Project description:The study of disease-associated immune biomarkers has revealed underlying pathogenic mechanisms and provided diagnostic and prognostic values in numerous clinical settings. Accurate immune profiling of normal age and sex demographics is crucial for understanding the relevance of disease biomarkers. The incidence of age-associated diseases in the elderly and prevalence of diseases in specific genders may be explained by these normal phenotypic variation in the cellular immune system. Here we use microarrays of cluster of differentiation (CD) antibodies to immunophenotype populations of peripheral blood mononuclear cells (PBMCs) from healthy adult blood donors. The data revealed a set of statistically significant age- and gender-dependent expression patterns across the study population. We identified functional differences in immune biomarkers on PBMC associated with major innate, adaptive and inflammatory immune functions. The CD array data supported established observations using flow cytometric methods on age-associated changes in immune biomarkers, while also identifying novel markers associated with age and/or gender. The data demonstrates the utility of CD antibody arrays in providing a systematic and quantitative basis for understanding the development and progression of diseases that have an age-dependent and gender-specific etiology. Blood samples were collected from healthy male and female blood donors acrooss various age groups, predominantly Caucasians, from the Australian Red Cross Blood Service, Sydney, Australia, and from elderly males enrolled in the Concord Health and Aging in Men Project (CHAMP) at Concord Hospital, Sydney, Australia. PBMCs were fractionated from whole blood and applied to a cell capture CD antibody microarray. The microarray was then scaned by a DotScan™ slide reader (Medsaic; Eveleigh, NSW, Australia). The number of immobilized cells was proportional to the light scattered at each antibody spot. Binding of PBMCs to each antibody dot was monitored by light scatter and averaged between the duplicate spots on each slide. We then performed statistical analysis to identify CD antigen markers that has an statistically significant association with age and/or gender

Project description:Purpose: Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer. A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases. Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale. Because of this, we performed an RNA-seq analysis of primary rat hepatocytes either expressing HBV or not over a series of time points to determine the global changes HBV has on primary hepatocyte physiology. Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes (PRH) over the course of 72hr. PRH were collected immediately after isolation (0hr), or 24hr, 48hr, or 72hr after plating. In addition, PRH were infected 24hr after plating with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV) and collected at 48hr after plating (24hr after infection) or 72hr after plating (48hr after infection). cDNA libraries were sequenced using the Illumina NextSeq 500 platform to generate either 1x75bp reads. Reads were mapped using the STAR aligner, and output BAMs were further analyzed in R using the GenomicAlignments package, to quantify number of reads per transcript, and DESeq2, to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample. Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 48hr and 72hr. In addition, we noted considerable change to the hepatocyte transcriptome as a direct result of the isolation/plating procedure, regardless of the presence of HBV. Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes.

Project description:Purpose: Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer. A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases. Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale. Because of this, we performed an RNA-seq analysis of primary rat hepatocytes either expressing HBV or not over a series of time points to determine the global changes HBV has on primary hepatocyte physiology. Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes (PRH) over the course of 72hr. PRH were collected immediately after isolation (0hr), or 24hr, 48hr, or 72hr after plating. In addition, PRH were infected 24hr after plating with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV) and collected at 48hr after plating (24hr after infection) or 72hr after plating (48hr after infection). cDNA libraries were sequenced using the Illumina NextSeq 500 platform to generate either 1x75bp reads. Reads were mapped using the STAR aligner, and output BAMs were further analyzed in R using the GenomicAlignments package, to quantify number of reads per transcript, and DESeq2, to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample. Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 48hr and 72hr. In addition, we noted considerable change to the hepatocyte transcriptome as a direct result of the isolation/plating procedure, regardless of the presence of HBV. Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes. mRNA profiles of cultured primary rat hepatocytes were generated, in triplicate, using the Illumina NextSeq 500 platform from freshly isolated cells (0hr), 24hr, 48hr, or 72hr after plating, and with or without expression of HBV 48hr or 72hr after plating.

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