Microbiome analysis, the immune response and transplantation in the era of next generation sequencing.

The human gastrointestinal tract, skin and mucosal surfaces are inhabited by a complex system of bacteria, viruses, fungi, archaea, protists, and eukaryotic parasites with predominance of bacteria and bacterial viruses (bacteriophages). Collectively these microbes form the microbiota of the microecosystem of humans. Recent advancement in technologies for nucleic acid isolation from various environmental samples, feces and body secretions and advancements in shotgun throughput massive parallel DNA and RNA sequencing along with 16S ribosomal gene sequencing have unraveled the identity of otherwise unknown microbial entities constituting the human microecosystem. The improved transcriptome analysis, technological developments in biochemical analytical methods and availability of complex bioinformatics tools have allowed us to begin to understand the metabolome of the microbiome and the biochemical pathways and potential signal transduction pathways in human cells in response to microbial infections and their products. Also, developments in human whole genome sequencing, targeted gene sequencing of histocompatibility genes and other immune response associated genes by Next Generation Sequencing (NGS) have allowed us to have a better conceptualization of immune responses, and alloimmune responses. These modern technologies have enabled us to dive into the intricate relationship between commensal symbiotic and pathogenic microbiome and immune system. For the most part, the commensal symbiotic microbiota helps to maintain normal immune homeostasis besides providing healthy nutrients, facilitating digestion, and protecting the skin, mucosal and intestinal barriers. However, changes in diets, administration of therapeutic agents like antibiotics, chemotherapeutic agents, immunosuppressants etc. along with certain host factors including human histocompatibility antigens may alter the microbial ecosystem balance by causing changes in microbial constituents, hierarchy of microbial species and even dysbiosis. Such alterations may cause immune dysregulation, breach of barrier protection and lead to immunopathogenesis rather than immune homeostasis. The effects of human microbiome on immunity, health and disease are currently under intense research with cutting edge technologies in molecular biology, biochemistry, and bioinformatics along with tremendous ability to characterize immune response at single cell level. This review will discuss the contemporary status on human microbiome immune system interactions and their potential effects on health, immune homeostasis and allograft transplantation.

Description and analysis of patients and outcomes following third-time heart transplantation: An analysis of the United Network for Organ Sharing database from 1985 to 2017.

BACKGROUND: Following second heart transplantation (HTx), some patients experience graft failure and require third-time heart transplantation. Little data exist to guide decision-making with regard to repeat retransplantation in older patients. METHODS: We performed a retrospective cohort analysis of patients receiving a third HTx, as identified in the United Network for Organ Sharing (UNOS) database from 1985 to 2017. RESULTS: The study cohort consisted of N = 60 patients, with an average age of 29 with a standard deviation of ±18 years. Overall survival for the cohort at 1, 5, and 10 years is 83%, 64%, and 44%, respectively. The rate of third-time HTxs has steadily increased in all age groups. Patients older than 50 years now account for 18.3% of all third-time HTxs. Although this group demonstrated longer average previous graft survival, after third HTx they demonstrate significantly poorer survival outcomes compared to third-time HTx recipients younger than 21 (P = 0.05). Age over 50, BMI over 30, and diabetes were all found to be independent risk factors for decreased survival following third HTx. CONCLUSIONS: We describe trends in patients undergoing third HTx. We highlight subsets of such recipients who exhibit decreased survival.

Development of a bead-based immunoassay to routinely measure vimentin autoantibodies in the clinical setting.

INTRODUCTION: Vimentin is an intermediate filament protein generally expressed in the cytosol of many adult cell types, including leukocytes, fibroblasts and endothelial cells. Several tissue and/or injury-specific isoforms of vimentin are known to exist that may trigger autoimmune responses due to aberrant structural or conformational variations. Such scenarios include allograft rejection and certain autoimmune diseases, such as rheumatoid arthritis. The primary objective for this study was to develop a Luminex immunobead assay to quantitate circulating levels of vimentin antibodies and, secondarily, to appraise the feasibility of these autoantibodies as a biomarker for clinical diagnosis. METHODS: Recombinant human vimentin was conjugated to MagPlex® beads using standard carbodiimide/NHS chemistry and coupling efficiency tested and assay parameters determined using a commercial anti-vimentin polyclonal antibody. A limited number of serum samples (n=71) were then tested to evaluate the diagnostic value for future biomarker development efforts. RESULTS: Findings from repeated testing of three distinct batches of assays provide assay range parameters of 0.18-15µg/mL, median inter-assay recovery parameter within 1% of completion, and inter-assay variation (%CV) at 7%. The assay was found to be stable at several conditions with less than 5% loss in a month. Preliminary evaluation of the assay demonstrates significantly (p=0.022) higher circulating levels of anti-vimentin antibodies in 51 cases of renal allograft rejection relative to 20 cases of age-matched controls. CONCLUSION: A direct capture assay for vimentin autoantibodies was developed and analytically validated. Preliminary evaluation of this assay against patient materials was promising and justifies additional testing with larger cohorts in future studies.