Jielin Zhang

Cytomegalovirus Infection Causes an Increase of Arterial Blood Pressure

Cytomegalovirus (CMV) infection is a common infection in adults (seropositive 60–99% globally), and is associated with cardiovascular diseases, in line with risk factors such as hypertension and atherosclerosis. Several viral infections are linked to hypertension, including human herpes virus 8 (HHV-8) and HIV-1. The mechanisms of how viral infection contributes to hypertension or increased blood pressure are not defined. In this report, the role of CMV infection as a cause of increased blood pressure and in forming aortic atherosclerotic plaques is examined. Using in vivo mouse model and in vitro molecular biology analyses, we find that CMV infection alone caused a significant increase in arterial blood pressure (ABp) (p<0.01∼0.05), measured by microtip catheter technique. This increase in blood pressure by mouse CMV (MCMV) was independent of atherosclerotic plaque formation in the aorta, defined by histological analyses. MCMV DNA was detected in blood vessel samples of viral infected mice but not in the control mice by nested PCR assay. MCMV significantly increased expression of pro-inflammatory cytokines IL-6, TNF-α, and MCP-1 in mouse serum by enzyme-linked immunosorbent assay (ELISA). Using quantitative real time reverse transcriptase PCR (Q-RT-PCR) and Western blot, we find that CMV stimulated expression of renin in mouse and human cells in an infectious dose-dependent manner. Co-staining and immunofluorescent microscopy analyses showed that MCMV infection stimulated renin expression at a single cell level. Further examination of angiotensin-II (Ang II) in mouse serum and arterial tissues with ELISA showed an increased expression of Ang II by MCMV infection. Consistent with the findings of the mouse trial, human CMV (HCMV) infection of blood vessel endothelial cells (EC) induced renin expression in a non-lytic infection manner. Viral replication kinetics and plaque formation assay showed that an active, CMV persistent infection in EC and expression of viral genes might underpin the molecular mechanism. These results show that CMV infection is a risk factor for increased arterial blood pressure, and is a co-factor in aortic atherosclerosis. Viral persistent infection of EC may underlie the mechanism. Control of CMV infection can be developed to restrict hypertension and atherosclerosis in the cardiovascular system.

Silencing p21 Waf1/Cip1/Sdi1 expression increases gene transduction efficiency in primitive human hematopoietic cells

Adult hematopoietic and other tissue stem cells have highly constrained cell cycling that limits their susceptibility to standard gene therapy vectors, which depend upon chromosomal integration. Using cytokine cocktails to increase transduction efficiency often compromises subsequent stem cell function in vivo. We previously showed that p21Waf1/Cip1/Sdi1 (p21) mediates stem cell quiescence in vivo and decreasing its expression ex vivo leads to an expansion of stem cell pool in vivo. Here, we report that application of p21 specific siRNA increased the gene transduction efficiency in hematopoietic stem cells while preserving cell multipotentiality. Both types of siRNA, synthesized siRNA and transcribed shRNA, reduced p21 expression in target cells by 85–98%. The effect of RNAi in these cells was transient and the level of p21 mRNA returned to base line 14–28 days after siRNA treatment. This brief interval of reduction, however, was sufficient to increase transduction efficiency to two- to four-fold in cell cultures, and followed by a seven- to eight-fold increase in mice. The RNAi treated, lentivector-transduced CD34+ cells retained multipotentiality as assessed in vitro by colony formation assay and in vivo by NOD/SCID mouse transplantation assay. Reduction of p21 resulted in an increased chromosomal integration of lentivector into target cellular DNA. Taken together, both synthesized and transcribed siRNA knocked down p21 expression in human CD34+ hematopoietic stem/progenitor cells. Silencing p21 expression increased gene transduction efficiency and vector integration while retaining stem cell multipotentiality. Thus, RNAi targeting of p21 is a useful strategy to increase stem cell gene transfer efficiency. Decreasing p21 expression transiently while increasing gene-transfer vector integration may ultimately facilitate clinical applications of gene therapy.

Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Nuclear Localization Mediates Early Viral mRNA Expression

ABSTRACT An important aspect of the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection is the ability of the virus to replicate in the host vigorously without a latent phase and to kill cells with a dynamic turnover of 1.8 × 109 cells/day and 10.3 × 109 virions/24 h. The transcription of HIV-1 RNA in acute infection occurs at two stages; the transcription of viral spliced mRNA occurs early, and the transcription of viral genomic RNA occurs later. The HIV-1 Tat protein is translated from the early spliced mRNA and is critical for HIV-1 genomic RNA expression. The cellular transcription factors are important for HIV-1 early spliced mRNA expression. In this study we show that virion nucleocapsid protein (NC) has a role in expression of HIV-1 early spliced mRNA. The HIV-1 NC migrates from the cytoplasm to the nucleus and accumulates in the nucleus at 18 h postinfection. Mutations on HIV-1 NC zinc fingers change the pattern of early viral spliced mRNA expression and result in a delayed expression of early viral mRNA in HIV-infected cells. This delayed HIV-1 early spliced mRNA expression occurs after proviral DNA has been integrated into the cellular genome, as shown by a quantitative integration assay. These results show that virion NC plays an important role in inducing HIV-1 early mRNA expression and contributes to the rapid viral replication that occurs during HIV-1 infection.

Human Immunodeficiency Virus Type 1 RNA in Peripheral Blood Mononuclear Cells of Patients Receiving Prolonged Highly Active Antiretroviral Therapy

The levels of early spliced mRNA and genomic RNA of human immunodeficiency virus (HIV) type 1 in peripheral blood mononuclear cells (PBMC) of 14 patients who were receiving highly active combination antiretroviral therapy for > or =116 weeks were determined. The level of viral genomic RNA was below the level of detection in the plasma of these patients (<50 copies/mL), but cell-associated viral tat, rev, and nef mRNA were detected in 86% (12 of 14) of the patients. Cell-associated viral genomic RNA was detected in 57% (8 of 14) of the patients. Early viral spliced mRNA was detected in the PBMC of all patients who had positive results of testing for HIV-1 genomic RNA, and the level of viral genomic RNA in these patients was 34-2214 copies per 10(6) cells.

A Passive Immunotherapy, PEHRG214, in Patients Infected with Human Immunodeficiency Virus: A Phase I Study

(PE)HRG214 (HRG) is a polyclonal antibody preparation produced by immunization of goats with purified human immunodeficiency virus (HIV) antigens. In this phase I study, HRG was administered intravenously as a single dose (1, 2, 4, 8, or 16 mg/kg) to 18 HIV-1-infected patients with CD4 cell counts >/=50 cells/microL and virus loads >/=500 copies/mL. The most frequent adverse event was a transient rash, which appeared to be both dose- and CD4 cell count-dependent. At the 16 mg/kg level, median half-life was 68.4 h, and median C(max) was 392 microg/mL, a level well above that which inhibits HIV in vitro. At that dose level, median and maximum decreases in HIV-1 RNA levels at day 8 were 0.24 log(10) and 0.58 log(10), respectively, and, at day 29, were 0.24 log(10 ) and 2.2 log(10), respectively. HRG, administered as a single dose, is reasonably well tolerated and achieves adequate plasma concentrations.

Hematopoietic stem and progenitor cells in HIV/AIDS and immune reconstitution

The human immunodeficiency virus type 1 (HIV-1) causes an acquired immunodeficiency syndrome (AIDS). HIV-1 infects human immune cells, specifically CD4+ lymphocytes, which leads to AIDS and undermines reconstitution of immunity. The unique challenges of HIV/AIDS have triggered multidisciplinary investigators to study the virology of the pathogen and the biology of the host cells, especially the interactions of HIV-1 with T-lymphocytes, macrophages, and hematopoietic stem and progenitor cells (HSPC) [1-8]. To study the possibility that HIV-1 can infect multipotent progenitor cells and cause cell death and latent infection in cellular reservoirs, Carter et al. [9] have used different clones of the DNA plasmid, p89.6 to infect CD34+ multipotent hematopoietic progenitor cells (HPCs). They have found that some HPCs were infected in cell culture and rapidly killed. Carter et al. conclude that these findings enhance the understanding of HIV bone marrow pathology and the mechanisms by which HIV causes persistent infection.

Hematopoietic Stem and Immune Cells in Chronic HIV Infection.

Hematopoietic stem cell (HSC) belongs to multipotent adult somatic stem cells. A single HSC can reconstitute the entire blood system via self-renewal, differentiation into all lineages of blood cells, and replenishment of cells lost due to attrition or disease in a persons lifetime. Although all blood and immune cells derive from HSC, immune cells, specifically immune memory cells, have the properties of HSC on self-renewal and differentiation into lineage effector cells responding to the invading pathogens. Moreover, the interplay between immune memory cell and viral pathogen determines the course of a viral infection. Here, we state our point of view on the role of blood stem and progenitor cell in chronic HIV infection, with a focus on memory CD4 T-cell in the context of HIV/AIDS eradication and cure.

Naive and Memory CD4+ T Cells in HIV Eradication and Immunization

TO THE EDITOR—The recent article by Lewis and colleagues [1] on age and CD4 count at initiation of antiretroviral therapy (ART) in human immunodeficiency virus (HIV)–infected children is an important contribution. The study shows that memory CD4 T cells (TMC) increase more rapidly than naive CD4 T cells, and that the current guidelines on initiating ART may not optimize longterm immunological health of patients. If the study had included more data on the substudy of TMC, they might have observed the important contribution of memory cells to immune reconstitution. We suggest that a further analysis on the memory subpopulation could provide strong evidence on deciding when to initiate ART and, therefore, might result in long-term immunological health. It is well known that following the resolution of infection, or after successful vaccination, most virus-specific effector CD4 T cells die. This leaves a small population of memory T cells, which ensures that the frequency of virus-specific T cells is greater than it was before priming. In HIV infection, however, after the first viremia, fewer HIV-specific TMC are left, due to viral direct infection of these cells, in addition to programmed effector cell death. Thus, the population of TMC in the first and second viremia determines the anti-HIV activity in an individual, whether in children or adults or in progressors or nonprogressors [2–7]. Studying the activity of TMC during ART, as shown in this study, and coordinating TMC activity with the early viremia will be a key to deciding when to initiate ART to benefit patients by reconstitution of anti-HIV immunity. TMC orchestrate innate, humoral, and cellular immunity to clear the invading pathogen. Compared to native CD4 cells, TMC have superior capacity to launch a protective response against the invading pathogen in respect to speed, specificity, and recruiting B and CD8 cells against a specific pathogen. Following the programmed fate of immune cells, after a rapid and effective antiviral response (as in the first viremia of HIV infection), infection is resolved and the majority of effector CD4 T cells die, leaving a much smaller population of memory CD4 TMC that persist [4–7]. This process occurs rapidly, since the immune system is poised to abruptly shift from the effector mode to the more benign memory mode shortly after the resolution of a primary response. This rapid shift can itself be pathogenic, however, depending on the clonal development and the specific repertoire of a TMC pool. A balanced or more benign TMC population may never develop in this short interval, provided that the naive T-cell pool is exhausted or destroyed, as occurs in HIV infection. Determining the status of TMC in an HIV infection, particularly in the first and second period of viremia, allows evaluation of the potential contribution of these cells to anti-HIV immunity. Upon encountering a previously met pathogen, TMC orchestrate multilevel immune responses to destroy a targeted pathogen. The memory of T MC underpins this multilevel antiviral immunity. The secondary effectors derived from TMC precursors are more capable of mediating direct antiviral activity than primary effectors derived from naive CD4Tcells [8]. Importantly, the two types of effector cells, derived from TMC or naive T cells, are distinguishable functionallyandphenotypically.How toutilize the memory of TMC to restore or to reconstitute patient anti-HIV immunity after ART is not only a keystone for efficacy of ART but also for protective vaccination against and eradication of HIV. Despite its complexity, TMC is probably the best-understood immune cell with stem cell properties [9, 10]. Although the population of TMC diminishes with time and may require boosting, our knowledge and techniques developed from the study of hematopoietic stem cells and humanized mouse models may lead to rapid progress in harnessing the memory of TMC to reconstitute patient anti-HIV immunity, and to develop AIDS vaccines that synergize with ART to eradicate HIV.

Toward a Cure: Does Host Immunity Play a Role?

The decades of research on HIV and AIDS has contributed to an explosion of knowledge on immune responses. Pivoting on it, we reveal a host genome’s immune system that is defined as epigenetic immunity, utilizing it to protect eukaryotic DNA against the HIV infection to reprogram the patient’s immunity and to develop successful vaccines toward a cure for AIDS. ABSTRACT Three decades of research on human immunodeficiency virus (HIV) and AIDS reveal that the human body has developed through evolution a genome immune system embodying epigenetic regulation against pathogenic nucleic acid invasion. In HIV infection, this epigenetic regulation plays a cardinal role in HIV RNA production that silences HIV transcription at a molecular (RNA) level, controls viral load at a cellular (biological) level, and governs the viremic stage of AIDS at the clinical (patient) level. Even though the human genome is largely similar among humans and HIV is a single viral species, human hosts show significant differences in viral RNA levels, ranging from cell to organ to individual and expressed as elite controllers, posttreatment controllers, and patients with AIDS. These are signature biomarkers of typical epigenetic regulation whose importance has been shunted aside by interpreting all of AIDS pathogenesis by the known properties of innate and adaptive immunity. We propose that harnessing the host genome immune system, defined as epigenetic immunity, against HIV infection will lead toward a cure.