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Patients with Wiskott–Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346and NCT01347242), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

After initial acceptance and on-line publication it was decided that this article warranted translation into English. This English translation has been published in Gastroentérologie Clinique et Biologique, 34/11, pp. 590–605 (http://dx.doi.org/10.1016/j.gcb.2010.09.008) and to avoid unnecessary duplication this version of the article has been withdrawn.

This study provides an update on multidisciplinary staffing and clinical activity in Australian specialist persistent pain services. Of the 109 services identified, 57 responded, met inclusion criteria and completed a study-specific questionnaire detailing service characteristics, staff resources, and clinical activities. Where possible, data were compared between the ‘Waiting in Pain’ (WIP) investigations (WIP-I: Dec’08–Jan’10, WIP-II: Jul’16–Feb’18). WIP-II found more pain services (Level 1 centres, rural services) and more full-time equivalent (FTE) staffing (overall, psychiatry, psychology, occupational therapy) than WIP-I. Although Level 1 centres employed more FTE staff (overall, medical) than Level 2 clinics, staffing was comparable when considered relative to clinical activity and this was stable over time for most disciplines. Clinical activity in metropolitan and rural services also remained stable, as did rural service staffing (type, FTE), suggesting that newer clinics replicated existing models. WIP-II highlighted greater diversity in group structures than WIP-I and an associated mean .02FTE allied health staff/patient seen (WIP-I = .03 FTE). Staffing (amounts, types) did not change significantly over time when considered relative to clinical activity, supporting the conclusion that these are workable clinical structures. However, changes in group format (duration, staffing) suggest a shift towards lower-intensity programmes that require less allied health staffing to deliver.

T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precursors arrested at specific differentiation stages, including an ‘early-cortical’ thymic maturation arrest characterized by expression of cytoplasmic TCRβ but no surface T-cell receptor (TCR) and frequent ectopic expression of the TLX1/3 NK-like homeotic proteins (NKL). We designed a TCRα VJC PCR to identify clonal TCRα rearrangements in 32% of 127 T-ALLs, including 0/52 immature/TCRγδ lineage cases and 41/75 (55%) TCRαβ lineage cases. Amongst the latter, TCRα rearrangements were not identified in 30/54 (56%) of IMβ/pre-αβ early-cortical T-ALLs, of which the majority (21/30) expressed TLX1/3. We reasoned that the remaining T-ALLs might express other NKL proteins, so compared transcript levels of 46 NKL in T-ALL and normal thymic subpopulations. Ectopic overexpression of 10 NKL genes, of which six are unreported in T-ALL (NKX2-3, BARHL1, BARX2, EMX2, LBX2 and MSX2), was detectable in 17/104 (16%) T-ALLs. Virtually all NKL overexpressing T-ALLs were TCRα unrearranged and ectopic NKL transcript expression strongly repressed Eα activity, suggesting that ectopic NKL expression is the major determinant in early-cortical thymic T-ALL maturation arrest. This immunogenetic T-ALL subtype, defined by TCRβ VDJ but no TCRα VJ rearrangement, is associated with a favorable outcome in GRAALL-treated adult T-ALLs.

The tumour suppressor gene PTENis commonly altered in T-cell acute lymphoblastic leukaemia but its prognostic impact is still debated. We screened a cohort of 573 fully characterised adult and paediatric T-cell acute lymphoblastic leukaemia (T-ALL) patients for genomic PTENabnormalities. PTEN-inactivating mutations and/or deletions were identified in 91 cases (16%), including 18% of paediatric (49/277) and 14% of adult cases (42/296). Thirty-four patients harboured only mutations, 12 cases demonstrated only large deletions and 9 only microdeletions. About 36 patients had combined alterations. Different mechanisms of PTENinactivation predicted differences in the clinical outcome for both adult and paediatric patients treated according to the GRAALL03/05 and FRALLE2000 protocols. Whereas large deletions predicted lower 5-year overall survival (P=0.0053 in adults, P=0.001 in children) and disease-free survival (P=0.0009 in adults, P=0.0002 in children), mutations were not associated with a worse prognosis. The prognostic impact of PTEN loss is therefore linked to the underlying type of genomic abnormality, both in adult and paediatric T-ALLs, demonstrating that detailed analysis of the type of abnormality type would be useful to refine risk stratification.

The thymus is the major site for normal and leukemic T-cell development. The dissection of the molecular determinants of T-cell survival and differentiation is paramount for the manipulation of healthy or transformed T cells in cancer (immuno)therapy. Casein kinase 2 (CK2) is a serine/threonine protein kinase whose anti-apoptotic functions have been described in various hematological and solid tumors. Here we disclose an unanticipated role of CK2 in healthy human thymocytes that is selective to the γδ T-cell lineage. γδ thymocytes display higher (and T-cell receptor inducible) CK2 activity than their αβ counterparts, and are strikingly sensitive to death upon CK2 inhibition. Mechanistically, we show that CK2 regulates the pro-survival AKT signaling pathway in γδ thymocytes and, importantly, also in γδ T-cell acute lymphoblastic leukemia (T-ALL) cells. When compared with healthy thymocytes or leukemic αβ T cells, γδ T-ALL cells show upregulated CK2 activity, potentiated by CD27 costimulation, and enhanced apoptosis upon CK2 blockade using the chemical inhibitor CX-4945. Critically, this results in inhibition of tumor growth in a xenograft model of human γδ T-ALL. These data identify CK2 as a novel survival determinant of both healthy and leukemic γδ T cells, and may thus greatly impact their therapeutic manipulation.

PTEN is a protein phosphatase that is crucial to prevent the malignant transformation of T-cells. Although a numerous mechanisms regulate its expression and function, they are often altered in T-cell acute lymphoblastic leukaemias and T-cell lymphomas. As such, PTENinactivation frequently occurs in these malignancies, where it can be associated with chemotherapy resistance and poor prognosis. Different Ptenknockout models recapitulated the development of T-cell leukaemia/lymphoma, demonstrating that PTEN loss is at the center of a complex oncogenic network that sustains and drives tumorigenesis via the activation of multiple signalling pathways. These aspects and their therapeutic implications are discussed in this review.

Perkinsus marinus is responsible for a chronic disease (Dermo) of the Eastern oyster, Crassostrea virginica. In order to simulate the in vivo environment more closely, a chemically defined medium (JL-ODRP-3) was supplemented with tissue homogenate extracts or plasma from oysters possessing varying degrees of susceptibility to P. marinus infection. In media supplemented with extracts from highly susceptible oysters (C. virginica), P. marinus cells secreted elevated amounts of a set of low molecular weight serine proteases (LMP: 30–45 kDa) as assessed by enhanced digestion within gelatin-substrate SDS–PAGE gels. Oyster species of low susceptibility (C. gigas and C. ariakensis) did not exhibit this ability to upregulate P. marinus LMP expression. Oyster extract supplementation also led to pronounced changes in P. marinus cellular morphology, such that the cells were comparable to those observed within naturally infected oysters.

Thymic epithelial malignant diseases are extremely rare in children. The authors report a 12-year-old white girl admitted for a polymetastatic tumor of the anterior mediastinum. Tumor proliferation was typical of an undifferentiated thymic carcinoma. A close link between Epstein-Barr virus (EBV) and the tumor was established by a high titer of anti-VCA IgA and the presence of EBV RNA and DNA in the tumor. In addition, monoclonal viral episomes were present in tumor cells, indicating that EBV infection was an early event in the oncogenic process. The patient died despite resection, irradiation, and chemotherapy.

To define the junctional diversity of T-cell antigen receptor delta gene rearrangements in fresh T-acute lymphoblastic cells and to correlate cell phenotype with the coding potential of rearrangements, we determined the junctional nucleotide sequences of 13 T-cell antigen receptor delta gene rearrangements involving the preferentially rearranged V (V delta 1) and J (J delta 1) segments using in vitro gene amplification and direct sequencing. We showed that, as in gamma delta+ cell lines, extensive junctional diversity exists in these clones and that this diversity is due both to random nucleotide deletions/additions and to the use of at least two D delta segments. We also showed that a high percentage of these rearrangements are potentially translatable (7:13) and that such functional rearrangements occur in both surface CD3+ and CD3- cells. Comparison of alpha beta versus gamma delta surface expression demonstrates that all CD3+ T acute lymphoblastic leukemias with a functional V delta 1-J delta 1 rearrangement express a surface gamma delta receptor and are recognized by the anti-delta monoclonal antibody delta TCS1, whereas a control CD3+ gamma delta+ leukemic case that had not undergone V delta 1 rearrangement was delta TCS1-. In addition, expression of this monoclonal antibody is not restricted by V gamma or C gamma usage or by the covalent or noncovalent link between gamma and delta chains.

Prolymphocytic leukemia (PLL) is a chronic lymphoproliferative disorder, characterized by prominent splenomegaly, prolymphocytes accounting for more than 55% of circulating lymphocytes, and short-term survival. To better characterize the nature of the cellular origin in this disease, we analyzed lg heavy chain variable region (VH) genes in eleven cases of de novo PLL Leukemic cells expressed a skewed repertoire characterized by predominant use of the V3 family members (73%), with preferential use of the V3–23 gene (50% of the VH3 genes). All sequences from expressed VH genes diverged from their putative germline counterpart, and in eight cases the divergence was greater than 5%. In seven cases, which expressed the V3–23 gene and VH4 family members, nucleotide substitutions could be confidently attributed to somatic mutations. The type and distribution of these mutations clearly indicated that in three cases the cells had been subjected to an antigen selection process. Taken together, these results suggest that B- PLL cells display a skewed repertoire of lg VH regions and probably represent, at least in some instances, expansion of postgerminal center cells that have undergone antigen driven selection.

Background & Aims: The etiology of refractory sprue is unclear. To gain insight into its pathogenesis, the phenotype and T-cell receptor (TCR) gene rearrangement status of intestinal lymphocytes were analyzed in a group of patients with clinical or biological features of celiac disease but either initially or subsequently refractory to a gluten-free diet. Methods: Intestinal biopsy specimens were obtained from 26 adults: 6 patients with refractory sprue, 7 patients with active celiac disease, and 13 normal controls. The phenotype of intestinal lymphocytes was studied by immunohistochemistry and, in 3 patients with refractory sprue, by cytometry of lymphocytes purified from intestinal biopsy specimens. TCR rearrangements were assessed by studying TCR@cV-J junctional regions from DNA extracted from intestinal biopsy specimens and purified intestinal lymphocytes. Results: In the 6 patients with refractory sprue, but not in normal controls or patients with active celiac disease, the intestinal epithelium was massively infiltrated by small lymphocytes that lacked CD8, CD4, and TCR, contained intracytoplasmic but not surface CD3@e chains, and exhibited restricted TCR@c gene rearrangements. Conclusions: Refractory sprue is associated with an abnormal subset of intraepithelial lymphocytes containing CD3@e and restricted rearrangements of the TCR@c chain but lacking surface expression of T-cell receptors. GASTROENTEROLOGY 1998;114:471-481

In order to dissect the correlation between aberrant TAL1 basic-helix-loop-helix (b-HLH) expression and the exclusive development of T cell acute lymphoblastic leukemias (T-ALL) of the TCRαβ lineage, we have assessed the ability of class A b-HLH proteins to regulate the TCRα and δ enhancers. We demonstrate that E47S binds to TCRα but not to TCRδ E-boxes in vitro. Despite this, neither E2-5 nor HEB transactivate the TCRα enhancer in NIH 3T3, nor did Id1 modify endogenously driven TCRα [αE1-4] activity in a TCRαβ cell line. We also demonstrate that TAL-1 inhibits both binding of E47S to αE3 and αE4 and endogenous transactivation of the TCRα enhancer. Comparison of the activity of the minimal [αE1-2] fragment, which contains no E-boxes, with the accessory [αE3-4] fragment, which contains two, suggested some contribution from the latter to TCRα enhancer activity in HPB-ALL. TCR [αE1-2] activity was partially (40%) inhibited by TAL1 but not all by Id1. In contrast, [αE3-4] activity was almost completely inhibited by TAL1 (80%) and slightly reduced by Id1 (15%). These data demonstrate that class A b-HLH regulation of the TCRα enhancer E-boxes differs from their B lymphoid Igμ counterparts and suggest a novel mechanism on transcriptional inhibition by TAL1, which may be, at least partly, independent of E-box-mediated activation, as we currently recognize it. They also clearly demonstrate that the restriction of TAL1 deregulation to T-ALL of the TCRαβ lineage is not due to induction of TCRα enhancer activity by the TAL1 protein.

The cellular interactions involved in the platelet response after immunoglobulin infusion in acute and chronic idiopathic thrombocytopenic purpura (ITP) are unknown. There have been a number of theories including the competitive inhibition of platelet-binding to macrophages by the preferential sequestration of immunoglobulin coated red cells. We report a study to examine this hypothesis. Adult acute and chronic patients were given infusions of immunoglobulin at a rate of 0.4 g/kg body weight, daily for 5 days. Serum haptoglobin, lactate dehydrogenase and the absolute reticulocyte counts were monitored and no significant change in any value was seen during the period of study. A red cell survival was performed on four of the patients and no increase in the rate of red cell clearance occurred during the infusion period. We conclude from this that in these patients no significant degree of haemolysis was provoked by the infusion although this does not preclude this as a mechanism of action in some individuals.

To determine the incidence of MLL rearrangement in acute myeloid leukemia (AML) French-American-British (FAB) type M1 and to evaluate optimal screening strategies for the characterization of such abnormalities, we analyzed specimens from 41 patients with AML by Southern blotting with two MLL genomic probes and compared the capacities of reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent in situ hybridization (FISH) to identify the types of rearrangement found in AML M1 with those observed in AML M5. MLL rearrangement was found in 6 of 29 (20%) AML M1 and 6 of 10 AML M5 cases. RT-PCR characterization of 11 cases showed four MLL self- fusions, four MLL-AF6, two MLL-AF9, including a novel AF9 breakpoint, and one uncharacterized t(11:19). Only 5 of 10 MLL-rearranged cases tested demonstrated karyotypic 11q23 abnormalities. FISH analysis of nine cases with an MLL-specific yeast artificial chromosome (YAC) confirmed the cytogenetic abnormalities in two cases, clarified them in one, and did not detect six cases, including three MLL self-fusions, one case with a probable MLL-rearranged subclone not represented karyotypically, and twoMLL-AF6. A whole chromosome 11 paint detected one of these MLL-AF6, and an AF6 cosmid demonstrated that the other was probably due to insertion of a submicroscopic portion of chromosome 6, including part of AF6, into an apparently normal chromosome 11. We conclude that MLL rearrangements are common in adult AML M1, that MLL self-fusion and MLL-AF6 are the most frequent types of abnormalities, and that RT-PCR is preferable to 11q23 FISH analysis for their characterization.

To characterize the T cell receptor (TCR) repertoire expressed by the V delta 1+ gamma/delta T cell population, we have studied the V delta 1-J delta 1 junctional sequences from peripheral blood samples of healthy donors. We show that, surprisingly, this repertoire is restricted in most healthy adults, with a donor-specific and relatively stable pattern, whereas this repertoire remains unrestricted in infants, and is similar to that of thymocytes. These data contrast with the general assumption that the junctional repertoire of V delta 1+ gamma/delta T cells is extensive, and strongly suggest that peripheral recruitment of V delta 1+ cells bearing particular TCR occurs in humans during the postnatal stage.

Hydroxyethyl starch (HES 450.000/0.7; Hespan 6.0 g/100 ml) was compared with standard crystalloid solutions in postoperative volume replacement in 20 patients undergoing routine orthopaedic surgery. The HES group showed no clinical evidence of haemorrhage and no laboratory evidence of significant haemostatic defects as assessed by standard coagulation tests, platelet aggregation and fibrinogen concentrations. There was a slight shortening in the thrombin time and a smaller increase in postoperative FVIII RAg and FVIII RCof levels in the HES group. HES is a safe and effective volume expander for postoperative use.

Chronic B cell lymphocytosis with binucleated lymphocytes (LWBL) is a recently described entity. The lymphocytosis is stable over years and atypical binucleated lymphocytes are detected on peripheral blood smears. Further investigation has shown a predominance in females, a polyclonal increase in serum IgM and HLA-DR7 expression in most cases. In almost all cases cytogenetic studies with classical culture conditions have not shown any abnormality.We describe 7 patients with LWBL associated with an abnormal karyotype. An additional i(3q) was found in 6 cases. Premature chromosome condensation (PCC) was detected in all 7 cases. As both abnormalities are rarely present in other benign or malignant proliferations, we suggest a strong correlation between LWBL and i(3q) and/or PCC.

The t(1;19) in B-lineage ALL is classically associated with a FAB LI/L2 phenotype and the expression of cIg. Recent reports have demonstrated immuno-phenotypic and molecular heterogeneity among cases demonstrating apparently identical karyotypic abnormalities. We report 5 cases of t(1; 19) with cytological features resembling an L3 (Burkitt-like) phenotype, suggesting that accurate assessment of these cases requires detailed correlation of cytological, immunological and molecular characteristics.

In order to dissect the correlation between aberrant TAL1 basic-helix-loop-helix (b-HLH) expression and the exclusive development of T cell acute lymphoblastic leukemias (T-ALL) of the TCRalphabeta lineage, we have assessed the ability of class A b-HLH proteins to regulate the TCRalpha and delta enhancers. We demonstrate that E47S binds to TCRalpha but not to TCRdelta E-boxes in vitro. Despite this, neither E2-5 nor HEB transactivate the TCRalpha enhancer in NIH 3T3, nor did Id1 modify endogenously driven TCRalpha [alphaE1-4] activity in a TCRalphabeta cell line. We also demonstrate that TAL1 inhibits both binding of E47S to aE3 and aE4 and endogenous transactivation of the TCRalpha enhancer. Comparison of the activity of the minimal [alphaE1-2] fragment, which contains no E-boxes, with the accessory [aE3-4] fragment, which contains two, suggested some contribution from the latter to TCRalpha enhancer activity in HPB-ALL. TCR [alphaE1-2] activity was partially (40%) inhibited by TAL1 but not at all by Id1. In contrast, [alphaE3-4] activity was almost completely inhibited by TAL1 (80%) and slightly reduced by Id1 (15%). These data demonstrate that class A b-HLH regulation of the TCRalpha enhancer E-boxes differs from their B lymphoid Igmicro counterparts and suggest a novel mechanism of transcriptional inhibition by TAL1, which may be, at least partly, independent of E-box-mediated activation, as we currently recognize it. They also clearly demonstrate that the restriction of TAL1 deregulation to T-ALL of the TCRalphabeta lineage is not due to induction of TCRalpha enhancer activity by the TAL1 protein.

A highly malignant human T-cell receptor (TCR) gamma/delta+ T-cell leukemia was shown to have a productive rearrangement of the TCR delta locus on one chromosome 14 and a novel t(8;14)(q24;q11) rearrangement involving the J delta 1 gene segment on the other chromosome 14. Chromosome walking coupled with pulsed-field gel electrophoretic (PFGE) analysis determined that the TCR J delta 1 gene fragment of the involved chromosome was relocated approximately 280 kb downstream of the c-myc proto-oncogene locus found on chromosome band 8q24. This rearrangement was reminiscent of the Burkitt's lymphoma variants that translocate to a region identified as the pvt-1 locus. Sequence comparison of the breakpoint junctions of interchromosomal rearrangements in T-cell leukemias involving the TCR delta-chain locus revealed novel signal-like sequence motifs, GCAGA(A/T)C and CCCA(C/G)GAC. These sequences were found on chromosome 8 at the 5' flanking site of the breakpoint junction of chromosome 8 in the TCR gamma/delta leukemic cells reported here and also on chromosome 1 in T-cell acute lymphocytic leukemia patients carrying the t(1;14)(p32;q11) rearrangement. These results suggest that (i) during early stages of gamma delta T-cell ontogeny, the region 280 kb 3' of the c-myc proto-oncogene on chromosome 8 is fragile and accessible to the lymphoid recombination machinery and (ii) rearrangements to both 8q24 and 1p32 may be governed by novel sequence motifs and be subject to common enzymatic mechanisms.

Comparison of human astrocytes (1181N1 line) and sheep choroid plexus cells (SCP) infected with visna virus (V-1181N1 and V-SCP) shows that the mode of virus assembly and the morphology and size of the free virions are identical in the 2 systems. V-SCP cells usually cytolyse soon after infection; V-1181N1 cells do not. This is considered to be due to inter ference with membrane assembly in the former. Syncytial giant cells from both V-1181N1 and V-SCP cultures have a much looser attachment to the growth surface than their un infected parents. This is shown to be associated with a loss of certain microfibrillar arrays close to the plasmalemma. Uninfected SCP cultures have cilia and desmosomes and retain these after infection till cytolysis; 1181N1 cells do not have these structures, but have a well developed microtubular system, which the SCP cells lack.

Moscovici, Carlo(University of Colorado Medical Center, Denver), and E. H. Macintyre. Effect of avian myeloblastosis virus in the Japanese quail. J. Bacteriol. 92:1141–1149. 1966.—Avian myeloblastosis virus (AMV) induced a spectrum of neoplasms in Japanese quail (Coturnix coturnix japonica) which was similar to that observed in the chicken, with one exception: the total absence of acute myeloblastic leukemia in quail. Studies in vivo as well as in vitro suggested that the cause for this difference may be ascribed to the heterogeneity of AMV and to the genetic makeup of the quail cell.

N6,O2′-dibutyryl cyclic AMP (DBcAMP) (3× 10-4 M) causes reproducible changes in the morphology, growth rate and terminal cell density of 3 different types of human tumour cells grown in culture. Human tumour astrocyte lines 1181N1 and EH-118MG show generalized ablation of membrane systems when grown in the presence of this cyclic AMP analogue. This is not consistent with differentiation; it may be a toxic effect but possibly represents the response of these cell types to DBcAMP. These cells also grow more slowly and to lower terminal cell densities as a function of the concentration of DBcAMP. Although the growth kinetics demonstrate a return of these malignant cells to a state of density-dependent growth, micrographic analyses of intercellular relationships reveal that the cells still grow in overlapping patterns. Thus as judged by the commonly accepted criteria, DBcAMP does not induce contact inhibition of growth. All observed changes induced by the presence of DBcAMP are rapidly and completely reversed upon its removal from the growth medium. Human neuroblasts (NBI) grown in the presence of DBcAMP show irreversible morphological changes and the rate and extent of cell division is reduced. There is marked hyperplasia of intracytoplasmic microfilaments, which is considered consistent with differentiation.

Human tumour astrocytes (118MG) were exposed in vitro to the Engelbreth-Holm strain of Rous sarcoma virus at a multiplicity of infection of one. Morphological transformation of the cells in 60-mm plates was complete in 9-11 weeks after viral exposure. The transformed cells (EH-118MG) grew as a monolayer, upon which nests of rounded cells developed. From these nests, rounded cells shed into the medium and in turn on seeding formed a monolayer and repeated the cycle. The transformed cells contained the viral group-specific antigen in the cytoplasm, and produced Rous sarcomas in chickens. No complete virus was demonstrated in the mammalian cells. Electron micrographs of the parent 118MG and transformed EH-118MG cells gave further details of the structure of cytoplasmic protrusions which were formed in some of the free EH-1 18MG but not in the 118MG cells. Their nature is completely unknown.

N6,O2′-dibutyryl cyclic AMP (DBcAMP) (3× 10-4 M) causes reproducible changes in the morphology, growth rate and terminal cell density of 3 different types of human tumour cells grown in culture. Human tumour astrocyte lines 1181N1 and EH-118MG show generalized ablation of membrane systems when grown in the presence of this cyclic AMP analogue. This is not consistent with differentiation; it may be a toxic effect but possibly represents the response of these cell types to DBcAMP. These cells also grow more slowly and to lower terminal cell densities as a function of the concentration of DBcAMP. Although the growth kinetics demonstrate a return of these malignant cells to a state of density-dependent growth, micrographic analyses of intercellular relationships reveal that the cells still grow in overlapping patterns. Thus as judged by the commonly accepted criteria, DBcAMP does not induce contact inhibition of growth. All observed changes induced by the presence of DBcAMP are rapidly and completely reversed upon its removal from the growth medium. Human neuroblasts (NBI) grown in the presence of DBcAMP show irreversible morphological changes and the rate and extent of cell division is reduced. There is marked hyperplasia of intracytoplasmic microfilaments, which is considered consistent with differentiation.

The question of which fluid should be used to augment a falling blood volume has been investigated in several animal models and numerous clinical trials. The results do not indicate an ideal approach. Generally electrolyte solutions are not as good as the other products. Because of the demands on the transfusion service for plasma procurement whole blood is in limited supply. However, whole blood should be available for large acute haemorrhages in excess of 2 l. The synthetic macromolecular colloids are probably as good as albumen solutions and fall into two clinical categories. The first group increase the blood volume by an amount slightly greater than the volume infused but are rapidly eliminated so their clinical effects remain for only two to three hours. Of these solutions the modified gelatins are probably the colloid of choice. For a more prolonged volume expansion of up to 24–36 h the larger macromolecules are more ideal but with a less potent initial oncotic effect [7]. Hydroxyethyl starch seems the most suitable of these substances. Albumen solutions are considerably more expensive but, otherwise have properties of volume expansion intermediate to the two colloid groups and limiting side effects of similar overall frequency. The final choice depends on local availability, cost effectiveness and personal clinical experience.

CHRONIC infection with measles virus is causally related to the human syndrome of subacute sclerosing panencephalitis (SSPE)1–5, and means of reproducing that condition in experimental animals are being sought6–10. Measles carrier states in vitrohave been established in some human11–13and rodent14,15cell systems. We now report the establishment of measles carrier states in human astrocyte cultures of normal brain, and in a tumour astrocyte line, as well as the emergence of fine structural features which bear a close resemblance to findings in the brain of some patients with SSPE16.

Secondary therapy-related, acute lymphoblastic leukemia (S-ALL) is less common than its myeloblastic counterpart. S-ALL with MLL gene rearrangements have only been reported on six previous occasions. Only three of these had t(4;11)(q21;23) S-ALL with MLL-AF4 fusion transcript has only been reported in one earlier case.In this report a rare case of S-ALL with MLL-AF4 transcript is described in a 36 year old woman treated for breast carcinoma with chemotherapy which included the topoisomerase II inhibitor, VP-16. The precise incidence of MLL gene rearrangement in S-ALL still remains to be clarified.

The CEBPA gene codes for the C/EBPa transcription factor which regulates hematopoietic stem cell homeostasis and orientates towards myeloid differentiation and against a T lymphoid fate. CEBPA silencing by promoter hypermethylation (CEBPAmeth) has recently been identified (Wouters, Blood 2007) in a minor subset of AML, all of which demonstrated T lymphoid features (expression of CD7 and CD3 transcripts, TCRG rearrangement and frequent NOTCH1 mutations (NOTCH1m)). These AML may be closer to immature T-ALLs, with CEBPa silencing and NOTCH1m determining lineage orientation. Although expression of CD7 and cytoplasmic CD3 (cCD3) are considered as defining markers in T-ALL, CD7 is commonly expressed in AML and reproducible multicenter detection of low level cCD3, when performed, can be difficult. We have classified CD7+, cCD3+ T-ALLs on the basis of their TCR profiles: immature cases arrested prior to TCRB VDJ rearrangement include TCRgd lineage precursors and can be divided into those with no TCR rearrangement (IM0), TCRd only rearrangement (IMD) or TCRd and TCRg rearrangement (IMG). The CALM-AF10 fusion transcript is found in both AML and T-ALL, where it is restricted to the TCRgd lineage. We initially searched for in CEBPAmeth by methylation specific PCR in 5 AML with downregulated CEBPand confirmed that the 3 cases (FAB-M0/M1) with CEBPAmeth expressed CD3, CD7 and/or TCRg rearrangement. Only 1/49 AML M0, all of which were cCD3 negative, demonstrated CEBPAmeth.We then searched for CEBPAmeth and mutation and NOTCH1m in 18 IM T-ALL (4 aged <15y; 5 IM0, 5 IMD, 8 IMG) compared to 6 adults with TCRB VDJ+, TCRab lineage T-ALL. No CEBPA mutations were found but 11/24 (56%) demonstrated CEBPAm and 10/23 (43%) NOTCH1m. Both correlated with IMG T-ALL, when 6/8 demonstrated CEBPAmeth and NOTCH1m and a seventh only NOTCH1m, compared to only 4/10 CEBPAmeth and 1/10 NOTCH1m in IM0/D cases, with no overlap. In ab-lineage T-ALLs 2/6 demonstrated NOTCH1m, one of which was also CEBPAmeth, and notably expressed TLX1/HOX11. CEBPAmeth cases were more frequently CALM-AF10+ (5/10), CD2-, CD123/IL3R-, HOXA9 transcript+ and IgH DJ rearranged but did not differ from non-CEBPAmeth T-ALL in CD34, CD5, CD117, CD13/33, CD79a or CD56 expression. As such CEBPAmeth is rare in cCD3- M0 AML but common in cCD3+ immature T-ALL, independently of myeloid antigen expression. CEBPAmeth and NOTCH1m in IM T-ALL are preferentially associated with cases having undergone TCRG rearrangement but are not found in the majority of more immature cases, demonstrating that CEBPAmeth is not necessary for CD7 and cCD3 expression, within a T-ALL context. It also suggests that CALM-AF10 should be searched for in CEBPAmeth AML, raises issues regarding AML vs. ALL protocol assignement and the use of demethylating agents in IMG T-ALLs and suggests that the potentially misleading “biphenotypic leukaemia” label be reassessed.

The CEBPAgene codes for the C/EBPa transcription factor which regulates hematopoietic stem cell homeostasis and orientates towards myeloid differentiation and against a T lymphoid fate. CEBPAsilencing by promoter hypermethylation (CEBPAmeth) has recently been identified (Wouters, Blood 2007) in a minor subset of AML, all of which demonstrated T lymphoid features (expression of CD7 and CD3 transcripts, TCRG rearrangement and frequent NOTCH1mutations (NOTCH1m)). These AML may be closer to immature T-ALLs, with CEBPa silencing and NOTCH1mdetermining lineage orientation. Although expression of CD7 and cytoplasmic CD3 (cCD3) are considered as defining markers in T-ALL, CD7 is commonly expressed in AML and reproducible multicenter detection of low level cCD3, when performed, can be difficult. We have classified CD7+, cCD3+ T-ALLs on the basis of their TCR profiles: immature cases arrested prior to TCRB VDJ rearrangement include TCRgd lineage precursors and can be divided into those with no TCR rearrangement (IM0), TCRd only rearrangement (IMD) or TCRd and TCRg rearrangement (IMG). The CALM-AF10 fusion transcript is found in both AML and T-ALL, where it is restricted to the TCRgd lineage. We initially searched for in CEBPAmethby methylation specific PCR in 5 AML with downregulated CEBPand confirmed that the 3 cases (FAB-M0/M1) with CEBPAmethexpressed CD3, CD7 and/or TCRg rearrangement. Only 1/49 AML M0, all of which were cCD3 negative, demonstrated CEBPAmeth.We then searched for CEBPAmethand mutation and NOTCH1min 18 IM T-ALL (4 aged <15y; 5 IM0, 5 IMD, 8 IMG) compared to 6 adults with TCRB VDJ+, TCRab lineage T-ALL. No CEBPAmutations were found but 11/24 (56%) demonstrated CEBPAmand 10/23 (43%) NOTCH1m. Both correlated with IMG T-ALL, when 6/8 demonstrated CEBPAmethand NOTCH1mand a seventh only NOTCH1m, compared to only 4/10 CEBPAmethand 1/10 NOTCH1min IM0/D cases, with no overlap. In ab-lineage T-ALLs 2/6 demonstrated NOTCH1m, one of which was also CEBPAmeth, and notably expressed TLX1/HOX11. CEBPAmethcases were more frequently CALM-AF10+ (5/10), CD2-, CD123/IL3R-, HOXA9 transcript+ and IgH DJ rearranged but did not differ from non-CEBPAmethT-ALL in CD34, CD5, CD117, CD13/33, CD79a or CD56 expression. As such CEBPAmethis rare in cCD3- M0 AML but common in cCD3+ immature T-ALL, independently of myeloid antigen expression. CEBPAmethand NOTCH1min IM T-ALL are preferentially associated with cases having undergone TCRG rearrangement but are not found in the majority of more immature cases, demonstrating that CEBPAmethis not necessary for CD7 and cCD3 expression, within a T-ALL context. It also suggests that CALM-AF10 should be searched for in CEBPAmethAML, raises issues regarding AML vs. ALL protocol assignement and the use of demethylating agents in IMG T-ALLs and suggests that the potentially misleading “biphenotypic leukaemia” label be reassessed.

T-LL are rare immature T lymphomas defined by frequent mediastinal involvement, expression of cCD3 and/or CD2 and <25% bone marrow involvement. Their prognosis is strikingly different in children (5 yr. survival approximately 90%) compared to adults (approximately 55%), but no reliable prognostic indicators exist. The extent to which T-LL differ biologically from T-ALL is not known. To adress this question, we performed paraffin TMA immunophenotyping, BIOMED 2 TCR genotyping (Southern and PCR) and RQ-PCR quantification of pTa, RAG1, HOXA5/A9, TLX1/3, LMO1/2, LYL1, TAL1, SIL-TAL, CALM-AF10 and NUP214-ABL (fusion) transcripts in a retrospective series of 44 cCD3+ T-LL (12 aged<16 y; 32 adults; 25 lymphnodes, 13 mediastinal biopsies and 6 pleural effusions). Mediastinal imaging was used to distinguish cases with bulky thymic masses from those with predominant lymphnode involvement. We used detection of TCRB VDJ clonal rearrangement to identify two distinct categories of T-LL: TCRB VDJ positive cases (n=29) and TCRB VDJ negative cases (n=15). TCRB VDJ+ expressed CD2 70%, CD5 95%, CD1a 45%, CD4/8 DP 55%, CD8 SP 25%, CD34 10 % and CD117 18%, pTa and RAG1 transcripts, TCRG end-stage rearrangement and predominant TCRD deletion, in keeping with cases arrested during beta-selection. This category included 90% of pediatric cases but only 56% of adults. 85% corresponded to thymic disease and bone marrow involvement was rare. In contrast, TCRB VDJ - cases were CD1a, CD4, and CD8 negative, with variable expression of CD2 50%, CD5 60%, CD34 40% and CD117 10% and low or absent pTa and RAG1. They include 2 cases with complete TCRD and TCRG rearrangement, but the majority either demonstrate no clonal TCRG or TCRD rearrangement (n=8) or only incomplete TCRD rearrangement (n=4). As such, they correspond to immature cells which could include T lymphoid, NK and/or dendritic precursors. This category was frequent in adults (44%) but rare in children (10%) and was associated with lymphnode disease (75%) and frequent bone marrow involvement (50%). Oncogenic transcripts were analysed in 8 children and 26 adults. TLX1 was over-expressed in 3 adult TCRB VDJ+ cases. HOXA5 and HOXA9 were over-expressed in 7 cases, including the 2 CALM-AF10 cases, 4 TCRB VDJ+ cases and the single case with NUP214-ABL. Only 1 case had LMO1 and TAL1 over expression and none were SIL-TAL1 or TLX3 positive. LMO2 and LYL1 expression was restricted to TCRB VDJ-T-LL. As for adult T-ALLs, initial response to induction was better in adult TCRB VDJ+ T-LL. In conclusion, pediatric T-LL is a predominantly thymic disease arrested during beta-selection but with different oncogenic profiles to T-ALLs arrested at the same stage. Adult T-LL includes approximately 55% of cases with similar characteristics, with the rest corresponding to immature tumors of lymphnode origin, frequent partial bone marrow involvement and a probable inferior response to ALL type induction. TCRB clonality detection represents a simple method which will allow rapid distinction of these T-LL categories and prospective evaluation of their response in therapeutic trials. Transmission of appropriate diagnostic material to specialised diagnostic plateforms will allow better understanding of T-LL, particularly identification of the lineage potential of TCRB VDJ negative cases.

We have previously reported that ex vivo retroviraly-mediated gc gene transfer into CD34 (+) bone marrow precursor cells led to the correction of the immunodeficiency in 9 out of 10 patients with X-linked severe combined immunodeficiency. Follow-up now reaches more that 6 years for the first 2 treated patients. Patients’immune function has been restored. The distribution of both TCR Vb family usage and TCR Vb CDR3 length still reveals a broadly diversified T cell repertoire. Moreover 6 years after treatment the thymus is still seeded by transduced progenitor cells as attested by the presence of TRECS in peripheral blood RTE. Among these patients, three (P4, P5 and P10) developed at 30 to 34 months after gene therapy a monoclonal T cell proliferation requiring a chemotherapy. P4 received also an allogenic HSCT from a MUD but died 26 months after the occurence of the lymphoproliferation. For P5 and P10, chemotherapy has led to an overall control of the clonal proliferation. These two patients are doing well and P5 is off treatment with a good immunological recovery. Genetic analysis of the blastic cells showed that in the two first cases the vector had integrated within or upstream of the LMO2 locus causing an insertional activation of LMO2 transcription. The last case revealed the involvement of several targeted sites, but their exact contribution to the lymphoproliferation is still under investigation. The repeated involvment of LMO2 as a site of vector integration in the proliferating T-cells points to an insertional activation of this gene as at least one of the causes of the oncogenic process. However, the long latency observed in all cases (> 30 months) suggests that additional “hits” have been required for overt desease. Synergy with gc expression and thereby induced proliferative signals (explaining occurrence in SCID-X1 patients only) is the most obvious hypothesis which we are trying to analyse in a mouse model. A deep analysis of retroviral integration patterns has been performed on patients’PBMCS by LAM-PCR to estimate the frequency of potentially harmful integration events and to assess the risk factors associated with the LTR’s strong enhancer effect of the MLV-based retroviral vector. 708 unique integration sites (IS) have been obtained from all analysed patients post-gene therapy and among them, 577 could be mapped unequivocally to the human genome. *Most of these insertions (63%) are located in the vicinity of 10kb or within the coding sequence of a known gene*. A significant peak of insertion frequency is related closely to the transcription strart site *among the 577 IS, 43 are common integration sites. Among the latter, we found out a high selection of genes involved in human oncogenic process.

We have previously reported that ex vivo retroviraly-mediated gc gene transfer into CD34 (+) bone marrow precursor cells led to the correction of the immunodeficiency in 9 out of 10 patients with X-linked severe combined immunodeficiency. Follow-up now reaches more that 6 years for the first 2 treated patients. Patients'immune function has been restored. The distribution of both TCR Vb family usage and TCR Vb CDR3 length still reveals a broadly diversified T cell repertoire. Moreover 6 years after treatment the thymus is still seeded by transduced progenitor cells as attested by the presence of TRECS in peripheral blood RTE. Among these patients, three (P4, P5 and P10) developed at 30 to 34 months after gene therapy a monoclonal T cell proliferation requiring a chemotherapy. P4 received also an allogenic HSCT from a MUD but died 26 months after the occurence of the lymphoproliferation. For P5 and P10, chemotherapy has led to an overall control of the clonal proliferation. These two patients are doing well and P5 is off treatment with a good immunological recovery. Genetic analysis of the blastic cells showed that in the two first cases the vector had integrated within or upstream of the LMO2 locus causing an insertional activation of LMO2 transcription. The last case revealed the involvement of several targeted sites, but their exact contribution to the lymphoproliferation is still under investigation. The repeated involvment of LMO2 as a site of vector integration in the proliferating T-cells points to an insertional activation of this gene as at least one of the causes of the oncogenic process. However, the long latency observed in all cases (> 30 months) suggests that additional “hits” have been required for overt desease. Synergy with gc expression and thereby induced proliferative signals (explaining occurrence in SCID-X1 patients only) is the most obvious hypothesis which we are trying to analyse in a mouse model. A deep analysis of retroviral integration patterns has been performed on patients'PBMCS by LAM-PCR to estimate the frequency of potentially harmful integration events and to assess the risk factors associated with the LTR's strong enhancer effect of the MLV-based retroviral vector. 708 unique integration sites (IS) have been obtained from all analysed patients post-gene therapy and among them, 577 could be mapped unequivocally to the human genome. *Most of these insertions (63%) are located in the vicinity of 10kb or within the coding sequence of a known gene*. A significant peak of insertion frequency is related closely to the transcription strart site *among the 577 IS, 43 are common integration sites. Among the latter, we found out a high selection of genes involved in human oncogenic process.

Coupling of the substituted 2‐acetylresorcinols (I) with the phenacyl bromide (II) followed by in situ cyclization gives the 2‐benzoylbenzofurans (III).