A Precision Medicine Approach Uncovers a Unique Signature of Neutrophils in Patients With Brushite Kidney Stones.

INTRODUCTION: We have previously found that papillary histopathology differs greatly between calcium oxalate and brushite stone formers (SF); the latter have much more papillary mineral deposition, tubular cell injury, and tissue fibrosis. METHODS: In this study, we applied unbiased orthogonal omics approaches on biopsied renal papillae and extracted stones from patients with brushite or calcium oxalate (CaOx) stones. Our goal was to discover stone type-specific molecular signatures to advance our understanding of the underlying pathogenesis. RESULTS: Brushite SF did not differ from CaOx SF with respect to metabolic risk factors for stones but did exhibit increased tubule plugging in their papillae. Brushite SF had upregulation of inflammatory pathways in papillary tissue and increased neutrophil markers in stone matrix compared with those with CaOx stones. Large-scale 3-dimensional tissue cytometry on renal papillary biopsies showed an increase in the number and density of neutrophils in the papillae of patients with brushite versus CaOx, thereby linking the observed inflammatory signatures to the neutrophils in the tissue. To explain how neutrophil proteins appear in the stone matrix, we measured neutrophil extracellular trap (NET) formation-NETosis-and found it significantly increased in the papillae of patients with brushite stones compared with CaOx stones. CONCLUSION: We show that increased neutrophil infiltration and NETosis is an unrecognized factor that differentiates brushite and CaOx SF and may explain the markedly increased scarring and inflammation seen in the papillae of patients with brushite stones. Given the increasing prevalence of brushite stones, the role of neutrophil activation in brushite stone formation requires further study.

Deep sequencing and analyses of miRNAs, isomiRs and miRNA induced silencing complex (miRISC)-associated miRNome in primary human chondrocytes.

MicroRNAs, a group of small, noncoding RNAs that post-transcriptionally regulate gene expression, play important roles in chondrocyte function and in the development of osteoarthritis. We characterized the dynamic repertoire of the chondrocyte miRNome and miRISC-associated miRNome by deep sequencing analysis of primary human chondrocytes. IL-1ß treatment showed a modest effect on the expression profile of miRNAs in normal and osteoarthritis (OA) chondrocytes. We found a number of miRNAs that showed a wide range of sequence modifications including nucleotide additions and deletions at 5' and 3' ends; and nucleotide substitutions. miR-27b-3p showed the highest expression and miR-140-3p showed the highest number of sequence variations. AGO2 RIP-Seq analysis revealed the differential recruitment of a subset of expressed miRNAs and isoforms of miRNAs (isomiRs) to the miRISC in response to IL-1ß, including miR-146a-5p, miR-155-5p and miR-27b-3p. Together, these results reveal a complex repertoire of miRNAs and isomiRs in primary human chondrocytes. Here, we also show the changes in miRNA composition of the miRISC in primary human chondrocytes in response to IL-1ß treatment. These findings will provide an insight to the miRNA-mediated control of gene expression in the pathogenesis of OA.

An effective and efficient method of transfecting primary human chondrocytes in suspension.

Human chondrocytes accumulate an ECM-rich matrix by secreting matrix macromolecules during monolayer culture, which makes them difficult to transfect efficiently. Here we report a non-viral based protocol to transfect the primary human chondrocytes with high efficiency in suspension. Chondrocyte cultures were digested using Pronase and Collagenase and transfected in suspension. Transfection efficiencies of more than 80% were achieved routinely using the protocol described. The viability of siRNA transfected or un-transfected chondrocytes was not affected and resulted in 80-90% knockdown of the target mRNA levels. This protocol may be useful in gene knockdown, and ectopic overexpression studies in chondrocytes.

Histone Deacetylase Inhibitor Vorinostat (SAHA) Suppresses IL-1ß-Induced Matrix Metallopeptidase-13 Expression by Inhibiting IL-6 in Osteoarthritis Chondrocyte.

Osteoarthritis (OA) is the most common whole-joint disease and is characterized by progressive loss of the cartilage matrix. Matrix metallopeptidase-13 (MMP-13) is a highly active and an abundantly expressed protease in OA cartilage and chondrocytes and degrades type II collagen and proteoglycans. We investigated the mechanism of MMP-13 suppression by histone deacetylase inhibitor vorinostat (SAHA). OA chondrocytes were obtained from knee cartilage after enzymatic digestion and treated with IL-1ß in the absence or presence of various histone deacetylase inhibitors. Gene expression was quantified using quantitative RT-PCR. Protein expression and chromatin modifications were determined by Western immunoblotting using specific antibodies. The effect of IL-6 on the expression of MMP-13 was determined by treating chondrocytes with recombinant IL-6 or by IL6 knockdown using IL6-specific siRNA. We found that SAHA is a potent suppressor of IL-1ß-induced MMP-13, tumor necrosis factor-α, and other catabolic marker expression in OA chondrocytes. Interestingly, SAHA rescued the COL2A1 and ACAN expression in OA chondrocytes that was down-regulated by IL-1ß. Of importance is our finding that IL-6-stimulated MMP-13 expression was independent of IL-1ß stimulation and was blocked by SAHA, suggesting that SAHA inhibits IL-6 signaling in OA chondrocytes. Taken together, our results suggest that SAHA could be used as a therapeutic agent for the management of OA.

miR-139 modulates MCPIP1/IL-6 expression and induces apoptosis in human OA chondrocytes.

IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3' untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 'seed sequence', therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1ß in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the 'seed sequence' located in the 3' UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes IL-6 expression in OA. Therefore, targeting miR-139 could be therapeutically beneficial in the management of OA.

Overexpression of ß-catenin and cyclinD1 predicts a poor prognosis in ovarian serous carcinomas.

UNLABELLED: Ovarian serous cancer is the most common subtype of epithelial ovarian cancer, and is the leading cause of death from gynecologic cancer. There is an important need for exploration of diagnostic and prognostic markers for this disease. ß-catenin and cyclinD1 play central roles in the tumorigenesis for certain cancers. The role of ß-catenin and cyclinD1 in diagnosis and prognosis of ovarian serous carcinoma is uncertain. In the present study, the expression of ß-catenin and cyclinD1 was examined in 60 ovarian serous carcinomas patients with immunohistochemical staining. The relationship between expression of ß-catenin and cyclinD1 and FIGO stage, pathological grade was analyzed. Kaplan-Meier survival function was used to analyze the prognosis. Overexpression of ß-catenin is more often detected in patients with FIGO stage III and IV than in those with stage I, and II (P=0.003). No significant relationship was found between expression of ß-catenin and pathological grade (P=0.817). Positive expression of ß-catenin related to lower survival rate (P=0.034). The expression of cyclinD1 had no relationship with FIGO stage (P=0.829). Overexpression of cyclinD1 was positively to pathological grade (P=0.017) and survival rate (P=0.009). There is a significantly positive relationship between expression of ß-catenin and cyclinD1 (P=0.014). No statistical significance was found between expression of ß-catenin and cyclinD1 and other pathological parameters. CONCLUSIONS: Expression of ß-catenin and cyclinD1 may be used as predict markers for poor prognosis.

Modulation of ten-eleven translocation 1 (TET1), Isocitrate Dehydrogenase (IDH) expression, α-Ketoglutarate (α-KG), and DNA hydroxymethylation levels by interleukin-1ß in primary human chondrocytes.

5-Hydroxymethylcytosine (5-hmC) generated by ten-eleven translocation 1-3 (TET1-3) enzymes is an epigenetic mark present in many tissues with different degrees of abundance. IL-1ß and TNF-α are the two major cytokines present in arthritic joints that modulate the expression of many genes associated with cartilage degradation in osteoarthritis. In the present study, we investigated the global 5-hmC content, the effects of IL-1ß and TNF-α on 5-hmC content, and the expression and activity of TETs and isocitrate dehydrogenases in primary human chondrocytes. The global 5-hmC content was found to be ∼0.1% of the total genome. There was a significant decrease in the levels of 5-hmC and the TET enzyme activity upon treatment of chondrocytes with IL-1ß alone or in combination with TNF-α. We observed a dramatic (10-20-fold) decrease in the levels of TET1 mRNA expression and a small increase (2-3-fold) in TET3 expression in chondrocytes stimulated with IL-1ß and TNF-α. IL-1ß and TNF-α significantly suppressed the activity and expression of IDHs, which correlated with the reduced α-ketoglutarate levels. Whole genome profiling showed an erasure effect of IL-1ß and TNF-α, resulting in a significant decrease in hydroxymethylation in a myriad of genes including many genes that are important in chondrocyte physiology. Our data demonstrate that DNA hydroxymethylation is modulated by pro-inflammatory cytokines via suppression of the cytosine hydroxymethylation machinery. These data point to new mechanisms of epigenetic control of gene expression by pro-inflammatory cytokines in human chondrocytes.

Clinicopathological features of myxopapillary ependymoma.

Myxopapillary ependymoma (MPE) is a rare and distinct variant of ependymoma with a tendency for local recurrence and metastasis. Its clinicopathological spectrum is heterogenous, underscoring the need to understand and characterize MPE for better diagnosis and treatment. The purpose of this study was to explore the tumor biology and assess the management of patients with MPE. Tumors from a cohort of 19 patients were analyzed by light microscopy, electron microscopy, immunohistochemistry and fluorescence in situ hybridization (FISH). Clinical characteristics, therapeutic options and clinical follow-up data were also analyzed. Back pain was the most common presenting symptom. The main pathological morphology observed was papillae embedded in a myxoid background, but other rare morphologies were also present. Immunostaining revealed epidermal growth factor receptor (EGFR) expression in four MPE, while FISH for EGFR was negative. No correlation between tumor recurrence and EGFR overexpression was found. Ultrastructural examination revealed adherens junctions and intracytoplasmic lumina with microvilli. Patients with gross-total resection (GTR) had no tumor recurrence (p=0.021). Also, patients with subtotal resection (STR) followed by radiotherapy showed a higher local control rate than patients with STR alone (p=0.043). The diagnosis of MPE should be made considering the histology, immunohistochemistry, imaging studies and anatomical site. GTR of the tumor or STR followed by radiotherapy are more likely to avoid tumor recurrence than STR alone. Based on our findings, there is no correlation between tumor recurrence and EGFR expression.

Osteolytic myxopapillary ependymoma with marked hyaline degeneration in a 72-year-old male: A case report.

Myxopapillary ependymomas (MPEs) are uncommon and account for ∼15% of all ependymomas. The current study presents a case of rare spinal MPE with abnormal hyaline degeneration. The patient was a 72-year-old male with a 10-month history of lower back pain. Magnetic resonance imaging revealed a mass involving the L4 and L5 vertebrae with local bone destruction. The tumor was completely resected. Histologically, the majority of the tumor exhibited low cellularity. A marked change in hyaline was observed in the blood vessels and stroma. In specific areas, the tumor showed reticular or tubular patterning embedded in hyaline materials. The tumor cells were cuboidal to columnar in shape with strong immunostaining for glial fibrillary acidic protein and S-100. A fluorescence in situ hybridization analysis for amplification of the epidermal growth factor receptor gene was negative. The results of pathological and immunohistochemical studies were consistent with the ependymal nature of neoplastic cells.

TSA downregulates Wilms tumor gene 1 (Wt1) expression at multiple levels.

The Wilms tumor gene WT1 encodes a zinc-finger transcription factor that is inactivated in a subset of pediatric kidney cancers. During embryogenesis, WT1 is expressed in a time- and tissue-specific manner in various organs including gonads and kidney but also in the hematopoietic system. Although widely regarded as a tumor suppressor gene, wild-type WT1 is overexpressed in a variety of hematologic malignancies, most notably in acute lymphoblastic leukemia as well as myelodysplastic syndromes. Reduction of WT1 expression levels leads to decrease of proliferation and apoptosis of leukemic cells, suggesting that in certain contexts WT1 might act as an oncogene. We show here that histone deacetylase inhibitors like Trichostatin A (TSA) can promptly and dramatically downregulate Wt1 expression levels in different cell lines. This effect was mostly due to the cessation of transcription and was mediated by sequences located in intron 3 of Wt1. In addition, TSA also caused enhanced degradation of the Wt1 protein by the proteasome. This was at least in part due to induction of the ubiquitin-conjugating enzyme UBCH8. Thus, downregulation of Wt1 expression might contribute to the beneficial effects of histone deacetylase inhibitors that are currently used in clinical trials as cancer therapeutics.