Feasibility and outcome of haploidentical SCT in pediatric high-risk hematologic malignancies and Fanconi anemia in Uruguay.

In total, 17 pediatric patients with hematologic malignancies (n=14) and Fanconi anemia (FA) (n=3) underwent haploidentical SCT with T-cell depletion. The patients were conditioned with reduced-intensity regimens, and CYA was used for GVHD prophylaxis. Successful engraftment occurred in 16 patients (94%). One patient failed to achieve a primary engraftment. Another patient rejected the first SCT after 10 weeks and had a successful second transplant. Of all engrafted patients, only one developed severe acute GVHD. Ten patients were alive at a median follow-up of 18 months (range, 5-62 months). The 5-years' OS was 53.8%. The three patients with FA are currently well with full-donor chimerism at 16, 6 and 5 months post transplant, respectively. The OS of 14 patients with high-risk hematologic malignancies was 47.6%. Three patients died as a result of post transplant leukemia relapse. CMV infection, GVHD and organ injury were other causes of mortality. Haploidentical SCT was found to be an alternative feasible treatment in Uruguay for patients who need allogenic transplantation but lack an HLA-identical family donor. It should be considered as an early option in FA patients before transformation or significant exposure to blood products.

Estudio sobre la miocardiopatía hipertrófica: aspectos clínicos, ecocardiográficos, hemodinámicos y angiocardiográficos en una serie de treinta pacientes / Hypertronic mycardiopathy study: clinical, ecocardiographic, ecographic, hemodynamic and angiographic aspects in series of thirty patients

Se hace la descripción clínica de la miocardiopatía hipertrófica en el medio venezolano. El estudio versó sobre los aspecto clínicos, ecocardiográficos, hemodinámicos y angiocardiográficos de esta entidad, el cual fue realizado en una serie de treinta pacientes, observados durante el lapso de 7 años (1978-1985) en nuestro país. El espectro clínico de la afección fue muy amplio, el cual abarcó desde formas asintomáticas hasta formas con sintomatología de severidad progresiva. El diagnóstico clínico se fundamentó en una historia clínica sugestiva, a veces de carácter familiar, con una signología en la que sobresalen las alteraciones del pulso, el hallazgo de un choque apexiano anormal (bífido o trífido), el latido presistólico y los soplos relacionados con la obstrucción del tracto de salida y la insufiencia valvular mitral. La enfermedad se caracterizó por un cuadro clínico de evolución progresiva. El diagnóstico clínico fue corroborado por la presencia de dos alteraciones ecocardiográficas significativas: 1) la hipertrofia septal asimétrica, 2) el movimiento sistólico anterior de la valva anterior de la válvula mitral o de ambos. En el estudio hemodinámico por la presencia de un gradiente de presión intraventricular en reposo o provocado y en la angiocardiografía por la constatación de la hipertrofia septal y de los músculos papilares. Se desconoce la prevalencia de la afección en escala mundial y nacional. Es necesario realizar estudios sobre el aspecto familiar y las alteraciones genéticas que presenta la afección en nuestro medio. Los estudios patológicos, realizados en el Instituto de Anatomía Patológica, UCV, han permitido corroborar las alteraciones estructurales características de la afección en el material patológico de nuestro medio

Interallelic complementation at the Drosophila melanogaster gastrulation defective locus defines discrete functional domains of the protein.

The gastrulation defective (gd) locus encodes a novel serine protease that is involved in specifying the dorsal-ventral axis during embryonic development. Mutant alleles of gd have been classified into three complementation groups, two of which exhibit strong interallelic (intragenic) complementation. To understand the molecular basis of this interallelic complementation, we examined the complementation behavior of additional mutant alleles and sequenced alleles in all complementation groups. The data suggest that there are two discrete functional domains of Gd. A two-domain model of Gd suggesting that it is structurally similar to mammalian complement factors C2 and B has been previously proposed. To test this model we performed SP6 RNA microinjection to assay for activities associated with various domains of Gd. The microinjection data are consistent with the complement factor C2/B-like model. Site-directed mutagenesis suggests that Gd functions as a serine protease. An allele-specific interaction between an autoactivating form of Snake (Snk) and a gd allele altered in the protease domain suggests that Gd directly activates Snk in a protease activation cascade. We propose a model in which Gd is expressed during late oogenesis and bound within the perivitelline space but only becomes catalytically active during embryogenesis.

Autoproteolysis and feedback in a protease cascade directing Drosophila dorsal-ventral cell fate.

Three serine protease zymogens, Gastrulation defective (GD), Snake (Snk) and Easter (Ea), and a nerve growth factor-like growth factor ligand precursor, Spaetzle, are required for specification of dorsal- ventral cell fate during Drosophila embryogenesis. The proteases have been proposed to function in a sequential activation cascade within the extracellular compartment called the perivitelline space. We examined biochemical interactions between these four proteins using a heterologous co-expression system. The results indicate that the three proteases do function in a sequential activation cascade, that GD becomes active and initiates the cascade and that interaction between GD and Snk is sufficient for GD to cleave itself autoproteolytically. The proteolytically active form of Ea cleaves GD at a different position, revealing biochemical feedback in the pathway. Both GD and Snk bind to heparin-Sepharose, providing a link between the pipe-defined ventral prepattern and the protease cascade. Our results suggest a model of the cascade in which initiation is by relief from inhibition, and spatial regulation of activity is due to interaction with sulfated proteoglycans.

Simultaneous PML/RARalpha and AML1/ETO expression with t(15;17) at onset and relapse with only t(8;21) in an acute promyelocytic leukemia patient.

We report a patient with acute promyelocytic leukemia with the common translocation (15;17) and PML-RARAalpha fusion gene. In relapse, blasts showed typical FAB M2 morphologic features, and the karyotype was 45,X, -Y,t(8;21). A reexamination of the leukemic cells at diagnosis revealed that an AML1-ETO fusion gene was also present at that time without cytogenetic evidence of t(8;21). In relapse, only t(8;21) was detected. Two different clones were identified by cytogenetic standard techniques. The association of two common translocations supervening in the same time in the same cells could not be established.

Correlaciones anatomoclínicas en la enfermedad de chagas / Correlation anatomoclínical in the chagas disease

La Miocardiopatía Chagásica Crónica constituye la forma de miocardiopatía dilatada más frecuentemente encontrada en este trabajo el cual se basa en un material de 14.872 necropsias realizadas durante un período de más de 40 años. En las etapas precoces de la fase indeterminada el corazón chagásico puede encontrarse macroscópicamente normal, como es el caso de pacientes fallecidos súbitamente. Luego cabe observar tres tipos de remodelación ventricular en la fase crónica (concéntrica, ambigua y excéntrica) las cuales suministran la base patológica para explicar la secuencia de las manifestaciones clínicas observadas en la fase crónica

Evaluation of hematopoietic progenitors in hematopoietic progenitor cell transplants. CD34+ dose effect in marrow recovery. Retrospective analysis in 38 patients.

Our main goal was to evaluate the CD34+ dose in patients undergoing haemotopoietic stem celltransplantation and its results in terms of recovery of neutrophile and platelet counts, transfusion requirements, days of fever, antibiotic requirements and length of hospital stay. We studied 38 consecutive patients with haematological malignancies transplanted at our Department, from Feb. 96 through Sept. 98. The CD34+ cell quantification technique was standardized, using a modification of the ISAGHE 96 protocol. Patients were sorted into three groups according to the CD34+ count administered: a) between 3 and 5 x 10(6) cells/kg; b) between 5 and 10 x 10(6) cells/kg; c) > 10 x 10(6) CD34+ cells/kg. As a secondary end point, results were assessed according to the number of aphereses required to arrive at the target count of CD34+, separating those patients that required only 1 or 2 aphereses versus those requiring 3 or more. Finally, an analysis was made of the results of transplantation comparing the different sources of stem cells (PBSC versus PBSC + B.M.). The best results were obtained in the group with cells between 3 and 5 x 10(6) CD34+. No statistically significant advantages were found in the group with cells over 5. The supra-optimal dose of more 10 x 10(6) would yield no additional beneficial results, while they can imply a greater infusion of residual tumor cells. The number of aphereses had no impact on engraftment. Results obtained with PBSC transplants were better than those with BM+PBSC in terms of neutrophile and platelet recovery. The number of CD34+ cells remains the main element in stem cell transplantation to evaluate the haematopoietic recovery after engraftment. Minimum and optimum yields remain unclear. Centers should establish their own optimal dose based on local methodologies and outcomes, maximizing costs and benefits.

High-throughput screening assay for helicase enzymes.

We have developed a novel 96-well microtiter plate high-throughput screening filtration assay for the detection of helicase activity. In this paper we present data for detection of helicase activity of the UL5/8/52 helicase-primase complex from herpes simplex virus 1 (HSV1). The assay involves the detection of radioactively labeled oligonucleotide annealed to a single-stranded circular DNA following capture of the annealed complex on silica beads. We have screened over 200,000 samples containing small organic molecules and natural products and identified T157602, a two-amino thiazole, as a specific inhibitor of HSV replication.

Inhibition of herpes simplex virus replication by a 2-amino thiazole via interactions with the helicase component of the UL5-UL8-UL52 complex.

With the use of a high-throughput biochemical DNA helicase assay as a screen, T157602, a 2-amino thiazole compound, was identified as a specific inhibitor of herpes simplex virus (HSV) DNA replication. T157602 inhibited reversibly the helicase activity of the HSV UL5-UL8-UL52 (UL5/8/52) helicase-primase complex with an IC50 (concentration of compound that yields 50% inhibition) of 5 microM. T157602 inhibited specifically the UL5/8/52 helicase and not several other helicases. The primase activity of the UL5/8/52 complex was also inhibited by T157602 (IC50 = 20 microM). T157602 inhibited HSV growth in a one-step viral growth assay (IC90 = 3 microM), and plaque formation was completely prevented at concentrations of 25 to 50 microM T157602. Vero, human foreskin fibroblast (HFF), and Jurkat cells could be propagated in the presence of T157602 at concentrations exceeding 100 microM with no obvious cytotoxic effects, indicating that the window between antiviral activity and cellular toxicity is at least 33-fold. Seven independently derived T157602-resistant mutant viruses (four HSV type 2 and three HSV type 1) carried single base pair mutations in the UL5 that resulted in single amino acid changes in the UL5 protein. Marker rescue experiments demonstrated that the UL5 gene from T157602-resistant viruses conferred resistance to T157602-sensitive wild-type viruses. Recombinant UL5/8/52 helicase-primase complex purified from baculoviruses expressing mutant UL5 protein showed complete resistance to T157602 in the in vitro helicase assay. T157602 and its analogs represent a novel class of specific and reversible anti-HSV agents eliciting their inhibitory effects on HSV replication by interacting with the UL5 helicase.

Spatial regulation of Drosophila snake protease activity in the generation of dorsal-ventral polarity.

Positional information along the dorsal-ventral axis of the Drosophila embryo is acquired through a signal transduction pathway which employs a extracellular protease cascade. The sequential activation of serine protease zymogens results in the ventrally localized production of a ligand in the perivitelline space of the embryo. Snake is one of several serine proteases which function in generating the ventralizing signal. Here, we investigate the biochemical properties of Snake in vivo and in vitro using recombinant forms of the protease. Wild-type Snake zymogen completely rescues embryos from snake null females when microinjected into the perivitelline space. Biochemical evidence for a covalently associated two-chain form of the activated protease is presented. The contribution of the activation peptide region to zymogen activation was addressed using site-directed mutagenesis. The phenotypic rescue properties of an autoactivated form of Snake reveal that the covalently associated proenzyme polypeptide chain suppresses a dominant effect associated with the activated catalytic chain alone. Recombinant active catalytic chain was produced and found to be short lived as a recombinant protein. These results suggest a model in which the proenzyme polypeptide both stabilizes and targets the Snake catalytic chain to a ventrally localized activation complex within the perivitelline space.

Mutational analysis of the Drosophila snake protease: an essential role for domains within the proenzyme polypeptide chain.

Two genes involved in the generation of dorsoventral asymmetry in the developing Drosophila melanogaster embryo, snake and easter, encode the zymogen form of serine proteases. Mutant alleles of snake were cloned and sequenced revealing two types of lesions: point mutations which alter the amino acid sequence (snk073 and snkrm4) and point mutations which alter the splicing (snk229 or snk233) of intron 1 of the mRNA from the normal 3' end of the intron to a cryptic site. snake mutant embryos derived from homozygous mothers can be fully rescued by injection of RNA transcripts of the wild-type snake cDNA. RNA phenotypic rescue and site-directed mutagenesis experiments indicate that snake requires the serine, histidine and aspartic acid of the catalytic triad for normal activity. Deletion experiments show that an acidic proenzyme domain is required for snake rescue activity to be uniformly distributed throughout the embryo. A second proenzyme domain, called the disulfide knot, appears to be essential for normal regulation of activity of the snake catalytic chain. Transcripts encoding only the proenzyme polypeptides of either snake or easter can dorsalize wild type embryos. We propose a model in which the proenzyme determinants of both the snake and easter enzymes mediate interaction between the serine proteases and other components of the dorsal-ventral patterning system.

Radionuclide evaluation of left-ventricular function in chronic Chagas' cardiomyopathy.

Left-ventricular ejection fraction (LVEF) and abnormalities of regional wall motion (WMA) were studied by means of radionuclide ventriculography in 41 patients prospectively diagnosed as having chronic Chagas' disease. Thirteen patients were asymptomatic (ASY), 16 were arrhythmic (ARR), and 12 had congestive heart failure (CHF). Mean LVEF was normal in ASY (0.64 +/- 0.06) but markedly depressed in CHF (0.28 +/- 0.08). Regional WMAs were minimal in ASY and their severity increased in ARR. Most CHFs (75%) had diffuse hypokinesia of the left ventricle. The region most frequently affected was the infero-apical (63%). Seven patients had a distinct apical aneurysm. Correlation between radionuclide and contrast ventriculography data was good in 17 patients. For LVEF, r = 0.90. For WMA there was agreement between the two techniques in 77% of 65 segments compared. Best agreement occurred with infero-apical lesions (88%), and worst with septal (69%). Selective coronary arteriography showed normal arteries in all patients. Therefore, chronic Chagas' heart disease joins ischemic heart disease as a cause of regional WMA.

Diagnosis of Chagas' cardiomyopathy. Non-invasive techniques.

The natural history of Chagas' disease and its manifestations when the heart is involved are detailed clinically and pathologically. Three phases are recognized: the acute phase, lasting from 1-3 months, the latent phase, which may last from 10-20 years, and the chronic phase, which has the most serious manifestations. This phase is subdivided into three clinical stages. An analysis of the varied cardiac manifestations on 235 patients is included.