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Pathology resulting from human immunodeficiency virus (HIV) infection is driven by protracted inflammation; the primary loss of CD4+ T cells is caused by activation-driven apoptosis. Recent studies of nonhuman primates (NHPs) have suggested that during the acute phase of infection, antiviral mucosal immunity restricts viral replication in the primary infection compartment. These studies imply that HIV achieves systemic infection as a consequence of a failure in host antiviral immunity. Here, we used high-dose intrarectal inoculation of rhesus macaques with simian immunodeficiency virus (SIV) SIVmac251 to examine how the mucosal immune system is overcome by SIV during acute infection. The host response in rectal mucosa was characterized by deep mRNA sequencing (mRNA-seq) at 3 and 12 days postinoculation (dpi) in 4 animals for each time point. While we observed a strong host transcriptional response at 3 dpi, functions relating to antiviral immunity were absent. Instead, we observed a significant number of differentially expressed genes relating to cell adhesion and reorganization of the cytoskeleton. We also observed downregulation of genes encoding members of the claudin family of cell adhesion molecules, which are coexpressed with genes associated with pathology in the colorectal mucosa, and a large number of noncoding transcripts. In contrast, at 12 dpi the differentially expressed genes were enriched in those involved with immune system functions, in particular, functions relating to T cells, B cells, and NK cells. Our findings indicate that host responses that negatively affect mucosal integrity occur before inflammation. Consequently, when inflammation is activated at peak viremia, mucosal integrity is already compromised, potentially enabling rapid tissue damage, driving further inflammation. [ABSTRACT FROM AUTHOR]

: ObjectiveEx vivo expansion of primitive hematopoietic cells remains of interest for gene therapy and transplantation. Previous studies reported loss of repopulating activity following culture of cells for more than 4–7 days in the presence of cytokines or stromal cells. In the current study, we investigated whether prolonged culture and transduction in the presence of the carboxy-terminal portion of fibronectin (FN) could maintain or expand retrovirally transduced repopulating hematopoietic stem cells (HSCs).: MethodsThe impact of culture and transduction on rhesus macaque CD34+ peripheral blood stem cells (PBSCs) was assessed in the presence of FN and stimulatory cytokines. A competitive repopulation design using up to three retroviral vectors allowed direct comparison of repopulating activity between cells transduced and cultured for 4 days vs 10 days.: ResultsIn the first animal, all cells were cultured and transduced for 10 days, with one vector used on days 0–4 and a second on days 4–10. There was stable long-term marking from both vectors, indicating that cells cycling both early and late could engraft. In three animals, we compared cells that were cryopreserved following a 4-day transduction to cells that were continued in culture for an additional 6 days. Total marking derived from the 10-day expanded cells was significantly higher than marking from the 4-day cultured cells.: ConclusionsThese results suggest that culture on FN support allows prolonged ex vivo maintenance and even expansion of transduced repopulating stem cells. [Copyright &y& Elsevier]

To better understand Simian betaretrovirus ( SRV) seropositivity in virus-negative macaques, we transfused blood from SRV-infected or suspect donors into immunosuppressed naive recipients. Our results do not support typical SRV1-5 infection as the cause, but provide evidence for several possibilities including serological artifact, new/different SRV, or an endogenous virus. [ABSTRACT FROM AUTHOR]

An altered intestinal microbiome during chronic human immunodeficiency virus (HIV) infection is associated with mucosal dysfunction, inflammation, and disease progression. We performed a preclinical evaluation of the safety and efficacy of fecal microbiota transplantation (FMT) as a potential therapeutic in HIV-infected individuals. Antiretroviral-treated, chronically simian immunodeficiency virus (SIV)-infected rhesus macaques received antibiotics followed by FMT. The greatest microbiota shift was observed after antibiotic treatment. The bacterial community composition at 2 weeks post-FMT resembled the pre-FMT community structure, although differences in the abundances of minor bacterial populations remained. Immunologically, we observed significant increases in the number of peripheral Th17 and Th22 cells and reduced CD4+ T cell activation in gastrointestinal tissues post-FMT. Importantly, the transplant was well tolerated with no negative clinical side effects. Although this pilot study did not control for the differential contributions of antibiotic treatment and FMT to the observed results, the data suggest that FMT may have beneficial effects that should be further evaluated in larger studies. [ABSTRACT FROM AUTHOR]

Given the critical role of mucosal surfaces in susceptibility to infection, it is imperative that effective mucosal responses are induced when developing efficacious vaccines and prevention strategies for infection. Modulating the microbiota in the gastrointestinal (GI) tract through the use of probiotics (PBio) is a safe and well-tolerated approach to enhance mucosal and overall health. We assessed the longitudinal impact of daily treatment with the VSL#3 probiotic on cellular and humoral immunity and inflammation in healthy macaques. PBio therapy resulted in significantly increased frequencies of B cells expressing IgA in the colon and lymph node (LN), likely because of significantly increased LN T follicular helper cell frequencies and LN follicles. Increased frequencies of IL-23+ APCs in the colon were found post-PBio treatment, which correlated with LN T follicular helper cells. Finally, VSL#3 significantly downmodulated the response of TLR2-, TLR3-, TLR4-, and TLR9-expressing HEK293 cells to stimulation with Pam3CSK4, polyinosinic-polycytidylic acid, LPS, and ODN2006, respectively. These data provide a mechanism for the beneficial impact of PBio on mucosal health and implicates the use of PBio therapy in the context of vaccination or preventative approaches to enhance protection from mucosal infection by improving immune defenses at the mucosal portal of entry. [ABSTRACT FROM AUTHOR]

Gene transfer of O6-methylguanine-DNA-methyltransferase (MGMT) into HSCs results in efficient in vivo selection of murine HSCs after treatment with temozolomide (TMZ) and O6- benzylguanine (BG). In addition, HOXB4 coexpression is associated with an increase in HSC selection efficiency using a DHFR drug selection system in mice. Based on these results, we have studied whether the MGMT and HOXB4 selection systems are efficacious in a rhesus macaque autologous transplantation model. SIV vectors were constructed incorporating either MGMT alone or MGMT together with HOXB4 in a GFP or YFP backbone. In our first transplant case, we purified CD34+ cells from mobilized bone marrow cells and split the graft into two parts: half of the cells were transduced with a SIV MGMT-GFP vector and half were transduced with a SIV MGMT-HOXB4-GFP vector. In our second and third transplant cases, half of the CD34+ cells were transduced with a SIV MGMT- YFP vector and the other half with a SIV MGMT-HOXB4-GFP vector. During the early engraftment period, there was a slight advantage (2-fold maximal) for HOXB4 transduced cells relative to the “MGMT-only” vector. However, by 1 month post- transplantation, there was no difference in marking between the two vectors, suggesting HOXB4 had little effect on long-term repopulating cells. The first animal has been treated with 7 courses of TMZ/BG, starting with a fixed dose of 120 mg/m2 for BG and 170 mg/m2/day for 5 days for TMZ. The dose of TMZ was escalated on each course, and well tolerated by the animal. For the 7th course, the animal received 450 mg/m2 of TMZ with BG, a significant dose increase relative to the pediatric maximum tolerated dose (185 mg/ m2 without BG in irradiated patients). This increase in dose intensity was verified by formal pharmacokinetic measurements. After each course of drug treatment, there was a rapid increase in GFP marking in all lineages. The highest marking achieved after last course of TMZ/BG treatment was 91% for myeloid cells and 50% for lymphoid lineages. However, the proportion of GFP+ cells subsequently declined and leveled off at 13% for granulocytes, 17% for B cells and 36% for T cells. The second animal had been treated with two courses of TMZ/BG and there was about 7-fold increase in GFP marking in granulocytes and a 3 and 8-fold increase in B, T cells respectively. Altogether, we have shown that TMZ selection was occurring mainly in progenitor cells with limited self-renewal capacity and to a lesser degree at the HSC level as evidence by increased baseline GFP marking. We were also able to achieve a significant increase in dose intensity in the first animal, and to successfully administer multiple courses of drug treatment. These results suggest a clinical strategy for hematopoietic protection in cancer treatment. We are now treating these animals with BCNU plus BG to determine if HSC selection efficiency is increased relative to TMZ. Lastly, we did not see evidence of HSC amplification with HOXB4 expression, suggesting that the robust selection seen in mice is relatively species specific.Molecular Therapy (2006) 13, S29–S29; doi: 10.1016/j.ymthe.2006.08.085 [ABSTRACT FROM AUTHOR]

An abstract of the article "A Rhesus Macaque Transplant Model for Drug Selection of Hematopoietic Stem Cells Transduced with MGMT Vectors," by Yan Shou and colleagues is presented.

Recent studies have suggested a remarkable potential of adult stem cells from a variety of organs to give rise to cells of disparate organs, but evidence of such potential at a clonal level is lacking in most if not all studies to date. To assess directly the hematopoietic potential of muscle-derived cells in a relevant large animal, we initiated retroviral-tagging studies in the rhesus macaque to allow tracking at the clonal level by integration site analysis. Four rhesus macaques underwent transplantation with transduced muscle-derived cells after lethal irradiation followed by delayed infusion of an autologous hematopoietic graft. The first animal showed no evidence of hematopoietic recovery and, despite infusion of the backup hematopoietic graft, succumbed due to complications of prolonged cytopenias. In the remaining three animals, the overall contribution of retrovirally tagged muscle-derived cells toward hematopoiesis was exceedingly low. Retroviral integration site analysis among clonally derived muscle cells and bone marrow cells in vivo in one animal suggests a common source. These results demonstrate that harvesting disparate organs for cellular therapy is currently highly inefficient at best. [Copyright &y& Elsevier]

Recently, RD114 (feline endogenous retrovirus envelope protein)-pseudotyped retroviral particles have been shown to transduce human NOD/SCID repopulating cells efficiently. In this study, we compared directly transduction of repopulating cells with RD114-pseudotyped vector to that with standard amphotropic vector in the rhesus macaque model. G-CSF/SCF-mobilized CD34+ rhesus peripheral blood cells were cultured in the presence of SCF, Flt-3 ligand, and MGDF on Retronectin-coated flasks. To assess directly the ability of the two pseudotypes to transduce primitive cells, both vectors were added simultaneously to the target cells every 24 h, for a total of four exposures in 96 h. The cells were reinfused after the animals received 1000 cGy total body irradiation. At the end of transduction, gene marking efficiency of CFU was higher with amphotropic LNL6 vector (mean 88.4%) vs RD114-G1Na vector (mean 18.5%). After long-term engraftment in three animals, total neo gene marking levels were 4–5% in PBMNCs and 1.5–4% in granulocytes. The RD114-G1Na marking levels were consistently higher in granulocytes than in mononuclear cells, while amphotropic LNL6 marking levels were higher in PBMNCs than in granulocytes. The differential gene marking patterns suggest that RD114 and amphotropic vectors may target distinct progenitor or stem cell populations. There was no clear advantage for RD114-pseudotyped vectors in this predictive preclinical model in terms of overall long-term marking levels; however, optimization of transduction conditions by increasing m.o.i. or inducing the receptor could potentially improve results with this novel vector system. [Copyright &y& Elsevier]

The ability to efficiently transfer a gene into repopulating hematopoietic stem cells would create many therapeutic opportunities. We have evaluated the ability of particles bearing an alternative envelope protein, that of the feline endogenous virus (RD114), to transduce stem cells in a nonhuman primate autologous transplantation model using rhesus macaques. We have previously shown this pseudotyped vector to be superior to the amphotropic vector at transducing cells in umbilical cord blood capable of establishing hematopoiesis in immunodeficient mice. Gene transfer efficiency as reflected by the number of genetically modified cells in hematopoietic tissues varied among the five monkeys studied from low levels (<1%) in three animals to much higher levels in two (20–60%). An animal that exhibited extremely high levels for several weeks was found by vector genome insertion site analysis to have reconstitution predominantly with a single clone of cells. This variability among animals is in keeping with computer simulations of reconstitution with limiting numbers of stem cells genetically modified at about 10% efficiency. Our studies provide insights into the biology of hematopoietic reconstitution and suggest approaches for increasing stem cell targeted gene transfer efficiency. [Copyright &y& Elsevier]

The ability to efficiently transduce hematopoietic stem and progenitor cells under serum-free conditions would be desirable for safety and standardization of clinical gene therapy protocols. Using rhesus macaques, we studied the transduction efficiency and engraftment ability of CD34-enriched SCF/G-CSF mobilized progenitor cells (PBSC) transduced with standard amphotropic marking vectors under serum-free and serum-containing conditions. Supernatants were collected from producer cells 16 hours after serum-free medium or medium containing 10% fetal calf serum was added. Vector titers were approximately two- to threefold higher when producer cells were cultured in serum-containing medium. However, retroviral transduction of rhesus CFU-GM was improved using serum-free vector-containing medium. For analysis of engraftment with transduced cells, three macaques had CD34+ peripheral blood stem cells split into two fractions for transduction. One fraction was transduced using serum-free vector-containing medium, and the other fraction was transduced using standard serum-containing medium. The two fractions were re-infused simultaneously following total body irradiation. In all three animals, there was equivalent marking from both vectors for 7–9 months post-transplantation. These data are encouraging regarding the removal of serum-containing medium from clinical hematopoietic cell transduction protocols, given the lack of a detrimental effect on transduction and engraftment with transduced cells. [Copyright &y& Elsevier]