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doi:10.4172/jaa.100002

Summary:Since the incidence of cytomegalovirus (CMV) infections after hematopoietic stem cell transplantation (HSCT) may depend on the intensity of the pretreatment, we studied the incidence of CMV infections after reducedintensity compared to myeloablative conditioning. A total of 82 patients with matched related or unrelated donors were prospectively monitored for CMV infections after HSCT by CMV-PCR techniques, CMV-antigenemia and clinical observation. A total of 45 patients received reduced-intensity conditioning consisting of fludarabine, busulfan and ATG and 37 patients received myeloablative conditioning. Leukocyte engraftment occurred after a median of 15 vs 18 days (P ¼ 0.012) and platelet engraftment after 12 days vs 20 days (P ¼ 0.001), respectively. Acute graft-versus-host disease (GVHD) grade II-IV was observed in 58 vs 54% patients (P ¼ 0.737), respectively. The onset and peak values of CMV-antigenemia and DNAemia and the incidence of CMV infections did not differ statistically significantly between the two treatment groups. Multivariate analysis confirmed CMV seropositivity of the recipient (P ¼ 0.035), acute GVHD II-IV (P ¼ 0.001) but not the type of conditioning as significant risk factors for CMVantigenemia. In conclusion, the kinetics of CMV-antigenemia and DNAemia and the incidence of CMV infections were not statistically different in patients who received HSCT after reduced-intensity conditioning with fludarabine, busulfan and ATG compared to myeloablative conditioning.

Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.

In the present study we explored systematically the influence of human interleukin-3 (IL-3) on the cord blood (CB) cell-derived production of human hematopoietic cells in the bone marrow, blood, and spleen of chimeric nonobese/severe combined immunodeficient mice ((NOD/SCID) mice. CB mononuclear cells and MACS-enriched CB CD34 + cells were injected into irradiated NOD/SCID mice. The mice were additionally transplanted with a stably transfected rat fibroblast cell line expressing the human IL-3 gene (Rat-IL-3) constitutively, or with the nontransfected rat fibroblast cell line as a control (Rat-1). Rat-IL-3 mice displayed a higher engraftment of human hematopoietic cells in bone marrow, spleen, and peripheral blood compared with mice with Rat-1 cotransplantation. When we transplanted their total bone marrow cell population into secondary mice, surprisingly, mice transplanted with bone marrow cells from Rat-1 mice displayed a higher proportion of human hematopoietic cells compared with Rat-IL-3 mice. As expected, bone marrow cultures (BMCs) from Rat-IL-3 mice contained a higher proportion of human cells than Rat-1 bone marrow cells. However, when BMCs were passaged to new flasks, we observed a higher proportion of human cells in BMCs from Rat-1 mice compared with BMCs from Rat-IL-3 mice. IL-3 promotes the proliferation and differentiation of hematopoietic stem cells in chimeric bone marrow. In addition, IL-3 may play a role in the depletion of hematopoietic stem cells in chimeric bone marrow. In the absence of IL-3, the hematopoietic stem cells may remain in a quiescent state and proliferation can be induced by stimuli, including secondary transplantation or cell passage.

Two variants of human herpesvirus 6 (HHV-6) have been described, variants A and B (Yoshikawa, 2004). The present study analysed cryo-conserved plasma samples of 82 patients after allogeneic stem cell transplantation (SCT) for HHV-6 DNA by specific quantitative real-time polymerase chain reaction (PCR) for variant A and variant B in order to answer three questions: is haematopoietic engraftment delayed in patients with HHV-6A or HHV-6B infections during marrow aplasia? Are HHV-6A or HHV-6B infections associated with an increased risk for graft-versus-host disease (GVHD)? Which risk factors for HHV-6A or HHV-6B reactivation after allogeneic SCT can be delineated? Patients and methodsA total of 82 consecutive patients who received an allogeneic SCT in our unit between January 1998 and March 2001 were included in the analysis. Their characteristics are summarised in Table I. Supportive care and treatment of cytomegalovirus (CMV) infections were carried out in a standardised manner as published (Schetelig et al, 2003). Samples of heparinised blood, which had originally been taken for monitoring CMV infections, were analysed retrospectively. The procedures for the separation of plasma, and for the identification and quantification of HHV-6A and HHV-6B DNA in the plasma have been described elsewhere (Wilborn et al, 1994;Nitsche et al, 2001). HHV-6 infection was defined as the detection of HHV-6 DNA in plasma, early HHV-6 infections were defined as those occurring before day 28. Platelet and neutrophil engraftment were defined as the first of three subsequent days with unsupported platelet counts >20 · 10 9 /l or with neutrophil counts >0AE5 · 10 9 /l after SCT. GVHD was classified according to common clinical criteria (Przepiorka et al, 1995 SummaryThe clinical significance of human herpesvirus (HHV-6) infections after allogeneic stem cell transplantation (SCT) remains controversial. We analysed cryoconserved plasma samples from 82 patients after allogeneic SCT by quantitative polymerase chain reaction for HHV-6 variants A and B. Platelet engraftment was delayed in patients with HHV-6B infections but not with HHV-6A infections detected before day +28. In multivariate analysis early HHV-6B infections and the type of conditioning were associated with platelet engraftment. In conclusion, the two variants of HHV-6 should be studied separately; early infections with HHV-6B may contribute to delayed platelet engraftment after allogeneic SCT.

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