Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection

<p>Abstract</p> <p>Background</p> <p>Human embryonic stem (hES) cells hold considerable promise for cell replacement and gene therapies. Their remarkable properties of pluripotency, self-renewal, and tractability for genetic modification potentially allows for the production of sizeable quantities of therapeutic cells of the hematopoietic lineage. Dendritic cells (DC) arise from CD34+ hematopoietic progenitor cells (HPCs) and are important in many innate and adaptive immune functions. With respect to HIV-1 infection, DCs play an important role in the efficient capture and transfer of the virus to susceptible cells. With an aim of generating DCs from a renewable source for HIV-1 studies, here we evaluated the capacity of hES cell derived CD34+ cells to give rise to DCs which can support HIV-1 infection.</p> <p>Results</p> <p>Undifferentiated hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34+ HPCs which were subsequently grown in specific cytokine differentiation media to promote the development of DCs. The hES derived DCs (hES-DC) were subjected to phenotypic and functional analyses and compared with DCs derived from fetal liver CD34+ HPC (FL-DC). The mature hES-DCs displayed typical DC morphology consisting of veiled stellate cells. The hES-DCs also displayed characteristic phenotypic surface markers CD1a, HLA-DR, B7.1, B7.2, and DC-SIGN. The hES-DCs were found to be capable of antigen uptake and stimulating naïve allogeneic CD4+ T cells in a mixed leukocyte reaction assay. Furthermore, the hES-DCs supported productive HIV-1 viral infection akin to standard DCs.</p> <p>Conclusion</p> <p>Phenotypically normal and functionally competent DCs that support HIV-1 infection can be derived from hES cells. hES-DCs can now be exploited in applied immunology and HIV-1 infection studies. Using gene therapy approaches, it is now possible to generate HIV-1 resistant DCs from anti-HIV gene transduced hES-CD34+ hematopoietic progenitor cells.</p

The Determinants of Private Fixed Investment and the Relationship between Public and Private Capital Accumulation in Turkey

The purpose of this study is to analyse the determinants of private fixed investment spending in Turkey over the period 1970–96, which covers years of both financial repression and financial liberalisation. A reformulated neoclassical investment model and a reformulated flexible accelerator investment model have been tested for the Turkish economy. The results obtained support the accelerator principle and the crowding out hypothesis, that is, public and private sector investments have been found to be substitutes. Furthermore, the hypothesis that the volume of funds is as important as the cost of funds used in financing private fixed investment has been verified. On the other hand, the so-called McKinnon-Shaw hypothesis has not been completely verified because the effect of the medium-term real lending rate on private fixed investment has been found to be negative but statistically insignificant. Finally, the financial and liberalisation programmes that have been implemented since 1983 have not yet shown any noticeable positive effects on private investment.

Stable gene transfer of CCR5 and CXCR4 siRNAs by sleeping beauty transposon system to confer HIV-1 resistance

<p>Abstract</p> <p>Background</p> <p>Thus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB) transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy.</p> <p>Results</p> <p>Two well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP) reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5) into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3) or R5-tropic (BaL) HIV-1 strains, the respective transposed cells showed marked viral resistance.</p> <p>Conclusion</p> <p>SB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins and thus confer resistance against viral infection by restricting viral entry. These studies have demonstrated for the first time the utility of the non-viral SB system in conferring stable resistance against HIV infection and paved the way for the use of this system for HIV gene therapy studies.</p

HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector

BACKGROUND: RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4) and monocyte tropic (R5) viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. RESULTS: To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. CONCLUSIONS: Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy

The Determinants of Private Fixed Investment and the Relationship between Public and Private Capital Accumulation in Turkey

The purpose of this study is to analyse the determinants of private fixed investment spending in Turkey over the period 1970-96, which covers years of both financial repression and financial liberalisation. A reformulated neoclassical investment model and a reformulated flexible accelerator investment model have been tested for the Turkish economy. The results obtained support the accelerator principle and the crowding out hypothesis, that is, public and private sector investments have been found to be substitutes. Furthermore, the hypothesis that the volume of funds is as important as the cost of funds used in financing private fixed investment has been verified. On the other hand, the so-called McKinnon-Shaw hypothesis has not been completely verified because the effect of the medium-term real lending rate on private fixed investment has been found to be negative but statistically insignificant. Finally, the financial and liberalisation programmes that have been implemented since 1983 have not yet shown any noticeable positive effects on private investment

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy

BACKGROUND: Many novel studies and therapies are possible with the use of human embryonic stem cells (hES cells) and their differentiated cell progeny. The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications. Here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for AIDS. RESULTS: Undifferentiated normal and lentiviral vector transduced hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34 hematopoietic progenitor cells. The differentiated CD34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages. Phenotypic and functional analyses were carried out to compare these with that of fetal liver CD34 cell derived macrophages. As assessed by FACS analysis, the hES-CD34 cell derived macrophages displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR suggesting a normal phenotype. Tests evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation showed that these macrophages are also functionally normal. When infected with HIV-1, the differentiated macrophages supported productive viral infection. Lentiviral vector transduced hES cells expressing the transgene GFP were evaluated similarly like above. The transgenic hES cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation. CONCLUSION: Phenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells. Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies. Based on these results, it is also now feasible to transduce hES-CD34 cells with anti-HIV genes such as inhibitory siRNAs and test their antiviral efficacy in down stream differentiated cells such as macrophages which are among the primary cells that need to be protected against HIV-1 infection. Thus, the potential utility of hES derived CD34 hematopoietic cells for HIV-1 gene therapy can be evaluated

Output-inflation Trade-offs: The Latin American Experience

This paper investigates the natural rate hypothesis, using the Lucas and Hanson approaches for ten Latin American countries. The purpose of using two methods to test this hypothesis is to ascertain the robustness of the results to the underlying differences in the assumptions of these methods. The evidence strongly supports the natural rate hypothesis and the predictions of the Lucas model. The results of the Hanson method are in general consistent with the natural rate hypothesis, but they are not as conclusive as the results of the Lucas method. The evidence from the Hanson model suggests that the monetary growth predicted by past inflation performs better than the one predicted by past monetary growth.

Lentiviral transduction of Tar Decoy and CCR5 ribozyme into CD34+ progenitor cells and derivation of HIV-1 resistant T cells and macrophages

BACKGROUND: RNA based antiviral approaches against HIV-1 are among the most promising for long-term gene therapy. These include ribozymes, aptamers (decoys), and small interfering RNAs (siRNAs). Lentiviral vectors are ideal for transduction of such inhibitory RNAs into hematopoietic stem cells due to their ability to transduce non-dividing cells and their relative refractiveness to gene silencing. The objective of this study is to introduce an HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme into CD34+ hematopoietic progenitor cells via an HIV-based lentiviral vector to derive viral resistant progeny T cells and macrophages. RESULTS: High efficiency and sustained gene transfer into CD34+ cells were achieved with lentiviral vector constructs harboring either Tar decoy or Tar decoy in combination with CCR5 ribozyme. Cells transduced with these constructs differentiated normally into T-lymphocytes in vivo in thy/liv grafts of SCID-hu mice, and into macrophages in vitro in the presence of appropriate growth factors. When challenged in vitro, the differentiated T lymphocytes and macrophages showed marked resistance against HIV-1 infection. CONCLUSIONS: Viral resistant transgenic T cells and macrophages that express HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme could be obtained by lentiviral gene transduction of CD34+ progenitor cells. These results showed for the first time that expression of these anti-HIV-1 transgenes in combination do not interfere with normal thymopoiesis and thus have set the stage for their application in stem cell based gene therapy for HIV/AIDS

HIV-1 infection and CD4 T cell depletion in the humanized Rag2(-/-)γc(-/- )(RAG-hu) mouse model

BACKGROUND: The currently well-established humanized mouse models, namely the hu-PBL-SCID and SCID-hu systems played an important role in HIV pathogenesis studies. However, despite many notable successes, several limitations still exist. They lack multi-lineage human hematopoiesis and a functional human immune system. These models primarily reflect an acute HIV infection with rapid CD4 T cell loss thus limiting pathogenesis studies to a short-term period. The new humanized Rag2(-/-)γc(-/- )mouse model (RAG-hu) created by intrahepatic injection of CD34 hematopoietic stem cells sustains long-term multi-lineage human hematopoiesis and is capable of mounting immune responses. Thus, this model shows considerable promise to study long-term in vivo HIV infection and pathogenesis. RESULTS: Here we demonstrate that RAG-hu mice produce human cell types permissive to HIV-1 infection and that they can be productively infected by HIV-1 ex vivo. To assess the capacity of these mice to sustain long-term infection in vivo, they were infected by either X4-tropic or R5-tropic HIV-1. Viral infection was assessed by PCR, co-culture, and in situ hybridization. Our results show that both X4 and R5 viruses are capable of infecting RAG-hu mice and that viremia lasts for at least 30 weeks. Moreover, HIV-1 infection leads to CD4 T cell depletion in peripheral blood and thymus, thus mimicking key aspects of HIV-1 pathogenesis. Additionally, a chimeric HIV-1 NL4-3 virus expressing a GFP reporter, although capable of causing viremia, failed to show CD4 T cell depletion possibly due to attenuation. CONCLUSION: The humanized RAG-hu mouse model, characterized by its capacity for sustained multi-lineage human hematopoiesis and immune response, can support productive HIV-1 infection. Both T cell and macrophage tropic HIV-1 strains can cause persistent infection of RAG-hu mice resulting in CD4 T cell loss. Prolonged viremia in the context of CD4 T cell depletion seen in this model mirrors the main features of HIV infection in the human. Thus, the RAG-hu mouse model of HIV-1 infection shows great promise for future in vivo pathogenesis studies, evaluation of new drug treatments, vaccines and novel gene therapy strategies