Emergence of translocation t(9;11)-positive leukemia during treatment of childhood acute lymphoblastic leukemia

Therapy-related acute myeloid leukemia (t-AML) characterized by the t(9;11)(p22;q23) translocation is one of the most frequent secondary malignancies. The timing of the initiation of translocation and of development of the malignant t(9;11) clone during chemotherapy is presently unknown. In the present study, we backtracked bone marrow samples from three children during treatment for acute lymphoblastic leukemia (ALL). Two patients developed a t(9;11)-positive t-AML 19 and 30 months after therapy start, whereas the third patient, diagnosed with a rare t(9;11)-positive ALL, suffered from an ALL relapse 23 months after initial diagnosis. The genomic MLL-MLLT3 (MLL-AF9) fusion site was amplified by a multiplex, nested long-range PCR and used as a clonal marker for quantification of the MLL-MLLT3-positive cells during chemotherapy. The t(9;11)-positive clone was detectable 13 and 18 months after therapy start in both t-AML cases, which was 6-12 months before clinical diagnosis of the secondary malignancy. In the t(9;11)-positive ALL patient, the identical leukemic clone reoccurred during maintenance therapy after a short molecular remission, 8 months before clinically overt ALL relapse. The time course and characteristics of the genomic breakpoints in the present t-AML cases support the hypothesis of translocation formation as a result of defective breakage repair after topoisomerase II cleavage.     

Genetic ablation of FLRT3 reveals a novel morphogenetic function for the anterior visceral endoderm in suppressing mesoderm differentiation

During early mouse development, the anterior visceral endoderm (AVE) secretes inhibitor and activator signals that are essential for establishing the anterior–posterior (AP) axis of the embryo and for restricting mesoderm formation to the posterior epiblast in the primitive streak (PS) region. Here we show that AVE cells have an additional morphogenetic function. These cells express the transmembrane protein FLRT3. Genetic ablation of FLRT3 did not affect the signaling functions of the AVE according to the normal expression pattern of Nodal and Wnt and the establishment of a proper AP patterning in the epiblast. However, FLRT3^−/− embryos showed a highly disorganized basement membrane (BM) in the AVE region. Subsequently, adjacent anterior epiblast cells displayed an epithelial-to-mesenchymal transition (EMT)-like process characterized by the loss of cell polarity, cell ingression, and the up-regulation of the EMT and the mesodermal marker genes Eomes, Brachyury/T, and FGF8. These results suggest that the AVE acts as a morphogenetic boundary to prevent EMT and mesoderm induction in the anterior epiblast by maintaining the integrity of the BM. We propose that this novel function cooperates with the signaling activities of the AVE to restrict EMT and mesoderm induction to the posterior epiblast.     

Modification of T-cell receptor Vβ repertoire in response to allergen stimulation in peanut allergy

Background: Peanut is one of the most common foods causing allergic reactions and is the most common cause of fatal and near-fatal food-related anaphylaxis. Little is known of the immunologic mechanisms that underlie peanut allergy. Objectives: In this study we examined clonality of the T-cell response (TCR) to peanut in MHC class II identical, peanut allergy–discordant sibling pairs. Methods: Four sibling pairs were investigated. The TCR repertoire was analyzed before and after in vitro stimulation of PBMCs with crude peanut or PHA, as control for general/nonspecific reactivity. Eighteen TCR-Vβ families were examined by flow cytometry. Where significant differences in incidence of particular TCR-Vβ families were observed, PCR familyspecific cDNA amplification and gene scanning were performed. Results: After stimulation with peanut, no selective expansion of any TCR-Vβ subpopulation was observed with flow cytometry, in either the peanut-allergic or nonallergic siblings, with the exception of 1 peanut-allergic subject who demonstrated a significant increase of TCR-Vβ11⁺ cells (0.3%-5.9% of the total CD3⁺ cells). However, gene scanning revealed predominant single-size PCR products for TCRBV11 in all peanut-allergic subjects after peanut stimulation. TCRBV11 polyclo-nality was observed in allergic and nonallergic subjects before peanut stimulation and in nonallergic subjects after peanut stimulation. In comparison, all subjects, before and after stimulation with peanut, showed polyclonality for TCRBV2.Conclusions: Our results argue for clonal or oligoclonal TCRs to crude peanut and indicate that changes in the TCRBV11 subpopulation are restricted to peanut-allergic subjects after stimulation with crude peanut allergen. (J Allergy Clin Immunol 2001;107:1089-94.)     

Augmentation in the infectivity of SV40 virus DNA by amphotericin B methyl ester

Summary The methyl ester of amphotericin B (100 µg/ml) enhanced the infectivity of SV40 virus DNA for CV-1 cells up to 300-fold. Amphotericin B methyl ester may prove useful for augmenting cellular penetration of macromolecules and for probing biological mechanisms.With 2 Figures     

A microperfusion investigation of bicarbonate secretion by the rat submaxillary gland

Summary Bicarbonate transport in the rat submaxillary main duct has been studied by microperfusion. Bicarbonate was concentrated in the duct lumen against an electrochemical gradient and the equilibrium concentration was estimated to be 56.5 mEq/l±3.1 (S.E.M.,n=11). The secretory mechanism could not be inhibited by 6 mMolar cyanide although such concentrations caused marked inhibition of both net sodium efflux and net potassium influx. Bicarbonate secretion in the main duct was not inhibited by acetazolamide whether applied from the duct lumen or given intravenously. Similarly, the drug was without effect on bicarbonate excretion by the intact gland even when maximum excretory rates had been induced with carbachol. It was concluded that catalytic hydration of carbon dioxide to carbonic acid was not a rate-limiting step in the bicarbonate secretory process. The data did not permit a distinction to be made between a bicarbonate secretory processper se and a process of either H⁺ reabsorption or OH^– secretion.The parasympathomimetic agent, carbachol, when given parenterally was found to increase sharply the net influx of bicarbonate into the microperfused main duct as well as to reduce net sodium efflux and net potassium influx. Previously it had been postulated that final saliva was formed in two stages. First a plasma-like primary secretion was formed at a rate depending on the degree of stimulation, and second, the primary secretion was modified in the gland duct system by reabsorptive and secretory processes whose transport rates were presumed to be independent of the degree of stimulus. It now becomes necessary to postulate that stimulation can act on electrolyte transport at both primary and secondary levels; at present, however, no data are available to show whether appreciable net water influx can ever occur at the secondary level.Preliminary reports of this study have been presented to the Australian Physiological and Pharmacological Society [28, 31].     

Relationship between X-inactivation and clinical involvement in Fabry heterozygotes. Eleven novel mutations in the α-galactosidase A gene in the Czech and Slovak population

We have identified 21 different -galactosidase A gene (GLA) mutations in 22 unrelated Czech and Slovak families with Fabry disease. Eleven of these mutations were novel (point mutations D93N, A135V, D155H, G171R, Q280K, G360S, Q330X, splicing errors c.194ins14, c.801ins36 and deletions c.674_732del59, g.3405_6021del2617). Genotyping of family members for family-specific mutations revealed 55 heterozygotes that manifested clinical symptoms of different severity. To examine the contribution of X-inactivation skewing to disease manifestation in Fabry heterozygotes, we have adopted the Mainz severity scoring scheme and compared the score values with the X-inactivation status in 39 carriers in an age-dependent manner. The age-score trendline of Fabry females who had a predominantly inactivated X-chromosome bearing a wild-type GLA allele (10 of 38 females) was markedly steeper than in the rest of the cohort. One female carrier with an inactivated mutated allele had a low score value when compared to the other heterozygotes of the same age. These data suggest that X-inactivation is indeed a major factor determining the severity of clinical involvement in Fabry heterozygotes. There was a statistically significant difference between the severity score values of heterozygotes with random and non-random X-chromosome inactivation at the 5% level of significance. Further studies will show if the degree of the wildtype allele inactivation will be useful as a predictive marker of severity of phenotype in Fabry heterozygotes. Although the correlation between X-inactivation skewing and presentation of the disease in Fabry heterozygotes has previously been suggested in the literature, this report is among the first attempts to examine this relationship systematically.     

Results of unrelated cord blood transplant in fanconi anemia patients: risk factor analysis for engraftment and survival.

We retrospectively analyzed results of unrelated cord blood transplantation (UCBT) in 93 Fanconi anemia (FA) patients. Median age at transplantation was 8.6 years (1-45). The units transplanted were HLA-A, -B, or -DRB1 identical in 12 cases, 1 HLA mismatch in 35 cases, and 2 or 3 HLA differences in 45 cases. The median number of nucleated cells (NC) and CD34+ cells infused of recipient weight was 4.9x10(7)/kg and 1.9x10(5)/kg, respectively. Participating centers selected the preparative regimen of their choice, in 57 patients (61%), it included Fludarabine. Graft-versus-host disease (GVHD) prophylaxis consisted mostly of cyclosporine with prednisone. Cumulative incidence (CI) of neutrophil recovery was 60+/-5% at day +60. In multivariate analysis, Fludarabine containing regimen and NC infused>or=4.9x10(7)/kg were associated with higher probability of recovery. CI of grade II-IV acute and of chronic GVHD (aGVHD, cGVHD) was 32%+/-5% and 16%+/-4%, respectively. Overall survival (OS) was 40%+/-5%. In multivariate analysis, factors associated with favorable outcome were use of Fludarabine in the conditioning regimen, number of NC infused>or=4.9x10(7)/kg, and negative cytomegalovirus (CMV) serology in the recipient. In conclusion, factors easily modifiable such as donor selection and a Fludarabine-containing regimen can considerably improve survival in FA patients given a UCBT. These data are the basis for designing prospective protocols.