The Role of HIV-1 in Affecting the Proliferation Ability of HPCs Derived From BM.

HIV-1 causes chronic infection characterized by the depletion of CD4+ T lymphocytes and the development of AIDS. Current antiretroviral drugs inhibit viral spread, but they do not lead to a full immune recovery. Hematopoietic stem cells (HSCs) and multipotent hematopoietic progenitor cells (HPCs) give rise to all blood and immune cells, and in HIV infection, hematological abnormalities frequently occur in patients. Here, we used bone marrow samples from HIV-1-infected people to study the relationship between the proliferation ability of HSCs/HPCs and peripheral CD4+ T lymphocytes. Three indexes were used to reflect the proliferation ability of HSCs and HPCs: (1) colony-forming units of bone marrow mononuclear cells (BMMCs), (2) amplification of CD34+ cells purified from bone marrow mononuclear cells, (3) expression of HOXB4 and HOXA9 in CD34+ cells. We observed a direct correlation between peripheral number of CD4+ T lymphocytes and the HSCs/HPCs proliferation ability in our study. We also compared HIV-infected patients with or without antiretroviral therapy (ART). Our results demonstrated that after antiretroviral therapy, CD4+ T-cell recovery and HPCs proliferation ability are correlated. Our findings have implications in understanding whether bone marrow-derived HPCs can supplement for the loss of CD4+ T lymphocytes during HIV-1 infection.

Differentiation of human fetal multipotential neural progenitor cells to astrocytes reveals susceptibility factors for JC virus.

Viral infections of the central nervous system (CNS) are of increasing concern, especially among immunocompromised populations. Rodent models are often inappropriate for studies of CNS infection, as many viruses, including JC virus (JCV) and HIV, cannot replicate in rodent cells. Consequently, human fetal brain-derived multipotential CNS progenitor cells (NPCs) that can be differentiated into neurons, oligodendrocytes, or astrocytes have served as a model in CNS studies. NPCs can be nonproductively infected by JCV, while infection of progenitor-derived astrocytes (PDAs) is robust. We profiled cellular gene expression at multiple times during differentiation of NPCs to PDAs. Several activated transcription factors show commonality between cells of the brain, in which JCV replicates, and lymphocytes, in which JCV is likely latent. Bioinformatic analysis determined transcription factors that may influence the favorable transcriptional environment for JCV in PDAs. This study attempts to provide a framework for understanding the functional transcriptional profile necessary for productive JCV infection.

The generation and the manipulation of human multipotent adipose-derived stem cells.

In this chapter, we describe a method to isolate and to expand multipotent adipose-derived stem (hMADS) cells from human adipose tissue. We also describe culture conditions to differentiate them into adipocytes at a high rate. This culture system provides a powerful means for studying the first steps of human adipose cell development and a route for investigating effects of drugs on the biology of adipocytes. Finally, we provide a protocol to investigate gene function during proliferation and differentiation of hMADS cells by means of siRNA-mediated gene silencing approaches or forced expression by transducing hMADS cells permissive to infection with murine retrovirus vectors.

HIV-1 infects multipotent progenitor cells causing cell death and establishing latent cellular reservoirs.

HIV causes a chronic infection characterized by depletion of CD4(+) T lymphocytes and the development of opportunistic infections. Despite drugs that inhibit viral spread, HIV infection has been difficult to cure because of uncharacterized reservoirs of infected cells that are resistant to highly active antiretroviral therapy (HAART) and the immune response. Here we used CD34(+) cells from infected people as well as in vitro studies of wild-type HIV to show infection and killing of CD34(+) multipotent hematopoietic progenitor cells (HPCs). In some HPCs, we detected latent infection that stably persisted in cell culture until viral gene expression was activated by differentiation factors. A unique reporter HIV that directly detects latently infected cells in vitro confirmed the presence of distinct populations of active and latently infected HPCs. These findings have major implications for understanding HIV bone marrow pathology and the mechanisms by which HIV causes persistent infection.

[Inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation and its mechanisms].

OBJECTIVE: Cytomegalovirus (CMV) is the leading infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection. However, the exact pathogenesis of these brain abnormalities has not been fully elucidated. It has been reported that periependymitis, periventricular necrosis and calcification are the most frequent findings in the brains of congenital CMV infection. Because a number of multipotential neural stem cells (NSCs) have been identified from ventricular zone, it is possible that NSCs in this area are primary targets for viral infection, which seems to be primarily responsible for the generation of the brain abnormalities. Therefore, the objective of the present study was to investigate the effect and mechanism of murine cytomegalovirus (MCMV) infection on neural stem cells' differentiation in vitro and its role in the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. METHODS: NSCs were prepared from fetal BALB/c mouse and were infected with recombinant MCMV RM461 inserted with a report gene LacZ at 1 multiplicity of infection (MOI = 1). The effect of MCMV infection on neural stem cells' differentiation was observed by detecting the ratio of nestin, GFAP and NSE positive cells with immunohistochemistry and flow cytometry on day 2 postinfection. The effects of MCMV infection on gene expression of Wnt-1 and neurogenin 1 (Ngn1) related to neural differentiation were detected by RT-PCR. RESULTS: NSCs isolated from embryonic mouse brains strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes. At MOI = 1, the results of flow cytometry assay showed that nestin positive cells' proportion in the infection group [(62.2 +/- 1.8)%] was higher than that in the normal group [(37.2 +/- 2.4)%] (t = 4.62, P < 0.01). At the same time, the rates of GFAP and NSE positive cells' in the infection group were significantly lower than those in the normal group (P < 0.01). The scanning densities of Wnt-1 was 0.14 +/- 0.03 in the infection group while 0.32 +/- 0.04 in the control group (t = 7.21, P < 0.01). The scanning densities of Ngn1 were 0.09 +/- 0.01 and 0.21 +/- 0.02 in the two groups (t = 10.7, P < 0.01). CONCLUSIONS: These results suggest that MCMV infection could inhibit neuronal differentiation, which may be primary causes of disorders of brain development in congenital CMV infection. The decreased expression of Wnt-1 and Ngn1 may be involved in the inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation, which may lead to a new strategy for preventing and treating brain abnormalities caused by CMV infection through regulating these two signal pathways.

Interactions between c-Jun, nuclear factor 1, and JC virus promoter sequences: implications for viral tropism.

The infectious cycle of the human polyomavirus JC (JCV) is ultimately regulated in cellular nuclei at the level of viral protein expression and genomic replication. Such activity is prompted by interactions between variant nucleotide sequences within the JCV regulatory region (promoter) and cellular transcription factors that bind specific DNA consensus sites. In previous work we identified an NF-1 class member, NF-1X, as a critical transcription factor affecting the JCV cellular host range. Within variant JCV promoters, as well as other viral and cellular promoters, adjacently located NF-1 and AP-1 consensus sites are often found. The close proximity of these two binding sites suggests the opportunity for interaction between NF-1 and AP-1 proteins. Here, by electrophoretic mobility shift assays, we show temporal and dose-dependent interference by an AP-1 family member, c-Jun, upon NF-1 proteins binding an NF-1 consensus site derived from JCV promoter sequence. Moreover, as demonstrated by protein-protein interaction assays, we identify specific binding affinity independent of DNA binding between NF-1X and c-Jun. Finally, to compare the binding profiles of NF-1X and c-Jun on JCV promoter sequence in parallel with in vivo detection of viral activity levels, we developed an anchored transcriptional promoter (ATP) assay. With use of extracts from JCV-infected cells transfected to overexpress either NF-1X or c-Jun, ATP assays showed concurrent increases in NF-1X binding and viral protein expression. Conversely, increased c-Jun binding accompanied decreases in both NF-1X binding and viral protein expression. Therefore, inhibition of NF-1X binding by c-Jun appears to play a role in regulating levels of JCV activity.

[Effect of human cytomegalovirus on hematopoietic system].

OBJECTIVE: To investigate the mechanism and the suppression effect of human cytomegalovirus (HCMV) on hematopoietic system. METHODS: Semi-solid culture system was used to observe the effect of HCMV AD169 strain on colony forming unit granulocyte/macrophage (CFU-GM), CFU-erythroid (CFU-E), CFU-multipotent (CFU-Mix) and CFU-megakaryocyte (CFU-MK) growth. The techniques of in situ polymerase chain reaction (IS-PCR) and polymerase chain reaction (PCR) were used to demonstrate the existence of HCMV DNA in the colony cells of cultured CFU-GM, CFU-Mix, CFU-MK and CFU-E, respectively. The immediate early antigen (IEA) mRNA in CFU-MK and late antigen (LA) mRNA in CFU-E were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). HCMV early protein P52 was detected with immunohistochemical technique. RESULTS: HCMV AD169 suppressed the differentiation and proliferation of CFU-GM, CFU-E, CFU-Mix and CFU-MK in vitro significantly (P < 0.05). The suppression was dose-dependent. HCMV DNA was successfully detected in CFU-GM, CFU-Mix, CFU-MK colony cells from viral infection groups by IS-PCR, and was detected in CFU-E by PCR, while it was negative in blank control or mock control groups. CFU-MK colony cells expressed HCMV IEA mRNA with the size of 340 bp in virus infection groups of 10(3) plague forming unit (PFU), 10(4) PFU and 10(5) PFU, respectively. The HCMV LA mRNA was detected by RT-PCR and was 263 bp long in positive control group of HCMV-infected human embryonic fibroblasts. The expression of HCMV LA mRNA in CFU-E was negative. The early protein P52 of HCMV in 10(4) PFU group was also identified by immunohistochemical staining. CONCLUSION: HCMV AD169 strains inhibited the differentiation and proliferation of CFU-GM, CFU-E, CFU-Mix and CFU-MK by the infection of the hematopoietic progenitors. HCMV might cause the suppression of hematopoiesis by direct infection, which is thought to be one of the reasons of HCMV infection associated with thrombocytopenia, neutropenia and anemia.

E26 leukemia virus converts primitive erythroid cells into cycling multilineage progenitors.

Acute chicken leukemia retroviruses, because of their capacity to readily transform hematopoietic cells in vitro, are ideal models to study the mechanisms governing the cell-type specificity of oncoproteins. Here we analyzed the transformation specificity of 2 acute chicken leukemia retroviruses, the Myb-Ets- encoding E26 virus and the ErbA/ErbB-encoding avian erythroblastosis virus (AEV). While cells transformed by E26 are multipotent (designated "MEP" cells), those transformed by AEV resemble erythroblasts. Using antibodies to separate subpopulations of precirculation yolk sac cells, both viruses were found to induce the proliferation of primitive erythroid progenitors within 2 days of infection. However, while AEV induced a block in differentiation of the cells, E26 induced a gradual shift in their phenotype and the acquisition of the potential for multilineage differentiation. These results suggest that the Myb-Ets oncoprotein of the E26 leukemia virus converts primitive erythroid cells into proliferating definitive-type multipotent hematopoietic progenitors.