Fermentation supernatant of Staphylococcus aureus drives catabolism in chondrocytes via NF-κB signaling mediated increase of cholesterol metabolism.

Septic arthritis induced by Staphylococcus aureus (S. aureus) causes irreversible cartilage degradation and subsequent permanent joint dysfunction. Recently, cartilage degradation in osteoarthritis is recognized to be associated with metabolic disorders. However, whether cholesterol metabolism is linked to septic arthritis pathology remains largely unknown. Here, we found that exposure to fermentation supernatant (FS) of S. aureus in chondrocytes resulted in a significant increase in expression of key modulators involved in cholesterol metabolism, including lectin-type oxidized low density lipoprotein receptor 1 (LOX1), cholesterol 25-hydroxylase (CH25H), 25- hydroxycholesterol 7α-hydroxylase (CYP7B1) as well as retinoic acid-related orphan receptor alpha (RORα), a binding receptor for cholesterol metabolites. We further demonstrated that enhancement of CH25H/CYP7B1/RORα axis resulted from FS exposure was mediated by activation of NF-κB signaling, along with upregulation in catabolic factors including matrix metallopeptidases (MMP3 and MMP13), aggrecanase-2 (ADAMTS5), and nitric oxide synthase-2 (NOS2) in chondrocytes. Exogenous cholesterol acts synergistically with FS in activating NF-κB pathway and increases cholesterol metabolism. While, the addition of tauroursodeoxycholic acid (TUDCA) which promotes cholesterol efflux, resulted in remarkable reduction of intracellular cholesterol level and restoration of balance between anabolism and catabolism in FS treated chondrocytes. Collectively, our data indicated that, in response to FS of S. aureus, NF-κB signaling activation coupled with increased cholesterol metabolism to stimulate catabolic factors in chondrocytes, highlighting cholesterol metabolism as a potential therapeutic target for treating septic arthritis.

Proteomic analysis reveals critical molecular mechanisms involved in the macrophage anti-spinal tuberculosis process.

Tuberculosis infection activates the autoimmune system. However, the role of host-pathogen interactions involved in Mycobacterium tuberculosis infection is unclear. In this study, we analyzed 6 spinal tuberculosis tissues and 6 herniated disc tissues by using liquid chromatography-tandem mass spectrometry coupled with tandem mass spectrometry, and immunohistochemical staining was performed for validating the results. We identified 42 differential immune-related proteins and 3 hub genes that are primarily localised in the tertiary granule and involved in biological processes such as cellular response to the presence of cadmium ions, regulation of ion transmembrane transport, transmembrane transport, and inflammatory responses. Genes encoding cytochrome B-245 beta chain (CYBB), matrix metallopeptidase 9 (MMP9), and C-X-C motif chemokine ligand 10 (CXCL10) were identified as the hub genes that exhibited anti-tuberculosis activity and were responsible for macrophage resistance against M. tuberculosis. In conclusion, CYBB, MMP9, and CXCL10 resist M. tuberculosis infection through chemotaxis and macrophage activation. Our results indicate that CYBB, MMP9, and CXCL10 could be considered as molecular targets for spinal tuberculosis treatment, which may significantly improve patients' quality of life and prognosis.

Sodium hypochlorite is more effective than chlorhexidine for eradication of bacterial biofilm of staphylococci and Pseudomonas aeruginosa.

PURPOSE: Periprosthetic infection is a common reason for surgical revision. Given the increasing resistance of bacteria to antibiotics (e.g., VRE, 4-MRGN) local antiseptic treatment is gaining in importance. However, no standard guideline-based treatment recommendation is yet available. The aim of this study was to investigate the effectiveness of sodium hypochlorite and chlorhexidine against bacterial biofilms. Furthermore, the toxicity of both antiseptics towards human chondrocytes was examined. METHODS: Human chondrocytes were isolated, cultivated and treated with sodium hypochlorite and chlorhexidine. The viability of cultures was assessed by determination of cell count, XTT and MTT ELISAs, and fluorescent staining with propidium iodide. Bacterial strains of Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa were added to liquid media and incubated overnight. After determination of bacterial concentrations polyethylene (PE) devices were inoculated with bacteria for 48 h until biofilms formed. The devices were then washed, treated with antiseptics for 2 and 5 min and subsequently spread on agar plates. RESULTS: Sodium hypochlorite is more effective than chlorhexidine in penetrating biofilms of S. aureus, S. epidermidis and P. aeruginosa. Both antiseptics are chondrotoxic, but sodium hypochlorite damages human chondrocytes less than chlorhexidine in vitro. CONCLUSIONS: The findings confirm the effectiveness of sodium hypochlorite and chlorhexidine against bacterial biofilms. Both antiseptics can be recommended for the treatment of periprosthetic infections. The toxic effects of sodium hypochlorite and chlorhexidine towards chondrocytes may mean there is a risk of damage to cartilage tissue. LEVEL OF EVIDENCE: Controlled experimental study.

Septic Arthritis/Osteomyelitis May Lead to Osteochondrosis-Like Lesions in Foals.

Failure of the cartilage canal blood supply leads to ischemic chondronecrosis which causes osteochondrosis, and osteochondral lesions. Osteochondrosis is a disease with a heritable component and usually occurs under aseptic conditions. Because bacteria can bind to growth cartilage and disrupt the blood supply in pigs and chickens, we considered whether this might play a role in development of equine osteochondrosis. The aim of this study was to examine whether bacteria are present in canals in the growth cartilage of foals with septic arthritis/osteomyelitis, and whether this is associated with osteochondrosis. The material consisted of 7 foals aged 9-117 days euthanized because of septic arthritis/osteomyelitis. The 7 cases had 16 lesions in growth cartilage that were evaluated histologically. Bacteria were present in cartilage canals in foals with septic arthritis/osteomyelitis. Portions of necrotic canals adjacent to bacteria frequently contained neutrophils, termed acute septic canals; or granulation tissue with neutrophils, termed chronic septic canals. Acute and chronic septic canals were associated with ischemic chondronecrosis in the articular-epiphyseal cartilage complex (AECC) of 5 cases and in the physis of 2 cases, and ossification was focally delayed in 5 of those 7 cases. Lesions occurred with and without adjacent osteomyelitis. Bacteria were present in cartilage canals and were associated with focal chondronecrosis in both the AECC and the physis. This establishes sepsis as a plausible cause of some osteochondral lesions in horses. It is recommended that horses with sepsis-related osteochondral lesions may be used for breeding without increasing the prevalence of OCD-predisposing genes in the population.

Chondrocyte Viability After a Simulated Blast Exposure.

OBJECTIVES: The effects of blast exposure have gained increasing interest in the military medical community with their continued occurrence on the battlefield. The impact of the direct and indirect energy imparted from blasts to hollow viscera, as well as closed head injuries, have been well studied. However, the injury to articular cartilage has not been investigated, despite previous correlations regarding the development of osteoarthritis. The purpose of this study was to assess the degree of injury to articular chondrocytes after exposure to a simulated blast overpressure wave. METHODS: Fresh juvenile porcine stifle joints were subjected to a simulated blast overpressure wave utilizing a custom fabricated blast simulator with compressed gases, within the reported range of observed battlefield blasts. Chondrocyte viability was assessed with live/dead staining using ethidium homodimer-2 and calcien acetoxymethylester stain and confocal laser scanning microscopy, calculated as a ratio of dead chondrocytes to live chondrocytes. Testing was performed at time points of 2, 4, and 8 hours after blast exposure and was compared with unblasted control samples. RESULTS: Chondrocyte viability decreased after exposure to a blast overpressure wave when compared with control samples. The amount of death was greater closer to the articular surface and dissipated with increasing tissue depth. Chondrocyte death increased with time after exposure. CONCLUSIONS: Chondrocyte death is present after exposure to a simulated blast wave. There is an inverse relationship between chondrocyte viability and the depth from the articular surface. Additional studies are needed to further characterize dose and time effects of blast exposure.

Porphyromonas gingivalis and its LPS differentially regulate the expression of peptidyl arginine deiminases in human chondrocytes.

Periodontitis, an inflammatory disease initiated by Gram-negative bacteria such as Porphyromonas gingivalis ( Pg), is considered as a risk factor for rheumatoid arthritis (RA). Our study aimed to determine the effect of Pg and its LPS on the expression of peptidyl arginine deiminase isotypes (PADs) in human primary chondrocytes (HC). HCs were infected with Pg and activated by its LPS (LPS- Pg). The mRNA expression levels of human PADs (1, 2, 3, 4 and 6) and bacterial enzyme (PADPg) were quantified by RT-qPCR. Cellular extracts served to measure the enzymatic activities of PADs and PADPg and to visualize the profiles of citrullinated proteins/peptides by Western blotting. Our data showed significant inhibitions of mRNA expressions of human PAD-2, PAD-3 and PAD-4 during infection of HC with live Pg. Activation of HC by LPS- Pg increased mRNA expressions of human PAD-2 and PAD-3. The PADPg enzymatic activity was significantly increased in only infected HC. Analysis of citrullinated proteins/peptides profiles revealed the occurrence of low molecular bands only in cellular extracts from HC infected with Pg. Our data showed that Pg and its LPS differentially regulate the expression of PADs in human chondrocytes and that Pg favors the apparition of new citrullinated proteins/peptides.

Mycoplasma and host interaction: In vitro gene expression modulation in Mycoplasma synoviae and infected chicken chondrocytes.

The complex interplay between Mycoplasma synoviae and chicken chondrocytes (CCH), which come into direct contact during infectious synovitis, has been examined at the level of gene expression. Our previous studies demonstrated a significant influence of M. synoviae on the level of CCH gene expression. Here, we show for the first time that in vitro co-cultivation of M. synoviae and CCH also induces upregulation of gene expression in this mycoplasma. We observed significantly increased expression of genes important for M. synoviae pathogenicity, including cysteine protease cysP, neuraminidase nanH, haemagglutinin vlhA, and the putative nuclease MS53_0284. Moreover, the pattern of gene expression was dependent on the infection environment. In CCH, significant changes in the expression of genes encoding catabolic enzymes of the cartilage extracellular matrix (cathepsins B, K and L, aggrecanase ADAM10, and matrix metalloproteinase MMP2) were demonstrated. Infection of CCH with M. synoviae also elevated the expression of the gene encoding peptidyl arginine deiminase, type III (PADI3), which is responsible for the post-translational citrullination of proteins.

IL-1ß mediating high mobility group box protein-1 expression in condylar chondrocyte during temporomandibular joint inflammation.

BACKGROUND: Temporomandibular joint (TMJ) osteoarthritis(OA)characterized with cartilage degen-eration is associated with inflammation. High mobility group box chromosomal protein-1(HMGB-1)is a potent mediator of inflammation and the trigger of OA. The expression of HMGB-1 in TMJ OA was uncovered, but the role of HMGB-1 in TMJ cartilage degeneration is not fully understood. In this study, the regulation of HMGB-1 in TMJ condylar cartilage was revealed. METHODS: A complete Freund's adjuvant (CFA)-induced TMJ inflammation animal model was employed and the expression of HMGB-1 was detected at 1st, 2nd, and 6th weeks by immunohistochemistry. TMJ condylar chondrocytes were incubated with IL-1ß (10 and 40 ng/ml) at 24, 48, and 72 h, and the translocation and protein level of HMGB-1 were evaluated by immunofluorescence and Western blot. RESULT: Nuclear HMGB-1 staining was predominantly located in chondrocytes of both the fibrosis and proliferative zones in healthy TMJ. 1st week and 2nd week after CFA injection, immunoreaction could be detected in the cytoplasms of HMGB-1-positive cells and cartilage matrix especially in hypertrophic zone. At 6th week after CFA injection, cartilage matrix expression was disappeared and the cytoplasm expression of HMGB-1 was very weak in hypertrophic zone. HMGB-1 was translocated from the nucleus to the cytoplasm at 48 h after incubated with IL-1ß (10 ng/ml and 40 ng/ml). The protein level of HMGB-1 was increased after stimulation and had a peak at 48 h. CONCLUSION: HMGB-1 might be associated with TMJ inflammation and OA. Insight into the role of HMGB-1 in TMJ inflammation is helpful to add the new knowledge into the pathogenesis of TMJ OA.

Phenotypic characterization of Mycoplasma synoviae induced changes in the metabolic and sensitivity profile of in vitro infected chicken chondrocytes.

In infectious synovitis caused by Mycoplasma synoviae chicken chondrocytes (CCH) may come into direct contact with these bacteria that are also capable of invading CCH in vitro. In this study, phenotype microarrays were used to evaluate the influence of Mycoplasma synoviae on the global metabolic activity of CCH. Therefore, CCH were cultured in the presence of 504 individual compounds, spotted in wells of 11 phenotype microarrays for eukaryotic cells, and exposed to Mycoplasma synoviae membranes or viable Mycoplasma synoviae. Metabolic activity and sensitivity of normal cells versus infected cells were evaluated. Metabolic profiles of CCH treated with viable Mycoplasma synoviae or its membranes were significantly different from those of CCH alone. CCH treated with Mycoplasma synoviae membranes were able to use 48 carbon/nitrogen sources not used by CCH alone. Treatment also influenced ion uptake in CCH and intensified the sensitivity to 13 hormones, 5 immune mediators, and 29 cytotoxic chemicals. CCH were even more sensitive to hormones/immune mediators when exposed to viable Mycoplasma synoviae. Our results indicate that exposure to Mycoplasma synoviae or its membranes induces a wide range of metabolic and sensitivity modifications in CCH that can contribute to pathological processes in the development of infectious synovitis.

Prediction of microbial infection of cultured cells using DNA microarray gene-expression profiles of host responses.

Infection by microorganisms may cause fatally erroneous interpretations in the biologic researches based on cell culture. The contamination by microorganism in the cell culture is quite frequent (5% to 35%). However, current approaches to identify the presence of contamination have many limitations such as high cost of time and labor, and difficulty in interpreting the result. In this paper, we propose a model to predict cell infection, using a microarray technique which gives an overview of the whole genome profile. By analysis of 62 microarray expression profiles under various experimental conditions altering cell type, source of infection and collection time, we discovered 5 marker genes, NM_005298, NM_016408, NM_014588, S76389, and NM_001853. In addition, we discovered two of these genes, S76389, and NM_001853, are involved in a Mycolplasma-specific infection process. We also suggest models to predict the source of infection, cell type or time after infection. We implemented a web based prediction tool in microarray data, named Prediction of Microbial Infection (http://www.snubi.org/software/PMI).

Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes.

The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans.

Results of the clinical donor case and quality system case workshops of the European Association of Tissue Banks annual meeting 2009.

The European Association of Tissue Banks (EATB) Donor Case Workshop and Quality System Case workshop are forums held within the program of the EATB Annual Congress. These workshops offer an opportunity to discuss and evaluate approaches taken to challenging situations, regarding donor selection and quality issues, and strengthen the professional tissue banking and regulatory networks across Europe. This report reflects some of the discussion at the congress workshops and also subsequent correspondence between the various individuals who submitted cases for discussion. The cases presented to the workshops demonstrate that the findings, their interpretation, deducted actions and preventive measures in tissue banks are not predictable. The varied responses and lack of consensus corroborate this and clearly indicate that operating procedures cannot comprehensively cover or prepare for all eventualities. For many of the issues raised there is a lack of information in the published literature. The workshops actively engage participants, representing a wide array of international expertise, in an informal, secure and enjoyable setting, which facilitates learning from peers and provides potential solutions to those submitting cases. By publishing a summary of the discussions, we hope to reach a wider audience and to stimulate individuals to undertake full literature reviews or research on some of the discussed subjects.

Induced apoptosis of chondrocytes by Porphyromonas gingivalis as a possible pathway for cartilage loss in rheumatoid arthritis.

The role of bacterial infections in the pathogenesis of rheumatoid arthritis (RA) has gained increasing interest. Patients with RA often exhibit periodontal disease, which is associated with pathogens like Porphyromonas gingivalis. The present study examines the direct effects of P. gingivalis on apoptosis of human chondrocytes (a feature of inflammatory joint diseases) as one can assume an interrelation of pathogenesis of RA and P. gingivalis infections. Primary chondrocytes were infected with P. gingivalis. Early apoptotic and dead cell analysis was performed using Annexin-V, 7AAD, and propidium iodide and examined by flow cytometry and fluorescence microscopy. Caspase activation and DNA fragmentation were determined by western blot analysis and TUNEL reaction. Flow cytometry and fluorescence microscopy demonstrated an increase of Annexin-V-positive early apoptotic chondrocytes after infection. Western blot showed upregulation of activated caspase-3 expression, and TUNEL reaction revealed considerable DNA fragmentation following infection. The data show that P. gingivalis promotes early and later stages of apoptosis of primary human chondrocytes, which might contribute to the joint damage seen in the pathogenesis of RA.

Mycoplasma synoviae invades non-phagocytic chicken cells in vitro.

Mycoplasma synoviae and Mycoplasma gallisepticum are major poultry pathogens, but their strains differ significantly in invasiveness and pathogenicity. Recent studies have demonstrated that M. gallisepticum invades chicken erythrocytes (CER) and chicken embryonic fibroblasts. The aim of this study was to determine whether M. synoviae also invades chicken cells. Using the gentamicin invasion assay, relative invasion frequency (RIF) of four M. synoviae strains was determined for CER, chicken embryonic cell line (CEC-32) and/or primary chicken chondrocytes (CCH). All tested strains of M. synoviae were capable of invading chicken cells within 24 h after infection. The type strain WVU 1853 showed significantly higher invasiveness in CER (RIF 7.5+/-1.5%) and CEC-32 (RIF 7.0+/-0.3%) than field strain ULB 02/T6 and M. gallisepticum strain R(low). Surprisingly, WVU 1853, which is capable of causing synovitis and arthritis in chickens, was less invasive for CCH with a RIF (1.2+/-0.3%) similar to that of R(low) (1.1+/-0.1%). This is the first study documenting the invasiveness of M. synoviae strains for non-phagocytic chicken cells.

Effects of Porphyromonas gingivalis on cell cycle progression and apoptosis of primary human chondrocytes.

BACKGROUND: It has been suggested that bacterial infections have a role in the pathogenesis of rheumatoid arthritis (RA). P gingivalis, a Gram-negative, anaerobic rod, is one of the major pathogens associated with periodontal disease. OBJECTIVE: To examine P gingivalis infection and its effects on cell cycle progression and apoptosis of human articular chondrocytes. METHODS: Primary human chondrocytes cultured in monolayers were challenged with P gingivalis. Infection and invasion of P gingivalis into chondrocytes was analysed by scanning electron microscopy, double immunofluorescence and by antibiotic protection and invasion assay. Cell cycle progression of infected chondrocytes was evaluated by flow cytometry. Also, cell apoptosis was visualised by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) of DNA strand breaks and by western blot analysis. RESULTS: Data showed that P gingivalis could adhere and infect primary human chondrocytes. After chondrocyte infection, intracellular localisation of P gingivalis was noted. Flow cytometry analyses demonstrated affected cell cycle progression, with an increase of the G(1) phase and a significant decrease of the G(2) phase after infection. In addition, increased apoptosis of P gingivalis-infected chondrocytes was visualised by TUNEL assay and by upregulation of caspase-3 protein expression. CONCLUSION: These data demonstrate that P gingivalis infects primary human chondrocytes and affects cellular responses, which might contribute to the tissue damage seen in the pathogenesis of rheumatoid arthritis.

Streptococcus pyogenes degrades extracellular matrix in chondrocytes via MMP-13.

Group A streptococcus (GAS) causes a wide range of human diseases, including bacterial arthritis. The pathogenesis of arthritis is characterized by synovial proliferation and the destruction of cartilage and subchondral bone in joints. We report here that GAS strain JRS4 invaded a chondrogenic cell line ATDC5 and induced the degradation of the extracellular matrix (ECM), whereas an isogenic mutant of JRS4 lacking a fibronectin-binding protein, SAM1, failed to invade the chondrocytes or degrade the ECM. Reverse transcription-PCR and Western blot analysis revealed that the expression of matrix metalloproteinase (MMP)-13 was strongly elevated during the infection with GAS. A reporter assay revealed that the activation of the AP-1 transcription factor and the phosphorylation of c-Jun terminal kinase participated in MMP-13 expression. These results suggest that MMP-13 plays an important role in the destruction of infected joints during the development of septic arthritis.

Borrelia burgdorferi BBB07 interaction with integrin alpha3beta1 stimulates production of pro-inflammatory mediators in primary human chondrocytes.

Borrelia burgdorferi, the causative agent of Lyme disease, activates multiple signalling pathways leading to induction of pro-inflammatory mediators at sites of inflammation. Binding of B. burgdorferi to integrin alpha(3)beta(1) on human chondrocytes activates signalling leading to release of several pro-inflammatory mediators, but the B. burgdorferi protein that binds integrin alpha(3)beta(1) and elicits this response has remained unknown. A search of the B. burgdorferi genome for a canonical integrin binding motif, the RGD (Arg-Gly-Asp) tripeptide, revealed several candidate ligands for integrins. In this study we show that one of these candidates, BBB07, binds to integrin alpha(3)beta(1) and inhibits attachment of intact B. burgdorferi to the same integrin. BBB07 is expressed during murine infection as demonstrated by recognition by infected mouse sera. Recombinant purified BBB07 induces pro-inflammatory mediators in primary human chondrocyte cells by interaction with integrin alpha(3)beta(1). This interaction is specific, as P66, another integrin ligand of B. burgdorferi, does not activate signalling through alpha(3)beta(1). In summary, we have identified a B. burgdorferi protein, BBB07, that interacts with integrin alpha(3)beta(1) and stimulates production of pro-inflammatory mediators in primary human chondrocyte cells.

Role of aggrecanase 1 in Lyme arthritis.

OBJECTIVE: Arthritis is one of the hallmarks of late-stage Lyme disease. Previous studies have shown that infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in degradation of proteoglycans and collagen in cartilage. B burgdorferi do not appear to produce any exported proteases capable of digesting proteoglycans and collagen, but instead, induce and activate host proteases, such as matrix metalloproteinases (MMPs), which results in cartilage degradation. The role of aggrecanases in Lyme arthritis has not yet been determined. We therefore sought to delineate the contribution of aggrecanases to joint destruction in Lyme arthritis. METHODS: We examined the expression patterns of aggrecanases 1 and 2 (ADAMTS 4 and 5, respectively) in B burgdorferi-infected primary human chondrocyte cell cultures, in synovial fluid samples from patients with active Lyme arthritis, and in the joints of mice by real-time quantitative reverse transcription-polymerase chain reaction and immunoblotting techniques. Bovine cartilage explants were used to determine the role of aggrecanases in B burgdorferi-induced cartilage degradation. RESULTS: ADAMTS-4, but not ADAMTS-5, was induced in human chondrocytes infected with B burgdorferi. The active forms of ADAMTS-4 were increased in synovial fluid samples from patients with active Lyme arthritis and were elevated in the joints of mice infected with B burgdorferi. Using cartilage explant models of Lyme arthritis, it appeared that the cleavage of aggrecan was predominantly mediated by "aggrecanases" rather than MMPs. CONCLUSION: The induction of ADAMTS-4 by B burgdorferi results in the cleavage of aggrecan, which may be an important first step that leads to permanent degradation of cartilage.

Identification of a TLR-independent pathway for Borrelia burgdorferi-induced expression of matrix metalloproteinases and inflammatory mediators through binding to integrin alpha 3 beta 1.

Borrelia burgdorferi stimulates a robust inflammatory response at sites of localization. Binding of borrelial lipoproteins to TLR-2 is one pathway important in the host response to B. burgdorferi. However, while TLR-2 is clearly important in control of infection, inflammation is actually worsened in the absence of TLR-2 or the shared TLR adapter molecule, MyD88, suggesting that there are alternative pathways regulating inflammation. Integrins are cell surface receptors that play an important role in cell to cell communications and that can activate inflammatory signaling pathways. In this study, we report for the first time that B. burgdorferi binds to integrin alpha(3)beta(1) and that binding of B. burgdorferi to this integrin results in induction of proinflammatory cytokines, chemokines, and end-effector molecules such as matrix metalloproteinases in primary human chondrocyte cells. Expression of these same molecules is not affected by the absence of MyD88 in murine articular cartilage, suggesting that the two pathways act independently in activating host inflammatory responses to B. burgdorferi. B. burgdorferi-induced alpha(3) signaling is mediated by JNK, but not p38 MAPK. In summary, we have identified a new host receptor for B. burgdorferi, integrin alpha(3)beta(1); binding of B. burgdorferi to integrin alpha(3)beta(1) results in the release of inflammatory mediators and is proposed as a TLR-independent pathway for activation of the innate immune response by the organism.

Expression and regulation of human beta-defensin-2 in osteoarthritic cartilage.

Defensins are antibiotic peptides that are involved in host defence at epithelial and mesenchymal surfaces. Previous studies have shown the induction of human beta-defensin-3 (HBD-3) in osteoarthritic joints, suggesting that these molecules have functions in addition to their ability to kill microbes. The aim of this study was to investigate the production of a further human beta-defensin, named HBD-2, in osteoarthritis (OA) and to determine its regulation by inflammatory cytokines. Healthy and osteoarthritic cartilage was assessed for HBD-2 expression by RT-PCR, immunohistochemistry, and ELISA. C28/I2 chondrocytes, primary chondrocytes, and cartilage explants were cultured for in vitro studies. After 24 h of stimulation with tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or IL-6, real-time RT-PCR and ELISA experiments were performed to evaluate the effect of these cytokines on the production of HBD-2. In contrast to healthy cartilage, HBD-2 expression was identified in most of the OA samples examined (eight of ten). Cytokines that are potentially involved in the pathogenesis of OA, namely TNF-alpha, IL-1, and IL-6, were transcriptional inducers of HBD-2 in cultured chondrocytes and cartilage explants in vitro, as measured by real-time RT-PCR and ELISA. These results illustrate the induction of HBD-2 in osteoarthritic cartilage and suggest that it is a further factor in the pathogenesis of OA. However, further studies are required to elucidate the role played by HBD-2 in osteoarthritic cartilage.