The value of primate models for studying human immunodeficiency virus pathogenesis

Abstract: Research on human immunodeficiency virus (HIV) infection is compromised by the obvious limitation in having for study only virus-infected individuals or those exposed to the virus. Steps involved in transmission or pathogenesis require planned experimentation. The identification of animal models of acquired immunodeficiency syndrome (AIDS) has therefore been helpful for evaluating phases of HIV pathogenesis. Of the seven subgenera of lentiviruses now recognized, two share the characteristics with HIV of a T cell tropism and the associated loss of CD4+ cells in the host associated with disease: the feline immunodeficiency virus (FIV) and the simian immunodeficiency virus (SIV) (Table 1). The other animal lentiviruses grow best in macrophages and their infection generally reflects clinical sequellae of infection of this cell type. This review addresses those features of SIV, HIV, and SHIV infections of non-human primates that illustrate the importance of the animal models of AIDS.     

Establishment of AIDS Animal Model with SIVmac239 Infected Chinese Rhesus Monkey

In the present research,two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4 T lymphocyte in peripheral blood,plasma viral loads,proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection,proviral DNA had been detected in PBMCs,and infectious SIVmac239 virus had been isolated from PBMCs. At the same period,the numbers of CD4 T lymphocytes were significantly decreased,and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover,antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches.     

SHIV transmission and susceptibility to re-exposure through social contact following vaccination with an HIV synthetic peptide-cocktail: a case study

Abstract: An effective vaccine against human immunodeficiency virus (HIV) should not only protect from infection and development of acquired immunodeficiency syndrome (AIDS), but also prevent potential transmission to naïve partners. We recently reported protection of rhesus macaques from chronic simian‐human immunodeficiency virus (SHIV) infection and AIDS by an HIV envelope peptide‐cocktail vaccine. In the present case study, we observed that one of the vaccinated females, with undetectable circulating virus, when housed in a pair with a naïve male, did not transmit the infection over a 35‐week period of social contact. Subsequent experimental challenge of the male with the same SHIV strain resulted in high‐level infection and transmission to its female cage‐mate. However, the virus was undetectable in the female by 12 weeks without further vaccination, validating the multivalent peptide cocktail vaccine approach in the SHIV‐rhesus model, and suggesting its potential utility as an HIV vaccine strategy for humans.     

Molecular typing of major histocompatibility complex class I alleles in the Indian rhesus macaque which restrict SIV CD8<Superscript>+</Superscript> T cell epitopes

The utility of the rhesus macaque as an animal model in both HIV vaccine development and pathogenesis studies necessitates the development of accurate and efficient major histocompatibility complex (MHC) genotyping technologies. In this paper, we describe the development and application of allele-specific polymerase chain reaction (PCR) amplification for the simultaneous detection of eight MHC class I alleles from the rhesus macaque (Macaca mulatta) of Indian descent. These alleles were selected, as they have been implicated in the restriction of CD8(+) T cell epitopes of simian immunodeficiency virus (SIV). Molecular typing of Mamu-A 01, Mamu-A 02, Mamu-A 08, Mamu-A 11, Mamu-B 01, Mamu-B 03, Mamu-B 04, and Mamu-B 17 was conducted in a high throughput fashion using genomic DNA. Our amplification strategy included a conserved internal control target to minimize false negative results and can be completed in less than 5 h. We have genotyped over 4,000 animals to establish allele frequencies from colonies all over the western hemisphere. The ability to identify MHC-defined rhesus macaques will greatly enhance investigation of the immune responses, which are responsible for the control of viral replication. Furthermore, application of this technically simple and accurate typing method should facilitate selection, utilization, and breeding of rhesus macaques for AIDS virus pathogenesis and vaccine studies.     

Age-dependent changes in T cell homeostasis and SIV load in sooty mangabeys

Sooty mangabeys ( Cercocebus atys ) showed age-dependent changes in T cell regeneration. Younger animals had a high percentage of CD4+ CD45RA+ T cells and a high concentration of T cell receptor excisional circles (TRECs) in peripheral blood, which indicated active thymopoiesis. In contrast, older animals had an increased T cell turnover, which suggested that most T cell production occurred in the periphery. In addition, the number of peripheral CD4+ T cells naturally decreased with age. Non-pathogenic SIVsm infection did not significantly change the T cell proliferation rate or the TREC concentration, though it did cause a moderate loss of peripheral CD4+ T cells. The viral load correlated negatively with age, which could be accounted for by the reduced availability of CD4+ target cells in older mangabeys. Thus, the number of susceptible target cells may be a limiting factor in natural SIV infection.     

A new in vitro model for stem cell differentiation and interaction

Development involves an interplay between various cell types from their birth to their disappearance by differentiation, migration, or death. Analyzing these interactions provides insights into their roles during the formation of a new organism. As a study tool for these interactions, we have created a model based on embryoid bodies (EBs) generated from mouse embryonic stem (mES) cells, which can be used to visualize the differentiation of mES cells into specific cell types while at the same time allowing controlled removal of this same cell population using an enzyme–prodrug approach. Cell-specific expression of Cre induces a switch of EGFP expression to LacZ. Furthermore, it leads to the expression of nitroreductase (NTR), which in combination with the prodrug CB1954 induces apoptosis. Here, we validate this model by showing expression of LacZ and NTR after Cre-mediated recombination. Additionally we show, as an example, that we can target the endothelial cells in EBs using the Tie-2 promoter driving Cre. Ablating Cre-expressing cells by adding CB1954 to the culture led to an abrogated vascular formation. This system can easily be adapted to determine the fate and interaction of many different cell types provided that there is a cell-type-specific promoter available.     

Mechanisms of protection induced by live attenuated simian immunodeficiency virus: III. Viral interference and the role of CD8+ T-cells and beta-chemokines in the inhibition of virus infection of PBMCs in vitro

In this study, we investigated whether a type of retroviral interference might be one mechanism that mediates the powerful protection induced by live attenuated SIVC8. Our results show that retroviral interference could be demonstrated between SIV and SHIV-HXBc2 in human T-cell lines chronically infected with either SIVC8 or SIVJ5. Lymphocytes from macaques infected with live attenuated SIVC8 were significantly less sensitive (P < 0.05) to in vitro infection by virulent SIVJ5 and SHIV-HXBc2 than were lymphocytes from naive controls. However, this significant difference in the sensitivity of lymphocytes to virus infection was not observed for more efficiently replicating viruses such as SHIVSF33 and SIVsm3. Virus growth was significantly enhanced (P < 0.01) by depletion of CD8+ T-cells, suggesting a role for these cells in the control of SIV replication, both in vitro and in vivo. We found that levels of the beta-chemokines regulated upon activation, normal T-cell expressed and secreted, macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta did not correlate with inhibition of virus replication. Taken together, our findings do not support the hypothesis that retroviral interference is the mechanism by which live attenuated SIVC8 induces protection.     

RNA-based gene therapy for the treatment and prevention of HIV: from bench to bedside

Gene therapy is considered a feasible approach for the treatment and prevention of HIV/AIDS. Targeting both viral genes and host dependency factors can interfere with the viral lifecycle and prevent viral replication. A number of approaches have been taken to target these genes, including ribozymes, aptamers, and RNAi based therapies. A number of these therapies are now beginning to make their way into clinical trials and providing proof of principle that gene therapy is a safe and realistic option for treating HIV. Here, we focus on those therapies that have progressed along the pipeline to preclinical and clinical testing.     

In situ stem cell therapy: novel targets, familiar challenges

Tissue engineering approaches for expanding, differentiating and engrafting embryonic or adult stem cells have significant potential for tissue repair but harnessing endogenous stem cell populations offers numerous advantages over these approaches. There has been rapid basic biological progress in the identification of stem cell niches throughout the body and the molecular factors that regulate their function. These niches represent novel therapeutic targets and efforts to use them involve the familiar challenges of delivering molecular medicines in vivo. Here we review recent progress in the use of genes, proteins and small molecules for in situ stem cell control and manipulation, with a focus on using stem cells of the central nervous system for neuroregeneration.     

Lessons for the stem cell discourse from the gene therapy experience

The discovery of human embryonic stem cells has pointed to the potential use of these cells in developing new approaches to therapy for many major human diseases. While there is general agreement that such applications are possible and will require a great deal of additional basic and preclinical research, some discussions of the therapeutic applications of human embryonic stem cells have been characterized by the kinds of exaggerations and elevated expectations that characterized the field of human gene therapy a decade ago. In the case of gene therapy, public perception of and confidence in the field were damaged by the hype. Most unfortunate of all, the hopes of patients and their advocates were disappointed. The eventual success of a gene therapy approach, albeit one still plagued by serious adverse events, has come through scientific advance and careful clinical application. The probable eventual use of human embryonic stem cells for therapy of human disease will also require thorough basic and clinical research, but that goal is endangered by the current level of inaccurate representations and undeliverable promises.     

In vitro and in vivo activity of soluble cross-linked uricase-albumin polymers: A model for enzyme therapy

The possibility of using soluble cross-linked enzyme-albumin polymers as a means of enzyme therapy for the treatment of certain enzyme deficiency diseases is investigated. The hyperuricemic Dalmatian coach hound is used as an experimental animal and the enzyme uricase (urate oxidase) as the administered enzyme. Chemically cross-linking uricase with an excess of canine albumin yields a soluble enzyme polymer that is significantly more heat stable and resistant to proteolytic activity than the native enzyme. Intravenous administration of similar amounts of enzyme in the native or polymeric form indicated that the “solubilized” enzyme survived in the circulation for a longer period of time (clearance half-time of 26 hours as opposed to 4 hours for the native enzyme) and was more effective in lowering plasma uric acid levels for longer periods. In vivo administration of the native enzyme lowered uric acid levels by about 35% with a return to normal levels with a half-time of about 24 hours. Subsequent injections of native uricase proved less effective and produced a severe hypersensitivity reaction following the third injection. No such adverse reactions or decreased activity of the administered “solubilized” uricase-albumin polymers were observed. The plasma uric acid levels were decreased by about 40% and only after 48 hours did the substrate levels begin to rise towards their resting levels.     

In vitro analysis of complement-dependent HIV-1 cell infection using a model system

Previous studies based on the use of human serum as a source of C have provided evidence for the C-dependent enhancement of cell infection by HIV-1. The present study was undertaken to distinguish C from other serum factors and to identify the proteins and the mechanisms involved in C-dependent cell infection by HIV-1. The classical C activation pathway was reconstituted from the proteins C1q, C1r, C1s, C4, C2, C3, factor H, and factor I; each were purified to homogeneity. A mixture of these proteins at physiological concentrations was shown to reproduce the ability of normal human serum to enhance the infection of MT2 cells by HIV-1 at low doses of virus. This enhancing effect was abolished when heat-inactivated serum and C2- or C3-depleted serum were used, and was restored upon addition of the corresponding purified proteins. A mixture of two synthetic peptides corresponding to positions 10-15 and 90-97 of human C receptor type 2 (CD21) as well as soluble CD4 both inhibited the C-dependent infection process. These data provide unambiguous evidence that HIV-1 triggers a direct activation of the classical C pathway in vitro and thereby facilitates the infection of MT2 cells at low doses of virus. These findings are consistent with a mechanism involving increased interaction between the virus opsonized by C3b-derived fragment(s) and the CD21 cell receptors and subsequent virus entry through CD4 receptors.     

Intestinal myofibroblasts: targets for stem cell therapy

The subepithelial intestinal myofibroblast is an important cell orchestrating many diverse functions in the intestine and is involved in growth and repair, tumorigenesis, inflammation, and fibrosis. The myofibroblast is but one of several α-smooth muscle actin-positive (α-SMA(+)) mesenchymal cells present within the intestinal lamina propria, including vascular pericytes, bone marrow-derived stem cells (mesenchymal stem cells or hematopoietic stem cells), muscularis mucosae, and the lymphatic pericytes (colon) and organized smooth muscle (small intestine) associated with the lymphatic lacteals. These other mesenchymal cells perform many of the functions previously attributed to subepithelial myofibroblasts. This review discusses the definition of a myofibroblast and reconsiders whether the α-SMA(+) subepithelial cells in the intestine are myofibroblasts or other types of mesenchymal cells, i.e., pericytes. Current information about specific, or not so specific, molecular markers of lamina propria mesenchymal cells is reviewed, as well as the origins of intestinal myofibroblasts and pericytes in the intestinal lamina propria and their replenishment after injury. Current concepts and research on stem cell therapy for intestinal inflammation are summarized. Information about the stem cell origin of intestinal stromal cells may inform future stem cell therapies to treat human inflammatory bowel disease (IBD).     

MicroRNAs from the Planarian Schmidtea mediterranea: a model system for stem cell biology

MicroRNAs (miRNAs) are approximately 22-nt RNA molecules that typically bind to the 3' untranslated regions of target mRNAs and function to either induce mRNA degradation or repress translation. miRNAs have been shown to play important roles in the function of stem cells and cell lineage decisions in a variety of organisms, including humans. Planarians are bilaterally symmetric metazoans that have the unique ability to completely regenerate lost tissues or organs. This regenerative capacity is facilitated by a population of stem cells known as neoblasts. Planarians are therefore an excellent model system for studying many aspects of stem cell biology. Here we report the cloning and initial characterization of 71 miRNAs from the planarian Schmidtea mediterranea. While several of the S. mediterranea miRNAs are members of miRNA families identified in other species, we also identified a number of planarian-specific miRNAs. This work lays the foundation for functional studies aimed at addressing the role of these miRNAs in regeneration, cell lineage decisions, and basic stem cell biology.