RESUMO
Major advances in the monitoring and treatment of viral infections after hematopoietic stem cell transplantation (HSCT) have been achieved over the last decade. The appropriate extent of viral monitoring and antiviral therapy remains controversial, and reports in pediatric patients receiving allogeneic unmanipulated hematopoietic stem cells (HSCs) are sparse. A total of 40 pediatric patients who underwent HSCT with either peripheral blood stem cells (PBSCs, n = 30) or bone marrow (BM; n = 10) were prospectively monitored every week for viral DNAemia (VDNA) by simultaneous detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV6), human adenovirus (ADV), and polyoma BK virus (BKV) using real-time TaqMan polymerase chain reaction (PCR). All patients received prophylactic acyclovir and preemptive ganciclovir (GCV) when 500 copies/microg DNA (EBV/HHV6) or >1 copy/microg DNA (CMV) were detected on 2 consecutive measurements. VDNA occurred in 25 of 40 recipients (CMV, 11/40 patients [28%]; EBV, 19/40 [48%]; HHV6, 2/40 [5%]; ADV/BKV, 1/40) and was found exclusively after neutrophil engraftment and in most cases up to day +100. Recurrent VDNA (P = .028) and (readily treatable) viral disease (P = .003) were observed predominantly in patients suffering from nonmalignant diseases, a cohort characterized by delayed lymphocyte engraftment. VDNA occurred more frequently in HLA-mismatched HSCT and in the 24 of 40 patients receiving antithymocyte globulin (ATG). The incidence of EBV, but not that of CMV, was increased in the ATG group. Yet, in these patients, viral loads of both EBV and CMV were higher, but with prompt initiation of preemptive GCV, no posttransplantation lymphoproliferative disorder or other life-threatening morbidities occurred. HHV6 was typically detected at low viral loads (<10(2) copies/microg DNA), with only 5% of HSC recipients fulfilling our HHV6 criteria for triggering GCV treatment. In multivariate analysis, ATG treatment, HLA mismatch, recipient CMV seropositivity, and stem cell source, but not severe acute graft-versus-host disease were identified as independent risk factors for VDNA. This comprehensive viral monitoring program with defined thresholds for initiation of preemptive GCV effectively prevents the development of critical viral disease, even in high-risk patients receiving ATG.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Complicações Pós-Operatórias/epidemiologia , Viroses/epidemiologia , Infecções por Adenoviridae/diagnóstico , Infecções por Adenoviridae/tratamento farmacológico , Infecções por Adenoviridae/epidemiologia , Infecções por Adenoviridae/prevenção & controle , Adolescente , Soro Antilinfocitário , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Vírus BK/isolamento & purificação , Transplante de Medula Óssea/estatística & dados numéricos , Criança , Pré-Escolar , DNA Viral/sangue , Feminino , Ganciclovir/uso terapêutico , Doença Enxerto-Hospedeiro/complicações , Neoplasias Hematológicas/cirurgia , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/prevenção & controle , Humanos , Lactente , Masculino , Transplante de Células-Tronco de Sangue Periférico/estatística & dados numéricos , Infecções por Polyomavirus/diagnóstico , Infecções por Polyomavirus/tratamento farmacológico , Infecções por Polyomavirus/epidemiologia , Infecções por Polyomavirus/prevenção & controle , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/tratamento farmacológico , Complicações Pós-Operatórias/prevenção & controle , Pré-Medicação , Estudos Prospectivos , Transplante Homólogo , Viremia/diagnóstico , Viremia/epidemiologia , Viroses/diagnóstico , Viroses/tratamento farmacológico , Viroses/prevenção & controle , Adulto JovemRESUMO
High throughput analysis using amplicon-based next-generation sequencing (NGS) of HLA class I genes in samples of registered stem cell donors of the German Stem Cell Donor Registry Düsseldorf revealed 151 novel variants. In addition, four new variants were identified in well-defined samples obtained from the UCLA International Cell Exchange program. New alleles included 37 HLA-A, 57 HLA-B, and 61 HLA-C variant alleles. All variants were confirmed by NGS of HLA-A, HLA-B, and HLA-C genes including the respective 5' and 3' untranslated regions as well as Sanger sequence analysis. Mainly, the variants encompass single nucleotide changes in intronic as well as exonic parts of the genes. We identified intronic variations in 114 new alleles, nonsynonymous nucleotide changes in 25 alleles, synonymous nucleotide changes in nine alleles, and three hybrid alleles. Four alleles carry exonic deletions or insertions resulting in frameshift of peptide translation. Two novel alleles of HLA-C were shown to result in splicing defects of the transcript. Two alleles showed exonic as well as intronic changes. Thirty-four of the new alleles were found in multiple samples.
Assuntos
Genes MHC Classe I , Variação Genética , Sistema de Registros , Células-Tronco/metabolismo , Doadores de Tecidos , Alelos , Sequência de Bases , Frequência do Gene , Alemanha , Humanos , Íntrons/genética , Los AngelesRESUMO
To determine the impact of high-resolution (HR) HLA typing with outcomes after UCBT, DNAs of 122 pairs were analysed for HLA class I and class II mismatches (MM) based on HR typing. For HLA-A, -B on low-resolution typing and -DRB1 on HR typing, the following MM situation resulted: no MM (13%), one MM (40%), two MM (36%), three MM (8%), four MM (3%). For A, B, C, DR and DQ based on HR typing the following MM occurred: No MM (4%), one MM (10%), two MM (15%), three MM (22%), four MM (25%), five MM (12%), six MM (6%), seven MM (3%), eight MM (2%). There was no significant association between number of MM (HR) for both HLA-A, -B and -DRB1 and HLA-A, -B, -C, -DRB1 and DQB1 and aGvHD grade II-IV. There was a trend that MM in class I HR were associated with neutrophil recovery; HLA-A locus typing analysed in HvG direction was associated with reduced cumulative incidence of engraftment (P=0.04), the same for C-KIR in HvG direction (P=0.01). No significant correlation was found between numbers of HLA-MM on the HR level with 2-year survival. The analysis shows that the degree of mismatching in UCBT is even higher than expected.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Sangue Fetal/citologia , Antígenos HLA/química , Imunofenotipagem/métodos , Sistema ABO de Grupos Sanguíneos , Adolescente , Adulto , Criança , Pré-Escolar , Citomegalovirus/genética , Feminino , Doença Enxerto-Hospedeiro , Antígenos HLA/biossíntese , Antígenos HLA-A/química , Antígenos HLA-B/química , Antígenos HLA-C/química , Antígenos HLA-DQ/química , Antígenos HLA-DR/química , Células-Tronco Hematopoéticas/citologia , Teste de Histocompatibilidade , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Neutrófilos/citologia , Fatores de Tempo , Condicionamento Pré-Transplante , Resultado do TratamentoRESUMO
Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.
Assuntos
Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Bases , Biomarcadores Tumorais , Primers do DNA , Proteínas de Fusão bcr-abl/normas , Humanos , Controle de QualidadeRESUMO
Oligonucleotide primer pairs specific for interleukins (IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, and IL-6, as well as for granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA/cDNA were synthesized in order to detect cytokine transcripts by reverse transcription and subsequent polymerase chain reaction (RT/PCR). Analysis of RNA preparations of the human bladder carcinoma cell line 5637 by this methodology reveals expression of mRNAs for IL-1 alpha, IL-1 beta, IL-6, G-CSF, M-CSF, and for GM-CSF, whereas mRNAs for IL-2, IL-3, IL-4, and IL-5 are not detectable. These results are in agreement with data obtained by classical methods. Thus, for the cytokines IL-2, IL-3, IL-4, and IL-5, it was not possible to detect a phenomenon described as 'illegitimate transcription,' defined as the low level transcription of any gene in any cell type. This finding is of importance for the applicability of mRNA phenotyping employing RT/PCR for the determination of mRNA expression patterns. For M-CSF mRNA detection, two oligonucleotide primer pairs had to be used to distinguish between the alpha-(pcCSF17) and beta-splicing forms and to overcome the problem of non-amplification of a larger fragment in the presence of a competing smaller one, defined here as 'incomplete positivity.' For G-CSF, IL-4, IL-2, and IL-5, RT/PCR reveals two fragments. Restriction enzyme analysis of the additional fragments suggests that they may arise from alternative splicing events. For G-CSF and IL-4, exons 3 and 2 seem to be spliced out, respectively. The additional fragments for IL-2 and IL-5 RT/PCR have not yet been further characterized, but the size of the fragments makes it seem probable that exons 2 and 3 are spliced out for IL-2 and IL-5, respectively. The biological role of these alternative mRNAs has yet to be determined.
Assuntos
Fatores Estimuladores de Colônias/genética , Interleucinas/genética , RNA Mensageiro/análise , Transcrição Gênica/genética , Neoplasias da Bexiga Urinária/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/metabolismoRESUMO
The expression of interleukin-4 (IL-4) mRNA of human peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA) for 15 hours was analyzed by reverse transcription and subsequent polymerase chain reaction (RT/PCR). These analyses revealed an additional smaller fragment that hybridizes with an IL-4 cDNA probe and an oligonucleotide that is specific for a fragment lacking the sequence encoded by exon 2. Sequencing of this fragment demonstrates that it is generated from an alternatively spliced transcript of the IL-4 gene with the sequence encoded by exon 2 being skipped. Skipping of exon 2 does not result in a frame shift but would delete part of the mature protein (48 bp coding for amino acid residues 22 to 37), including Cys24 but not a region directly involved in receptor binding. Differential splicing of other exons or exon combinations has not been observed. The data suggest that the alternatively spliced transcript is not generated by a splicing or PCR error and is not detectable solely because of the high sensitivity of RT/PCR, but in contrast, argue for a physiological role of the transcript and its potentially encoded protein.
Assuntos
Interleucina-4/genética , Processamento Alternativo , Sequência de Bases , Linhagem Celular , Éxons , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , RNA Mensageiro/genéticaRESUMO
One of the major questions in any gene therapy approach is the selection of the appropriate vector system. Here, the optimization of a gene transfer protocol for renal cell carcinoma using lipofection as a nonviral gene transduction system was evaluated. To select the promoter which gives the highest expression, different plasmids which are able to express Escherichia coli beta-galactosidase gene as a reporter gene under the control of different promoters were tested: human cytomegalovirus promoter (pCMVbeta), simian virus 40 promoter (pSVbeta), adenovirus promoter (ADbeta), and herpes simplex virus thymidine kinase promoter (TKbeta). The pCMVbeta revealed the highest expression of the beta-gal gene in the renal cell carcinoma (RCC) lines. Thus this CMV promoter was selected for the expression of the granulocyte-macrophage colony stimulator factor (GM-CSF) gene. Three different lipids (LipofectAmine, LipofectAce, and Lipofectin) were compared for their transduction efficiency, and the optimal conditions for quantitatively high lipofection rates were established. The consistently best results regarding gene expression as well as viability of the RCC lines were obtained when Lipofectin was used. Gene expression was monitored by a specific enzyme-linked immunosorbent assay and functionally validated by a cell proliferation test. The GM-CSF expression profile showed a peak at 48 hours after transfection and was still detectable after 5 days. Here the feasibility of efficient lipofection of the GM-CSF gene into RCC lines is demonstrated. Most importantly, considerable differences in the relative quantity of GM-CSF gene transfer into the different RCC lines was observed here. This may be of critical relevance for the design of any clinical gene transduction protocol in tumor cell vaccination attempts.
Assuntos
Carcinoma de Células Renais/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Neoplasias Renais/metabolismo , Transfecção/métodos , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Terapia Genética , Humanos , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Lipídeos , Regiões Promotoras GenéticasRESUMO
Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencing-based typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors.
Assuntos
Genes MHC da Classe II , Antígenos HLA-D/genética , Transplante de Células-Tronco Hematopoéticas , População Branca/genética , Alelos , Estudos de Coortes , Frequência do Gene , Ligação Genética , Alemanha , Antígenos HLA-DQ/genética , Cadeias beta de HLA-DQ , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Cadeias HLA-DRB3 , Cadeias HLA-DRB4 , Cadeias HLA-DRB5 , Haplótipos/genética , Teste de Histocompatibilidade , Humanos , Reação em Cadeia da PolimeraseRESUMO
Registries and cord blood banks around the world collect and store the HLA types of volunteers in order to identify matched unrelated donors for patients requiring hematopoietic stem cell transplantation. This task is complicated by the many formats in which HLA types are provided by the testing laboratories (types obtained by serology vs by DNA-based methods; high vs intermediate vs low resolution) and by the need to identify which of these diverse types are most likely to match the HLA assignments of a searching patient as closely as possible. Conversion of the assignments to 'search determinants' may be included within the algorithm used to select and prioritize a list of potentially suitable donors, either as an aid to matching or as a tool to optimize the performance of comparisons within large data files. The strategies used by registries to create search determinants are described. A set of search determinants, utilized by the National Marrow Donor Program, is provided as an example and is intended to initiate further discussion aimed at understanding the process used by each registry with the possibility of developing a standard process among registries worldwide.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Teste de Histocompatibilidade/métodos , Sistema de Registros , Doadores de Tecidos , Algoritmos , Histocompatibilidade , Humanos , Métodos , Guias de Prática Clínica como Assunto , Doadores de Tecidos/provisão & distribuiçãoRESUMO
In Germany allotransplantation of bone marrow or peripheral blood stem cells is presently performed by 34 different teams operating more or less independently. Thus, strategies of immunogenetic donor search, use of the various tissue typing techniques and policy on acceptable HLA mismatches in related and unrelated settings may vary considerably from one transplant centre to another. This paper summarises the results of the first German consensus meeting on immunogenetic donor search for bone marrow/peripheral blood stem cell grafting. The main goal of the participating transplant physicians and immunogeneticists was to define national standards for the above issues.
Assuntos
Transplante de Medula Óssea/normas , Transplante de Células-Tronco Hematopoéticas/normas , Doadores de Tecidos , Alemanha , Teste de Histocompatibilidade/normas , HumanosRESUMO
OBJECTIVE: Recent studies report the beneficial effect of HLA matching for long-term prognosis of penetrating keratoplasty (kp). This improvement of prognosis, however, has to be weighed against the additional time on the waiting list due to the search for a HLA-compatible graft. Reliable estimation of this additional waiting period is a prerequisite for informed consent on the waiting policy. METHODS: A mathematical model based on survival analysis and HLA haplotype frequencies was used to estimate time on the waiting list for each of 1,400 HLA-typed patients registered at the Lions Cornea Bank NRW. Additionally, the waiting period of each patient was retrospectively determined. Both values were tested for correlation. This analysis was performed for acceptance of up to two mismatches on HLA-A, -B and -DR. RESULTS: When accepting two, one and zero mismatches, median predicted waiting period was 1 +/- 6, 7 +/- 49 and 17 +/- 159 months respectively. Median waiting period in retrospective simulation was 1 +/- 3, 5 +/- 9 and 15 +/- 14 months. Correlation of values from the predictive formula and simulation was statistically significant (p < 0.0001). CONCLUSION: Predicted time on the waiting list is a valuable tool for management of HLA matching in kp.
Assuntos
Córnea/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Ceratoplastia Penetrante/imunologia , Listas de Espera , Sobrevivência de Enxerto/fisiologia , Teste de Histocompatibilidade , Humanos , Modelos Biológicos , Modelos Teóricos , Fatores de Tempo , Doadores de TecidosRESUMO
Protein C deficiency is an autosomally inherited disorder that is associated with a high risk of recurrent venous thrombosis. The authors have shown that temperature gradient gel electrophoresis (TGGE) is a simple and rapid screening method for the detection of mutations in the protein C gene. Samples from eleven patients with sequence defined point mutations in the promoter region of exon I, and in exons II, III, VII, VIII and IX were analysed by TGGE. In all cases the mutations were readily detected. The exons IV, V and VI were not submissive to TGGE analysis due to amplification difficulties. However, specific computer calculations predict a more general applicability of TGGE for the detection of any mutation in the protein C gene. The presented data establish the usefulness of TGGE as a simple and rapid screening method for the detection of hereditary mutations in the protein C gene.
Assuntos
Análise Mutacional de DNA , Eletroforese em Gel de Poliacrilamida/métodos , Proteína C/genética , Sequência de Bases , Éxons/genética , Genes , Testes Genéticos/métodos , Humanos , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Deficiência de Proteína C , Software , TemperaturaRESUMO
The influence of inorganic and organic forms of selenium (Se) on human antibody production was studied in a Pokeweed Mitogen (PWM)-driven in vitro system. Mitogen-stimulated peripheral blood mononuclear cells (PBMC) of eight healthy donors were cultured with different Se compounds at concentrations between 10(-3) and 10(-9) M. At high Se levels (10(-3)-10(-4) M), IgM and IgG production of all donors were strongly inhibited owing to reduced cell viability. However, in five of eight donors, low levels of Se enhanced IgG secretion. This was most effective in the presence of inorganic Se, whereas selenomethionine and selenocystine were less effective. In contrast to IgG, IgM synthesis was significantly reduced by low Se levels in five donors. No significant correlation between donor serum Se levels and antibody production in vitro was found. The addition of low levels of Se to PBMC, stimulated with PHA or PWM, showed no effect on proliferation, whereas a high concentration (5 x 10(-3) M) of sodium selenite and selenocystine suppressed proliferation owing to reduced cell viability. Thus, the present results show that Se supplementation can enhance human antibody production and, moreover, suggest some selectivity of Se action on human immune responses that may result in increased switching from IgM to IgG production.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Mitógenos de Phytolacca americana/farmacologia , Selênio/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Imunoglobulinas/biossíntese , Técnicas In Vitro , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Selênio/sangue , Timidina/metabolismoRESUMO
BACKGROUND: Since 1991 homologous penetrating limbokeratoplasty has been performed in 32 patients with severe limbal stem cell insufficiency following chemical/thermal burns. The long-term results considering the effects of HLA matching are presented for the first time. PATIENTS: All patients received systemic cyclosporin A and/or mycophenolate mofetil in the postoperative course. In 9 patients grafts with 0-1 HLA mismatches in the HLA A, B and DR loci, in 6 patients grafts with 2-6 mismatches and in 17 patients untyped grafts were used. Long-term clear graft survival was estimated according to Kaplan and Meier. RESULTS: Five years postoperatively, 50% of the grafts with 0-1 mismatches, 32% of the grafts with 2-6 mismatches and 18% of the untyped grafts were centrally clear (log-rank-test, p>0.05). CONCLUSIONS: Although statistically not significant, HLA matched grafts seem to deliver better results than untyped grafts in penetrating limbokeratoplasty. Improvement of matching strategies and immunosuppression may possibly further improve current results.
Assuntos
Queimaduras Químicas/cirurgia , Queimaduras Oculares/cirurgia , Ceratoplastia Penetrante/métodos , Limbo da Córnea/lesões , Complicações Pós-Operatórias/etiologia , Células-Tronco , Análise Atuarial , Adulto , Idoso , Contagem de Células , Queimaduras Oculares/induzido quimicamente , Feminino , Seguimentos , Sobrevivência de Enxerto , Antígenos HLA/genética , Teste de Histocompatibilidade , Humanos , Limbo da Córnea/citologia , Limbo da Córnea/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
BACKGROUND: Pluripotent unrestricted somatic stem cells (USSC) from UC blood can differentiate into hepatic cells in the in utero sheep model, resulting in 20% human albumin-producing parenchymal hepatic cells without cell fusion or tumor-formation events. Additionally, we have shown in vitro differentiation of USSC by hepatocyte growth factor and oncostatin M induction, causing changes in the gene expression towards the endodermal lineage. Positive glycogen synthase expression and a positive periodic acid-schiff reaction demonstrated a functional production of polysaccharides in the cells. METHODS: We describe the in vitro differentiation of USSC towards an endodermal pathway using different matrices, growth factors and organic substances. Also, co-cultures of USSC with primary cells of endodermal tissue were prepared to mimic the biologic niche. We investigated the effect of direct co-culture of USSC with primary rat hepatocytes or with sheep tissue of endodermal origin. Direct co-cultures were set up to ensure cell-cell contacts. For co-cultures without cell-cell contacts, transwell inlays with 1-microm membranes were used to separate the cells. Furthermore, the effect of endodermally conditioned medium was investigated. Changes in the gene expression patterns were analyzed by RT-PCR. RESULTS: We have shown that USSC can differentiate in vitro into an endodermal-like cell with a phenotype similar to hepatic cells. Differentiation of USSC with growth factors, retinoic acid, matrigel matrix and different co-cultures led to an increased expression of albumin and also to the detection of GSC, SOX 17, Cyp2B6, Cyp3A4, Gys2, HNF4a, ISL-1 and Nkx6.1. In addition, functional albumin secretion was observed. DISCUSSION: Although the differentiation assays demonstrated here produce only an immature hepatocyte-like cell, endodermaly differentiated USSC might be a useful alternative for cell replacement in the future.
Assuntos
Diferenciação Celular , Endoderma/citologia , Sangue Fetal/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Colágeno/efeitos dos fármacos , Meios de Cultivo Condicionados , Combinação de Medicamentos , Endoderma/efeitos dos fármacos , Sangue Fetal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Laminina/efeitos dos fármacos , Oncostatina M/farmacologia , Reação do Ácido Periódico de Schiff , Proteoglicanas/efeitos dos fármacos , Ratos , Albumina Sérica/metabolismo , Ovinos , Células-Tronco/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
BACKGROUND: Peripheral blood stem cell (PBSC) grafts are increasingly used for autologous and allogeneic haematopoietic stem cell transplantation (alloHSCT) with the aim to hasten neutrophil and platelet engraftment and thereby to reduce transplant-related complications due to infections, bleeding and graft failure. Based on the paucity of data on PBSC transplantation in children we performed a retrospective single-center analysis comparing the outcome of children receiving mobilized PBSC from human leukocyte antigen (HLA)-identical sibling donors to bone marrow (BM) transplant recipients. PATIENTS AND METHODS: Between 1996 and 2004, 16 children with haematologic malignancies and standard indication for alloHSCT underwent PBSC transplantation from HLA-identical sibling donors. The outcome of these children was compared to a historic control group of 19 bone marrow (BM) transplant recipients. Time to neutrophil engraftment, incidence of acute and chronic graft-versus-host disease (GvHD), relapse rate, transplant-related mortality, event-free and overall survival were analyzed. RESULTS: Neutrophil engraftment was achieved significantly faster after PBSC compared to BM transplantation with a median time to neutrophil engraftment of 11 (range: 8-21) and 19 (16-44) days for the PBSC and BM cohort, respectively (p < 0.001). Two of 19 (11 %) BM recipients did not achieve primary neutrophil engraftment and both patients died due to infectious complications. The rate of clinically significant acute GvHD > or = grade II was higher in the PBSC compared to the BM group (75 vs. 39 %; p = 0.045). Incidences of chronic GvHD (PBSC vs. BM: 60 vs. 44 %), death of disease (13 vs. 21 %) and death of complication (13 vs. 16 %) were comparable between both groups (p = ns). With a median follow up of 4.7 years (PBSC) and 10.2 years (BM) overall survival (PBSC vs. BM: 68.6 +/- 13.5 vs. 63.2 +/- 11.1 %; p = 0.65) and event-free survival (67.0 +/- 12.1 vs. 63.2 +/- 11.1 %; p = 0.80) is without demonstrable difference in both groups. CONCLUSIONS: Transplantation of PBSC compared to BM is associated with faster neutrophil engraftment and a higher rate of > or = grade II acute GvHD. As overall survival and event-free survival is similar when using PBSC and BM, PBSC is an alternative stem cell source for HLA-identical sibling transplantation. Further prospective analyses with higher number of patients stratified according to well established risk factors are required to define the precise role of both stem cell sources for children with haematologic malignancies.
Assuntos
Transplante de Medula Óssea , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco de Sangue Periférico , Adolescente , Adulto , Transplante de Medula Óssea/mortalidade , Criança , Pré-Escolar , Interpretação Estatística de Dados , Feminino , Doença Enxerto-Hospedeiro/epidemiologia , Transplante de Células-Tronco Hematopoéticas/mortalidade , Teste de Histocompatibilidade , Humanos , Incidência , Lactente , Masculino , Seleção de Pacientes , Transplante de Células-Tronco de Sangue Periférico/mortalidade , Estudos Retrospectivos , Irmãos , Análise de Sobrevida , Fatores de Tempo , Doadores de TecidosRESUMO
The present paper demonstrates the usefulness of the temperature gradient gel electrophoresis (TGGE) as a precise method for the rapid identification of HLA-DR full matched donors by molecular crossmatch analysis. The resulting TGGE fingerprints are highly diverse for different HLA-DR genotypes and identical for samples with identical HLA-DR type, as shown for a panel of 25 randomly selected PCR-SSO pretyped DNA samples. In addition, TGGE analysis of a panel of homozygous typing cell lines (HTCs) established, that even subtle differences in the DR4 subtypes are detectable by TGGE crossmatch analysis. Furthermore, TGGE crossmatch analysis of unrelated donor/patient pairs demonstrated, that only HLA-DR identical patient/donor combinations showed identical TGGE patterns. Thus crossmatch analysis by TGGE presents a novel basis for a rapid and safe unrelated bone marrow donor selection strategy. Only HLA class I identical donors which also show HLA-DR identity in the TGGE crossmatch test are selected for the final confirmation of the HLA class II genes by a suitable DNA subtyping method.
Assuntos
Transplante de Medula Óssea , Impressões Digitais de DNA , Eletroforese em Gel de Poliacrilamida/métodos , Antígenos HLA-DR/genética , Teste de Histocompatibilidade/métodos , Obtenção de Tecidos e Órgãos , Alelos , Genótipo , Humanos , Reação em Cadeia da Polimerase , Polimorfismo Genético , Desnaturação Proteica , Temperatura , Doadores de TecidosRESUMO
The human leucocyte antigen (HLA) class II compatibility of bone marrow donor and recipient is an essential prerequisite for the prevention of severe graft versus host disease and therefore for the successful outcome of bone marrow transplantation. In this study an efficient protocol was developed for the rapid analysis of the polymorphic HLA-DR gene locus, based on DNA-polymerase chain reaction (PCR) amplification of the variable exon II of the HLA class II DR genes and subsequent temperature gradient gel electrophoresis (TGGE). Computer-assisted melting map calculations were carried out to determine the melting behavior of the different HLA-DR fragments. Despite the high variability of the DR alleles on the nucleotide sequence level the calculations revealed a common melting domain structure of the different HLA-DR fragments, which was experimentally confirmed by perpendicular TGGE. On parallel TGGE, all samples were separated under the same electrophoretical conditions using a single PCR fragment without GC-clamp. TGGE was applied for the analysis of the DR alleles of numerous bone marrow receipt pairs and compared to the corresponding serological and DNA typing results. The TGGE patterns were found to be different for all samples with different HLA-DR typing results. Identical homoduplex and heteroduplex patterns occurred only in the case of complete genotypic HLA-DR identity as determined by direct sequencing of PCR products.
Assuntos
Transplante de Medula Óssea/imunologia , Genes MHC da Classe II , Doença Enxerto-Hospedeiro/imunologia , Antígenos HLA-DR/genética , Polimorfismo Genético , Alelos , Sequência de Bases , Medula Óssea/imunologia , Simulação por Computador , DNA/sangue , DNA/genética , DNA/isolamento & purificação , Primers do DNA , Eletroforese em Gel de Poliacrilamida/métodos , Éxons , Cadeias HLA-DRB1 , Humanos , Indicadores e Reagentes , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , TemperaturaRESUMO
This study addresses the question whether the different forms of autosomal dominant cerebellar ataxia (ADCA) are related to different ethnic/geographical regions in Europe. One mutation in families originating from Holland, Prussia and Italy has previously been localized to chromosome 6p (SCA1 locus), whereas the mutation in families of Iberic origin has been excluded from chromosome 6p. In a Danish five-generation pedigree with ADCA and in which previous HLA-serotyping had shown inconclusive linkage results, the present study shows unequivocal exclusion from the SCA1 locus, firstly through the use of the new, highly informative microsatellites D6S89 and D6S109, which closely flank the SCA1 locus, and secondly through the manifestation of disease in four pedigree members previously scored as unaffected. Additional molecular genetic analysis of the HLA DRbeta and F13A polymorphisms also argue against a cluster of ADCA genes on chromosome 6p. Since this study demonstrates the existence of non-SCA1 families and therefore heterogeneity in the North-European population, molecular family counselling remains restricted to the few known SCA1 families.
Assuntos
Ataxia Cerebelar/genética , Cromossomos Humanos Par 6 , Degenerações Espinocerebelares/genética , Ataxia Cerebelar/classificação , Ataxia Cerebelar/etnologia , DNA Satélite/análise , Dinamarca , Feminino , Genes Dominantes , Ligação Genética , Variação Genética , Antígenos HLA-DR/genética , Humanos , Masculino , Linhagem , Polimorfismo de Fragmento de Restrição , Degenerações Espinocerebelares/etnologiaRESUMO
We investigated the effects of murine rTNF-alpha, human rIL-1 beta, and rat rIFN-gamma in various concentrations and/or combinations on inducible nitric oxide (NO) production in primary cultures of rat aortic endothelial cells. Northern blot analysis of total RNA from induced and control cultures using the cloned mouse macrophage gene of inducible NO synthase as probe as well as polymerase chain reaction using a specific primer sequence gave a positive signal for activated cells only. A RNA approximately 4.4 kb of length similar to the inducible form of NO synthase in macrophages was labeled. The concentration of nitrite as a stable reaction product of NO in culture supernatants was determined 24 h after incubation with the various cytokines. IL-1 beta alone (40 to 1000 U/ml) induced formation of increasing amounts of nitrite with increasing concentrations of IL-1 beta present. Neither TNF-alpha alone (10 to 2000 U/ml) nor IFN-gamma alone 25 to 500 U/ml) showed significant effects on nitrite production. Simultaneous incubation with low concentrations of TNF-alpha (< or = 100 U/ml) and IL-1 beta abrogated the induction effect of IL-1 beta. Conversely, addition of high concentrations of TNF-alpha (> or = 500 U/ml) led to near maximal levels of nitrite formation even at lowest IL-1 beta concentrations (40 U/ml). In addition, simultaneous incubation of endothelial cells with IFN-gamma plus IL-1 beta and/or TNF-alpha led to near maximal NO production of endothelial cells, even at lowest IFN-gamma concentrations (25 U/ml). We hypothesize that the regulating effect of TNF-alpha may in vivo help to prevent local inflammatory responses from spreading to intact sites.