. © 2017 Wiley Periodicals, Inc.

Effect of iNOS inhibitor LNMMA along with antibiotics Chloramphenicol or Ofloxacin in murine peritoneal macrophages regulates S.aureus infection as well as inflammation: An in vitro study.

Death due to sepsis by S. aureus is rapidly increasing because of their potent weaponries against macrophage mediated killing. Macrophages serve as intracellular reservoirs of S. aureus. Although significant resources have been invested during the last decade in new treatments for sepsis, only antibiotic therapy has failed to improve outcomes. Moreover the host pathogen interaction resulted in host cell death triggering inflammation. So, successful therapy requires amalgamation of therapies to delineate pathogen along with providing protection to host cell. With this idea, LNMMA, the iNOS inhibitor is used along with antibiotics Ofloxacin or Chloramphenicol on S. aureus infected mouse peritoneal macrophage. ROS like H2O2, O2- production has been measured. NO inhibition by iNOS inhibitor and antioxidant levels has been analysed. COX2, TLR2 and iNOS expression along with proinflammatory cytokine level was studied. It was found that the use of iNOS inhibitor LNMMA along with antibiotics not only enhances bacterial clearance but also decreases proinflammatory responses in Staphylococcus aureus infected macrophages. Inhibition of TLR2 as well as COX2 has also been found in combined treatment groups. The use of iNOS inhibitor LNMMA plus Ofloxacin or Chloramphenicol pretreatment enhanced bacterial clearance by increasing ROS. Decreases in NO protect the cell from harmful peroxynitril as well as inflammatory damage by changes in iNOS, COX2 activity along with reduced proinflammatory cytokines like TNFα, IFNγ, IL1-ß etc. Changes in antioxidant level has been found. This in-vitro realm of augmented bacterial clearance and regulated inflammation may be considered as a novel and important therapeutic intervention.

Dog skin parasite load, TLR-2, IL-10 and TNF-α expression and infectiousness.

Visceral leishmaniosis is a zoonotic disease that is transmitted by Lutzomyia longipalpis sandflies. Dogs are the main peri-urban reservoir of the disease, and progression of canine leishmaniosis is dependent on the type of immune response elaborated against the parasite. Type 1 immunity is characterized by effective cellular response, with production of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α). In contrast, Type 2 immunity is predominantly humoral, associated with progression of the disease and mediated by anti-inflammatory cytokines such as interleukin 10 (IL-10). Although seemly important in the dynamics of leishmaniosis, other gene products such as toll-like receptor 2 (TRL-2) and inducible nitric oxide synthase (iNOS) exert unclear roles in the determination of the type of immune response. Given that the dog skin serves as a micro-environment for the multiplication of Leishmania spp., we investigated the parasite load and the expression of TLR-2, iNOS, IL-10 and TNF-α in the skin of 29 infected and 8 control dogs. We found that increased parasite load leads to upregulation of TLR-2, IL-10 and TNF-α, indicating that abundance of these transcripts is associated with infection. We also performed a xenodiagnosis to demonstrate that increased parasitism is a risk factor for infectiousness to sandflies.

Different effects of arginine vasopressin on high-mobility group box 1 expression in astrocytes isolated from stroke-prone spontaneously hypertensive rats and congenic SHRpch1_18 rats.

Stroke-prone spontaneously hypertensive rats (SHRSP/Izm) develop severe hypertension and astrocytic oedema following ischaemic stimulation. During ischaemic stress high-mobility group box 1 (Hmgb1) expression in astrocytes is induced, and subsequently potentiates deterioration of the brain due to ischaemic injury, which manifests as both cerebral inflammation and astrocytic oedema. Arginine vasopressin (AVP) induces brain injury and increases astrocytic swelling. After stroke, Hmgb1 and peroxiredoxin (Prx) are released at different times and activate macrophages in the brain via Toll-like receptors (Tlr2s). The purpose of this study was to examine whether AVP and/or hypoxia and reoxygenation (H/R) contribute to Hmgb1 regulation following ischaemic stroke. Thus, Hmgb1, Prx2 and Tlr2 expression levels in astrocytes isolated from Wistar Kyoto rats (WKY/Izm), spontaneously hypertensive rats (SHR/Izm), SHRSP/Izm and congenic rat strain SHRpch1_18 treated with AVP and/or H/R were compared. Gene and protein expression levels were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative PCR, and Western blot. mRNA expression of Hmgb1, Prx2 and Tlr2 induced by AVP was dose-dependent, and Hmgb1 and Prx2 expression was higher in SHR/Izm, SHRSP/Izm and SHRch1_18 than in WKY/Izm. Tlr2 expression with AVP was reduced in SHR/Izm compared to WKY/Izm. In SHRpch1_18, Hmgb1 expression increased after AVP plus H/R. AVP-modulated expression of Hmgb1 protein was reduced by the addition of the antioxidant N-acetylcysteine (NAC). These results suggest that oxidative stress by AVP enhanced expression of Hmgb1, Prx2 and Tlr2 in astrocytes. We hypothesize that regulation of Hmgb1 by AVP during H/R might be related to induction of inflammation and stroke in SHRSP/Izm and SHRpch1_18 rats.

Lactobacillus acidophilus alleviates the inflammatory response to enterotoxigenic Escherichia coli K88 via inhibition of the NF-κB and p38 mitogen-activated protein kinase signaling pathways in piglets.

BACKGROUND: A newly isolated L. acidophilus strain has been reported to have potential anti-inflammatory activities against lipopolysaccharide (LPS) challenge in piglet, while the details of the related inflammatory responses are limited. Here we aimed to analysis the ability of L. acidophilus to regulate inflammatory responses and to elucidate the mechanisms involved in its anti-inflammatory activity. RESULTS: The ETEC (enterotoxigenic Escherichia coli) K88-induced up-regulations of IL-1ß, IL-8 and TNF-α were obviously inhibited by L. acidophilus while IL-10 was significantly increased. Moreover, L. acidophilus down-regulated pattern recognition receptors TLR (Toll-like receptor) 2 and TLR4 expression in both spleen and mesenteric lymph nodes of ETEC-challenged piglets, in accompanied with the reduced phosphorylation levels of nuclear factor kappa B (NF-κB) p65 and mitogen-activated protein kinase (MAPK) p38 as well in spleen of ETEC-infected piglets. Furthermore, L.acidophilus significantly increased the expression of the negative regulators of TLRs signaling, including Tollip, IRAK-M, A20 and Bcl-3 in spleen of ETEC-challenged piglets. CONCLUSIONS: Our findings suggested that L. acidophilus regulated inflammatory response to ETEC via impairing both NF-κB and MAPK signaling pathways in piglets.