TNFα modulates protein degradation pathways in rheumatoid arthritis synovial fibroblasts

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint. The synovial lining consists of two main types of cells: synovial fibroblasts and macrophages. The macrophage-derived cytokine TNFα stimulates RA synovial fibroblasts to proliferate and produce growth factors, chemokines, proteinases and adhesion molecules, making them key players in the RA disease process. If proteins are not correctly folded, cellular stress occurs that can be relieved in part by increased degradation of the aberrant proteins by the proteasome or autophagy. We hypothesized that the activity of the protein degradation pathways would be increased in response to TNFα stimulation in RA synovial fibroblasts compared with control fibroblasts.

Methods

Endoplasmic reticulum (ER) stress markers were examined in synovial fibroblasts by immunoblotting and PCR. Use of the autophagy and proteasome protein degradation pathways in response to TNFα stimulation was determined using a combination of experiments involving chemical inhibition of the autophagy or proteasome pathways followed by immunoblotting for the autophagy marker LC3, measurement of proteasome activity and long-lived protein degradation, and determination of cellular viability.

Results

RA synovial fibroblasts are under acute ER stress, and the stress is increased in the presence of TNFα. Autophagy is the main pathway used to relieve the ER stress in unstimulated fibroblasts, and both autophagy and the proteasome are more active in RA synovial fibroblasts compared with control fibroblasts. In response to TNFα, the autophagy pathway but not the proteasome is consistently stimulated, yet there is an increased dependence on the proteasome for cell viability. If autophagy is blocked in the presence of TNFα, an increase in proteasome activity occurs in RA synovial fibroblasts but not in control cells.

Conclusions

TNFα stimulation of synovial fibroblasts results in increased expression of ER stress markers. Survival of synovial fibroblasts is dependent on continuous removal of proteins by both the lysosome/autophagy and ubiquitin/proteasome protein degradation pathways. Both pathways are more active in RA synovial fibroblasts compared with control fibroblasts. These results may provide a better understanding of the mechanism of TNFα on prolonging the survival of synovial fibroblasts in RA tissue.     

Evaluation of Xpert? MTB/RIF Assay in Induced Sputum and Gastric Lavage Samples from Young Children with Suspected Tuberculosis from the MVA85A TB Vaccine Trial

Objective

Diagnosis of childhood tuberculosis is limited by the paucibacillary respiratory samples obtained from young children with pulmonary disease. We aimed to compare accuracy of the Xpert^® MTB/RIF assay, an automated nucleic acid amplification test, between induced sputum and gastric lavage samples from young children in a tuberculosis endemic setting.

Methods

We analyzed standardized diagnostic data from HIV negative children younger than four years of age who were investigated for tuberculosis disease near Cape Town, South Africa [2009–2012]. Two paired, consecutive induced sputa and early morning gastric lavage samples were obtained from children with suspected tuberculosis. Samples underwent Mycobacterial Growth Indicator Tube [MGIT] culture and Xpert MTB/RIF assay. We compared diagnostic yield across samples using the two-sample test of proportions and McNemar’s χ² test; and Wilson’s score method to calculate sensitivity and specificity.

Results

1,020 children were evaluated for tuberculosis during 1,214 admission episodes. Not all children had 4 samples collected. 57 of 4,463[1.3%] and 26 of 4,606[0.6%] samples tested positive for Mycobacterium tuberculosis on MGIT culture and Xpert MTB/RIF assay respectively. 27 of 2,198[1.2%] and 40 of 2,183[1.8%] samples tested positive [on either Xpert MTB/RIF assay or MGIT culture] on induced sputum and gastric lavage samples, respectively. 19/1,028[1.8%] and 33/1,017[3.2%] admission episodes yielded a positive MGIT culture or Xpert MTB/RIF assay from induced sputum and gastric lavage, respectively. Sensitivity of Xpert MTB/RIF assay was 8/30[26.7%; 95% CI: 14.2–44.4] for two induced sputum samples and 7/31[22.6%; 11.4–39.8] [p = 0.711] for two gastric lavage samples. Corresponding specificity was 893/893[100%;99.6–100] and 885/890[99.4%;98.7–99.8] respectively [p = 0.025].

Conclusion

Sensitivity of Xpert MTB/RIF assay was low, compared to MGIT culture, but diagnostic performance of Xpert MTB/RIF did not differ sufficiently between induced sputum and gastric lavage to justify selection of one sampling method over the other, in young children with suspected pulmonary TB.

Trial Registration

ClinicalTrials.gov NCT00953927     

Partial physical and functional map of aMycobacterium aurum carotenogenesis operon

The genes controlling the biosynthesis of the carotenes inMycobacterium aurum were clustered in a 10.83-kb segment. Fragments generated by endonuclease digestions of the segment were cloned into a pHLD69 shuttle vector. The plasmids so constructed were used to transform a colorless (albino)M. aurum mutant (strain A₁₁), a brick-red mutant accumulating large amounts of lycopene (strain NgR9), the buff-coloredMycobacterium smegmatis MC²-155, and the buffcoloredMycobacterium tuberculosis H37Ra. From the endonuclease digestion patterns and the phenotypes of the transformed strains, the partial physical and functional maps of a carotenogenesis operon were established. This investigation also showed that the genes controlling the conversion of lycopene into the xanthophylls were not located in the 10.83-kb segment.     

Empirically derived maximal acceptable wait time for surgery to treat adolescent idiopathic scoliosis

Background

Prioritizing patients using empirically derived access targets can help to ensure high-quality care. Adolescent scoliosis can worsen while patients wait for treatment, increasing the risk of adverse events. Our objective was to determine an empirically derived access target for scoliosis surgery and to compare this with consensus-based targets

Methods

Two-hundred sixteen sequential patients receiving surgery for adolescent idiopathic scoliosis were included in the study. The main outcome was need for additional surgery. Logistic regression modeling was used to evaluate the relation between surgical wait times and adverse events and χ² analysis was used as the primary analysis for the main outcome.

Results

Of the 88 patients who waited longer than six months for surgery, 13 (14.8%) needed additional surgery due to progression of curvature versus 1.6% (2 of 128 patients) who waited less than six months for surgery (χ² analysis, p = 0.0001). Patients who waited longer than six months for surgery had greater progression of curvature, longer surgeries and longer stays in hospital. These patients also had less surgical correction than patients who waited less than six months for surgery (Wilcoxon–Mann–Whitney test, p = 0.011). All patients requiring additional surgeries waited longer than three months for their initial surgery. A receiver–operator characteristic curve also suggested a three-month wait as an access target. The adjusted odds ratio for an adverse event for each additional 90 days of waiting from time of consent was 1.81 (95% confidence interval 1.34–2.44). The adjusted odds ratio increased with skeletal immaturity and with the size of the spinal curvature at the time of consent.

Interpretation

A prolonged wait for surgery increased the risk of additional surgical procedures and other adverse events. An empirically derived access target of three months for surgery to treat adolescent idiopathic scoliosis could potentially eliminate the need for additional surgery by reducing progression of curvature. This is a shorter access target than the six months determined by expert consensus.Adolescent idiopathic scoliosis effects just over 2% of females aged 12–14 years.^1–3 Although only 10% of patients require surgery, spinal instrumentation and fusion for adolescent idiopathic scoliosis is the most common procedure done in pediatric orthopaedics.⁴ Patients who wait too long for scoliosis surgery may require additional surgery such as anterior release to achieve satisfactory correction of the spinal curvature. These patients may also need longer surgeries and may be at increased risk of complications such as increased blood loss, neurologic deficits or inadequate correction of the curvature.^5–14 Furthermore, as seen in other studies of wait times, patients and families can feel anxiety and prolonged suffering while waiting for treatment, which can negatively impact the quality of care.^15–19 Programs such as the Canadian Pediatric Surgical Wait Times Project have determined a maximal acceptable wait time for adolescent scoliosis through expert consensus (similar to how other surgical wait time targets have been determined).²⁰ Surprisingly, there has been little or no attention given to developing evidence-based access targets or maximal acceptable wait times for most treatments.²¹ The purpose of this study was to determine the maximal acceptable wait time for surgical correction of adolescent idiopathic scoliosis using an empirically based approach to minimize the possibility of adverse events related to progression of curvature.     

Rifampicin Mono-Resistance in Mycobacterium tuberculosis in KwaZulu-Natal,South Africa: A Significant Phenomenon in a High Prevalence TB-HIV Region

Setting

The dual epidemics of HIV-TB including MDR-TB are major contributors to high morbidity and mortality rates in South Africa. Rifampicin (RIF) resistance is regarded as a proxy for MDR-TB. Currently available molecular assays have the advantage of rapidly detecting resistant strains of MTB, but the GeneXpert does not detect isoniazid (INH) resistance and the GenoTypeMTBDRplus(LPA) assay may underestimate resistance to INH. Increasing proportions of rifampicin mono-resistance resistance (RMR) have recently been reported from South Africa and other countries.

Objective

This laboratory based study was conducted at NHLS TB Laboratory, Durban, which is the reference laboratory for culture and susceptibility testing in KwaZulu-Natal. We retrospectively determined, for the period 2007 to 2009, the proportion of RMR amongst Mycobacterium tuberculosis (MTB) isolates, that were tested for both RIF and INH, using the gold standard of culture based phenotypic drug susceptibility testing (DST). Gender and age were also analysed to identify possible risk factors for RMR.

Design

MTB culture positive sputum samples from 16,748 patients were analysed for susceptibility to RIF and INH during the period 2007 to 2009. RMR was defined as MTB resistant to RIF and susceptible to INH. For the purposes of this study, only the first specimen from each patient was included in the analysis.

Results

RMR was observed throughout the study period. The proportion of RMR varied from a low of 7.3% to a high of 10.0% [overall 8.8%]. Overall, males had a 42% increased odds of being RMR as compared to females. In comparison to the 50 plus age group, RMR was 37% more likely to occur in the 25–29 year age category.

Conclusion

We report higher proportions of RMR ranging from 7.3% to 10% [overall 8.8%] than previously reported in the literature. To avoid misclassification of RMR, detected by the GeneXpert, as MDR-TB, culture based phenotypic DST must be performed on a second specimen, as recommended by the SA NDOH TB guidelines as well as WHO. We suggest that two sputum samples should be obtained at the first visit. The second sputum sample should be stored at 4°C. The latter sample is then readily available for performing additional DST (phenotypic or genotypic) for 2nd lines drugs, resulting in a decreased waiting period for DST results to become available.     

Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load

High Ag load in chronic viral infections has been associated with impairment of Ag-specific T cell responses; however, the relationship between Ag load in chronic Mycobacterium tuberculosis infection and functional capacity of M. tuberculosis-specific T cells in humans is not clear. We compared M. tuberculosis-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads-that is, persons with latent M. tuberculosis infection (LTBI), with smear-negative pulmonary tuberculosis (TB), and smear-positive TB. Patients with smear-positive TB had decreased polyfunctional IFN-γ(+)IL-2(+)TNF-α(+) and IL-2-producing specific CD4 T cells and increased TNF-α single-positive cells, when compared with smear-negative TB and LTBI. TB patients also had increased frequencies of M. tuberculosis-specific CD8 T cells, compared with LTBI. M. tuberculosis-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear-positive TB, and correlated positively with ex vivo IFN-γ(+)IL-2(+)TNF-α(+) CD4 T cells, and inversely with TNF-α single-positive CD4 T cells. During 6 mo of anti-TB treatment, specific IFN-γ(+)IL-2(+)TNF-α(+) CD4 and CD8 T cells increased, whereas TNF-α and IFN-γ single-positive T cells decreased. These results suggest progressive impairment of M. tuberculosis-specific T cell responses with increasing mycobacterial load and recovery of responses during therapy. Furthermore, these data provide a link between specific cytokine-producing subsets and functional capacity of M. tuberculosis-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. These data have potentially significant applications for the diagnosis of TB and for the identification of T cell correlates of TB disease progression.     

Friction in metal-on-metal total disc arthroplasty: effect of ball radius

Total disc arthroplasty (TDA) can be used to replace a degenerated intervertebral disc in the spine. There are different designs of prosthetic discs, but one of the most common is a ball-and-socket combination. Contact between the bearing surfaces can result in high frictional torque, which can then result in wear and implant loosening. This study was designed to determine the effects of ball radius on friction. Generic models of metal-on-metal TDA were manufactured with ball radii of 10, 12, 14 and 16 mm, with a radial clearance of 0.015 mm. A simulator was used to test each sample in flexion-extension, lateral bending and axial rotation at frequencies of 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75 and 2 Hz under loads of 50, 600, 1200 and 2000 N, in new born calf serum. Frictional torque was measured and Stribeck curves were plotted to illustrate the lubrication regime in each case. It was observed that implants with a smaller ball radius showed lower friction and showed boundary and mixed lubrication regimes, whereas implants with larger ball radius showed boundary lubrication only. This study suggests designing metal-on-metal TDAs with ball radius of 10 or 12 mm, in order to reduce wear and implant loosening.     

General practice and pandemic influenza: a framework for planning and comparison of plans in five countries

Background

Although primary health care, and in particular, general practice will be at the frontline in the response to pandemic influenza, there are no frameworks to guide systematic planning for this task or to appraise available plans for their relevance to general practice. We aimed to develop a framework that will facilitate planning for general practice, and used it to appraise pandemic plans from Australia, England, USA, New Zealand and Canada.

Methodology/Principal Findings

We adapted the Haddon matrix to develop the framework, populating its cells through a multi-method study that incorporated the peer-reviewed and grey literature, interviews with general practitioners, practice nurses and senior decision-makers, and desktop simulation exercises. We used the framework to analyse 89 publicly-available jurisdictional plans at similar managerial levels in the five countries. The framework identifies four functional domains: clinical care for influenza and other needs, public health responsibilities, the internal environment and the macro-environment of general practice. No plan addressed all four domains. Most plans either ignored or were sketchy about non-influenza clinical needs, and about the contribution of general practice to public health beyond surveillance. Collaborations between general practices were addressed in few plans, and inter-relationships with the broader health system, even less frequently.

Conclusions

This is the first study to provide a framework to guide general practice planning for pandemic influenza. The framework helped identify critical shortcomings in available plans. Engaging general practice effectively in planning is challenging, particularly where governance structures for primary health care are weak. We identify implications for practice and for research.