The Application of 3base™ Technology to Diagnose Eight of the Most Clinically Important Gastrointestinal Protozoan Infections.

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. Other protozoan agents that have been implicated in gastroenteritis in humans include Cyclospora cayetanensis, Dientamoeba fragilis, Blastocystis hominis, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis. Genetic Signatures previously developed a 3base™ multiplexed Real-Time PCR (mRT-PCR) enteric protozoan kit (EP001) for the detection of Giardia intestinalis/lamblia/duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, and B. hominis. We now describe improvements to this kit to produce a more comprehensive assay, including C. cayetanensis, E. bieneusi, and E. intestinalis, termed EP005. The clinical performance of EP005 was assessed using a set of 380 clinical samples against a commercially available PCR test and other in-house nucleic acid amplification tests where commercial tests were not available. All methods provided at least 90% agreement. EP005 had no cross-reactivity against 82 organisms commonly found in the gut. The EP005 method streamlines the detection of gastrointestinal parasites and addresses the many challenges of traditional microscopic detection, resulting in cost savings and significant improvements in patient care.

Retrospective Proteomic Analysis of Cellular Immune Responses and Protective Correlates of p24 Vaccination in an HIV Elite Controller Using Antibody Arrays.

BACKGROUND: HIV p24 is an extracellular HIV antigen involved in viral replication. Falling p24 antibody responses are associated with clinical disease progression and their preservation with non-progressive disease. Stimulation of p24 antibody production by immunization to delay progression was the basis of discontinued p24 vaccine. We studied a therapy-naive HIV+ man from Sydney, Australia, infected in 1988. He received the HIV-p24-virus like particle (VLP) vaccine in 1993, and continues to show vigorous p24 antigen responses (>4% p24-specific CD4⁺ T cells), coupled with undetectable plasma viremia. We defined immune-protective correlates of p24 vaccination at the proteomic level through parallel retrospective analysis of cellular immune responses to p24 antigen in CD4⁺ and CD8⁺ T cells and CD14⁺ monocytes at viremic and aviremic phases using antibody-array. We found statistically significant coordinated up-regulation by all three cell-types with high fold-changes in fractalkine, ITAC, IGFBP-2, and MIP-1α in the aviremic phase. TECK and TRAIL-R4 were down-regulated in the viremic phase and up-regulated in the aviremic phase. The up-regulation of fractalkine in all three cell-types coincided with protective effect, whereas the dysfunction in anti-apoptotic chemokines with the loss of immune function. This study highlights the fact that induction of HIV-1-specific helper cells together with coordinated cellular immune response (p < 0.001) might be important in immunotherapeutic interventions and HIV vaccine development.

First Evidence for the Disease-Stage, Cell-Type, and Virus Specificity of microRNAs during Human Immunodeficiency Virus Type-1 Infection.

The potential involvement of host microRNAs (miRNAs) in HIV infection is well documented, and evidence suggests that HIV modulates and also dysregulates host miRNAs involved in maintaining the host innate immune system. Moreover, the dysregulation of host miRNAs by HIV also effectively interferes directly with the host gene expression. In this study, we have simultaneously evaluated the expression of host miRNAs in both CD4+ and CD8+ T-cells derived from HIV-positive (HIV+) individuals (viremic and aviremic individuals while receiving highly active antiretroviral therapy (HAART), therapy-naïve long-term non-progressors (LTNP), and HIV-negative (HIV-) healthy controls. miRNAs were run on Affymetrix V2 chips, and the differential expression between HIV+ and HIV- samples, along with intergroup comparisons, was derived using PARTEK software, using an FDR of 5% and an adjusted p-value < 0.05. The miR-199a-5p was found to be HIV-specific and expressed in all HIV+ groups as opposed to HIV- controls. Moreover, these are the first studies to reveal clearly the highly discriminatory miRNAs at the level of the disease state, cell type, and HIV-specific miRNAs.

Gene array data relevant to immunological and virological monitoring of human immunodeficiency virus type 1 infection.

PURPOSE OF REVIEW: Immunomonitoring technologies are not only fast changing, but also have already revolutionized the way we look at treatments, diagnosis, and prognosis of a given disease. The purpose of the review is to provide a recent update on the use or possible use of array-based data in immunomonitoring of HIV patients. RECENT FINDINGS: Since the inception of gene arrays, there has been a rapid surge in the development of a variety of array-based technologies, which comprise of gene and protein expression platforms. These have been instrumental in studying various immunological and genomic aspects of HIV disease at the subcellular level. SUMMARY: Gene and protein array technologies are ideal for a high-throughput multiplexing of large datasets in determining the difference between diseased and nondiseased and pretreatment and posttreatment stages of HIV patients. Therefore, these technologies have the potential to revolutionize HIV immunomonitoring, treatment, diagnosis and prognosis. Although the array-based technologies have not yet replaced conventional immunomonitoring, the data coming out from high-throughput transcriptomic and proteomic studies and its global integration will lead to developing new generation of candidates and tools for immunomonitoring of HIV disease.

Transcriptome analysis of primary monocytes shows global down-regulation of genetic networks in HIV viremic patients versus long-term non-progressors.

Despite significant contributions of monocytes to HIV persistence, the genomic basis of HIV-infection of monocytes and its association with plasma viremia remain elusive. To understand HIV interactions with monocytes during disease progression, monocytic transcriptomes from long-term non-progressors (LTNP), HIV+ patients with viral load <1000, with viral load >1000, and seronegative controls were analyzed using Illumina microarray. Differentially expressed genes were identified (fold change >2; adjusted p<0.05) and GSEA between HIV+ groups demonstrated that the down-regulation of the pathways including Toll-like receptor (TLR) signaling, cytokine-cytokine receptor interaction, cell cycle and apoptosis was significantly associated with the viremic groups, whereas their up-regulation with the LTNP group. The down-regulation of TLR pathway in the viremic patients was exemplified by the decreased expression of TLR with the subsequent tuning down of MAPK, NF-κB, JAK-STAT, and IRF cascades. These data provide the first transcriptomic distinction between HIV+ progressors and LTNPs based on primary monocytes.