Objective The non resolving character of synovial inflammation in rheumatoid arthritis (RA) is a conundrum. To identify the contribution of fibroblast-like synoviocytes (FLS) to the perpetuation of synovitis, we investigated the molecular mechanisms that govern the TNFα-driven inflammatory program in human FLS. Methods FLS obtained from synovial tissues of patients with RA or osteoarthritis were stimulated with TNFα and assayed for gene expression and cytokine production by qPCR and ELISA. NF-κB signaling was evaluated using Western blotting. Histone acetylation, chromatin accessibility, and NF-κB p65 and RNA polymerase II (Pol II) occupancy at the IL6 promoter were measured by chromatin immunoprecipitation and restriction enzyme accessibility assays. Results In FLS, TNFα induced prolonged transcription of IL6 and progressive accumulation of IL-6 protein over four days. Similarly, induction of CXCL8/IL-8, CCL5/RANTES, MMP1 and MMP3 mRNA after TNFα stimulation was sustained for several days. This contrasted with the macrophage response to TNFα, which characteristically involved a transient increase in the expression of pro-inflammatory genes. In FLS, TNFα induced prolonged activation of NF-κB signaling and sustained transcriptional activity indicated by increased histone acetylation, chromatin accessibility, and p65 and Pol II occupancy at the IL6 promoter. Furthermore, FLS expressed low levels of the feedback inhibitors ABIN3, IRAK-M, SOCS3 and ATF3 that terminate inflammatory responses in macrophages. Conclusions TNFα signaling is not effectively terminated in FLS, leading to an uncontrolled inflammatory response. The results suggest that prolonged and sustained inflammatory responses by FLS, in response to synovial TNFα, contribute to the persistence of synovial inflammation in RA.

BACKGROUND AND OBJECTIVES. Mitochondrial DNA (mtDNA) mutations, inherited and somatically acquired, are common in clinical prostate cancer. We have developed model systems designed to study specific mtDNA mutations in controlled experiments. Because prostate cancer frequently metastasizes to bone we tested the hypothesis that mtDNA mutations enhance prostate cancer growth and survival in the bone microenvironment. METHODS. The pathogenic nucleotide position (np) 8993 mDNA mutation was introduced into PC3 prostate cancer cells by cybrid formation. Wild-type and mutant cybrids were grown as nude mouse subcutaneous xenografts with or without bone stromal cell co-inoculation. Cybrids were also grown in the intratibial space. Tumor growth was assayed by direct tumor measurement and luciferase chemiluminescence. Gene expression was assayed using cDNA microarrays confirmed by real time PCR, western blot analysis and immunohistochemistry. RESULTS. Cybrids with the 8,993 mtDNA mutation grew faster than wild-type cybrids. Further growth acceleration was demonstrated in the bone microenvironment. A 37 gene molecular signature characterized the growth advantage conferred by the mtDNA mutation and bone microenvironment. Two genes of known importance in clinical prostate cancer, FGF1 and FAK, were found to be substantially upregulated only when both mtDNA mutation and bone stromal cell were present. CONCLUSIONS. The ATP6 np 8,993 mtDNA mutation confers a growth advantage to human prostate cancer that is most fully manifest in the bone microenvironment. The identification of specific molecular alterations associated with mtDNA mutation and growth in bone may allow new understanding of prostate cancer bone metastasis.

Objectives This study aimed to [1] confirm that nonobese adolescents with polycystic ovary syndrome (PCOS) have higher anti-Mullerian hormone (AMH) than controls; [2] examine the relationship of AMH with PCOS features and hormonal profile; and [3] approximate an AMH value that discriminates between adolescents with PCOS and controls. Design Case-control study. Setting Subspecialty ambulatory clinic. Patients Thirty-one nonobese adolescent girls (age 13–21 years), 15 with PCOS diagnosed using the National Institutes of Health (NIH) criteria and 16 healthy control subjects. Subjects and controls were comparable for body mass index z-score, age and ethnicity. Main outcome measure(s) AMH in PCOS subjects and control groups, correlation of AMH with hormonal parameters. Results AMH was higher in PCOS subjects (4.4 ±3.4 ng/mL) than in controls (2.4 ±1.3 ng/mL), when adjusted for menstrual age. In the entire group (PCOS and controls), AMH correlated with androgens, ovarian size and the presence of polycystic ovary (PCO) appearance. There was no difference in average ovarian size between PCOS (7.1 ±2.6 cm3) and controls (6.7 ±1.8 cm3). PCOS subjects were 1.49 times more likely to have AMH >3.4 ng/mL (confidence interval 0.98–2.26 ng/mL). Conclusions Our data suggest that AMH may be a useful adjunct in the diagnosis of PCOS in adolescents.

Objective Type I IFNs have emerged as potential activators of the IFN signature and elevated STAT1 expression in RA synovium, but mechanisms that induce synovial IFN expression are unknown. Recently, TNFα was shown to induce a delayed IFN response in macrophages. Thus, we tested whether TNFα, classically thought to activate inflammatory NF-κB target genes in RA, also contributes to the ‘IFN signature’ in RA synovial macrophages. Methods Synovial fluid macrophages purified from patients with rheumatoid arthritis (n=24) and spondyloarthopathies (SpA) (n=18) were lysed immediately after isolation or cultured ex vivo in the absence or presence of blockade of endogenous type I IFN or TNFα. Expression of IFN-inducible target genes was measured by qPCR and ELISA. Results Expression of an IFN signature and STAT1 in RA synovial macrophages was suppressed when type I IFNs or TNFα were blocked, whereas TNFα blockade did not affect expression of IFN response genes or STAT1 in SpA synovial macrophages. RA synovial fluid suppressed the IFN signature in RA synovial macrophages, and in TNFα-, IFNα- and IFNβ-stimulated control macrophages. Type I IFNs suppressed expression of IL-8 and MMP9 in RA synovial macrophages and in TNFα-stimulated control macrophages. Conclusions Our findings identify a new function for TNFα in RA synovitis by implicating TNFα as a major inducer of the RA synovial IFN response. The results suggest that the expression of IFN response genes in RA synovium is regulated by interplay between TNFα and opposing homeostatic factors expressed in the synovial microenvironment.

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