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Adenovirus infection remains an important cause of mortality after bone marrow transplantation (BMT). Currently no efficient antiviral treatment is known. Thus, testing new modalities of early diagnosis and treatment is a crucial objective. Adenovirus infection is defined by the combination of symptoms and the isolation of virus from the source of clinical symptoms. The involvement of two or more organs and the presence of virus in blood cultures define disseminated disease. Seven children with a median age of 7 years received bone marrow transplantation for leukemia. All received an unrelated graft without T cell depletion. Adenovirus was sought in blood, urine and biopsy specimens using PCR and culture. Analysis of biopsy specimens included systematic immunohistochemistry. Cidofovir treatment was initiated as soon as biopsy revealed the histopathological signs of adenovirus. Cidofovir was given at 5 mg/kg once weekly for 3 weeks then every 2 weeks. Six patients had diarrhoea and one patient had cystitis. Adenovirus infection and disseminated disease were diagnosed in four cases and three cases, respectively. In six cases, serotype A31 was isolated from gastrointestinal biopsy and in two cases serotypes B2 and C6 were detected in blood and urine. Cidofovir treatment was associated with clinical improvement of diarrhoea, cystitis and fever in five patients, in whom the virus became undetectable in cultures and PCR analyses despite the persistence of immunodeficiency. The median follow-up was 360 days after BMT (240-570). One child died of invasive aspergillosis and another of disseminated adenovirus after interruption of cidofovir therapy. Further studies in immunocompromised patients will be needed to extend these promising results concerning the role of cidofovir in adenovirus infection.

Improvements in HLA-typing by DNA-based methods now allow accurate genotyping of unrelated bone marrow (UBM) donors and recipients and also definition of haplotypes. In this regard, B*5002 has been predicted in linkage disequilibrium with Cw*0602, DRB1*0406 and DQB1*0402 based on the frequency of allele coexistence. Here, we confirm this assumption by HLA genotyping of four informative families and three unrelated individuals. In the four families, the extended haplotype HLA-B*5002, -Cw*0602, -DRB1*0406, DQB1*0402 can be definitely assigned by the mode of heritance. Furthermore, this association of alleles was also found in the three B*5002 unrelated individuals. Knowledge of the frequent linkage disequilibrium of these rare alleles can improve UBM donor search strategies.

Resume Ces dernieres annees, les medecins de l'hopital pediatrique Robert-Debre avaient la perception que l'activite liee a la drepanocytose augmentait. Objectifs Notre etude avait deux objectifs: decrire l'evolution de la prise en charge des patients drepanocytaires suivis, puis etre capable d'en determiner les causes si l'on mettait en evidence une prise en charge plus lourde pour l'hopital. Patients et methodes Une enquete descriptive retrospective a ete menee, couplant deux approches: d'une part, une comparaison de l'ensemble des enfants drepanocytaires suivis en 1992 avec l'ensemble de ceux suivis en 1996, du point de vue de leur profil phenotypique et de leurs hospitalisations (frequence, duree et type de complications) et, d'autre part, une description de revolution des complications et hospitalisations dans la cohorte suivie de 1992 a 1996. Les enfants ont ete reperes grâce aux listes etablies par le ≪ centre de la drepanocytose ≫, qui coordonne le suivi de tous les patients hospitalises dans l'hopital. Les donnees etudiees proviennent du systeme d'information de l'hopital, ainsi que des dossiers individuels medicaux, infirmiers, et du service social. Resultats Les resultats de cette enquete montrent une augmentation de l'activite liee a la drepanocytose pour l'hopital etudie; elle se traduit par une multiplication des hospitalisations et par une charge de travail plus importante pour les equipes soignantes. Cette augmentation d'activite est liee a la fois a un recrutement plus important, a un accroissement du taux d'hospitalisations non programmees et a une augmentation de l'incidence des complications aigues chez les enfants suivis. Ceci peut s'expliquer en partie par le vieillissement de la cohorte. Par ailleurs, la charge de travail du personnel soignant semble avoir augmente, a en juger notamment par la prescription plus frequente de morphiniques.

Gene transduction into immature hematopoietic cells collected at birth from the umbilical cord could be useful for the treatment of genetic or acquired disorders of the hematopoietic system diagnosed during pregnancy. The SCID-hu mouse is a convenient model to investigate T- cell lineage gene therapy, since it allows replication of human intrathymic T-cell development. CD34+ cells isolated from cord blood were cocultured with CRIP MFG-murine CD2 (mCD2) cells that produce recombinant retroviruses encoding the mCD2 antigen, a cell surface marker easily detectable by flow cytometry. After 3 and 4 days in coculture, a mean of 19% and 39% human hematopoietic cells, respectively, expressed the mCD2 antigen. CD34+ cells cocultured for 4 days were used to reconstitute human fetal thymus implanted in SCID mice. Five to 10 weeks later, the mCD2 antigen was detected on approximately 10% of human thymocytes repopulating the thymic grafts in four of nine SCID mouse chimeras. Vector genomes were detected in graft cell DNA by Southern blot. Analysis of vector integration indicated that positive cells were of polyclonal origin in three animals and predominantly monoclonal in the other one. Our data show that foreign genes can be transduced into CD34+ cord blood cells endowed with T-cell differentiation potential, and suggest strategies for T-cell lineage gene therapy in the neonate.

The spontaneous proliferation and the effects of 8 various growth factors (GF) were evaluated on leukemic cells from 27 patients with B-lineage ALL. Two groups of ALLs were distinguished. ALLs from group I (21 patients) exhibited a low spontaneous proliferative rate and were stimulated by IL-3 + IL-7 +/- SCF and/or LIF, while ALLs from group II (6 patients) had a high spontaneous proliferative rate and did no longer require this combination of GFs for proliferation. No effect of bFGF, IGF-I, IL-10 and IL-11 alone or in combination, was observed. Such differences in the behaviour of B-ALLs indicated that the GF requirement of ALL blasts was not related to the presence of serum in the culture nor to the pattern of reactivity of ALL blasts for B lymphoid markers or CD34 antigen. Furthermore, we showed in 1/9 cases that high proliferation might be due to an overexpression of the bcl-2 proto-oncogene and to the acquisition of an autocrine secretion.

1. Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the catabolism of thiopurine drugs, which are used to treat cancer patients and organ transplant recipients. Because TPMT activity is polymorphic and under genetic control, large interindividual variations in the immunosuppressive activity and toxicity of these drugs may, at least in part, be inherited. 2. We have developed a specific h.p.l.c. method for measuring 6-methyl mercaptopurine formed from 6-mercaptopurine (6-MP) in red blood cell lysates during the TPMT assay procedure. In blinded assays of 55 samples from adult blood donors, the results of the h.p.l.c. method correlated with those of the radiochemical reference method (r = 0.83, P < 0.001). 3. Using this h.p.l.c. assay, we tested the effect of known inhibitors of TPMT activity (syringic acid, p-anisic acid and tropolone) in vitro and showed that they were highly inhibitory. We also found that drugs often administered concomitantly with 6-MP (prednisone, prednisolone, 6-methylprednisolone, cyclophosphamide, methotrexate, and trimethoprim-sulphamethoxazole) had little or no effect on TPMT activity in vitro. 4. In a group of 300 French individuals, TMPT activity was highly variable, ranging from 4.7 to 35.3 nmol h-1 ml-1 of packed red blood cells (nmol h-1 ml-1 PRBC) with a mean value of 19.3 +/- 4.9. TMPT activity was not influenced by sex. 5. This sensitive and reproducible h.p.l.c. assay for TPMT activity in red blood cells may prove useful for prospective clinical studies designed to optimise dosage regimens of thiopurine drugs (detection limit for 6-methyl mercaptopurine is 5 ng ml-1, intra- and inter-assay variations are 6.8 and 8.2%, respectively).

The pharmacokinetics of 6-mercaptopurine, including cerebrospinal-fluid (CSF) distribution, and the erythrocyte 6-thioguanine nucleotide concentrations were determined in children randomised to receive intravenous mercaptopurine for acute lymphoblastic leukaemia (ALL), according to the EORTC protocol ALL n.58881.After 1 month of oral treatment at a dose of 50 mg.m-2.day-1, the pharmacokinetic parameters were determined after the first i.v. administration of 1 g.m-2 (bolus dose of 0.2 g.m-2 followed by an 8-h infusion of 0.8 g.m-2) in 11 patients: systemic clearance was 23.02 1.h-1, volume of distribution was 0.75 l.kg-1, and elimination half-life was 1.64 h. The erythrocyte thioguanine concentrations were measured in the same 11 patients and increased significantly between the beginning and the end of infusion (10 pmol x 10(8) packed RBC) or within 24 h of infusion (223 pmol x 10(8) packed RBC). The CSF concentration was 3.78 mumol.1(-1), 1-6 h after the beginning of infusion (n = 28) and the CSF to plasma ratio was 0.15 (n = 16). In patients receiving the oral dose of 50-165 mg.m-2.day-1 of 6-mercaptopurine, CSF concentrations were below 0.18 mumol.1(-1), 1-24 h after drug intake (n = 67), and the CSF to plasma ratio was not calculated.Following the i.v. administration of 6-mercaptopurine, we observed high CSF concentrations of 6-mercaptopurine and an acute increase of erythrocyte thioguanine nucleotide concentrations. The clinical trial (EORTC protocol ALL n[symbol: see text]5881), comparing the oral and i.v. administrations of mercaptopurine, will demonstrate if the i.v. administration reduces the incidence of CNS relapses.

The treatment of children with acute lymphoblastic leukaemias (ALL) according to EORTC 58951 protocol is divided into 3 phases (induction, consolidation, maintenance). During the induction phase, patients are randomly assigned to receive either prednisone or dexamethasone (associated with anticancer drugs). The response to corticotherapy is evaluated by the quantification of the residual disease (RD) at day 35 of treatment and has an major impact on prognosis and treatment: patients with an RD lower than 10−2/100 000 cells. The distribution of CYP3A genotypes was : *1/*1: 2,7%, *1/*3: 18,8% and *3/*3: 78,5%, in aggrement with already published data. CYP3A5*2 and *6 mutations were not found There was no difference in the response to corticosteroid treatment according to individual CYP3A5 genotypes or to the type of corticosteroid. Our results did not evidence an impact of CYP3A5 on the initial response to corticosteroids. Clinical Pharmacology & Therapeutics (2004) 75, P20–P20; doi: 10.1016/j.clpt.2003.11.075

Resume La decontamination digestive totale offre une alternative aux mesures prophylactiques de lutte contre les infections qui accompagnent les traitements immunosuppresseurs des enfants atteints de leucemie algue. Trente-deux enfants atteints de leucemie aigue, en periode d'aplasie therapeutique et beneficiant ou non d'une decontamination digestive ont fait l'objet d'une surveillance bacteriologique. La decontamination digestive ne semble pas diminuer la frequence des infections. Cependant, les germes responsables d'accidents infectieux chez les enfants decontamines appartiennent surtout aux bacteries de l'environnement alors qu'ils sont le plus souvent d'origine digestive, donc potentiellement plus dangereux, chez les enfants non decontamines. Ces resultats nous conduisent a n'appliquer une decontamination intestinale qu'aux formes graves de leucemie aigue.

Resume La granulomatose chronique septique, qui se caracterise par une anomalie constitutionnelle des cellules phagocytaires s'accompagne frequemment d'infections a Aspergillus . Parmi 37 enfants atteints, 14 ont presente une infection aspergillaire pulmonaire (13 cas), et/ou osteoarticulaire. Le tableau clinique se caracterise par son evolution torpide paucisymptomatique. L'extension parietale d'une atteinte parenchymateuse pulmonaire semble toujours constituer un facteur de mauvais pronostic. Le diagnostic de certitude repose sur l'isolement de l' Aspergillus par le lavage alveolaire et la biopsie, alors que les methodes serologiques et l'intradermoreaction a l' Aspergillus ne permettent pas de diagnostic certain. L'Amphotericine B prescrit pendant de nombreux mois, represente le traitement principal, plus ou moins associe a d'autres antimycosiques. Depuis 2 ans, aucune infection aspergillaire n'a ete observee chez 23 enfants recevant un traitement phophylactique par Ketoconazole.

Summary CMV infection is one of the major infection after bone marrow transplantation. CMV viremia was systematically studied in 66 patients with aplastic anemia or leukemia undergoing BMT. 57% patients had CMV viremia with a frequency peak between 7 and 9 weeks after transplant. Clinical symptoms found during viremia were pancytopenia, fever, cytolytic hepatitis. Interstitial pneumonitis was found only in 4 cases. In 3 cases, viremia was not associated with clinical symptoms. Survival was identical to the group of patients without viremia. Viremia was positively associated with the presence of high anti-CMV antibody titer in donor or recipient before transplant, or to a lymphocyte proliferative response against CMV antigens in donor or recipient before BMT. Granulocyte transfusions increased the frequency of CMV viremia. CMV infection was significantly associated with acute and chronic graft versus host disease. The relation showed between these parameters and viremia provides a basis for an accurate diagnosis of CMV infection and a better background for the study of prophylactic or curative treatment of CMV infection.

AIDS is reported in a 14 year old hemophiliac B. This patient was treated on demand and used 800 F IX IU per kg B.W. and per year. He presented with severe opportunistic infection (Toxoplasmosis) and profound impairment of cellular immunity. He suffered of chronic active hepatitis and he was chronic HBs Ag carrier. A retrovirus (LAV) was isolated from his cultured T lymphocytes. He had no other identified risk factor than severe hemophilia B. The occurrence of AIDS in hemophiliacs suggests a relationship between the transfusion of blood products and the disease. The cause of AIDS is unknown. A possible relationship between LAV and AIDS is discussed.

We report on six cases of 6p rearrangement in various haematological malignancies. On reviewing the literature, we assume 6p rearrangements to be secondary anomalies in both myeloid and lymphoid malignancies, and confirm it to be strongly associated with -5/del(5q) in myelodysplastic syndromes.

In the present study we describe one CD2+CD3+ clone termed DS6 which expressed neither CD4 nor CD8 differentiation antigens and failed to react with WT31, a monoclonal antibody directed against the T cell antigen receptor alpha/beta heterodimer. This clone was isolated from peripheral blood T lymphocytes of a patient with a prolonged immunodeficiency after allogeneic bone marrow transplantation. Normal-sized T cell gamma gene transcripts were detected in DS6 by northern analysis, whereas no mature beta or alpha chain mRNA were found. The rearrangement of TCR beta chain genes and T cell gamma genes was analysed. While in DS6, TCR beta chain genes remain in germinal configuration, and a unique pattern of monoallelic T cell gamma gene rearrangement was observed. The rearrangement involves the recently described V gamma 5 segment and the J gamma 1 joining segment, which is located upstream of the C gamma 1 constant region. To determine the molecular structure present on DS6, an immunoprecipitation was performed with monoclonal anti-CD3 antibody and a rabbit antiserum raised against gamma protein. We have observed, in association with the CD3 complex, a 90 kDa structure which under reducing conditions resolves into three subunits of 45, 40 and 37 kDa. We demonstrated that the rabbit anti-gamma serum only immunoprecipitates the two lower bands. The upper band corresponds to a presently undefined T cell receptor chain. Next, we showed the non major histocompatibility complex (MHC)-restricted cytolytic activity exhibited by these CD3+CD4-CD8- cloned T cells and inhibition of the natural killer (NK)-like activity by the anti-CD3 monoclonal antibody. The triggering of CD2 or CD3 molecules increased IL-2 receptor expression on DS6 but failed to induce cell proliferation. This contrasts with recent results obtained with gamma-expressing T cell clones and illustrates the functional heterogeneity of the cells bearing the second T cell receptor.

Two kinds of T-cell antigen receptor (TCRs) α:β and γ:δ, have so far been identified as independently expressed on the cell surface of distinct subsets of T cells in association with the CD3 (T3) complex (1,3). The surface expression of these TCR was systematically analyzed in peripheral lymphocytes (PBL) from 27 patients (pts) having received allogeneic bone marrow transplantation for leukemia. A predominant emergence of circulating CD3+ lymphocytes— 40% of PBL—lacking α:β heterodimer was demonstrated in 3 pts. In two pts, we showed that these cells expressed γ chains associated to CD3. In one pt, functional analyses indicated that this γ:δ subset representing 40% of PBL may have played a role in a profound immunodeficiency state which occurred six months after grafting. Molecular studies of γ:δ cells indicated that these circulating cells preferentially expressed a single Vγ segment of the T-cell receptor γ gene (TRG): Vγ5 for pt 1 and Vγ9 for pt 2.

Thirty-seven patients with chronic granulocytic leukemia have been treated with supralethal chemoradiotherapy followed by transplantation of bone marrow from HLA-identical donors. All patients showed engraftment, and the Philadelphia chromosome (PH1) disappeared in each case. Four patients had syngeneic grafts before blast crisis and are still alive; 2 are in remission not maintained by therapy, and 2 others are receiving chemotherapy after having relapsed in the chronic phase. Thirty-three patients had allogeneic grafts; only 2 received the grafts during blast crisis, and neither is a long-term survivor. Of the 13 patients who had grafts in the accelerated phase, 6 died of complications related to the transplantation, and 1 died after a myeloblastic relapse. Thus 6 patients are in unmaintained remission with a median follow-up of 13 months. Eighteen patients received grafts in the chronic phase. All 10 survivors are in unmaintained remission with a median follow-up of 14 months; in this group, no patient has relapsed. The granulocytic hyperplasia of the chronic phase can be more effectively ablated than established blastic leukemia. The mortality rate of transplant-related complications must be weighted against the typical rate of progression of chronic granulocytic leukemia. Although a longer follow-up period is needed for full evaluation, bone marrow transplantation may now be offered to patients in the chronic phase in an attempt to achieve long-term survival or cure of more than one-half of these patients.

Resume L'infection a cytomegalovirus est la cause la plus frequente des infections lethales apres greffe de moelle osseuse. Une viremie est observee dans 50 % des cas lorsque les patients ou les donneurs sont sero-positifs avant la greffe. La pneumonie interstitielle a CMV survient dans 10 a 20 % des cas avec une mortalite de l'ordre de 85 %. L'infection a CMV est liee a la reactivation d'un virus latent dans les cellules de l'hote ou a la transmission d'un virus par le donneur de moelle ou les transfusions sanguines. Le traitement de la pneumonie a CMV a toujours ete tres decevant. Neanmoins, les etudes recentes semblent montrer l'interet d'un traitement prophylactique ou d'un traitement precoce des infections. L'utilisation des gammaglobulines hyperimmunes donne des resultats contradictoires et dans notre equipe, n'a amene aucune amelioration dans l'incidence des viremies et des pneumonies. La selection des donneurs de moelle ou de sang seronegatifs est utile mais uniquement si le receveur est seronegatif. Des etudes cliniques preliminaires semblent montrer l'utilite des nouveaux agents antiviraux (DHPG Synthex ou BWB 759 U Wellcome), analogues de l'Acyclovir ou d'un autre produit : le Phosphonoformate. Chacun de ces produits a une activite in vitro sur le CMV : in vivo les resultats sont plus nuances. Ils semblent efficaces lorsqu'ils sont utilises precocement au cours des viremies. Par contre, dans les formes les plus avancees, la mortalite par pneumonie interstitielle reste importante. Les rechutes surviennent frequemment, posant le probleme d'un traitement d'entretien.

GROWTH AND ENDOCRINE FUNCTION AFTER BONE MARROW TRANSPLANTATION (BMT) WITH OR WITHOUT TOTAL BODY IRRADIATION (TBI)