Biochemical characteristics of point mutated Capra hircus lysosome α-mannosidase.

Locoweeds, a type of poisonous weedare, are widely distributed throughout the world and have a significant impact on the development of herbivore animal husbandry. Swainsonine (SW), the main toxin in locoweeds, can competitively inhibit lysosomes α-mannosidase (LAM) in animal cells, resulting in α-mannosidosis. However, the specifics of the interaction between SW and LAM are still unclear. Here, we used molecular docking to predicte the interaction points between SW and LAM, built mutated lysosomes α-mannosidase (LAMM), and analyzed its biochemical properties changes in presumption points. The Trp at the 28th position and the Tyr at the 599th position of the LAM were interaction point candidates, and the above two amino acids in Capra hircus LAM (chLAM), were successfully mutated to glycine by constructing recombinant yeast GS115/PIC9K- LAMM. The results showed that the sensitivity of Capra hircus LAMM (chLAMM), to SW decreased significantly compared with wild-type LAM, the enzyme activity of LAM decreased approximately threefold, the optimum temperature of LAMM decreased from 55°C to 50°C, the optimum pH value increased from 4.5 to 5.0, and the effects of Mn2+, Fe3+, Al3+, Co2+, Cr3+, and ethylenediaminetetraacetic acid (EDTA) on LAM enzyme activity before and after point mutation changed significantly. These findings help us better understanding the molecular mechanism of the interaction mechanism between SW and chLAM, and provide new reference for solving locoweeds poisoning.

An update of knowledge on the regulatory role of Treg cells in apical periodontitis.

Apical periodontitis (AP) is a prevalent infectious and inflammatory disorder that involves inflammation of periapical tissues and the disintegration of alveolar bone. AP may eventually lead to tooth loss if not timely treated. This disease is caused by pathogenic bacteria in the necrotic pulps and root canals, thereby triggering responses from the innate and adaptive immune system of the periapical tissues. Regulatory T (Treg) cells play a major role in maintaining immune homoeostasis and immunological self-tolerance; however, these only account for roughly 5%-10% of human peripheral CD4+ T cells. Several studies have examined the possible role and underlying mechanism of Treg cells in different inflammatory and autoimmune disorders to facilitate the development of novel treatments for these diseases. Recent studies have indicated that Treg cells may gather at the sites of infection, thus limiting the generation of immune responses and bone resorption in the periapical area. This review will summarize studies regarding the presence and regulatory role of Treg cells in AP.

Role of Calprotectin as a Biomarker in Periodontal Disease.

Periodontal disease (PD) is a common infectious and inflammatory disease characterised by inflammation of tissues surrounding and supporting the teeth and destruction of the associated alveolar bone, eventually resulting in tooth loss. This disease is caused by periodontopathic bacteria in plaque biofilm and resultant innate and adaptive immune responses in periodontal tissues. Calprotectin (CLP) is a calcium-binding protein of the S-100 protein family and is found to be induced by activated granulocytes, monocytes, and epithelial cells. CLP has been shown to play an important role in numerous inflammatory diseases and disorders. Increasing evidence indicates that CLP is involved in the progression of PD, and its levels may be associated with disease severity and outcome of periodontal treatments. This review will summarise recent studies regarding the presence, regulation, and function of CLP in PD. The findings indicate that CLP may be an effective biomarker for diagnosis and treatment for the PD.

Elevated leptin levels in temporomandibular joint osteoarthritis promote proinflammatory cytokine IL-6 expression in synovial fibroblasts.

BACKGROUND: Leptin, through binding to its special receptor (Ob-Rb), has potent effects on immunity and inflammation. This study measured the levels of leptin in the synovial fluid of patients with temporomandibular joint osteoarthritis (TMJ-OA) and healthy controls, determined the expression of Ob-Rb and explored the effects and signalling pathways involved in leptin-induced proinflammatory cytokine interleukin (IL)-6 production in TMJ synovial fibroblasts (TMJ-SFs). METHODS: Synovial fluid samples were obtained from 16 patients with TMJ-OA and seven healthy controls. Leptin levels were measured in synovial fluid using enzyme-linked immunosorbent assay (ELISA). Ob-Rb expression was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot in cultured TMJ-SFs. Small interfering RNA (siRNA) was transfected into the TMJ-SFs to down-regulate the expression of Ob-Rb. qRT-PCR and ELISA were used to determine the levels of proinflammatory cytokine IL-6 in leptin-stimulated TMJ-SFs. The involved signalling pathways that mediate the leptin-stimulated production of IL-6 were investigated using specific signalling inhibitor analyses. RESULTS: Compared with healthy controls, patients with TMJ-OA had significantly higher concentrations of leptin in their synovial fluid. The expression levels of Ob-Rb mRNA and proteins were detected in the TMJ-SFs. Leptin can stimulate the mRNA and protein expression of IL-6 in TMJ-SFs by binding with Ob-Rb. The leptin-induced production of IL-6 by the TMJ-SFs significantly decreased after exposure to siRNA targeting Ob-Rb. Inhibiting JAK2/STAT3, p38 MAPK or PI3K/Akt substantially decreased leptin-induced IL-6 production. CONCLUSION: Leptin may up-regulate IL-6 production in vitro by binding with Ob-Rb in TMJ-SFs via the activation of the JAK2/STAT3, p38 MAPK or PI3K/Akt signalling pathways.

Leptin induces IL-6 and IL-8 expression through leptin receptor Ob-Rb in human dental pulp fibroblasts.

OBJECTIVE: Leptin, through binding to its special receptor (Ob-Rb), has potent effects on immunity and inflammation. This study aimed to investigate the expression of leptin receptor Ob-Rb in human dental pulp fibroblasts (HDPFs) and the effects of leptin on the production of proinflammatory cytokines of IL-6 and IL-8 by HDPFs. METHODS: Ob-Rb expression was determined by quantitative real-time PCR (real-time PCR), Western blot and immunofluorescence analyses in cultured HDPFs. Small interfering RNA (siRNA) was transfected into HDPFs to down-regulate the expression of Ob-Rb. Real-time PCR and enzyme-linked immunosorbent assay (ELISA) were used to determine the proinflammatory cytokines of IL-6 and IL-8 levels in leptin-stimulated HDPFs. The involved signalling pathways that mediate the leptin-stimulated production of proinflammatory cytokines were investigated using Western blot and specific signalling inhibitor analyses. RESULTS: The expression levels of Ob-Rb mRNA and protein were detected in HDPFs. Leptin could stimulate mRNA and protein expression of IL-6 and IL-8 in HDPFs in a concentration-dependent and time-dependent manner. Transfection with siRNA targeting Ob-Rb resulted in remarkable reduction of IL-6 and IL-8 expressions by HDPFs. In accordance with the enhanced expression of proinflammatory cytokines, leptin stimulation resulted in rapid phosphorylation of STAT3, p38 MAPK, ERK and Akt in HDPFs. Inhibiting JAK2/STAT3, p38 MAPK or PI3K/Akt substantially decreased leptin-induced IL-6 production, whereas blocking ERK and p38 MAPK substantially suppressed IL-8 production from leptin-stimulated HDPFs. CONCLUSIONS: Leptin may up-regulate IL-6 and IL-8 production through binding with Ob-Rb in HDPFs via the activation of different intracellular signalling pathways.

Leptin Levels in the Synovial Fluid of Patients With Temporomandibular Disorders.

PURPOSE: This study was conducted to measure concentrations of leptin and interleukin-6 (IL-6) in the synovial fluid (SF) of 38 patients with temporomandibular disorders (TMDs) and 7 healthy controls and to analyze the correlation between leptin and IL-6. PATIENTS AND METHODS: Patients with TMDs were divided into 3 subgroups according to imaging and clinical findings: displaced disc with reduction (DDR; n = 12), displaced disc without reduction (DDNR; n = 13), and osteoarthritis (OA; n = 13). SF samples were collected, and leptin and IL-6 levels were measured by enzyme-linked immunosorbent assay. RESULTS: No relevant difference in leptin level was found between the control group and the DDR or DDNR group, whereas the OA group presented a higher leptin concentration than all other groups. IL-6 concentrations were markedly higher in all patient groups than in the control group. Levels were markedly higher in the OA group than in the DDR or DDNR group, but no relevant differences were found between the DDR and DDNR groups. No relevant correlation was found between leptin and IL-6 concentrations. CONCLUSION: Distinct changes in leptin and IL-6 concentrations in the SF occurred at different stages of TMDs, suggesting their potential role in the pathogenesis of TMDs.

Upregulation of IL-6 expression in human salivary gland cell line by IL-17 via activation of p38 MAPK, ERK, PI3K/Akt, and NF-κB pathways.

BACKGROUND: The human salivary gland (HSG) cell line has so far been used as in vitro models for study of the influence of cytokines and pharmacologic agents on salivary glands, as well as a model system for inflammation in Sjögren's syndrome (SS). This study aimed to determine the effect of IL-17 on IL-6 production and the underlying molecular mechanism regulated by the HSG cell line. METHODS: Immunofluorescence analyses, RT-PCR, and Western blot were conducted to evaluate the IL-17 receptor (IL-17R) expression in cultured HSG cells. Real-time PCR and ELISA were then utilized to establish the mRNA and protein levels of IL-6 in IL-17-stimulated HSG cells. Western blot, flow cytometry, immunofluorescence, and inhibitor analyses were conducted to elucidate the involved signaling pathways. RESULTS: The HSG cells reliably expressed the IL-17R mRNA and its encoded surface-bound protein. The expression of IL-6 mRNA and protein was upregulated by stimulation of HSG cells with IL-17; this effect was impeded by IL-17- or IL-17R-neutralizing antibodies. IL-17 stimulation ended up with the fast phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), Akt, and translocation of nuclear factor-kappaB (NF-κB) in the HSG cells. p38 MAPK, Akt, and NF-κB inhibitors significantly subdued IL-6 generation in HSG cells stimulated by IL-17. PD98059, an ERK inhibitor, decreased IL-6 generation under low dose of IL-17 but not with high dose. CONCLUSIONS: The HSG cells expressed IL-17R and reacted to IL-17 to generate IL-6 via the stimulation of ERK, p38 MAPK, Akt, and NF-κB signaling pathways.

IL-17R activation of human periodontal ligament fibroblasts induces IL-23 p19 production: Differential involvement of NF-κB versus JNK/AP-1 pathways.

Interleukin (IL)-23 is an essential cytokine involved in the expansion of a novel CD4(+) T helper subset known as Th17, which has been implicated in the pathogenesis of periodontitis recently. This study hypothesised that Th17 signature cytokine IL-17 may target specialised human periodontal ligament fibroblasts (hPDLFs) for production of IL-23 p19, a key subunit of IL-23. Primary hPDLFs had steady expression of IL-17 receptor (IL-17R) mRNA and surface-bound protein. IL-17 was capable of stimulating the expression of IL-23 p19 mRNA and protein in cultured hPDLFs, which was attenuated by IL-17 or IL-17R neutralising antibodies. In accordance with the enhanced expression of IL-23 p19, IL-17 stimulation resulted in rapid activation of Akt, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK), nuclear factor-kappaB (NF-κB), and activator protein-1 (AP-1) in hPDLFs. Inhibitors of Akt, p38 MAPK, ERK 1/2, or NF-κB significantly suppressed, whereas blocking JNK and AP-1 substantially augmented IL-23 p19 production from IL-17-stimulated hPDLFs. Moreover, IL-17-initiated NF-κB activation was blocked by Akt, p38 MAPK, or ERK 1/2 inhibition, while AP-1 activity was specifically abrogated by JNK inhibition. Thus, these results provide evidence that hPDLFs are a target of Th17, and that IL-17 appears to up-regulate the expression of IL-23 p19 via a homeostatic mechanism involving Akt-, p38 MAPK-, and ERK 1/2-dependent NF-κB signalling versus the JNK/AP-1 pathway. Taken together, our findings suggest that disruption of the interaction between IL-17 and IL-23 may be a potential therapeutic approach in the treatment of periodontitis.

Boundary-lubricating ability and lubricin in synovial fluid of patients with temporomandibular joint disorders.

PURPOSE: This study was conducted to measure the boundary-lubricating ability and lubricin concentration of synovial fluid (SF) from patients with different stages of temporomandibular joint disorders (TMDs) and establish relationships between them. PATIENTS AND METHODS: According to the imaging and clinical findings, TMD patients were divided into 3 subgroups: displaced disc with reduction, displaced disc without reduction, and osteoarthritis. The boundary-lubricating ability of SF was determined by the coefficient of friction (COF) of SF in vitro with a friction apparatus. The lubricin concentrations were quantified by enzyme-linked immunosorbent assays. RESULTS: The COF of SF in TMD patients was significantly higher than that of healthy control subjects, but no observed difference was found among patient subgroups. Furthermore, a significant decline in lubricin concentrations was found in the group with osteoarthritis, whereas there was no significant change in the other groups. However, a significant correlation was not found between the COF and the lubricin concentrations in our study. CONCLUSIONS: These findings showed that distinct changes in lubricin and boundary-lubricating ability in the SF occurred with different stages of TMDs.

Immunohistochemical localization of IL-17 in induced rat periapical lesions.

Interleukin (IL)-17 is a member of a novel family of proinflammatory cytokines produced almost exclusively by a newly recognized subclass of activated T cells called "Th17" cells. From an endodontic perspective, IL-17 potently regulates cells of the innate immune system, serving as an important bridging molecule between the adaptive and innate immune systems. The purpose of this study was to investigate the immunohistochemical localization of IL-17 during the development of periapical lesions in rats. Periapical lesions developed within 28 days after mandibular first molar pulp exposure in Sprague-Dawley rats. The animals were randomly sacrificed at 0, 7, 14, 21, and 28 days after pulpal exposure. The jaws that contained the first molar were obtained and routinely prepared for histologic analysis, immunohistochemistry, and enzyme histochemistry. From day 0 to day 28, the number of IL-17-positive cells and neutrophils ascended and peaked on day 28. Osteoclast numbers substantially multiplied from day 0 to day 14 and then gradually decreased from day 14 to day 28. In addition, the osteoclast decrease contrasted with the increased number of IL-17-positive cells and neutrophils. These findings showed that IL-17 could be observed and might possibly be involved in the inflammatory response and bone resorption of periapical tissues as well as associated with periapical lesion pathogenesis.

Effect of an estrogen-deficient state and alendronate therapy on bone loss resulting from experimental periapical lesions in rats.

The aim of the research was to evaluate the impact of an estrogen-deficient state and alendronate (ALD) therapy on bone loss resulting from experimental periapical lesions in rats. Periapical lesions were induced on ovariectomized (OVX) and sham-ovariectomized (Sham) rats. After sample preparation, histologic and radiographic examination for periapical bone loss area and an enzyme histochemical test for tartrate-resistant acid phosphatase (TRAP) were performed. The results showed that OVX significantly increased bone loss resulting from periradicular lesions. After daily subcutaneous injection of ALD, the bone loss area and the number of TRAP-positive cells (osteoclasts) were reduced. These findings suggested that alendronate may protect against increased bone loss from experimental periapical lesions in estrogen-deficient rats. Given recent recognition of adverse effects of bisphosphonates, including an increased risk for osteonecrosis, the findings from this study should not be interpreted as a new indication for ALD treatment. However, they may offer insight into understanding and predicting outcomes in female postmenopausal patients already on ALD therapy for medical indications.