Yuko Osugi

Ganciclovir is effective for prophylaxis and treatment of human herpesvirus-6 in allogeneic stem cell transplantation

Human herpesvirus 6 (HHV-6) infection and disease are serious complications of allogeneic hematopoietic stem cell transplantation (allo-SCT). Ganciclovir (GCV) is effective against HHV-6 in vitro but the antiviral susceptibility of HHV-6 has not been well characterized in vivo. We retrospectively compared the HHV-6 reactivation rate in pediatric allo-SCT recipients with and without GCV prophylaxis. The HHV-6 reactivation rate at 3 weeks after allo-SCT in patients without prophylactic GCV administration was significantly higher than that in those receiving prophylactic GCV (11/28 vs 0/13, P < 0.01). Five of 36 patients without prophylactic GCV showed clinical manifestations including skin rash, interstitial pneumonitis, persistent thrombocytopenia, enterocolitis and thrombotic microangiopathy, respectively. HHV-6-associated symptoms were observed in one of the 13 patients receiving prophylactic GCV. This patient showed fever, diarrhea and graft rejection concomitantly with a sudden increase of HHV-6 DNA copy number. Patients who received GCV for treatment of HHV-6 infection showed an improvement in symptoms and/or decrease of HHV-6 copy number. Thus, GCV is effective for treating HHV-6 disease after allo-SCT in vivo.

Thrombotic microangiopathy associated with reactivation of human herpesvirus-6 following high-dose chemotherapy with autologous bone marrow transplantation in young children

Thrombotic microangiopathy (TMA) is a serious complication of BMT. Several factors are important in the etiology of TMA, such as cyclosporin A, GVHD, irradiation, intensive conditioning chemotherapy and infection, which cause damage to vascular endothelial cells leading to activation of these cells. We describe two young children with TMA following high-dose chemotherapy with autologous BMT. Development of TMA was accompanied by reactivation of HHV-6, which was identified by both an increase in the copy number of HHV-6 DNA in the peripheral blood and a significant increase in antibody titers to HHV-6. Thus, it was suggested that reactivation of HHV-6 together with high-dose chemotherapy played an important role in the pathogenesis of TMA in these patients. Since HHV-6 is known to infect vascular endothelial cells, and CMV which is virologically closely related to HHV-6, has been reported to be a pathogen that causes TMA, infection with HHV-6 of vascular endothelial cells may induce TMA via damage and activation of these cells.

Age-related changes in surface antigens on peripheral lymphocytes of healthy children.

The age‐related changes in proportion of various subsets within lymphocytes were investigated in cord blood and peripheral blood from healthy children and adults. The percentages of T and B cells did not show age‐related changes, whereas natural killer (NK) cells increased significantly with age. Within lymphocytes or the CD3+ T cell population the proportion of CD45RAbright+ lymphocytes decreased and that of CD45RO+ cells increased, while that of CD45RAdim+ cells showed no age‐related change. Within lymphocytes, the percentage of CD45RAbright+ CD4+ cells decreased, together with a decline of that of CD4+ cells. The proportions of CD45RAbright+ CD8+ cells and S6F1bright+ CD8+ cells increased with age, and the age‐dependent increase of the proportion of CD8+ cells seems to be mainly attributable to the increases in these subsets. The CD45RAdim+ CD4+ and CD45RAdim+ CD8+ cells co‐expressing CD45RO at a low level nevertheless showed no age‐related changes. In γδ T cells, both δTCS1+ and δTCS1‐ T cells increased with age, but the δTCS1‐γδ T cells increased more than the δCS1+ subset. Among lymphocytes, the percentages of CD20+, CD21+ and CD22+ cells remained similar, with no age‐related changes, but the proportion of CD5+ cells within lymphocytes or B cells decreased. The proportions of CD16+ NK cells among lymphocytes increased with age, and this change was attributable to the increase of CD56+ cells.

Mixed phenotype of blasts in acute megakaryocytic leukaemia and transient abnormal myelopoiesis in Down's syndrome

Blasts from eight cases with acute megakaryoblastic leukaemia (AMKL) and seven with transient abnormal myelopoiesis in Downs syndrome (TAM) were investigated to clarify their phenotypic characteristics. CD41 and CD7 were the most frequently expressed in both disorders. CD41 was positive in six TAM and five AMKL cases, and CD7 was positive in five TAM and five AMKL cases, respectively. CD33 was detected in four TAM and five AMKL cases. Other myeloid‐lineage associated antigens such as CD13 and CD11b could not be found in TAM but were expressed in five AMKL cases. Interestingly, CD56, a neural adhesion molecule, was expressed in three of four TAM and one of five AMKL cases. Cytoplasmic CD3 antigen was also noted in three of five examined cases.

Double-conditioning regimens consisting of thiotepa, melphalan and busulfan with stem cell rescue for the treatment of pediatric solid tumors

Major dose-limiting factors of high-dose thiotepa (TEPA) and melphalan are life-threatening mucositis and neurotoxicity. To administer a maximum dose of these drugs safely and to obtain a maximum anti-cancer effect, a double-conditioning regimen with a single grafting, two cycles of administration of a combination of TEPA (300–600u2009mg/m2) plus melphalan (70–150u2009mg/m2) with a 1-week interval was attempted in 20 patients with pediatric advanced or chemotherapy-resistant solid tumors (seven rhabdomyosarcoma, four hepatoblastoma, three neuroblastoma and four other malignancy). Combinations of TEPA plus melphalan/busulfan (Bu) (8–10u2009mg/kg) and TEPA plus Bu were given to four and two patients with brain tumors, respectively. In an additional two patients, three cycles of drug administration were performed. According to the results of the dose-escalating study, the maximum tolerable doses of TEPA and melphalan for children aged 2 years old or older were 1000u2009mg/m2 and 280u2009mg/m2, respectively. Mucositis was dose-limiting. Renal toxicity was also dose-limiting in young children (<2 years old). there were two treatment-related deaths (7%) (fungal pneumonia and renal tubular acidosis). among 13 patients who received high-dose chemotherapy during cr, 10 are alive with no evidence of disease (15–59u2009months, median: 35 months) and in 13 evaluable patients without cr, six are alive without regrowth of the disease (14–59 months, median: 39 months). thus, these novel conditioning regimens allowed us to increase the dose intensity to nearly the maximum for each drug and seemed to reduce adverse effects compared to previously reported regimens with these drugs. with regard to the effect on outcome, the results of this study seem to be encouraging, but a further study on a larger number of patients is required.

Serum levels of soluble adhesion molecules in stem cell transplantation-related complications.

To assess the involvement of vascular endothelial cell activation and damage in stem cell transplantation (SCT)-related complications, such as acute and chronic GVHD and thrombotic microangiopathy (TMA), we investigated the changes in serum levels of soluble forms of vascular cell adhesion molecule-1 (sVCAM-1) and E-selectin (sE-selectin) in SCT. The soluble form of intercellular adhesion molecule-1 (sICAM-1) was also analyzed. In patients with acute GVHD (grades II–IV), serum levels of sE-selectin and sICAM-1 increased around onset of GVHD (day 30). While the increase of sE-selectin levels was transient, sICAM-1 levels remained high until day 60. In patients with extensive chronic GVHD, sVCAM-1 as well as sE-selectin levels significantly increased. The appearance of clinical symptoms was preceded by elevations of sVCAM-1 and sE-selectin levels on day 60, and sICAM-1 levels on days 30 and 60. For the analysis of TMA, to exclude the influence of GVHD, serum levels were measured in auto-SCT patients. Around the onset of TMA, sVCAM-1 and sE-selectin levels significantly increased in patients with TMA without an increase of sICAM-1 levels. These findings support the notion that activation and injury of endothelium are commonly involved in the pathogenesis of acute and chronic GVHD and TMA. Bone Marrow Transplantation (2001) 27, 977–982.

Human herpesvirus 6 ganciclovir-resistant strain with amino acid substitutions associated with the death of an allogeneic stem cell transplant recipient

BACKGROUNDnViral resistance to antiviral drugs can cause serious complications in immunosuppressed patients. We isolated from an allogeneic stem cell transplant (SCT) recipient an antiviral-resistant human herpesvirus 6 (HHV-6) strain with mutations that caused amino acid substitutions.nnnOBJECTIVEnTo study the impact of mutations in the U38 and U69 genes of the ganciclovir (GCV)-resistant HHV-6 strain associated with the death of the SCT recipient.nnnSTUDY DESIGNnViruses were obtained from blood taken during symptomatic disease. Mutations in the genes for U69 protein kinase and U38 DNA polymerase were analyzed and the effects of the U69 mutations on GCV resistance were assayed using a recombinant baculovirus system.nnnRESULTSnIncreasing HHV-6 antigenemia occurred after 2-3 months of preemptive GCV therapy, followed by symptomatic HHV-6 disease that ended in fatal fungus-related septic shock. The HHV-6 strain isolated from the patient was 100-fold more resistant to GCV than was a wild-type strain. New mutations were found in HHV-6 genes U38 (P462S and A565V) and U69 (L202I and L213I). The mutation of U38 P462S corresponds to a mutation in the UL54 gene (P522S) of a GCV-resistant HCMV. The U69 mutations did not alter GCV sensitivity in baculovirus GCV-resistant assay system.nnnCONCLUSIONSnDrug-resistant mutations arising during preemptive therapy may complicate post-transplant HHV-6 disease in SCT recipients. The increased copy number during GCV treatment of this new GCV-resistant HHV-6 strain correlated with mutations in the U69 and U38 genes. Since the kinase mutation did not alter sensitivity to GCV when tested in the in vitro system, it is likely that the substitutions in the polymerase related to GCV resistance.

p53 gene mutations in pleuropulmonary blastomas

Pleuropulmonary blastoma (PPB) is rarechildhoodtumororiginating fromeitherlung orpleura. Although several cytogenetic changes, such as trisomy 2, trisomy 8, and loss of 17p material, have been reported, evidence of gene mutations is still lacking. Pathologically, PPB shares similarities with rhabdomyosarcoma in which p53 mutations are frequently detected. Possible implication of p53 mutations in PPB was investigated. PPBs of 3 patients were analyzed for occurrence of p53 mutations by using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method, and the nature of mutations was confirmed by direct sequencing. Two PPBs were confirmed to harbor p53 mutations. One was a Val to Leu substitution at codon 173, and another was a ArgArg to TrpCys substitution at codons 282 and 283. In each tumor, only the mutated allele was detected, suggesting inactivation of p53. Both patients with mutations had fatal outcome, while the remaining patient in whom no mutation was detected is disease free for 3 years after completion of treatment. The results raise the possibility that p53 inactivation can occur as a nonrandom genetic change involving the pathogenesis and outcome of PPB. Further studies in a larger series are necessary to clarify these matters.

Monitoring of Human Cytomegalovirus Infections in Pediatric Bone Marrow Transplant Recipients by Nucleic Acid Sequence-Based Amplification

In the diagnosis of human cytomegalovirus (HCMV) infection, it is very important to distinguish symptomatic from asymptomatic infection. The nucleic acid sequence-based amplification (NASBA) technique was compared with single and nested polymerase chainreaction (PCR) methods. For NASBA detection, the beta2.7 transcript was chosen as a target because of its abundant active HCMV-specific expression. Of 20 pediatric bone marrow transplant (BMT) recipients, 8 developed HCMV-related clinical symptoms. The clinical sensitivities and specificities were 50% and 100% for single PCR, 100% and 67% for nested PCR, and 100% and 83% for NASBA, respectively. Follow-up of HCMV infections in pediatric BMT recipients showed that NASBA could both detect viral transcript prior to the onset of clinical symptoms and reflect clinical improvement due to antiviral therapy. These data suggest that NASBA should be useful for both predicting HCMV disease development and monitoring the effect of antiviral therapy.

Allogeneic peripheral stem cell transplantation using positively selected CD34 + cells from HLA-mismatched donors

We examined five children who underwent allogeneic peripheral stem cell transplantation (PSCT) using positively selected CD34+ cells from three or two loci-mismatched donors. CD34+ cells mobilized from peripheral blood were separated by immunomagnetic beads. CD34+ cells at 2.2–6.2u2009×u2009106/kg were transplanted into three patients with refractory leukemia, a patient with relapsed medulloblastoma and a patient with Fanconi’s anemia following a conditioning regimen which included irradiation, alkylating agents and antithymocyte globulin treatment. The number of infused CD3+ cells included in grafts was 2.3–22.7u2009×u2009104/kg. Four patients achieved engraftment and hematopoietic reconstitution (>5u2009×u2009108/l of neutrophils on day 10 or 11). Graft rejection was observed in the patient with Fanconi’s anemia, but a rapid engraftment was obtained after second PSCT. Although no prophylactic agents other than ATG (included in the conditioning regimen) were used, greater than grade I acute GVHD was not observed, but limited chronic GVHD was observed in two patients. The two patients with leukemia relapsed on days 103 and 210, respectively, and the patient with medulloblastoma died of disease on day 159. The patient with Fanconi’s anemia died of fungal infection. CMV and HHV-6 diseases developed in four and two patients, respectively. Thus, although SCT using positively selected peripheral CD34+ cells may be an alternative approach for overcoming graft rejection and GVHD from HLA- mismatched donors, persistent immune deficiency attributing to extremely low numbers of T cells in grafts can potentially lead to reactivation of herpes viruses.