Tissue Reservoirs Research Articles

Polyomavirus JC in the Context of Immunosuppression: A Series of Adaptive, DNA Replication-Driven Recombination Events in the Development of Progressive Multifocal Leukoencephalopathy

Polyomavirus JC (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML), a demyelinating infection of oligodendrocytes in the brain. PML, a frequently fatal opportunistic infection in AIDS, has also emerged as a consequence of treatment with several new immunosuppressive therapeutic agents. Although nearly 80% of adults are seropositive, JCV attains an ability to infect glial cells in only a minority of people. Data suggest that JCV undergoes sequence alterations that accompany this ability, and these changes can be derived from an archetype strain by mutation, deletion, and duplication. While the introductory source and primary tissue reservoir of JCV remain unknown, lymphoid cells have been identified as potential intermediaries in progression of JCV to the brain. This review is focused on sequence changes in the noncoding control region (NCCR) of the virus. We propose an adaptive mechanism that involves a sequential series of DNA replication-driven NCCR recombination events involving stalled DNA replication forks at NCCR palindromic secondary structures. We shall describe how the NCCR sequence changes point to a model in which viral DNA replication drives NCCR recombination, allowing JCV adaptation to different cell types in its progression to neurovirulence.

In Vivo Anti-HIV Activity of the Heparin-Activated Serine Protease Inhibitor Antithrombin III Encapsulated in Lymph-Targeting Immunoliposomes

Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Several serpins have been reported to modulate HIV pathogenesis, or exhibit potent anti-HIV activity in vitro, but the efficacy of serpins as therapeutic agents for HIV in vivo has not yet been demonstrated. In the present study, we show that heparin-activated antithrombin III (hep-ATIII), a member of the serpin family, significantly inhibits lentiviral replication in a non-human primate model. We further demonstrate greater than one log10 reduction in plasma viremia in the nonhuman primate system by loading of hep-ATIII into anti-HLA-DR immunoliposomes, which target tissue reservoirs of viral replication. We also demonstrate the utility of hep-ATIIII as a potential salvage agent for HIV strains resistant to standard anti-retroviral treatment. Finally, we applied gene-expression arrays to analyze hep-ATIII-induced host cell interactomes and found that downstream of hep-ATIII, two independent gene networks were modulated by host factors prostaglandin synthetase-2, ERK1/2 and NFκB. Ultimately, understanding how serpins, such as hep-ATIII, regulate host responses during HIV infection may reveal new avenues for therapeutic intervention.

HAART roll‐out in the new fiscal and economic environment

There are 27 approved antiretroviral drugs and combinations of these drugs clearly change the natural history of HIV infection, dramatically decreasing HIV‐related morbidity and mortality and promoting healthy survival. Despite the proven benefits, we continue to consider basic strategic questions about antiretroviral therapy: When should we start ART? What regimen to start? When should we change ART? What ART regimen should we change to?Also, despite the benefits of treatment, some antiretroviral regimens may be inconvenient, toxic, and/or have suboptimal antiretroviral activity, particularly against drug‐resistant viruses. Thus, newer compounds are needed that continue to improve convenience and tolerability, reduce toxicity, and improve antiretroviral activity, particularly against drug‐resistant viruses. Additionally, new drugs may better penetrate tissue reservoirs (e.g. genital tract, central nervous system) or exploit new targets with new mechanisms of action.There are a number of HIV drugs in development currently. These include a new pharmacokinetic “boosting” agent and newer antiretroviral agents in a number of existing classes, including new nucleoside reverse transcriptase inhibitors, non‐nucleoside reverse transcriptase inhibitors, protease inhibitors, entry inhibitors, and integrase inhibitors. In addition there are drugs with new mechanisms of action in development, including the CD4 attachment inhibitors. The clinical use of the newer agents will depend on the results of clinical trials, and the timeline for development and availability.

A Single-Amino-Acid Change in Murine Norovirus NS1/2 Is Sufficient for Colonic Tropism and Persistence

Human norovirus (HuNoV) is the major cause of acute nonbacterial gastroenteritis worldwide but has no clear animal reservoir. HuNoV can persist after the resolution of symptoms, and this persistence may be essential for viral maintenance within the population. Many strains of the related murine norovirus (MNV) also persist, providing a tractable animal model for studying norovirus (NoV) persistence. We have used recombinant cDNA clones of representative persistent (CR6) and nonpersistent (CW3) strains to identify a domain within the nonstructural gene NS1/2 that is necessary and sufficient for persistence. Furthermore, we found that a single change of aspartic acid to glutamic acid in CW3 NS1/2 was sufficient for persistence. This same conservative change also caused increased growth of CW3 in the proximal colon, which we found to be a major tissue reservoir of MNV persistence, suggesting that NS1/2 determines viral tropism that is necessary for persistence. These findings represent the first identified function for NoV NS1/2 during infection and establish a novel model system for the study of enteric viral persistence.

The subcutaneous adipose tissue reservoir of functionally active stem cells is reduced in obese patients

It has been demonstrated that the adipose tissue, a highly functional metabolic tissue, is a reservoir of mesenchymal stem cells. The potential use of adipose-derived stem cells (ADSCs) from white adipose tissue (WAT) for organ repair and regeneration has been considered because of their obvious benefits in terms of accessibility and quantity of available sample. However, the functional capability of ADSCs from subjects with different adiposity has not been investigated. It has been our hypothesis that ADSCs from adipose tissue of patients with metabolic syndrome and high adiposity may be functionally impaired. We report that subcutaneous WAT stromal vascular fraction (SVF) from nonobese individuals had a significantly higher number of CD90+ cells than SVF from obese patients. The isolated ADSCs from WAT of obese patients had reduced differentiation potential and were less proangiogenic. Therefore, ADSCs in adipose tissue of obese patients have lower capacity for spontaneous or therapeutic repair than ADSCs from nonobese metabolically normal individuals.

Towards a cure for HIV: a long road ahead

Plasma viremia can be effectively suppressed and maintained below the limits of detection for extended periods of time in most human immunodeficiency virus (HIV)–infected individuals receiving antiretroviral therapy (ART). However, it has not been possible to eradicate HIV by ART alone, likely due in part to the persistence of various viral reservoirs in lymphoid tissues. In this regard, the existence of latently infected, resting CD4+ T cells carrying replication-competent HIV has posed one of the major challenges to the long-term control or eradication of HIV in infected individuals on ART. Consequently, there has been considerable focus on therapeutic strategies to reactivate the latent viral reservoir using various agents, such as cytokines, histone deacetylase inhibitors, and mitogens, under the assumption that these cells would die due to HIV induced cytopathic effects and antiretroviral drugs would prevent spread of infection. However, such approaches have shown no clinical benefit to date. Moreover, it also has become clear that HIV persists in subsets of CD4+ T cells in blood and lymphoid tissues of infected individuals receiving ART. Recent data from our laboratory will be discussed which will include potential mechanisms of HIV persistence and prospects for eradication and new therapeutic approaches in HIV-infected individuals receiving effective ART.

Plasma HIV-1 RNA Levels During Antiretroviral Therapy: How Low Is Low Enough?

Plasma HIV-1 RNA Levels During Antiretroviral Therapy: How Low Is Low Enough?

New tools for quantifying HIV-1 reservoirs: plasma RNA single copy assays and beyond.

Quantification of plasma HIV-1 RNA below the limit of FDA-approved assays by a single copy quantitative PCR assays (SCA) has provided significant insights into HIV-1 persistence despite potent antiretroviral therapy as well as a means to assess the impact of therapeutic strategies, such as treatment intensification, on residual viremia. In this review, we discuss insights gained from plasma HIV-1 RNA SCA and highlight the need for additional assays to characterize better the cellular and tissue reservoirs of HIV-1. Accurate, reproducible, and sensitive assays to quantify HIV-1 reservoirs, before and after therapeutic interventions, are essential tools in the quest for a cure of HIV-1 infection.

Global HIV/AIDS Clinical and Translational Pharmacology

Global HIV/AIDS Clinical and Translational Pharmacology

Relationship between body mass index and serum 25-hydroxyvitamin D stronger among Caucasians than African Americans in NHANES adults 2001-2006

The rapid decline in circulating levels of serum 25-hydroxyvitamin D in both African Americans and Caucasians in the US population remains unexplained, and appears to parallel the obesity epidemic. The cross sectional data on 7349 Caucasian and African American adults between 21 and 69 years of age from the continuous National Health and Nutrition Examination Study (NHANES) 2001 to 2006 were used to model by race, with smoothing functions, the true relationship between body mass index (BMI), waist circumference and serum 25-hydroxyvitamin D in men and women. Weighted regressions of determinants of serum 25-hydroxyvitamin D levels were analyzed. Serum 25-hydroxyvitamin D is negatively associated with BMI linearly above an inflection point at 20 kg/m2 and positively associated below a BMI of 20 kg/m2. The gender- and age-adjusted regression coefficients of BMI on 25-hydroxyvitamin D levels demonstrate a 50% lower coefficient (r = –0.18 ng/ml/ BMI unit) among African Americans than among Caucasians (r = –0.36 ng/ml/BMI unit). These relationships were as great in men as in women and were replicated when waist circumference was used as a surrogate for visceral fat levels. The extent to which BMI is a strong predictor of serum 25-hydroxyvitamin D levels depends upon BMI being greater than 20 kg/m2. The hockey stick shape of the BMI 25-hydroxyvitamin D relationship needs to be taken into account when adjusting serum values for BMI. Both this inflection and reduction in serum levels of 25-hydroxyvitamin D in the population may be due to sequestration in an increasing adipose tissue reservoir. The interpretation of serum 25-hydroxyvitamin D levels as a biomarker of vitamin D adequacy requires appropriate adjustment of body fat mass.

Impact of treatment with raltegravir during primary or chronic HIV infection on RNA decay characteristics and the HIV viral reservoir

Eradication of HIV-1 is prevented by the formation of viral reservoirs in peripheral blood, lymphoid tissues and other sanctuary sites. In most patients, rebound upon treatment cessation is prompt. We assessed whether early treatment with raltegravir can impact on the formation of the viral reservoir. We conducted an open-label, nonrandomized study, and assessed in detail the decay characteristics of HIV-1 RNA in plasma, HIV DNA in CD4 T cells and colon tissue biopies (CTBs) in 16 treatment-naive patients during either primary (PHI, n = 8) or chronic (CHI, n = 8) HIV-1 infection after treatment with raltegravir and Truvada for 52 weeks. HIV-1 RNA decreased rapidly with treatment in all patients; first and second phase levels were lower in PHI patients with no appreciable difference in residual viremia between the two groups at 52 weeks. Episomal HIV-1 DNA increased sharply in both groups with peak levels at 3-4 weeks. Total HIV-1 DNA levels were reduced in both groups with similar kinetics, but were markedly lower in PHI patients after 52 weeks. Integrated HIV-1 DNA levels were significantly lower at baseline in PHI patients and this difference widened on treatment. Finally, total HIV-1 DNA decayed substantially in both groups in CTB. Treatment with raltegravir resulted in a large number of abrogated integration events, reflected by the increase of episomal HIV-1 DNA after treatment initiation. Levels of total and integrated HIV-1 DNA were lower in PHI patients at the end of the study period.

Latent HIV-1 Infection of Resting CD4 + T Cells in the Humanized Rag2 −/− γ c −/− Mouse

Persistent human immunodeficiency virus type 1 (HIV-1) infection of resting CD4⁺ T cells, unaffected by antiretroviral therapy (ART), provides a long-lived reservoir of HIV infection. Therapies that target this viral reservoir are needed to eradicate HIV-1 infection. A small-animal model that recapitulates HIV-1 latency in resting CD4⁺ T cells may accelerate drug discovery and allow the rational design of nonhuman primate (NHP) or human studies. We report that in humanized Rag2⁻/⁻ γ(c)⁻/⁻ (hu-Rag2⁻/⁻ γ(c)⁻/⁻) mice, as in humans, resting CD4⁺ T cell infection (RCI) can be quantitated in pooled samples of circulating cells and tissue reservoirs (e.g., lymph node, spleen, bone marrow) following HIV-1 infection with the CCR5-tropic JR-CSF strain and suppression of viremia by ART. Replication-competent virus was recovered from pooled resting CD4⁺ T cells in 7 of 16 mice, with a median frequency of 8 (range, 2 to 12) infected cells per million T cells, demonstrating that HIV-1 infection can persist despite ART in the resting CD4⁺ T cell reservoir of hu-Rag2⁻/⁻ γ(c)⁻/⁻ mice. This model will allow rapid preliminary assessments of novel eradication approaches and combinatorial strategies that may be challenging to perform in the NHP model or in humans, as well as a rigorous analysis of the effect of these interventions in specific anatomical compartments.

Prior Virus Exposure Alters the Long-Term Landscape of Viral Replication during Feline Lentiviral Infection

We developed a feline model of lentiviral cross-species transmission using a puma lentivirus (PLV or FIVPco) which infects domestic cats but does not cause disease. Infection with PLV protects cats from CD4+ T-cell decline caused by subsequent infection with virulent feline immunodeficiency virus (FIV). Previous studies implicate innate immune and/or cellular restriction mechanisms for FIV disease attenuation in PLV-infected cats. In this study, we evaluated viral infection and cytokine mRNA transcription in 12 different tissue reservoirs approximately five months post infection. We quantitated tissue proviral load, viral mRNA load and relative transcription of IL-10, IL-12p40 and IFNγ from tissues of cats exposed to FIV, PLV or both viruses and analyzed these parameters using a multivariate statistical approach. The distribution and intensity of FIV infection and IFNγ transcription differed between single and co-infected cats, characterized by higher FIV proviral loads and IFNγ expression in co-infected cat tissues. Variability in FIV mRNA load and IFNγ was significantly more constrained in co-infected versus singly infected cat tissues. Single-infected:co-infected ratios of FIV mRNA load compared to FIV proviral load indicated that active viral transcription was apparently inhibited during co-infection. These results indicate that previous PLV infection increases activation of tissue innate immunity and constrains the ability of FIV to productively infect tissue reservoirs of infection for months, independent of FIV proviral load, supporting a model in which innate immunity and/or modulation of target cell susceptibility play a key role in PLV-induced protection from FIV disease.

Phase I trial of 7-hydroxystaurosporine and fludararbine phosphate: in vivo evidence of 7-hydroxystaurosporine induced apoptosis in chronic lymphocytic leukemia

This is a phase I study of 7-hydroxystaurosporine (UCN-01) and fludararbine monophosphate (FAMP) in relapsed lymphoma. UCN-01 alone was administered in cycle 1 and with FAMP in cycles 2–6. FAMP was escalated in cohorts from 1 to 5 days. UCN-01 and FAMP pharmacokinetics and apoptosis of malignant lymphocytes was evaluated. Eighteen patients were enrolled. Standard FAMP with UCN-01 was tolerated without dose-limiting toxicity (DLT) and those seen were common to either agent alone. One patient died due to Stevens-Johnson syndrome. Seven of 18 patients responded. No pharmacological effect of UCN-01 by FAMP was noted. Lymphocytosis occurred in 15 of 18 patients following UCN-01 to paradoxically increase circulating tumor cells. UCN-01 induced apoptosis in six of eight patients with chronic lymphocytic leukemia (CLL). UCN-01 does not increase FAMP toxicity. Transient lymphocytosis followed by apoptosis occurs with UCN-01. Mobilization from tissue reservoirs may play a role in the induction of cell death in malignant lymphocytes.

Greater diversity of HIV DNA variants in the rectum compared to variants in the blood in patients without HAART

The gut-associated lymphoid tissue represents the largest reservoir of HIV-1. Improving knowledge of this reservoir by studying the diversity of viral population is a key step towards understanding the pathogenesis and dynamics of HIV. Obtaining samples is difficult and little information is available on gut viral quasispecies during the course of infection in humans. The aim of this study was to characterize rectal viral strains and their diversity and to investigate the relationships between the rectal tissue reservoir and viral variants in the blood. Phylogenetic analyses were performed on the env sequences for rectal HIV DNA, blood HIV DNA, and HIV RNA clones, with maximum-likelihood and neighbor-joining methods on seven patients. Genetic diversity was assessed. Higher diversity of HIV DNA clones was noted in the rectum compared to blood in four out of five patients without HAART. Viral diversity was present in the rectum from time of the primary infection. Similar degrees of diversity were observed in the rectum and blood during HAART. Rectal and blood HIV variants were interspersed partially or totally in the seven patients. A certain number of rectal HIV DNA clones were clustered together in six patients. These results suggest that variants in the rectum were more heterogeneous than variants in the blood from patients without HAART, probably because the activated milieu of gut-associated lymphoid tissue may provide an improved environment for viral replication, and indicate exchange of viral populations between blood and rectal tissues, reflecting the dynamics of HIV during course of infection.

Establishment of a lung cancer biobank of a southern chinese population.

There is an increasing need for establishment of biobanks for human cancer, which may facilitate basic and clinical researches as well as the development of novel approaches to early diagnostics, prevention and treatment, including personalized medicine. Herein we report the establishment of a lung cancer biobanks using biological samples from a lung cancer patient population in Southern China. Since 2007, we have collected lung cancer tissue from 1,054 patients with lung cancer, from whom 11,895 frozen tumor and normal matched tissues, 4,899 tissue paraffin blocks, and 3,562 blood serum samples were accumulated. The information on clinical manifestations, laboratory tests, and follow-up was maintained by an independent information management system, including three data sets. The primary data set included frozen tissue specimens, formalin-fixed paraffin-embedded tissues, blood specimens and clinical and long-term follow-up data. The secondary data set included DNA, RNA and proteins extracted from corresponding tissue specimens. And the tertiary data set contained improved genome, RNA and proteomics correlation analysis with relevant clinical and long-term follow-up data. The lung cancer biobanks is accessible to academic research and public services. To our best knowledge, this biobanks represents the first lung cancer tissue reservoir in China and should facilitate the basic and clinical research of this disease and developing diagnostic markers and novel therapeutic modalities.

Evaluation of the Roche COBAS® TaqMan® HIV-1 test for quantifying HIV-1 RNA in infected cells and lymphoid tissue

Lymphoid tissue is the main reservoir of HIV-1 in infected individuals. In this study the COBAS® TaqMan® HIV-1 test was evaluated for use with the High Pure System (HPS), for quantifying HIV-1 RNA in infected cells and lymphoid tissue specimens. Serial dilutions of 8E5–LAV1 infected T-cells into SUP-T1 cells and 44 tonsil specimens were examined. Some modifications of the test were required, such as the removal of residual DNA and the HIV-1 RNA output copies were adjusted to the sample input and expressed as HIV-1 RNA copies/μg of total RNA. The Roche COBAS® TaqMan HIV-1® (HPS) test proved to be a robust, sensitive, specific and reproducible method for quantifying HIV-1 RNA in infected cells and lymphoid tissue. Linearity and reproducibility were observed in serial dilutions of 8E5–LAV1 infected T-cells (R2>0.86). High reproducibility was found in clinical tonsil specimens (Wilcoxon test p>0.05). rDNase I treatment was essential to avoid false positives caused by residual HIV-1 DNA, mainly in tonsil specimens obtained from infected patients receiving effective antiretroviral treatment. Probit analysis determined the limit of detection as 22HIV-1 RNA copies/μg of total RNA. The Roche COBAS® TaqMan® HIV-1 (HPS) test thereby proved to be a helpful tool for measuring the HIV-1 viral load in infected cells and lymphoid tissue reservoirs.

Eradication of HIV from the brain: reasons for pause

Recent advances in HIV research have made it possible to scientists to consider approaches to eradicate HIV infection from T cell reservoirs. These include either engineering cells that would be resistant to HIV infection of suppression of HIV replication with intensified antiretroviral therapy followed by activation of HIV reservoirs and elimination of these cells by cytotoxic immune responses. Several variations of this theme are being considered. Careful consideration needs to be given to the viral reservoirs in the brain, where it may infect cell types such as macrophages, microglia and astrocytes. The life cycle of the virus in these cells is distinct compared to that in T cells and thus the antiviral approaches may not be effective in fully suppressing viral replication. Even small amounts of viral proteins produced by these cells can be neurotoxic. And if T cells in the periphery are restored before impacting the brain reservoirs these cells may enter the brain causing a T cell mediated encephalitis. This articlediscusses thefundamentalquestionsthatneedto beaddressedwithregards tothe biology of the virus in the brain before successful attempts to eradicate the virus can be made. 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins AIDS 2011, 25:000‐000

Efficacy of Tat-Conjugated Ritonavir-Loaded Nanoparticles in Reducing HIV-1 Replication in Monocyte-Derived Macrophages and Cytocompatibility with Macrophages and Human Neurons

Human immunodeficiency virus (HIV)-1 targets mononuclear phagocytes (MP), which disseminate infection to organs such as brain, spleen and lymph. Thus MP, which include microglia, tissue macrophages and infiltrating monocyte-derived macrophages (MDM), are important target of anti-HIV-1 drug therapy. Most of the currently used antiretroviral drugs are effective in reducing viral loadin the periphery but cannot effectively eradicate infection from tissue reservoirs such as brain MP. HIV-1 infection of the central nervous system can lead to a wide variety of HIV-1-associated neurocognitive disorders. In this study, we demonstrate that ritonavir-loaded nanoparticles (RNPs) are effective in inhibiting HIV-1 infection of MDM. Reduced infection is observed in multiple read-out systems including reduction of cytopathic effects, HIV-1 p24 protein secretion and production of progeny virions. Furthermore, the RNPs retained antiretroviral efficacy after being removed from MDM cultures. As HIV-1-infected cells in the brain are likely to survive for a long period of time, both acute and chronic infection paradigms were evaluated. Tat-peptide-conjugated RNPs (Tat-RNP) were effective in both short-term and long-term HIV-1-infected MDM. Importantly, we confirm that delivery of RNPs, both with and without Tat-peptide conjugation, is toxic neither to MDM nor to neural cells, which may be bystander targets of the nanoformulations. In conclusion, Tat-NPs could be a safe and effective way of delivering anti-HIV-1 drugs for controlling viral replication occurring within brain MP.

Drought effects on water relations in beech: The contribution of exchangeable water reservoirs

The exceptional soil drought and heat wave that occurred in Europe in summer 2003 provided a good opportunity to analyze the response of forest ecosystems to extreme climatic conditions. This work aimed at studying the functioning of exchangeable water reservoirs of beech trees under climatic and edaphic constraints, in a 37-year-old beech stand located in north-eastern France. We characterised the impact of drought on seasonal variations in water fluxes at the tree (sap flow measurements) and the forest (eddy covariance measurements) scales and estimated (i) the daily water storage capacity of beech trees as the difference between stand-scaled sap flow and water vapour flux over the stand and (ii) the contribution of exchangeable water in tree reservoirs to the total tree transpiration, under non-limiting soil water (summer 2002) or severe soil drought conditions (summer 2003). In parallel, daily variations in trunk circumference were analyzed to estimate the contribution of elastic tissues as a compartment for water storage to tree transpiration. Stand transpiration was strongly reduced by soil water shortage (up to 80% at the peak of drought in August 2003). From the beginning of August 2003, we observed daytime contraction of stem circumference, with only partial, or even no night recovery, suggesting that trees were less and less able to refill the elastic and strongly depleted reservoirs. Even if those elastic reservoirs were active throughout the season, the corresponding volume of water withdrawn for tree transpiration remained very low (maximum 1% of the daily transpiration). Thus, elastic tissue reservoirs play a minor role in the total water budget of beech trees. The amount of water depleted daily from the whole reservoirs was much higher than the water extracted from elastic tissues. Furthermore, the contribution of total exchangeable water within trees to the transpiration stream increased during the dry period (from a few percent to 67% at the peak of the drought) and was positively correlated with soil water shortage. Our results thus clearly demonstrated the strong sensitivity of beech to both climate and drought and the major role of whole tree water reservoirs to maintain leaf transpiration under severe drought.