Abstract
Herpesvirus infections are common in childhood and lead to latency in the host. During immunosuppression, for example bone marrow transplantation, reactivation of human herpesvirus 6 (HHV-6) may cause bone marrow failure and may consequently cause the death of the patient. In the present thesis an in vivo model of human hematopoiesis, the NOD/SCID mouse, was applied to investigate the influence of HHV-6 on the repopulation and differentiation potential of hematopoietic pluripotent stem cells.
First, sensitive detection assays for HHV-6A and HHV-6B were developed. These assays include the detection of HHV-6 specific proteins using monoclonal antibodies and the amplification of HHV-6 DNA and RNA by PCR. Moreover, a quantitative real-time PCR assay was developed to determine HHV-6 DNA load. These assays were finally evaluated in clinical studies.
To ensure the validity of the mouse model, it had to be demonstrated, that HHV-6 is not pathogenic for NOD/SCID mice. After infection of cell free HHV-6 or HHV-6 infected cord blood mononuclear cells (CB-MNC), no pathological changes or variation in the blood cell count could be observed when compared to control mice. Neither a persistent nor a latent infection could be shown by antigen or DNA detection. Finally, the infection of murine cells in vitro was ineffective. These data suggest, that HHV-6 does not replicate in murine cells and is therefore not pathogenic for NOD/SCID mice.
The NOD/SCID mouse model was optimized to obtain maximal engraftment of human cells. The transplantation of CB-MNC or CB-CD34+ cells resulted in multi-lineage hematopoiesis in the mouse bone marrow, blood and organs. The supplementation of human IL-3 lead to increased engraftment and production of myeloid cells compared to lymphoid cells. IL-3 supplementation was essential after CD34+-cell transplantation. Under optimal conditions engraftment rates of up to 90% of human cells in mouse bone marrow could be obtained. Using long-term bone marrow cultures and repeated transplantations, it could be demonstrated that, in addition to the proliferative effect of IL-3, the repopulating potential of stem cells was reduced. In the absence of IL-3, the stem cells remained in a quiescent state and proliferation could only be induced by stimuli like secondary transplantation or cell passage.
In addition to the production of hematopoietic cells, non-hematopoietic (NHC) cells could be detected in the bone marrow of CD34+-cell transplanted mice. Obviously, precursor NHC cells with stromal characteristics were present in the CD34+-cell fraction. The formation of NHC was not affected by IL-3 supplementation.
Infection of CB-MNC with HHV-6A resulted in a reduced engraftment. This effect was observed even when infection was performed 12 days post-transplantation. The formation of a multi-lineage hematopoiesis remained unchanged, no single cell type was suppressed by the infection. This reduced engraftment was shown to be coupled to virus replication, since inactivated virus had no effect. However, HHV-6B had no effect on the engraftment.
In CD34+-cell transplantation experiments, no effect could be observed with HHV-6A and HHV-6B when infection was performed before or on the first days after transplantation. Only when infection was done at day 12 post-transplantation, was an effect observed as with CB-MNC transplantation..
To investigate whether the formation of NHC can be influenced by HHV-6 in vitro, culture conditions were developed to differentiate CD34+-cells into NHC. The presence of HHV-6B lead to a decreased growth of NHC in general. Using quantitative real-time RT-PCR assays for endothelial cell-related genes, a reduced expression of the VEGF-2 receptor gene (KDR) and the von Willebrandt factor gene could be shown. HHV-6A had no effect in this setting.
The results presented here lead to the following hypothesis: The homing and engraftment of CD34+-cells is not influenced by HHV-6. HHV-6A susceptible cells have to be generated in the mouse by differentiation. Those HHV-6 susceptible cells are already present in the CB-MNC cell population. In these cells HHV-6 can survive and replicate affecting stem cell proliferation. |
