RESUMEN
Patients with hypomorphic mutations in the RAG1 or RAG2 gene present with either Omenn syndrome or atypical combined immunodeficiency with a wide phenotypic range. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but data are scarce. We report on a worldwide cohort of 60 patients with hypomorphic RAG variants who underwent HSCT, 78% of whom experienced infections (29% active at HSCT), 72% had autoimmunity, and 18% had granulomas pretransplant. These complications are frequently associated with organ damage. Eight individuals (13%) were diagnosed by newborn screening or family history. HSCT was performed at a median of 3.4 years (range 0.3-42.9 years) from matched unrelated donors, matched sibling or matched family donors, or mismatched donors in 48%, 22%, and 30% of the patients, respectively. Grafts were T-cell depleted in 15 cases (25%). Overall survival at 1 and 4 years was 77.5% and 67.5% (median follow-up of 39 months). Infection was the main cause of death. In univariable analysis, active infection, organ damage pre-HSCT, T-cell depletion of the graft, and transplant from a mismatched family donor were predictive of worse outcome, whereas organ damage and T-cell depletion remained significant in multivariable analysis (hazard ratio [HR] = 6.01, HR = 8.46, respectively). All patients diagnosed by newborn screening or family history survived. Cumulative incidences of acute and chronic graft-versus-host disease were 35% and 22%, respectively. Cumulative incidences of new-onset autoimmunity was 15%. Immune reconstitution, particularly recovery of naïve CD4+ T cells, was faster and more robust in patients transplanted before 3.5 years of age, and without organ damage. These findings support the indication for early transplantation.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Recién Nacido , Humanos , Donantes de Tejidos , Linfocitos T , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Diagnóstico Precoz , Costo de Enfermedad , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/etiología , Estudios Retrospectivos , Donante no Emparentado , Acondicionamiento PretrasplanteRESUMEN
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. NCT01347346 and 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.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas , Lentivirus/genética , Síndrome de Wiskott-Aldrich/terapia , Adolescente , Adulto , Niño , Preescolar , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Humanos , Lactante , Resultado del Tratamiento , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/inmunología , Adulto JovenRESUMEN
Sperm parameters are known to be impaired in men with sickle cell disease (SCD). Although treatment with hydroxyurea (HU) has an impact on sperm quality, sperm preservation is impossible before puberty. This study's primary objective was to analyze and compare sperm parameters in male patients with SCD exposed (or not) to HU before puberty. Twenty-six sperm samples from 15 patients (median age, 17 years; range, 16-23) treated with HU during childhood were compared with 46 samples from 23 HU-naïve patients (20 years; 16-24). The median age at HU initiation was 6 years (1-14 years), the median duration of HU treatment was 4 years (0.5-10), and the mean dose of HU was 22.4 ± 3.7 mg/kg per day. Although we observed substantial quantitative and qualitative semen abnormalities in all patients, there were no significant differences in semen volume, sperm concentration, total sperm count, or spermatozoa motility, morphology, and vitality between the HU-exposed and HU-naïve groups. At the time of the semen analysis, 100% of the patients in the HU-exposed group and 52% of the patients in the HU-naïve group received transfusion therapy. The specific effect of HU on spermatogenesis in very young infants and the putative value of transfusion for reversing the toxicity of HU warrant further investigation.
Asunto(s)
Anemia de Células Falciformes/tratamiento farmacológico , Antidrepanocíticos/efectos adversos , Hidroxiurea/efectos adversos , Infertilidad Masculina/inducido químicamente , Pubertad , Espermatogénesis/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Síndrome Torácico Agudo/epidemiología , Síndrome Torácico Agudo/etiología , Adolescente , Factores de Edad , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/fisiopatología , Anemia de Células Falciformes/terapia , Antidrepanocíticos/administración & dosificación , Antidrepanocíticos/uso terapéutico , Arteriopatías Oclusivas/epidemiología , Arteriopatías Oclusivas/etiología , Transfusión Sanguínea , Niño , Preescolar , Terapia Combinada , Humanos , Hidroxiurea/administración & dosificación , Hidroxiurea/uso terapéutico , Lactante , Masculino , Recuento de Espermatozoides , Motilidad Espermática/efectos de los fármacos , Adulto JovenRESUMEN
Clinically useful pre-transplant predictive factors of acute graft-versus-host-disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT) are lacking. We prospectively analyzed HSC graft content in CD34+, NK, conventional T, regulatory T and invariant natural killer T (iNKT) cells in 117 adult patients before allo-SCT. Results were correlated with occurrence of aGVHD and relapse. In univariate analysis, iNKT cells were the only graft cell populations associated with occurrence of aGVHD. In multivariate analysis, CD4- iNKT/T cell frequency could predict grade II-IV aGVHD in bone marrow and peripheral blood stem cell (PBSC) grafts, while CD4- iNKT expansion capacity was predictive in PBSC grafts. Receiver operating characteristic analyses determined the CD4- iNKT expansion factor as the best predictive factor of aGVHD. Incidence of grade II-IV aGVHD was reduced in patients receiving a graft with an expansion factor above versus below 6.83 (9.7 vs 80%, P<0.0001), while relapse incidence at two years was similar (P=0.5).The test reached 94% sensitivity and 100% specificity in the subgroup of patients transplanted with human leukocyte antigen 10/10 PBSCs without active disease. Analysis of this CD4- iNKT expansion capacity test may represent the first diagnostic tool allowing selection of the best donor to avoid severe aGVHD with preserved graft-versus-leukemia effect after peripheral blood allo-SCT.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Células T Asesinas Naturales/inmunología , Donantes de Tejidos , Enfermedad Aguda , Femenino , Enfermedad Injerto contra Huésped/diagnóstico , Humanos , Masculino , Células T Asesinas Naturales/metabolismo , Periodo Preoperatorio , Pronóstico , Índice de Severidad de la Enfermedad , Trasplante HomólogoRESUMEN
Artemis is a factor of the non-homologous end joining pathway involved in DNA double-strand break repair that has a critical role in V(D)J recombination. Mutations in DCLRE1C/ARTEMIS gene result in radiosensitive severe combined immunodeficiency in humans owing to a lack of mature T and B cells. Given the known drawbacks of allogeneic hematopoietic stem cell transplantation (HSCT), gene therapy appears as a promising alternative for these patients. However, the safety of an unregulated expression of Artemis has to be established. We developed a transgenic mouse model expressing human Artemis under the control of the strong CMV early enhancer/chicken beta actin promoter through knock-in at the ROSA26 locus to analyze this issue. Transgenic mice present a normal development, maturation and function of T and B cells with no signs of lymphopoietic malignancies for up to 15 months. These results suggest that the over-expression of Artemis in mice (up to 40 times) has no deleterious effects in early and mature lymphoid cells and support the safety of gene therapy as a possible curative treatment for Artemis-deficient patients.
Asunto(s)
Endonucleasas/genética , Linfopoyesis , Linfocitos T/citología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN , Endonucleasas/uso terapéutico , Terapia Genética , Humanos , Cambio de Clase de Inmunoglobulina/genética , Linfopoyesis/genética , Ratones , Ratones Transgénicos , Inmunodeficiencia Combinada Grave/terapia , Linfocitos T/inmunologíaRESUMEN
Reticular dysgenesis is a human severe combined immunodeficiency that is primarily characterized by profound neutropenia and lymphopenia. The condition is caused by mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of mitochondrial AK2 protein expression. AK2 regulates the homeostasis of mitochondrial adenine nucleotides (ADP, ATP and AMP) by catalyzing the transfer of high-energy phosphate. Our present results demonstrate that AK2-knocked-down progenitor cells have poor proliferative and survival capacities and are blocked in their differentiation toward lymphoid and granulocyte lineages. We also observed that AK2 deficiency impaired mitochondrial function in general and oxidative phosphorylation in particular - showing that AK2 is critical in the control of energy metabolism. Loss of AK2 disrupts this regulation and leads to a profound block in lymphoid and myeloid cell differentiation.
Asunto(s)
Adenilato Quinasa/genética , Leucopenia/genética , Linfocitos/enzimología , Mitocondrias/genética , Neutrófilos/enzimología , Inmunodeficiencia Combinada Grave/genética , Células Madre/enzimología , Nucleótidos de Adenina/metabolismo , Adenilato Quinasa/deficiencia , Antígenos CD34/genética , Antígenos CD34/metabolismo , Diferenciación Celular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HL-60 , Humanos , Leucopenia/enzimología , Leucopenia/patología , Linfocitos/patología , Mitocondrias/enzimología , Mitocondrias/patología , Mutación , Neutrófilos/patología , Fosforilación Oxidativa , Cultivo Primario de Células , Inmunodeficiencia Combinada Grave/enzimología , Inmunodeficiencia Combinada Grave/patología , Células Madre/patologíaRESUMEN
Gene therapy has effectively entered Medicine via the field of primary immunodeficiencies (PID). Because hematopoietic stem cells are accessible and because it was understood that genetic correction of lymphocyte progenitor cells carrying a genetic defect impairing differentiation, could result in the production of long-lived T lymphocytes, it was reasoned that ex vivo gene transfer in hematopoietic cells could lead to disease phenotype correction. Retroviral vectors were designed to ex vivo transduce such cells. This has indeed been shown to lead to sustained correction of the T cell immunodeficiency associated with two forms of severe combined immunodeficiencies (SCID) for now more than ten years. Occurrence in some patients of genotoxicity related to retroviral vectors integration close to and transactivation of oncogenes has led to the development of retroviral vectors devoid of its enhancer element. Results of recent trials performed for several forms of PID indeed suggest that their use is both safe and efficacious. It is thus anticipated that their application to the treatment of many more life threatening PID will be developed over the coming years.
Asunto(s)
Terapia Genética/métodos , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapia , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Terapia Genética/tendencias , Vectores Genéticos/genética , Humanos , Reproducibilidad de los Resultados , Retroviridae/genética , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
The correction of genetic mutations by homologous recombination is an attractive approach to gene therapy. We used the DNA double-strand breaks introduced by the site-specific endonuclease I-Sce1 as a means of increasing homologous recombination of an exogenous DNA template in murine hematopoietic stem cells (mHSCs). To develop this approach, we chose an Artemis knockout (Art(-/-)) mouse in which exon 12 of the Artemis gene had been replaced by an I-Sce1 recognition site. The I-Sce1 enzyme and the Artemis correction template were each delivered by a self-inactivating (SIN)-integrase-defective lentiviral vector (SIN-IDLV-CMV-ISce1 and SIN-IDLV-Art, respectively). Transduction of Art(-/-) mHSCs with the two vectors successfully reverted the Art(-/-) phenotype in 2 of our 10 experiments. Even though the potential for genotoxicity has yet to be evaluated, this new approach to gene editing appears to be promising. Improving the efficacy of this approach will require further technical work.
Asunto(s)
Endonucleasas/genética , Terapia Genética/métodos , Células Madre Hematopoyéticas/citología , Recombinación Homóloga/genética , Proteínas Nucleares/genética , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Endonucleasas/deficiencia , Vectores Genéticos , Lentivirus/genética , Ratones , Ratones Noqueados , Mutación , Proteínas Nucleares/deficiencia , Proteínas de Saccharomyces cerevisiae , Transducción GenéticaRESUMEN
Scarce data exist on allogeneic hematopoietic stem cell transplantation (HSCT) outcomes in hepatitis B virus (HBV)-naïve recipients from HBV-experienced donors. Long-term follow-up is herein reported for 17 allogeneic HSCT performed in 13 HBV-naïve children from HBc-antibodies-positive donors between 2006 and 2012. Four donors were HBs-antigen-positive, with detectable but low viremia in 2 cases (<2 log10IU/ml). HBV-DNA was undetectable in all transplanted cell products. Recipients' HBV prophylaxis consisted of pre-transplant vaccination, polyvalent immune globulins, specific anti-HBV immune globulins, and/or oral lamivudine in 3, 12, 8, and 8 children, respectively. No case of HBV transmission occurred based on negative close monitoring of recipients' HBV serology and plasma HBV-DNA during a median follow-up of 22 months. In case of undetectable viremia in the donor, prophylaxis with vaccination and/or immune globulins in the recipient seems to be sufficient and lamivudine prophylaxis might be unnecessary to prevent viral transmission. In case of undetectable viremia in the donor, a systematic screening of HBV DNA in the stem cell product might be unnecessary to confirm the low risk of viral transmission. Prior exposure to HBV in the donor should not be considered a contraindication to HSCT.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/normas , Anticuerpos contra la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Donantes de Tejidos/estadística & datos numéricos , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Estudios Retrospectivos , Viremia/sangreRESUMEN
We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TCbat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded low parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype was found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans.
Asunto(s)
Quirópteros/parasitología , ADN Protozoario/genética , Genes Protozoarios/genética , Filogenia , Trypanosoma cruzi/clasificación , Trypanosoma cruzi/genética , Tripanosomiasis/veterinaria , Animales , Brasil , Grupo Citocromo b/genética , ADN Ribosómico/genética , ADN Espaciador Ribosómico/genética , Genotipo , Histonas/genética , Cariotipificación , Ratones , Datos de Secuencia Molecular , Técnica del ADN Polimorfo Amplificado Aleatorio , Triatominae/parasitología , Trypanosoma cruzi/citología , Tripanosomiasis/parasitologíaRESUMEN
Trypanosoma rangeli infects several mammalian orders but has never confidently been described in Chiroptera, which are commonly parasitized by many trypanosome species. Here, we described trypanosomes from bats captured in Central Brazil identified as T. rangeli, T. dionisii, T. cruzimarinkellei and T. cruzi. Two isolates, Tra643 from Platyrrhinus lineatus and Tra1719 from Artibeus planirostris were identified as T. rangeli by morphological, biological and molecular methods, and confirmed by phylogenetic analyses. Analysis using SSU rDNA sequences clustered these bat trypanosomes together with T. rangeli from other hosts, and separated them from other trypanosomes from bats. Genotyping based on length and sequence polymorphism of PCR-amplified intergenic spliced-leader gene sequences assigned Tra1719 to the lineage A whereas Tra643 was shown to be a new genotype and was assigned to the new lineage E. To our knowledge, these two isolates are the earliest T. rangeli from bats and the first isolates from Central Brazil molecularly characterized. Rhodnius stali captured for this study was found infected by T. rangeli and T. cruzi.
Asunto(s)
Quirópteros/parasitología , ARN Lider Empalmado/genética , Trypanosoma/clasificación , Trypanosoma/aislamiento & purificación , Tripanosomiasis/veterinaria , Animales , Brasil , Análisis por Conglomerados , ADN Protozoario/química , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Genotipo , Datos de Secuencia Molecular , Filogenia , Polimorfismo Genético , Análisis de Secuencia de ADN , Homología de Secuencia , Trypanosoma/citología , Trypanosoma/genéticaRESUMEN
Trypanosomatids are widespread in several plant families and although most isolates have been classified as Phytomonas, other trypanosomatid genera can also infect plants. In order to assess the natural occurrence of non-Phytomonas trypanosomatids in plants we characterized 21 new trypanosomatid cultures, 18 from fruits and three from seeds of 17 plant species. The trypanosomatids from fruit and seeds were compared in terms of morphological, growth, biochemical and molecular features. The high diversity among the isolates permitted the classification of the new flagellates into the genera Crithidia and Leptomonas as well as Phytomonas. The data showed that natural fruit infection with non-Phytomonas trypanosomatids is more common than usually thought, being detected in 43% of the fruit isolates.
Asunto(s)
Frutas/parasitología , Semillas/parasitología , Trypanosomatina/clasificación , Animales , Crithidia/clasificación , Crithidia/citología , Crithidia/aislamiento & purificación , Crithidia/fisiología , Dermatoglifia del ADN , Técnica del Anticuerpo Fluorescente Indirecta , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Técnica del ADN Polimorfo Amplificado Aleatorio , Ribotipificación , Trypanosomatina/citología , Trypanosomatina/aislamiento & purificación , Trypanosomatina/fisiologíaRESUMEN
The defense response of Veneza zonata (Hemiptera: Coreidae) against three different trypanosomatid infections was assessed: (1) strain 714TD, a Leptomonas which has V. zonata as vector of a plant trypanosomatid, (2) strain 563TD, a Leptomonas isolated from the digestive tract of Euchistus heros (Hemiptera: Pentatomidae), and (3) Leishmania (L.) amazonensis, a human parasite that cannot infect V. zonata. Experiments with V. zonata hemolymph showed agglutination only of L. (L.) amazonensis culture forms and hemocytic recognition was more intense with this strain. L. (L.) amazonensis also activated the prophenoloxidase system, whereas strains 714TD and 563TD did not activate this system but rather seemed to inhibit phenoloxidase activity. No flagellates were seen in the digestive tract, hemolymph, or salivary glands in insects infected with L. (L.) amazonensis. The digestive tract, the hemolymph, and the salivary glands of insects fed on tomatoes inoculated with 714TD are sequentially invaded by the flagellate, which is inoculated in plants together with saliva. Insects fed on tomatoes inoculated with 563TD exhibited culture forms in the digestive tract (6 days after) and hemocoel (three additional days); however, they died 12 to 14 days after exposure. The salivary glands in insects inoculated in the hemocoel with 714TD strain are rapidly invaded, whereas those with 563TD culture forms died approximately 24 h after infection. Bacterial proliferation in the hemocoel and hemocyte surface blebbing were seen in insects infected only with 563TD strain as the probable pathogenic mechanism of insect death.
Asunto(s)
Hemípteros/parasitología , Hemolinfa/fisiología , Infecciones por Protozoos/fisiopatología , Trypanosomatina/fisiología , Animales , Interacciones Huésped-Parásitos , Técnicas In VitroRESUMEN
In the present study, we investigated the genetic variability among 49 new isolates of trypanosomatids from phytophagous Hemiptera by means of morphological characters, growth features, and biochemical (enzymes of ornithine-arginine cycle) and molecular markers (based on spliced-leader, and ribosomal genes). From 402 phytophagous insects dissected and examined for the presence of trypanosomatids, 228 species belonging to Pyrrhocoridae, Coreidae, Lygaeidae, and Pentatomidae families harbored trypanosomatids in their salivary glands, or digestive tubes. Among these insects, 211 carried promastigotes and only 17 had choanomastigote forms. The results show a strong association among morphology, growth features, and biochemical and molecular markers and reveal the genetic diversity of the isolates, which were assigned to Crithidia, Phytomonas, and Leptomonas; we found genetic polymorphism within all these genera, thus indicating high genetic variability among trypanosomatids from phytophagous insects.
Asunto(s)
Variación Genética , Hemípteros/parasitología , Plantas/parasitología , Trypanosomatina/genética , Animales , Arginina/metabolismo , Crithidia/aislamiento & purificación , Ornitina/metabolismo , Trypanosomatina/clasificación , Trypanosomatina/enzimología , Trypanosomatina/aislamiento & purificaciónRESUMEN
The Western blot method, using antigens from epimastigote forms of the Trypanosoma cruzi Y strain, was evaluated for the confirmatory diagnosis of Chagas' disease. Serum samples were obtained from 136 chagasic patients (Group I), 23 patients with inconclusive serologic results for Chagas' disease (Group II), 53 patients with other diseases (Group III), and 50 healthy individuals (Group IV). The Western blot results for Group I gave a confirmatory diagnosis of Chagas' diseases in 118 (86.80%), an indeterminate pattern of reactivity in 16 (11.76%), and a negative pattern in only two (1.47%). Of the samples from Groups II, III and IV, none had a positive result in the Western blot; 60.86%, 52.83%, and 16.0%, respectively, showed indeterminate results; and 39.13%, 47,16% and 84,0%, respectively, showed negative results. The Western blot method showed a sensitivity of 86.60%, a specificity of 100.0%, a positive predictive value of 100.0%, and a negative predictive value of 73.50%, and the concordance coefficient kappa was high (0.7789). The results suggest that the previous serologic results for Chagas' disease could be confirmed by Western blot for the detection of specific antibodies to T. cruzi antigenic fractions, which may reduce the medical, legal, and social consequences of an inconclusive serologic result for Chagas' disease and also underscore the need for additional studies for continued efforts in the development of an ideal standard confirmatory test for Chagas' disease.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Western Blotting/métodos , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/inmunología , Pruebas Serológicas/métodos , Trypanosoma cruzi/inmunología , Animales , Donantes de Sangre , Western Blotting/estadística & datos numéricos , Brasil , Estudios de Casos y Controles , Estudios de Evaluación como Asunto , Reacciones Falso Negativas , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Pruebas Serológicas/estadística & datos numéricosRESUMEN
The effects of adriamycin (ADM), arabinosyl-cytosine (ARA-C), and etoposide (VP16) were studied on human bone marrow mononucleated cells using colony formation in agar, a modified liquid culture system, and flow cytometry analysis of the cell cycle. Drug concentrations tested during a 1-h incubation ranged from 0.1 to 4 micrograms/ml for ADM, from 0.3 to 30 micrograms/ml for VP16, and from 10(-7) to 10(-3) M for ARA-C. Regression analysis of the dose-response curves was used to assess the drug concentration that inhibited 90% +/- 5% (LD90) of colony growth. LD90s were 0.4 microgram/ml for ADM, 20 micrograms/ml for VP16, and 10(-4) M for ARA-C. LD90-surviving cells were cultured in liquid medium for 3 weeks. Surviving cells over this time were 13% of the control for ADM, 22% for VP16, and 95.7% for ARA-C. Although cells decreased drastically in ADM- and VP16-treated samples, granulocyte-macrophage colony-forming units (CFU-GM) per 10(5) surviving cells rose to twice the control for ADM, to 60% for VP16, and to 150% for ARA-C. Flow cytometry analysis of the cell cycle was performed at day 0 and at day 4 after treatment with the LD90 dose. It showed a rapid and reversible effect of ARA-C on cells in the S-phase, whereas the action of VP16 concerned all cells, regardless of their cycle phase. We conclude that the direct effects of the three drugs on CFU-GM in agar are poorly predictive of hematopoietic reconstitution capacity, except for VP16. Liquid culture gives a much more accurate appraisal of the long-term damage and recovery due to anticancer drugs.
Asunto(s)
Células de la Médula Ósea , Citarabina/farmacología , Doxorrubicina/farmacología , Etopósido/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Ciclo Celular , Supervivencia Celular , Medios de Cultivo , Granulocitos/citología , Granulocitos/efectos de los fármacos , Humanos , Monocitos/citología , Monocitos/efectos de los fármacosRESUMEN
In a consecutive series of 65 patients treated by allogeneic bone marrow transplantation, we have compared the efficacy on prevention of bacterial infection of total gut decontamination with oral non absorbable antibiotics or with oral absorbable broad spectrum antibiotics. On day-8, all patients were randomly allocated to one group: Group I received orally pefloxacin 400 mg/day and penicillin 3 M UI/day. Group II received capsules containing cephalothin 250 mg, gentamicin 20 mg and bacitracin 150 UI. Each patient received 9 to 12 capsules per day according to body weight. Patients less than 5 years old or with severe hepatic abnormalities were excluded from the study. All patients were treated in LAF room with usual precautions of asepsis. They received sterile food. In addition, they were on ketoconazole for prophylaxis of fungal infections and acyclovir for prevention of herpes infections. Antibiotics were started on day-8 before bone marrow transplantation and stopped 15 days after discharge from the hospital. 32 patients were allocated to Group I and 33 in Group II. There was no difference between both groups according to age, sex, diagnosis. After transplant, the one year survival and the complications were similar in both groups. The median time of decontamination was 65 days, the mean number of days of agranulocytosis was 20 days, the mean number of days of fever was 7 days in both groups. The compliance was better in Group I but the treatment had to be modified in 9 patients of Group I because of liver abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)