Regulation of subchondral bone osteoblast metabolism by cyclic compression.

OBJECTIVE: Recent data have shown that abnormal subchondral bone remodeling plays an important role in osteoarthritis (OA) onset and progression, and it was suggested that abnormal mechanical pressure applied to the articulation was responsible for these metabolic changes. This study was undertaken to evaluate the effects of cyclic compression on osteoblasts from OA subchondral bone. METHODS: Osteoblasts were isolated from sclerotic and nonsclerotic areas of human OA subchondral bone. After 28 days, the osteoblasts were surrounded by an abundant extracellular matrix and formed a resistant membrane, which was submitted to cyclic compression (1 MPa at 1 Hz) for 4 hours. Gene expression was evaluated by reverse transcription-polymerase chain reaction. Protein production in culture supernatants was quantified by enzyme-linked immunosorbent assay or visualized by immunohistochemistry. RESULTS: Compression increased the expression of genes coding for interleukin-6 (IL-6), cyclooxygenase 2, RANKL, fibroblast growth factor 2, IL-8, matrix metalloproteinase 3 (MMP-3), MMP-9, and MMP-13 but reduced the expression of osteoprotegerin in osteoblasts in both sclerotic and nonsclerotic areas. Colα1(I) and MMP-2 were not significantly affected by mechanical stimuli. Nonsclerotic osteoblasts were significantly more sensitive to compression than sclerotic ones, but after compression, differences in messenger RNA levels between nonsclerotic and sclerotic osteoblasts were largely reduced or even abolished. Under basal conditions, sclerotic osteoblasts expressed similar levels of α5, αv, ß1, and ß3 integrins and CD44 as nonsclerotic osteoblasts but 30% less connexin 43, an important mechanoreceptor. CONCLUSION: Genes involved in subchondral bone sclerosis are mechanosensitive. After compression, nonsclerotic and sclerotic osteoblasts expressed a similar phenotype, suggesting that compression could be responsible for the phenotype changes in OA subchondral osteoblasts.

Curcuma longa and Boswellia serrata Extracts Modulate Different and Complementary Pathways on Human Chondrocytes In Vitro: Deciphering of a Transcriptomic Study.

Objectives: Curcuma longa (CL) and Boswellia serrata (BS) extracts are used to relieve osteoarthritis symptoms. The aim of this in vitro study was to investigate their mechanisms of action at therapeutic plasmatic concentrations on primary human osteoarthritic (OA) chondrocytes. Methods: BS (10-50 µg/ml) and CL (0.4-2 µg/ml corresponding to 1-5 µM of curcumin) were evaluated separately or in combination on primary chondrocytes isolated from 17 OA patients and cultured in alginate beads. Ten patients were used for RNA-sequencing analysis. Proteomic confirmation was performed either by immunoassays in the culture supernatant or by flow cytometry for cell surface markers after 72 h of treatment. Results: Significant gene expression modifications were already observed after 6 h of treatment at the highest dose of CL (2 µg/ml) while BS was significantly effective only after 24 h of treatment irrespective of the concentration tested. The most over-expressed genes by CL were anti-oxidative, detoxifying, and cytoprotective genes involved in the Nrf2 pathway. Down-regulated genes were principally pro-inflammatory cytokines and chemokines. Inversely, BS anti-oxidant/detoxifying activities were related to the activation of Nrf1 and PPARα pathways. BS anti-inflammatory effects were associated with the increase in GDF15, decrease in cholesterol cell intake and fatty acid metabolism-involved genes, and down-regulation of Toll-like receptors (TLRs) activation. Similar to CL, BS down-regulated ADAMTS1, 5, and MMP3, 13 genes expression. The combination of both CL and BS was significantly more effective than CL or BS alone on many genes such as IL-6, CCL2, ADAMTS1, and 5. Conclusion: BS and CL have anti-oxidative, anti-inflammatory, and anti-catabolic activities, suggesting a protective effect of these extracts on cartilage. Even if they share some mechanism of action, the two extracts act mainly on distinct pathways, and with different time courses, justifying their association to treat osteoarthritis.

Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus.

The lactic acid bacterium Streptococcus thermophilus is widely used for the manufacture of yogurt and cheese. This dairy species of major economic importance is phylogenetically close to pathogenic streptococci, raising the possibility that it has a potential for virulence. Here we report the genome sequences of two yogurt strains of S. thermophilus. We found a striking level of gene decay (10% pseudogenes) in both microorganisms. Many genes involved in carbon utilization are nonfunctional, in line with the paucity of carbon sources in milk. Notably, most streptococcal virulence-related genes that are not involved in basic cellular processes are either inactivated or absent in the dairy streptococcus. Adaptation to the constant milk environment appears to have resulted in the stabilization of the genome structure. We conclude that S. thermophilus has evolved mainly through loss-of-function events that remarkably mirror the environment of the dairy niche resulting in a severely diminished pathogenic potential.

Patients' Expectations Impact Their Satisfaction following Total Hip or Knee Arthroplasty.

INTRODUCTION: The objective of this study was to assess the number and magnitude of preoperative expectations and to correlate them with the degree of satisfaction expressed one year after Total Hip Arthroplasty (THA) or Total Knee Arthroplasty (TKA), in patients with severe and painful osteoarthritis (OA). MATERIALS AND METHODS: Preoperative expectations (within 20 days prior to surgery) and postoperative satisfaction (one year after the intervention) were measured using the previously validated French version of the Hospital for Special Surgery Hip or Knee Replacement Expectations Survey. Postoperative satisfaction was measured using a specific scale, following the same methodology as that used for the assessment of expectations. Prediction of the satisfaction of the patients was performed using multivariate linear regression modelling. RESULTS: A total of 138 patients (80 THA and 58 TKA) completed the two parts of the study. The expectations score (mean ± SD) (range 0-100) was 72.58 ± 12.63 before THA and 69.10 ± 13.72 before TKA (p = 0.13). The number of expectations expressed was 14.34 ± 1.32 (out of a potential maximum of 18) before THA and 14.70 ± 2.29 (out of a potential maximum of 19) before TKA. After 1 year, THA generated a significantly higher degree of satisfaction compared to TKA (69.70 ± 14.46 v 60.44 ± 17.54, p<0.001) (range 0-100). The pre-operative expectations score was the single best positive predictor of the post-surgery satisfaction assessment both for TKA and THA. CONCLUSION: Patients undergoing total joint arthroplasty for end-stage OA have a high level of expectations, before both THA and TKA. While both types of interventions significantly improve essential and non-essential activities, the rate of satisfaction is significantly greater post THA. Preoperative expectations are a major contributor to the final degree of satisfaction, one year after surgery. These results re-emphasize the need for an optimal preoperative interaction between health care providers and patients, to allow patients a chance to foresee a reasonable outcome after TJA.

Development and validation of the French version of a tool assessing patient's expectations in lower limb osteoarthritis.

OBJECTIVE: The Hospital for Special Surgery (HSS) Hip Replacement Expectations Survey and Knee Replacement Expectations Survey are validated tools developed to measure patients' preoperative expectations for hip and knee arthroplasty. These instruments have possible uses in both daily practice and research. Our objective was to assess the test-retest reliability and the construct validity of the French version of the surveys. METHODS: Patients scheduled for total hip (n = 82) or knee replacement (n = 61) aged 38-90 years were included. All completed the HSS Hip or Knee Replacement Expectations Survey and the Expectation WOMAC to determine concurrent validity. The test-retest reliability was assessed using the intraclass coefficient correlation (ICC), the Bland and Altman Method and the coefficient of variation; the internal consistency was assessed by the Cronbach α coefficient. The construct validity was investigated using the Pearson correlation coefficient and floor and ceiling effects by percentage frequency of lowest or highest possible score achieved by respondents. RESULTS: 143 patients scheduled for hip or knee arthroplasty were included. The reliability was excellent between the test and the rested total score, with an ICC of 0.902 (0.853-0.936) and CV of 4.06% for the French Hip Replacement Expectations Survey and 0.865 (0.786-0.917) and CV of 7.7% for the French Knee Replacement Expectations Survey, without bias. The Cronbach α coefficient was 0.72 for hip Survey and 0.82 for knee Survey showing a good internal consistency. Pearson correlation coefficients of 0.45 and 0.48 between Expectations WOMAC and HSS, respectively for hip Survey and knee Survey, were observed but with systematic bias. The lowest possible score was not reported by any patient and only three patients (3.66%) scheduled for hip arthroplasty reported the highest possible score. CONCLUSIONS: The French version of the HSS Hip or Knee Replacement Expectations Survey is a reliable and valid questionnaire and compares favourably with the original English version. Therefore, this new version may help French-speaking clinicians to evaluate expectations before lower limb arthroplasty.

Genome-based in silico detection of putative manganese transport systems in Lactobacillus plantarum and their genetic analysis.

Manganese serves an important function in Lactobacillus plantarum in protection against oxidative stress and this bacterium can accumulate Mn(2+) up to millimolar levels intracellularly. Although the physiological role of Mn(2+) and the uptake of this metal ion have been well documented, the only uptake system described so far for this bacterium is the Mn(2+)- and Cd(2+)-specific P-type ATPase (MntA). Recently, the genome of L. plantarum WCFS1 has been sequenced allowing in silico detection of genes potentially encoding Mn(2+) transport systems, using established microbial Mn(2+) transporters as the query sequence. This genome analysis revealed that L. plantarum WCFS1 encodes, besides the previously described mntA gene, an ABC transport system (mtsCBA) and three genes encoding Nramp transporters (mntH1, mntH2 and mntH3). The expression of three (mtsCBA, mntH1 and mntH2) of the five transport systems was specifically derepressed or induced upon Mn(2+) limitation, supporting their role in Mn(2+) homeostasis in L. plantarum. However, in contrast to previous reports, mntA expression remains below detection levels in both Northern and real-time RT-PCR analysis in both Mn(2+) excess and starvation conditions. Growth of WCFS1 derivatives mutated in mntA, mtsA or mntH2, or both mtsA and mntH2 appears unaffected under Mn(2+) excess or Mn(2+) limitation. Moreover, intracellular Mn(2+) concentrations remained unaltered in these mutants compared to the wild-type. This may suggest that this species is highly adaptive in response to inactivation of these genes or, alternatively, that other transporters that have not yet been identified as Mn(2+) transporters in bacteria are involved in Mn(2+) homeostasis in L. plantarum.

Autolysis of Lactococcus lactis is increased upon D-alanine depletion of peptidoglycan and lipoteichoic acids.

Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase (alr) mutant is strictly dependent on an external supply of D-Ala to be able to synthesize peptidoglycan and to incorporate D-Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when D-Ala is removed at mid-exponential growth. AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of D-Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the D-alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that D-alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking D-Ala has been reported in other bacterial species to have an improved capacity for autolysin binding. Mutation of dltD in L. lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, D-Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis. Second, reduced amounts of D-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.

Improved adaptation to cold-shock, stationary-phase, and freezing stresses in Lactobacillus plantarum overproducing cold-shock proteins.

We have investigated the effect of overproducing each of the three cold shock proteins (CspL, CspP, and CspC) in the mesophilic lactic acid bacterium Lactobacillus plantarum NC8. CspL overproduction transiently alleviated the reduction in growth rate triggered by exposing exponentially growing cells to cold shock (8 degrees C), suggesting that CspL is involved in cold adaptation. The strain overproducing CspC resumed growth more rapidly when stationary-phase cultures were diluted into fresh medium, indicating a role in the adaptation and recovery of nutritionally deprived cells. Overproduction of CspP led to an enhanced capacity to survive freezing.

Cold shock induction of the cspL gene in Lactobacillus plantarum involves transcriptional regulation.

Fragments of the cspL promoter region were fused to the gusA reporter and reintroduced into Lactobacillus plantarum cells, either on multicopy plasmids or through single-copy chromosomal integration. beta-Glucuronidase activity and primer extension data demonstrate that the cspL promoter is induced in response to cold shock and that multicopy constructs quench the induction of the resident cspL gene.

Enhanced mucosal delivery of antigen with cell wall mutants of lactic acid bacteria.

The potential of recombinant lactic acid bacteria (LAB) to deliver heterologous antigens to the immune system and to induce protective immunity has been best demonstrated by using the C subunit of tetanus toxin (TTFC) as a model antigen. Two types of LAB carriers have mainly been used, Lactobacillus plantarum and Lactococcus lactis, which differ substantially in their abilities to resist passage through the stomach and to persist in the mouse gastrointestinal tract. Here we analyzed the effect of a deficiency in alanine racemase, an enzyme that participates in cell wall synthesis, in each of these bacterial carriers. Recombinant wild-type and mutant strains of L. plantarum NCIMB8826 and L. lactis MG1363 producing TTFC intracellularly were constructed and used in mouse immunization experiments. Remarkably, we observed that the two cell wall mutant strains were far more immunogenic than their wild-type counterparts when the intragastric route was used. However, intestinal TTFC-specific immunoglobulin A was induced only after immunization with the recombinant L. plantarum mutant strain. Moreover, the alanine racemase mutant of either LAB strain allowed induction of a much stronger serum TTFC-specific immune response after immunization via the vagina, which is a quite different ecosystem than the gastrointestinal tract. The design and use of these mutants thus resulted in a major improvement in the mucosal delivery of antigens exhibiting vaccine properties.

Efficient secretion of the model antigen M6-gp41E in Lactobacillus plantarum NCIMB 8826.

Four Lactobacillus strains (Lb. plantarum NCIMB 8826, Lb. paracasei LbTGS1.4, Lb. casei ATCC 393 and Lb. fermentum KLD) were tested for their ability to produce and secrete heterologous proteins. These strains were first screened with an alpha-amylase reporter under the control of a set of expression or expression/secretion signals from various lactic acid bacteria. With most of the constructions tested, the level of extracellular production was highest in Lb. plantarum NCIMB 8826, and lowest in Lb. paracasei LbTGS1.4. These two strains were next assayed using a model antigen consisting of the N-terminal part of the M6 protein from Streptococcus pyogenes fused to the linear epitope ELDKWAS from human immunodeficiency virus gp41 protein. Secretion of this heterologous protein was inefficient in Lb. paracasei LbTGS1.4, which accumulated a large intracellular pool of the unprocessed precursor, whereas Lb. plantarum NCIMB 8826 was able to secrete the antigen to a level as high as 10 mg l-1.

Use of the alr gene as a food-grade selection marker in lactic acid bacteria.

Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.