Polyomavirus BK (BKV) infection is an important clinical problem in kidney transplantation. Viremia complicates 10%-30% of patients while 1%-10% of patients develop BKV nephropathy (BKVN). 1 No effective antiviral treatment is available. Reduction in immunosuppression to enhance antiviral immunity increases the incidence of rejection. This leads to an approximately 2-fold increase in graft loss. 2 An increased understanding of BKV biology is essential to develop more effective therapies for BKVN. Host cell gene expression in BKV nephropathy has been addressed by transcriptomics as well as proteomics studies. 3-5 These have shown an inflammatory and profibrotic milieu in infected tissues and provide a molecular basis for the progressive tissue injury that is seen in this clinical setting. However, small RNA regulation of BKV induced gene expression directly in renal tubular epithelial cells is not well understood. Therefore, this study performed microRNA (miRNA) profiling of BKV-infected cell cultures with a view to enhance our understanding of BKV pathogenesis and potentially identify new targets for therapy. miRNAs are short RNA molecules 18-22 nucleotides long that are not translated into protein. 6 Their function is to silence target messenger RNA by base pairing complementary sequences, which results in cleavage or stabilization of the mRNA. In addition, translational repression of mRNA can occur at level of ribosomes. The BKV genome encodes a primary miRNA transcript, which is digested by Abstract Polyomavirus BK (BKV) is an important pathogen in kidney transplant patients.Regulation of BKV encoded microRNAs (miRNAs) is not well understood. Therefore, tubular epithelial cells infected with BKV were examined for changes in small RNA expression. The observed changes were further evaluated by real-time PCR and RNAseq analysis of renal allograft biopsies. BKV-miR-B1-5p and BKV-miR-B1-3p showed a 1000-fold increase over 12 days but did not prevent cell lysis. Downregulation of host miR-10b and miR-30a could be confirmed on all three platforms evaluated.Whereas, the BKV genome expressed more 3p than 5p miRNA species, the reverse was true for the human genome. Decreased expression of TP53INP2, and increased expression of BCL2A1, IL-6, IL8 and other proinflammatory cytokines were shown in biopsies with BKV nephropathy. No change in expression was seen in miR-10a dependent expression of NKG2D ligands ULBP3, MICA, or MICB. In conclusion, BKV infection results in regulation of cellular genes regulated by and possibly amenable to therapies targeting miR-10 and miR-30.

Rosai-Dorfman disease (RDD) is an uncommon histiocytic proliferative disorder that can present in nodal, extranodal, or, extremely rarely, in central nervous system (CNS)-restricted form. RDD is characterized histologically as a non-Langerhans cell histiocytosis composed of atypical CD68 /S-100 /CD1a macrophages demonstrating prominent emperipolesis and effacement of the surrounding tissue. Previously thought to represent a reactive process, recent studies have raised the possibility that RDD and other histiocytic lesions, including Erdheim-Chester and Langerhans cell histiocytosis, are clonal processes linked to somatic mutations in the mitogen-activated protein (MAP) kinase pathway. Herein, we present a fatal case of RDD isolated to the CNS and used a next-generation targeted gene panel and Sanger sequencing to uncover a pathogenic deletion in the β3-αC loop of the kinase domain in exon 12 of BRAF. This mutation, previously described in melanoma and Langerhans cell histiocytosis, represents the first BRAF mutation of this kind identified in RDD. These findings support the idea that RDD is a neoplastic condition and raise the possibility that inhibitors of the MAP kinase pathway may be effective in RDD. Ann Neurol 2018;83:147-152.

Tobacco smoke exposure stimulates the expression of genes that are likely to be involved in the metabolism of its combustion products such as polycyclic aromatic hydrocarbons (PAH). Four of the smoke induced genes are aldo-keto reductases (AKR), enzymes that metabolically activate PAH to PAH o-quinones. Alternatively, PAHs are metabolized to (±)-anti-diol epoxides, such as (±)-anti-benzo[a]pyrene diol epoxide ((±)-anti-BPDE)), by the combined action of P4501A1/1B1 and epoxide hydrolase. (±)-anti-BPDE forms DNA adducts directly, while PAH o-quinones cause DNA damage by oxidative stress through a futile redox cycle. To address the role of AKRs in PAH cytotoxicity, we compared the cytotoxicity of PAH metabolites and the effects of overexpressing AKR1A1 in lung cells. (±)-anti-BPDE and B[a]P-7,8-trans-dihydrodiol, an intermediate in (±)-anti-BPDE metabolism, are toxic to A549 cells at concentrations with an IC50 of ~2 μM. In contrast, the PAH o-quinone B[a]P-7,8-dione was about 10-fold less toxic to A549 cells with an IC50 > 20 μM. Similar differences in cytoxicity was observed with two other PAH o-quinones (benz[a]anthracene-3,4-dione and 7,12-dimethylbenz[a]anthracene-3,4-dione) compared with their respective diol-epoxide counterparts (BA-3,4-diol-1,2-epoxide and DMBA-3,4-diol-1,2-epoxide). In addition, both anti-BPDE and B[a]P-7,8-trans-dihydrodiol induced p53 expression ~6 hours post-treatment at concentrations as low as 1 μM consistent with extensive DNA damage. B[a]P-7,8-dione treatment did not induce p53 but generated reactive oxygen species (ROS) in A549 cells and induced the expression of oxidative response genes in H358 cells. We also observed that overexpression of AKR1A1 in H358 cells, which otherwise have low levels of AKR expression, protected cells 2–10 fold from the toxic effects of B[a]P-7,8-trans-dihydrodiol. These data suggest that overexpression of AKRs may protect lung cancer cells from the acute toxic effects of PAH.

BackgroundRecent work using DNA microarrays has suggested that genes related to DNA replication, RNA polymerase assembly, and pathogen recognition receptors can serve as surrogate tissue biomarkers for polyomavirus BK nephropathy (BKPyVN).MethodsWe have examined this premise by looking for differential regulation of these genes using a different technology platform (RNA-seq) and an independent set 25 biopsies covering a wide spectrum of diagnoses.ResultsRNA-seq could discriminate T cell–mediated rejection from other common lesions seen in formalin fixed biopsy material. However, overlapping RNA-seq signatures were found among all disease processes investigated. Specifically, genes previously reported as being specific for the diagnosis of BKPyVN were found to be significantly upregulated in T cell–mediated rejection, inflamed areas of fibrosis/tubular atrophy, as well as acute tubular injury.ConclusionsIn conclusion, the search for virus specific molecular signatures is confounded by substantial overlap in pathogenetic mechanisms between BKPyVN and nonviral forms of allograft injury. Clinical heterogeneity, overlapping exposures, and different morphologic patterns and stage of disease are a source of substantial variability in “Omics” experiments. These variables should be better controlled in future biomarker studies on BKPyVN, T cell–mediated rejection, and other forms of allograft injury, before widespread implementation of these tests in the transplant clinic.

Silent subtype III pituitary adenomas (SS-3) are clinically nonfunctional adenomas that are more aggressive in terms of invasion and risk of recurrence than their conventional null cell counterparts. We previously showed that these tumors can be distinguished by immunohistochemistry based on the identification of a markedly enlarged and fragmented Golgi apparatus. To understand the molecular correlates of differential aggressiveness, we performed whole transcriptome sequencing (RNAseq) on 4 SS-3 and 4 conventional null cell adenomas. The genes that were highly upregulated in all the SS-3 adenomas included 2 secreted proteins involved in the suppression of T-lymphocyte activity, i.e., ARG2 (multiple testing adjusted padj = 1.5 × 10-3) and SEMA3A (padj = 3.3 × 10-3). Highly downregulated genes in all the SS-3 adenomas included HLA-B (padj = 3.3 × 10-6), suggesting reduced antigen presentation by the adenoma to cytotoxic T-cells. Quantitative RT-PCR of these genes performed on the adenoma samples supported the RNAseq results. We also found a relative decrease in the overall concentration of T-lymphocytes in the SS-3 tumors. These results suggest that SS-3 adenomas actively suppress the immune system and raise the possibility that they may be treatable with immune checkpoint inhibitors or nonspecific cancer immunotherapies.