Expression of osteopontin messenger RNA and protein in rheumatoid arthritis: effects of osteopontin on the release of collagenase 1 from articular chondrocytes and synovial fibroblasts

OBJECTIVE: Osteopontin (OPN) is an extracellular matrix protein that has been implicated in the interactions between tumor cells and host matrix, including those involved in invasion and spread of tumor cells. Because joint destruction in rheumatoid arthritis (RA) is mediated by the invasive growth of synovial tissue through its attachment to cartilage, we examined the expression of OPN in the synovia of patients with RA and the effect of OPN on the production of collagenase 1 in rheumatoid synovial fibroblasts and articular chondrocytes. METHODS: The expression of OPN messenger RNA (mRNA) and protein in synovia from 10 RA patients was examined by in situ hybridization and immunohistochemistry. Synovial fibroblasts from RA patients and articular chondrocytes from patients without joint disease were cultured in the presence of various concentrations of OPN, and levels of collagenase 1 in the culture supernatants were measured by enzyme-linked immunosorbent assay. RESULTS: The expression of OPN mRNA and protein was observed in 9 of 10 specimens obtained from patients with RA. OPN was expressed in the synovial lining and sublining layer and at the interface of cartilage and invading synovium. Double labeling revealed that the majority of OPN-expressing cells were positive for the fibroblast-specific enzyme prolyl 4-hydroxylase and negative for the macrophage marker CD68, while only a few, single OPN-expressing cells were positive for CD68 at sites of synovial invasion into cartilage. OPN staining was not observed in lymphocytic infiltrates or leukocyte common antigen (CD45)-positive cells. Three of 3 cultures of human articular chondrocytes secreted detectable basal amounts of collagenase, with a dose-dependent increase upon OPN stimulation, while synovial fibroblast cultures produced much lower levels of collagenase, with only 2 of 4 fibroblast cultures responding in a dose-dependent manner. CONCLUSION: These findings suggest that OPN produced by synovial fibroblasts in the synovial lining layer and at sites of cartilage invasion not only mediates attachment of these cells to cartilage, but also contributes to matrix degradation in RA by stimulating the secretion of collagenase 1 in articular chondrocytes.     

Comparison of targeted next-generation sequencing and Sanger sequencing for the detection of <Emphasis Type="Italic">PIK3CA</Emphasis> mutations in breast cancer

Background

Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha, PIK3CA, is one of the most frequently mutated genes in breast cancer, and the mutation status of PIK3CA has clinical relevance related to response to therapy.The aim of our study was to investigate the mutation status of PIK3CA gene and to evaluate the concordance between NGS and SGS for the most important hotspot regions in exon 9 and 20, to investigate additional hotspots outside of these exons using NGS, and to correlate the PIK3CA mutation status with the clinicopathological characteristics of the cohort.

Methods

In the current study, next-generation sequencing (NGS) and Sanger Sequencing (SGS) was used for the mutational analysis of PIK3CA in 186 breast carcinomas.

Results

Altogether, 64 tumors had PIK3CA mutations, 55 of these mutations occurred in exons 9 and 20. Out of these 55 mutations, 52 could also be detected by Sanger sequencing resulting in a concordance of 98.4 % between the two sequencing methods. The three mutations missed by SGS had low variant frequencies below 10 %. Additionally, 4.8 % of the tumors had mutations in exons 1, 4, 7, and 13 of PIK3CA that were not detected by SGS. PIK3CA mutation status was significantly associated with hormone receptor-positivity, HER2-negativity, tumor grade, and lymph node involvement. However, there was no statistically significant association between the PIK3CA mutation status and overall survival.

Conclusions

Based on our study, NGS is recommended as follows: 1) for correctly assessing the mutation status of PIK3CA in breast cancer, especially for cases with low tumor content, 2) for the detection of subclonal mutations, and 3) for simultaneous mutation detection in multiple exons.

     

Dietary Interventions for Heart Failure in Older Adults: Re-Emergence of the Hedonic Shift

Dietary non-adherence to sodium restriction is an important contribution to heart failure (HF) symptom burden, particularly in older adults. While knowledge, skills, and attitudes toward sodium restriction are important, sodium intake is closely linked to the ability to taste salt. The ‘hedonic shift’ occurs when sodium restriction induces changes in an individual’s salt taste that lower subsequent salt affinity. Older adults often have compromised salt taste and higher dietary salt affinity due to age-related changes. Older HF patients may have additional loss of salt taste and elevated salt appetite due to comorbid conditions, medication use, and micronutrient or electrolyte abnormalities, creating a significant barrier to dietary adherence. Induction of the hedonic shift has the potential to improve long-term dietary sodium restriction and significantly impact HF outcomes in older adults.     

The impact of prior heart failure hospitalizations on long-term mortality differs by baseline risk of death

BackgroundHospitalizations for decompensated heart failure (HF) are thought to increase long-term mortality. However, previous reports focus on newly hospitalized HF patients or clinical trial populations and do not always adjust for baseline mortality risk. We hypothesized that the number of HF hospitalizations within the prior 12 months would improve overall mortality risk stratification, particularly in otherwise “low-risk” HF inpatients.MethodsWe studied 2221 HF patients admitted to 14 Michigan community hospitals during 2002-2004. We estimated 1-year mortality using the multivariable (Enhanced Feedback For Effective Cardiac Treatment [EFFECT]) model and classified patients as low (EFFECT <90), moderate (90-120), and high risk (>120). We used logistic regression and stratified Cox proportional hazard modeling to explore the overall EFFECT model performance and the influence of HF hospitalizations within the prior 12 months on mortality risk.ResultsThe EFFECT model adequately predicted and stratified for 1-year mortality (odds ratio 1.35 [95% confidence interval (CI), 1.30-1.40] per 10 points, P <.001, C-statistic 0.698), with low-, moderate-, and high-risk group mortality 18%, 35%, and 58%, respectively. The number of prior HF hospitalizations only modestly improved overall discrimination (C-statistic 0.704, P = .04). However, in low-risk patients the number of prior HF hospitalizations progressively increased the hazard for 1-year mortality (none: mortality 13%; 1: mortality 20%, hazard ratio [HR] 1.50 (95% CI, 0.86-2.60), P = .15; 2 or 3: mortality 27%, HR 2.24 (95% CI, 1.39-3.60); P = .001; 4 or more: mortality 31%, HR 2.80 (95% CI, 1.70-4.63); P <.001; P <.001 for trend). There was no consistent relationship between prior HF hospitalizations and 1-year mortality in moderate- or high-risk HF patients.ConclusionIn otherwise “low-risk” HF inpatients, a history of 2 or more HF hospitalizations within the prior 12 months markedly increases 1-year mortality risk. This easily obtained information could help allocate specialized HF resources to the subset of “low-risk” patients most likely to benefit.