Highly active antiretroviral therapy (HAART) in individuals infected with HIV-1 often lowers plasma viremia to below detection limits. However, cessation of therapy in such individuals results in rebound of virus replication, indicating that HIV-infected cells persist. Resting, memory CD4 T cells in the blood and lymph nodes comprise the major reservoir for persistent HIV infection. To devise new efficient strategies targeted toward the eradication of the latently infected HIV reservoir, a better understanding of the molecular and cellular basis for viral latency is needed. Dr. Spina's research group has developed a unique in vitro T cell model to study HIV latency. In my thesis project, I have used and modified this cell model to investigate: 1) the cellular proliferation and activation requirements for the development of latently infected CD4 cells, and 2) the transcriptional activity of the HIV provirus in a nonproductive, persistent infection. Results from the first research phase demonstrated that HIV infection immediately prior to T cell stimulation resulted in production of the greatest number of latently infected cells. Infected cells that did not divide, or divided only a few times, following stimulation went on to form the latently infected cell pool. The vast majority of acutely infected, activated CD4 cells were not able to survive multiple rounds of cell division in combination with the cytopathic effects of HIV. Rather, the subset of CD4 cells that exhibited minimal activation, in the presence of fully-activated and productively infected T cells, survived with latent HIV infection. In the second phase of research, a sensitive qRT-PCR assay was used to examine the transcriptional activity of the HIV provirus in resting, infected CD4 cells. Multiple different species of HIV mRNA were found, with unspliced gag transcripts being the most abundant followed by singly-spliced env, total multiply-spliced, nef, and tat species. Detection of viral RNA transcription in latently infected T cells from our in vitro model has raised the possibility that HIV latency is not maintained by a simple passive mechanism, but may involve active interactions between viral products and cell processes that influence viral latency and reactivation.

This volume summarizes recent advances in understanding the mechanisms of HIV-1 latency, in characterizing residual viral reservoirs, and in developing targeted interventions to reduce HIV-1 persistence during antiretroviral therapy. Specific chapters address the molecular mechanisms that govern and regulate HIV-1 transcription and latency; assays and technical approaches to quantify viral reservoirs in humans and animal models; the complex interchange between viral reservoirs and the host immune system; computational strategies to model viral reservoir dynamics; and the development of therapeutic approaches that target viral reservoir cells. With contributions from an interdisciplinary group of investigators that cover a broad spectrum of subjects, from molecular virology to proof-of-principle clinical trials, this book is a valuable resource for basic scientists, translational investigators, infectious-disease physicians, individuals living with HIV/AIDS and the general public.

Human immunodeficiency virus (HIV-1) infection remains a global health issue with approximately 33 million people living with HIV-1 worldwide. While HIV-1 preferentially infects activated T cells, multiple cells, including dendritic cells (DC), can also be infected. In vitro, thymocytes and resting CD4+ T cells are relatively resistant to CCR5 (R5)-tropic HIV-1 infection. In comparison, thymocytes and resting CD4+ T cells found in lymphoid tissues are clearly infected, suggesting a requirement for interactions with other cells and/or soluble factors. We hypothesised that the interaction between DC and T cells within tissues can facilitate infection of thymocytes and resting T cells, which may lead to altered CD4+ T cell homeostasis and long term persistence of HIV-1. Here we demonstrate a role for DC in the: [1] enhancement of productive infection in the thymus; and [2] establishment of latency in resting CD4+ T cells. We first demonstrated that productive HIV-1 infection can be established in both thymic plasmacytoid DC (pDC) and myeloid DC (mDC) and that thymic pDC were able to efficiently transfer productive R5 HIV-1 infection to both mature CD3hi and immature CD3lo thymocytes, which were otherwise refractory to R5 virus. This efficient transfer may represent a pathway to early infection and impaired production of thymocytes and CD4+ T cells in HIV-1-infected individuals. We then examined interactions between DC and resting CD4+ T cells isolated from blood to determine if this interaction was critical for infection of resting CD4+ T cells and the establishment of latency. We established a unique in vitro model, using enhanced green fluorescent protein (EGFP)-reporter viruses and resting CD4+ T cells labelled with the proliferation dye SNARF, to study the establishment of latency in resting CD4+ T cells. We demonstrated post-integration latency in non-proliferating CD4+ T cells following co-culture with syngeneic DC, which was facilitated by mDC, but not pDC. This effect was enhanced in the presence of an additional microbial stimulus, SEB, and required both DC-T cell contact and soluble factors, secreted by the DC as a result of HIV-1 stimulation. By comparing the gene profiles of these latently infected CD4+ T cells with those of mock-infected CD4+ T cells, we observed the induction of multiple genes associated with cell cycle arrest and the inhibition of HIV-1 transcription, while genes required for active cell cycle and NF-kappaB activation were repressed. Our results suggest a possible pathway for mDC-induced latency in CD4+ T cells in which low levels of cell activation may allow for enhanced HIV-1 integration but subsequent blocks in transcription and cell proliferation prevent progression to productive infection. This novel model can be used to further understand the different mechanisms involved in the establishment of HIV-1 latency.In summary, we have demonstrated that DC are important both in the spread of productive HIV-1 infection and the establishment of HIV-1 latency. An improved understanding of the interactions between DC and T cells, in the presence of HIV-1, may identify novel approaches to overcome the reduced thymic output of CD4+ T cells and eliminate the HIV-1 reservoir.

The process of HIV latency establishment and maintenance is not clearly understood. Homeostatic proliferation (HSP) is a major mechanism by which long-lived naive and memory CD4 T cells are maintained in vivo. HSP also contributes to the persistence of HIV latent reservoir. Furthermore, HIV-infected naive CD4 T cells cultured under HSP condition are refractory to reactivation, in contrast to TCR-activated memory CD4 T cells. This might be due to the suggested post-transcriptional block in naive HSP-cultured cells. Here we compared a transcriptomic signature of naive and memory CD4 T cells. Among differentially expressed genes that may influence HIV latency, we identified Schlafen 12 (SLFN12) as an interesting candidate for a potential HIV restriction factor. Our results showed that SLFN12 establishes post-transcriptional block in HIV infected cells and thus inhibits both, HIV production as well as its reactivation from latently infected cells. These findings may help to better understand the mechanisms underlying HIV latency and its reversal in HSP-maintained naive CD4 T cells. All together it might contribute to the design of novel HIV eradication strategies.

DNA Methylation and Complex Human Disease reviews the possibilities of methyl-group-based epigenetic biomarkers of major diseases, tailored epigenetic therapies, and the future uses of high-throughput methylome technologies. This volume includes many pertinent advances in disease-bearing research, including obesity, type II diabetes, schizophrenia, and autoimmunity. DNA methylation is also discussed as a plasma and serum test for non-invasive screening, diagnostic and prognostic tests, as compared to biopsy-driven gene expression analysis, factors which have led to the use of DNA methylation as a potential tool for determining cancer risk, and diagnosis between benign and malignant disease. Therapies are at the heart of this volume and the possibilities of DNA demethylation. In cancer, unlike genetic mutations, DNA methylation and histone modifications are reversible and thus have shown great potential in the race for effective treatments. In addition, the authors present the importance of high-throughput methylome analysis, not only in cancer, but also in non-neoplastic diseases such as rheumatoid arthritis. Discusses breaking biomarker research in major disease families of current health concern and research interest, including obesity, type II diabetes, schizophrenia, and autoimmunity Summarizes advances not only relevant to cancer, but also in non-neoplastic disease, currently an emerging field Describes wholly new concepts, including the linking of metabolic pathways with epigenetics Provides translational researchers with the knowledge of both basic research and clinic applications of DNA methylation in human diseases

This authoritative handbook covers all aspects of immunosenescence, with contributions from experts in the research and clinical areas. It examines methods and models for studying immunosenescence; genetics; mechanisms including receptors and signal transduction; clinical relevance in disease states including infections, autoimmunity, cancer, metabolic syndrome, neurodegenerative diseases, frailty and osteoporosis; and much more.