Survey of extracellular communication of systemic and organ-specific inflammatory responses through cell free messenger RNA profiling in mice

Inflammatory and immune responses are essential and dynamic biological processes that protect the body against acute and chronic adverse stimuli. While conventional protein markers have been used to evaluate systemic inflammatory response, the immunological response to stimulation is complex and involves modulation of a large set of genes and interacting signaling pathways of innate and adaptive immune systems. Therefore, there is a need for a non-invasive tool that can comprehensively evaluate and monitor molecular dysregulations associated with inflammatory and immune responses. Here we utilized cell-free messenger RNA (cf-mRNA) RNA-Seq whole transcriptome profiling to assess lipopolysaccharide (LPS) induced and JAK inhibitor modulated inflammatory and immune responses in mouse plasma samples. Considering that, both organ-specific recruitment of immune cells and organ resident bespoke immune cells contributes to restoration of organ homeostasis, we also examined LPS-induced gene-expression dysregulation of multiple organs to shed light on organ crosstalk. Cf-mRNA profiling displayed a pattern of systemic immune responses elicited by LPS and dysregulation of associated pathways. Moreover, attenuation of several inflammatory pathways, including STAT and interferon pathways, were observed following the treatment of JAK inhibitor. Lastly, we identified the dysregulation of liver-specific transcripts in cf-mRNA which reflected changes in the gene-expression pattern in this biological compartment. Collectively, using a preclinical model, we demonstrated the potential of plasma cf-mRNA profiling for systemic and organ-specific characterization of drug-induced molecular alterations that are associated with inflammatory and immune responses.

Urinary cell mRNA Profiling of Kidney Allograft Recipients: A Systematic Investigation of a Filtration Based Protocol for the Simplification of Urine Processing

Background. Kidney transplantation is a life-restorative therapy, but immune rejection undermines allograft survival. Urinary cell mRNA profiles offer a noninvasive means of diagnosing kidney allograft rejection, but urine processing protocols have logistical constraints. We aimed to determine whether the centrifugation-based method for urinary cell mRNA profiling could be replaced with a simpler filtration-based method without undermining quality. Methods. We isolated RNA from urine collected from kidney allograft recipients using the Cornell centrifugation-based protocol (CCBP) or the Zymo filter-based protocol (ZFBP) and compared RNA purity and yield using a spectrophotometer or a fluorometer and measured absolute copy number of transcripts using customized real-time quantitative PCR assays. We investigated the performance characteristics of RNA isolated using ZFBP and stored either at -80oC or at ambient temperature for 2 to 4 days and also when shipped to our Gene Expression Monitoring (GEM) Core at ambient temperature. We examined the feasibility of initial processing of urine samples by kidney allograft recipients trained by the GEM Core staff and the diagnostic utility for acute rejection, of urine processed using the ZFBP. Results. RNA purity (P=0.0007, Wilcoxon matched paired signed-ranks test ) and yield (P<0.0001) were higher with ZFBP vs. CCBP, and absolute copy number of 18S rRNA was similar (P=0.79) following normalization of RNA yield by reverse transcribing a constant amount of RNA isolated using either protocol. RNA purity, yield, and absolute copy numbers of 18S rRNA, TGF-β1 mRNA and microRNA-26a were not different (P>0.05) in the filtrates containing RNA stored either at -800C or at ambient temperature for 2 to 4 days or shipped overnight at ambient temperature. RNA purity, yield, and absolute copy numbers of 18S rRNA and TGF-β1 mRNA were also not different (P>0.05) between home processed and laboratory processed urine filtrates. Urinary cell levels of mRNA for granzyme B (P=0.01) and perforin (P=0.0002) in the filtrates were diagnostic of acute rejection in human kidney allografts. Conclusions. Urinary cell mRNA profiling was simplified using the ZFBP without undermining RNA quality or diagnostic utility. Home processing by the kidney allograft recipients, the stability of RNA containing filtrates at ambient temperature, and the elimination of the need for centrifuges and freezers represent some of the advantages of ZFBP over the CCBP for urinary cell mRNA profiling.

CD73 expression in normal, hyperplastic, and neoplastic thyroid: a systematic evaluation revealing CD73 overexpression as a feature of papillary carcinomas

CD73 converts AMP to adenosine, an immunosuppressive metabolite that promotes tumorigenesis. This study presents a systematic evaluation of CD73 expression in benign, hyperplastic, and neoplastic thyroid. CD73 expression was assessed by immunohistochemistry in 142 thyroid samples. CD73 was expressed in normal thyroid (3/6) and goiter (5/6), with an apical pattern and mild intensity. Apical and mild CD73 expression was also present in oncocytic cell adenomas/carcinomas (9/10; 5/8) and in follicular adenomas/carcinomas (12/18; 23/27). In contrast, papillary thyroid carcinomas featured extensive and intense CD73 staining (49/50) (vs. normal thyroid/goiter, p < 0.001). Seven of nine anaplastic carcinomas were CD73-positive with heterogeneous extensiveness of staining. Medullary and poorly differentiated carcinomas were mostly CD73-negative (1/6; 2/2). These results were corroborated by NT5E mRNA profiling. Papillary carcinomas feature enhanced CD73 protein and mRNA expression with distinct and intense staining, more pronounced in the invasive fronts of the tumors.