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     

Additive Contribution of HLA Class I Alleles in the Immune Control of HIV-1 Infection

Previous studies have identified a central role for HLA-B alleles in influencing control of HIV infection. An alternative possibility is that a small number of HLA-B alleles may have a very strong impact on HIV disease outcome, dominating the contribution of other HLA alleles. Here, we find that even following the exclusion of subjects expressing any of the HLA-B class I alleles (B*57, B*58, and B*18) identified to have the strongest influence on control, the dominant impact of HLA-B alleles on virus set point and absolute CD4 count variation remains significant. However, we also find that the influence of HLA on HIV control in this C-clade-infected cohort from South Africa extends beyond HLA-B as HLA-Cw type remains a significant predictor of virus and CD4 count following exclusion of the strongest HLA-B associations. Furthermore, there is evidence of interdependent protective effects of the HLA-Cw*0401-B*8101, HLA-Cw*1203-B*3910, and HLA-A*7401-B*5703 haplotypes that cannot be explained solely by linkage to a protective HLA-B allele. Analysis of individuals expressing both protective and detrimental alleles shows that even the strongest HLA alleles appear to have an additive rather than dominant effect on HIV control at the individual level. Finally, weak but significant frequency-dependent effects in this cohort can be detected only by looking at an individual''s combined HLA allele frequencies. Taken together, these data suggest that although individual HLA alleles, particularly HLA-B, can have a strong impact, HIV control overall is likely to be influenced by the additive effect of some or all of the other HLA alleles present.HIV-specific CD8⁺ T cells play a central role in resolution of primary viremia and the long-term suppression of viral replication (13). Supporting this notion is the observed correlation between possession of particular human leukocyte antigen (HLA) class I alleles and control of HIV, measured both directly by time-to-AIDS (5, 6) and indirectly via clinical markers of disease progression (viral load [VL] and CD4 count) (15, 26, 28). Specific HLA class I alleles have been associated with relatively successful control of viral replication and slow disease progression, most notably, alleles HLA-B*57 and HLA-B*27 (1, 7, 12, 15, 21, 23), and also with relatively ineffective control of viral replication and rapid disease progression [B*35(Px), B*5802, and B*18] (5, 15, 17, 23). In addition, general trends suggesting an HLA class I heterozygote advantage (5) and rare allele advantage (28) and, most recently, a correlation between levels of surface expression linked to certain HLA-Cw alleles (11, 27) and HIV control has also been described.Among the different HLA class I loci, the HIV-specific CD8⁺ T-cell responses restricted by HLA-B alleles are thought to play the central role in determining disease outcome: the majority of detectable HIV-specific CD8⁺ T-cell responses are restricted by HLA-B alleles (3, 15, 16), HLA-B-restricted responses typically express a more effective “polyfunctional” phenotype (14), the strongest HLA-associations with either slow or rapid progression are with HLA-B alleles (5, 10, 11, 15), and HLA-B-restricted CD8⁺ T cells exert the strongest selection pressure on the virus (15, 19, 24). However, whether this apparent association between HIV immune control and HLA-B is a general and causal trend or, rather, is biased by the coincidence that the strongest HLA associations with either extreme of disease control happen, by chance, to involve HLA-B alleles remains uncertain.In order to further investigate the correlation between HLA type and HIV infection control, we here examine a cohort now comprising >1,200 chronically HIV C-clade-infected, treatment-naïve subjects from Durban, South Africa, in an extended analysis following from our previous studies of a smaller cohort (15). We first address the question of whether the dominant role of HLA-B in this population compared to the roles of HLA-A or HLA-C results from the influence of HLA-B alleles in general or is dependent on a few known strong associations, such as that between HLA-B*57 alleles and low viremia. Second, in light of recent data (11, 27), we assess the impact of HLA-C alleles on HIV disease outcome and examine the effect of HLA haplotypes on observed HLA associations with disease control. Third, we investigate the question of whether the impact of certain HLA-B alleles on HIV outcome dominates that of other HLA-B alleles to negate the contribution of the latter or whether the impact of individual HLA alleles can be additive. Finally, we compare the impact of individual HLA alleles on HIV on immune control to the impact of heterozygote and rare allele advantage in this cohort.     

Electroencephalogram recordings from the olfactory bulb of juvenile (0 year) Atlantic cod in response to amino acids

Olfactory sensitivity of juvenile (0 year) Atlantic cod Gadus morhua to 20 L‐amino acids was studied by recording electroencephalograms (EEG) from the olfactory bulb. Leucine, methionine, asparagine, glutamine, alanine and threonine were highly stimulatory; proline, phenylalanine, aspartic acid and tryptophan were the least stimulatory. Threshold concentrations determined for four amino acids were 10⁻⁸ M for alanine, 10⁻⁷ M for arginine and leucine and 10⁻⁶ M for glutamic acid.