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1.
Clin Epigenetics ; 13(1): 61, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33757590

RESUMO

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the cardiac muscle, frequently caused by mutations in MYBPC3. However, little is known about the upstream pathways and key regulators causing the disease. Therefore, we employed a multi-omics approach to study the pathomechanisms underlying HCM comparing patient hearts harboring MYBPC3 mutations to control hearts. RESULTS: Using H3K27ac ChIP-seq and RNA-seq we obtained 9310 differentially acetylated regions and 2033 differentially expressed genes, respectively, between 13 HCM and 10 control hearts. We obtained 441 differentially expressed proteins between 11 HCM and 8 control hearts using proteomics. By integrating multi-omics datasets, we identified a set of DNA regions and genes that differentiate HCM from control hearts and 53 protein-coding genes as the major contributors. This comprehensive analysis consistently points toward altered extracellular matrix formation, muscle contraction, and metabolism. Therefore, we studied enriched transcription factor (TF) binding motifs and identified 9 motif-encoded TFs, including KLF15, ETV4, AR, CLOCK, ETS2, GATA5, MEIS1, RXRA, and ZFX. Selected candidates were examined in stem cell-derived cardiomyocytes with and without mutated MYBPC3. Furthermore, we observed an abundance of acetylation signals and transcripts derived from cardiomyocytes compared to non-myocyte populations. CONCLUSIONS: By integrating histone acetylome, transcriptome, and proteome profiles, we identified major effector genes and protein networks that drive the pathological changes in HCM with mutated MYBPC3. Our work identifies 38 highly affected protein-coding genes as potential plasma HCM biomarkers and 9 TFs as potential upstream regulators of these pathomechanisms that may serve as possible therapeutic targets.


Assuntos
Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/fisiopatologia , Proteínas de Transporte/genética , Metilação de DNA , Expressão Gênica , Genes Homeobox , Histonas/genética , Humanos , Mutação , Transcriptoma
2.
Eur J Med Res ; 24(1): 19, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971293

RESUMO

BACKGROUND: Skin biopsies are often used in daily practice for the diagnosis of acute (aGvHD) or chronic graft versus host disease (cGvHD). With the latest understanding in pathogenesis and new National Institute of Health (NIH) classifications for aGvHD and cGvHD, there is a need to evaluate the current prognostic value of histological grading cutaneous GvHD and its correlation to the clinical grade. METHODS: In a retrospective study with 120 skin biopsies (all taken for suspected GvHD) from 110 patients (all classified according to the NIH), biopsies were revised and graded, blinded for clinical information, for either acute of chronic features. Morphological grades were compared for concordance with the clinical grade and survival analyses were done for clinical and histological grading. RESULTS: Correlation for histologic vs. clinical grading was (very) poor for aGvHD and cGvHD (weighted κ - 0.038 and 0.0009, respectively). Patients with clinical aGvHD had worse prognosis compared to cGvHD. However, at time of biopsy neither clinical nor histological grading predicted the eventual survival for either aGvHD (p = 0.9739 and p = 0.0744, respectively) or cGvHD (p = 0.2149 and p = 0.4465, respectively). CONCLUSIONS: Confirming the diagnosis of GvHD is still a valuable reason for taking a skin biopsy, but this study shows that histologic grading of GvHD in the skin biopsy has no additional value for clinicians in current practice.


Assuntos
Doença Enxerto-Hospedeiro/patologia , Doença Aguda , Adolescente , Adulto , Idoso , Biópsia , Criança , Pré-Escolar , Doença Crônica , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Adulto Jovem
3.
BMC Cancer ; 17(1): 282, 2017 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-28424071

RESUMO

BACKGROUND: The identification of four Consensus Molecular Subtypes (CMS1-4) of colorectal cancer forms a new paradigm for the design and evaluation of subtype-directed therapeutic strategies. The most aggressive subtype - CMS4 - has the highest chance of disease recurrence. Novel adjuvant therapies for patients with CMS4 tumours are therefore urgently needed. CMS4 tumours are characterized by expression of mesenchymal and stem-like genes. Previous pre-clinical work has shown that targeting Platelet-Derived Growth Factor Receptors (PDGFRs) and the related KIT receptor with imatinib is potentially effective against mesenchymal-type colon cancer. In the present study we aim to provide proof for the concept that imatinib can reduce the aggressive phenotype of primary CMS4 colon cancer. METHODS: Tumour biopsies from patients with newly diagnosed stage I-III colon cancer will be analysed with a novel RT-qPCR test to pre-select patients with CMS4 tumours. Selected patients (n = 27) will receive treatment with imatinib (400 mg per day) starting two weeks prior to planned tumour resection. To assess treatment-induced changes in the aggressive CMS4 phenotype, RNA sequencing will be performed on pre- and post-treatment tissue samples. DISCUSSION: The development of effective adjuvant therapy for primary colon cancer is hindered by multiple factors. First, new drugs that may have value in the prevention of (early) distant recurrence are almost always first tested in patients with heavily pre-treated metastatic disease. Second, measuring on-target drug effects and biological consequences in tumour tissue is not commonly a part of the study design. Third, due to the lack of patient selection tools, clinical trials in the adjuvant setting require large patient populations. Finally, the evaluation of recurrence-prevention requires a long-term follow-up. In the ImPACCT trial these issues are addressed by including newly diagnosed pre-selected patients with CMS4 tumours prior to primary tumour resection, rather than non-selected patients with late-stage disease. By making use of the pre-operative window period, the biological effect of imatinib treatment on CMS4 tumours can be rapidly assessed. Delivering proof-of-concept for drug action in early stage disease should form the basis for the design of future trials with subtype-targeted therapies in colon cancer patients. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02685046 . Registration date: February 9, 2016.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Mesilato de Imatinib/uso terapêutico , Quimioterapia Adjuvante , Ensaios Clínicos Fase II como Assunto , Neoplasias Colorretais/patologia , Humanos , Estudos Multicêntricos como Assunto , Período Pré-Operatório , Prognóstico , Projetos de Pesquisa , Resultado do Tratamento
4.
Am J Transplant ; 17(1): 246-254, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27428759

RESUMO

Cardiac allograft vasculopathy (CAV) is a transplant pathology, limiting graft survival after heart transplantation. CAV arteries are surrounded by ectopic lymphoid structures (ELS) containing B cells and plasma cells. The aim of this study was to characterize the antigenic targets of antibodies produced in ELS. Coronary arteries and surrounding epicardial tissue from 56 transplant recipients were collected during autopsy. Immunofluorescence was used to identify antibody-producing plasma cells. Immunoglobulin levels in tissue lysates were measured by enzyme-linked immunosorbent assay and analyzed for donor-specific HLA antibodies by Luminex assay. Cytokine and receptor expression levels were quantified using quantitative polymerase chain reaction. Plasma cells in ELS were polyclonal and produced IgG and/or IgM antibodies. In epicardial tissue, IgG (p < 0.05) and IgM levels were higher in transplant patients with larger ELS than smaller ELS. In 4 of 21 (19%) patients with ELS, donor-specific HLA type II antibodies were detected locally. Cytokine and receptor expression (CXCR3, interferon γ and TGF-ß) was higher in large ELS in the epicardial tissue than in other vessel wall layers, suggesting active recruitment and proliferation of T and B lymphocytes. ELS exhibited active plasma cells producing locally manufactured antibodies that, in some cases, were directed against the donor HLA, potentially mediating rejection with major consequences for the graft.


Assuntos
Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto/imunologia , Transplante de Coração/efeitos adversos , Isoanticorpos/sangue , Isoanticorpos/imunologia , Tecido Linfoide/imunologia , Doadores de Tecidos , Aloenxertos , Feminino , Rejeição de Enxerto/patologia , Teste de Histocompatibilidade , Humanos , Masculino , Prognóstico , Fatores de Risco
6.
Neth Heart J ; 22(9): 391-5, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25172361

RESUMO

BACKGROUND: There is increasing interest in utilising novel markers of cardiovascular disease risk in patients with chronic heart failure (HF). Recently, it was shown that alpha-1-antichymotrypsin (ACT), an acute-phase protein and major inhibitor of cathpesin G, plays a role in the pathophysiology of HF and may serve as a marker for myocardial distress. OBJECTIVE: To assess whether ACT is independently associated with long-term mortality in chronic HF patients. METHODS: ACT plasma levels were categorised into quartiles. Survival times were analysed using Kaplan-Meier curves and Cox proportional hazards regression, without and with correction for clinically relevant risk factors, including sex, age, duration of HF, kidney function (MDRD), ischaemic HF aetiology and NT-proBNP. RESULTS: Twenty healthy individuals and 224 patients (mean age 71 years, 72 % male, median HF duration 1.6 years) with chronic HF were included. In total, 159 (71 %) patients died. The median survival time was 5.3 (95 % CI 4.5-6.1) years. ACT was significantly elevated in patients (median 433 µg/ml, IQR 279-680) in comparison with controls (median 214 µg/ml, IQR 166-271; p < 0.001). Cox regression analysis demonstrated that ACT was not independently related to long-term mortality in chronic HF patients (crude HR = 1.03, 95 % CI 0.75-1.41, p = 0.871; adjusted HR = 1.12, 95 % CI 0.78-1.60, p = 0.552), which was confirmed by Kaplan-Meier curves. CONCLUSION: ACT levels are elevated in chronic HF patients, but no independent association with long-term mortality can be established.

7.
Leukemia ; 26(8): 1821-8, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22395360

RESUMO

Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions.


Assuntos
Transplante de Células-Tronco Hematopoéticas/normas , Quimeras de Transplante/genética , Europa (Continente) , Marcadores Genéticos , Testes Genéticos/métodos , Testes Genéticos/normas , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Transplante Homólogo
8.
Crit Rev Oncol Hematol ; 80(3): 380-92, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21273092

RESUMO

BACKGROUND: Molecular techniques play an increasingly important role in breast cancer detection and help in the prediction of prognosis and treatment response. HER-2/neu predicts the sensitivity of breast tumors to trastuzumab and lapatinib. Presently there are several ways to assess HER2 status at the protein level (e.g. ELISA), at the RNA level (RT-PCR, microarray) and at the DNA level (fluorescence in situ hybridization, chromogenic in situ hybridization (ISH), silver in situ hybridization or multiplex ligation-dependent probe amplification). DESIGN: This paper provides an overview of new developments in HER2 testing. RESULTS: Although these techniques correlate well in comparative studies, discrepancies remain. Each technique has its own (dis)advantages and thus there is no real gold standard. Not surprisingly, there is no consensus at present on which of the protein- or gene-based techniques is superior, on the use of mono- or duo-probe ISH systems, nor on the use of manual or fully-automated staining- and scoring systems. CONCLUSION: Until large clinical trials clearly point out one strategy as the best predictive one for trastuzumab response, the choice for a testing strategy will probably be based on local preferences which consider both practical and economic issues. Standardization, proper internal and external quality control assessment, laboratory accreditation and automation of tissue processing (autostainers) and interpretation methods (image analysis) will play an increasingly important role in HER2 testing.


Assuntos
Neoplasias da Mama/diagnóstico , Detecção Precoce de Câncer/métodos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Detecção Precoce de Câncer/economia , Detecção Precoce de Câncer/normas , Feminino , Humanos , Metástase Neoplásica , Controle de Qualidade
9.
Bone Marrow Transplant ; 46(12): 1566-9, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21243031

RESUMO

GVHD remains a major problem in allo-SCT. We explored the presence of APC in skin biopsies of GVHD patients, using the IgG receptor CD64 expression as a hallmark for activated APC. By immunohistochemistry we demonstrated CD64 to be upregulated on host APC in skin biopsies of patients with acute GVHD and, less prominently, in chronic GVHD. Double staining for CD32 polymorphism revealed CD64-positive cells to be mainly of host origin. The majority of CD64-positive cells coexpressed CD68, indicating a macrophage phenotype. Given its very restricted cellular distribution, CD64 may represent an excellent target for APC-directed therapies in GVHD.


Assuntos
Regulação da Expressão Gênica , Doença Enxerto-Hospedeiro/metabolismo , Receptores de IgG/biossíntese , Dermatopatias/metabolismo , Pele/metabolismo , Doença Aguda , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/patologia , Doença Crônica , Feminino , Doença Enxerto-Hospedeiro/patologia , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Humanos , Masculino , Pele/patologia , Dermatopatias/patologia , Transplante de Células-Tronco , Transplante Homólogo
10.
Leukemia ; 24(8): 1462-9, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20535148

RESUMO

Relapse of pediatric acute lymphoblastic leukemia (ALL) remains the main cause of treatment failure after allogeneic stem cell transplantation (alloSCT). A high level of minimal residual disease (MRD) before alloSCT has been shown to predict these relapses. Patients at risk might benefit from a preemptive alloimmune intervention. In this first prospective, MRD-guided intervention study, 48 patients were stratified according to pre-SCT MRD level. Eighteen children with MRD level >or=1 x 10(-4) were eligible for intervention, consisting of early cyclosporine A tapering followed by consecutive, incremental donor lymphocyte infusions (n=1-4). The intervention was associated with graft versus host disease >or=grade II in only 23% of patients. Event-free survival in the intervention group was 19%. However, in contrast with the usual early recurrence of leukemia, relapses were delayed up to 3 years after SCT. In addition, several relapses presented at unusual extramedullary sites suggesting that the immune intervention may have altered the pattern of leukemia recurrence. In 8 out of 11 evaluable patients, relapse was preceded by MRD recurrence (median 9 weeks, range 0-30). We conclude that in children with high-risk ALL, immunotherapy-based regimens after SCT are feasible and may need to be further intensified to achieve total eradication of residual leukemic cells.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/cirurgia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Risco
11.
Eur J Surg Oncol ; 35(10): 1098-104, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19304440

RESUMO

AIMS: Despite surgical resection, pancreatic cancer carries a poor prognosis. In search for new molecular therapeutic targets, we investigated the expression of the HER-family and gene amplification of HER-2 in pancreatic adenocarcinomas of different stages. METHODS: Tissue of 45 resected patients was analyzed for all HER-family 1-4 expression by immunohistochemistry and HER-2 gene amplification was assessed by multiplex ligation-dependent probe amplification and chromogenic in situ hybridization. The type of surgery, location, stage and grade of the tumor, as well as involvement of the resection margins were correlated with HER-expressions and univariate and multivariate survival analysis performed. RESULTS: Normal pancreatic tissue lacked HER1-2 expression, but did show HER3-4 expression. In cancers, no membranous overexpression of HER-1 and HER-2 was seen nor gene amplification of HER-2 found. HER-3, HER-4 is physiologically expressed in the normal pancreas and loss of cytoplasmic HER-3 and HER-4 expression was seen in 33/45 (73%) and 8/45 (18%) of pancreatic cancers. Cytoplasmic HER-3 expression decreased from early to late stage (p=0.05). HER-4 expression was not associated with survival, stage or tumor grade. There were no statistically significant differences in HER1-4 expression between the papilla of Vater (n=13) and non-papilla cancers (n=32). Multivariate survival analysis showed only stage to be of independent prognostic value (p=0.015). CONCLUSIONS: HER-1 and HER-2 are not overexpressed in pancreatic cancers. HER-3 and HER-4 are expressed in the normal pancreas but expression is lost in pancreatic cancer. HER-targeted therapy in pancreatic cancer is not supported by HER-expression of the tumor.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Receptores ErbB/metabolismo , Genes erbB-2 , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Adulto , Idoso , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Técnicas de Amplificação de Ácido Nucleico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Análise de Sobrevida
12.
Am J Transplant ; 8(5): 1040-50, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18416740

RESUMO

Cardiac allograft vasculopathy (CAV) in heart transplantation (HTx) patients remains the major complication for long-term survival, due to concentric neointima hyperplasia induced by infiltrating mononuclear cells (MNC). Previously, we showed that activated memory T-helper-1 (Th-1) cells are the major component of infiltrating MNC in coronary arteries with CAV. In this study, a more detailed characterization of the MNC in human coronary arteries with CAV (n = 5) was performed and compared to coronary arteries without CAV (n = 5), by investigating MNC markers (CD1a, DRC-1, CD3, CD20, CD27, CD28, CD56, CD68, CD69, FOXP3 and HLA-DR), cytokines (IL-1A, 2, 4, 10, 12B, IFN-gamma, and TGF-beta1), and chemokine receptors (CCR3, CCR4, CCR5, CCR7, CCR8, CXCR3 and CX3CR1) by immunohistochemical double-labeling and quantitative PCR on mRNA isolated from laser microdissected layers of coronary arteries. T cells in the neointima and adventitia of CAV were skewed toward an activated memory Th-1 phenotype, but in the presence of a distinct Th-2 population. FOXP3 positive T cells were not detected and production of most cytokines was low or absent, except for IFN-gamma, and TGF-beta. This typical composition of T-helper cells and especially production of IFN-gamma and TGF-beta may play an important role in the proliferative CAV reaction.


Assuntos
Transplante de Coração/imunologia , Linfócitos T/imunologia , Células Th1/imunologia , Células Th2/imunologia , Transplante de Coração/patologia , Humanos , Memória Imunológica , Transplante Homólogo/imunologia , Transplante Homólogo/patologia
13.
Transpl Immunol ; 19(1): 64-8, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18346639

RESUMO

BACKGROUND: In the heart elevated levels of TNFalpha can cause lethal heart failure, like Dilated Cardiomyopathy (DCM). The level of TNFalpha production is in part determined by promoter gene polymorphisms. We investigated whether the TNFalpha promoter gene polymorphism is in this way involved in the outcome of end-stage heart failure and predicts whether patients require left ventricular assist device (LVAD) support or can be kept on medical therapy (MT)while awaiting heart transplantation (HTx). As most patients in this study received a heart transplant, the role of the TNFalpha polymorphisms in transplant rejection was studied as well. METHODS AND RESULTS: In twenty nine patients with DCM, 35 patients with Ischemic Heart Disease (IHD; both on MT), 26 patients on LVAD support and 61 cardiac transplant donors TNFalpha plasma level was detected by EASIA. In both patients groups high levels of TNFalpha plasma levels was observed however, in patients supported by LVAD this increase was much higher compared to patients on MT. Furthermore, this increase seems to be associated with the TNF 1 allele ('G' at position -308) instead of the TNF2 allele (A at position -308). The promoter polymorphisms at positions -238, -244 and -308 were observed by polymerase chain reaction and sequencing. Polymorphism at positions -238, -244 and -308 did not show any relevant differences between the groups. However, at position -308, a trend of a higher incidence of the TNF2 allele (an "A" at position -308) in DCM patients compared to donors was shown. The distribution of the TNF1 and TNF2 alleles was not different in patients on medical therapy compared to the patients supported by a LVAD. No association was found between patients' TNFalpha promoter gene polymorphism and rejection. However, patients that received a donor heart with the TNF2 allele developed more rejection episodes, compared to patients that received a donor heart with the TNF1 allele. CONCLUSION: TNFalpha levels are high in patients with end-stage heart failure on MT, but even higher in patients on LVAD support. These high TNFalpha plasma levels however, are not correlated with the TNF2 allele but seems to be associated with the TNF1 allele. Furthermore, in HTx the donor TNFalpha gene seem to play a more important role in severity of acute rejection than that of the patient.


Assuntos
Rejeição de Enxerto/genética , Insuficiência Cardíaca/metabolismo , Transplante de Coração , Coração Auxiliar , Fator de Necrose Tumoral alfa/sangue , Disfunção Ventricular Esquerda/metabolismo , Adulto , Alelos , Genótipo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Humanos , Pessoa de Meia-Idade , Polimorfismo Genético , Regiões Promotoras Genéticas , Fator de Necrose Tumoral alfa/genética , Disfunção Ventricular Esquerda/genética
14.
Bone Marrow Transplant ; 41(6): 563-9, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18037937

RESUMO

Cardiomyocytes are a stable cell population with only limited potential for renewal after injury. Tissue regeneration may be due to infiltration of stem cells, which differentiate into cardiomyocytes. We have analysed the influx of stem cells in the heart of patients who received either a gender-mismatched BMT (male donor to female recipient) or a gender-mismatched cardiac transplant (HTX; female donor to male recipient). The proportion of infiltrating cells was determined by Y-chromosome in situ hybridization combined with immunohistochemical cell characterization. In BM transplanted patients and in cardiac allotransplant recipients, cardiomyocytes of apparent BM origin were detected. The proportions were similar in both groups and amounted up to 1% of all cardiomyocytes. The number of stem cell-derived cardiomyocytes did not alter significantly in time, but were relatively high in cases where large numbers of BM-derived Y-chromosome-positive infiltrating inflammatory cells were present. The number of Y-chromosome-positive endothelial cells was small and present only in small blood vessels. The number of BM-derived cardiomyocytes in both BMT and HTX is not significantly different between the two types of transplantation and is at most 1%.


Assuntos
Transplante de Medula Óssea , Transplante de Coração , Células-Tronco Hematopoéticas/citologia , Miócitos Cardíacos/citologia , Autopsia , Cromossomos Humanos Y/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Microscopia Confocal , Quimeras de Transplante/genética
15.
Bone Marrow Transplant ; 39(5): 285-91, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17262061

RESUMO

The role of chimerism analysis as a prognostic indicator of relapse after hematopoietic stem cell transplantation (SCT) is controversial. We monitored chimerism status by short tandem repeat-based polymerase chain reaction (PCR) in T- and non-T-cell subsets and retrospectively evaluated clinical outcome in 96 patients with acute myeloid leukemia after myeloablative (MA) or reduced-intensity conditioning SCT. Fifty-six percent of 80 patients in the MA group demonstrated complete donor chimerism (CC) at all time points, whereas 6% had decreasing mixed chimerism (MC), 8% stable MC, 25% increasing MC and 3% increasing and decreasing MC. In 16 RIC patients, these percentages were 12, 50, 6, 6 and 19, respectively, together with 6% nonengraftment. Forty-three out of 96 patients experienced relapse. The last chimerism evaluation before relapse revealed increasing MC in only eight patients. In samples taken between 1 and 6 months post SCT, CC/decreasing MC was significantly related with a lower risk of relapse (31 versus 83%, P<0.000) and mortality (38 versus 83%, P<0.000) than with MC/increasing MC. However, the development of relapse was very rapid. Only very frequent monitoring of chimerism status by highly sensitive methods might identify impending relapse and allow early immunological intervention.


Assuntos
Transplante de Medula Óssea , Quimerismo , Leucemia Mieloide Aguda/genética , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase , Quimeras de Transplante/genética , Adolescente , Adulto , Idoso , Feminino , Humanos , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica/métodos , Valor Preditivo dos Testes , Recidiva , Transplante Homólogo
16.
Tissue Antigens ; 64(4): 452-61, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15361122

RESUMO

In cancer research, loss of heterozygosity (LOH), defined by microsatellite markers, is frequently used in the identification of gene loss. Especially, genomic alterations in the human leukocyte antigen (HLA) genes and the beta2-microglobulin (beta2m) gene on chromosome 15 are of interest regarding their function in the immune system. Because LOH analysis detects any allelic imbalance and not just allelic loss, we evaluated the LOH analysis in 11 head and neck squamous cell carcinoma (HNSCC) lesions using fluorescence in situ hybridization (FISH). The 11 tumors were selected out of 53 HNSCC lesions based upon beta2m LOH analysis and beta2m expression. Centromere 1 and 15 FISH were developed to determine the chromosome 15 copy number. Sequence-based mutation analysis of beta2m was conducted on tumors without beta2m expression; no mutations in the coding sequences were found. For five HNSCC lesions with LOH and beta2m expression, centromere 15 FISH indicated gain rather than loss. In the majority of the 11 HNSCC lesions, FISH showed centromere 1 and 15 heterogeneity throughout the tumor. Moreover, FISH indicated a more complex chromosome 1 and 15 distribution than could be concluded from microsatellite LOH analysis. Our results show that microsatellite LOH analysis does not represent the beta2m gene copy number and support the results obtained from comparative genomic hybridization (CGH) studies. Conclusions on genomic alterations in tumors cannot be based on LOH data only but depend on the results of immunohistochemical staining, FISH, and CGH.


Assuntos
Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 15/genética , Neoplasias de Cabeça e Pescoço/genética , Perda de Heterozigosidade/genética , Microglobulina beta-2/genética , Desequilíbrio Alélico/genética , Carcinoma de Células Escamosas/patologia , Cromossomos Humanos Par 1/genética , Ligação Genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Hibridização in Situ Fluorescente
17.
Transplant Proc ; 36(10): 3143-8, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15686714

RESUMO

INTRODUCTION: In vitro studies have shown that apoptotic cell death is triggered by a IL-2-dependent activation of the Fas-FasL pathway and that this pathway can be inhibited by FLIP. METHODS: To define whether FLIP regulates apoptotic death of graft infiltrating T-cells during IL-2-mediated rejection, we analyzed endomyocardial biopsies (EMB) from cardiac allograft recipients for CD3, DNA strand breaks (TUNEL assay), FLIP (mRNA and protein), and FasL mRNA expression. RESULTS: Apoptosis was present in CD3+ T-cell infiltrates. The number of TUNEL-stained mononuclear cells was inversely correlated with FLIP mRNA expression levels (P=.09). FLIP protein was present in 5% to 10% of the infiltrating cells and was constitutively produced by cardiomyocytes irrespective of the rejection grade. Rejection biopsies had elevated IL-2 and FasL mRNA expression levels compared to the expression levels before and after acute rejection (P=.03 and P=.11), while FLIP mRNA expression levels were significantly decreased during rejection (P=.05). CONCLUSION: Our results indicate that during the IL-2-induced rejection process, infiltrated T cells become more sensitive to apoptosis.


Assuntos
Apoptose , Transplante de Coração/patologia , Interleucina-2/fisiologia , Glicoproteínas de Membrana/genética , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD/análise , Sequência de Bases , Complexo CD3/análise , Dano ao DNA , Primers do DNA , Proteína Ligante Fas , Regulação da Expressão Gênica , Humanos , Marcação In Situ das Extremidades Cortadas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Linfócitos T/patologia , Transplante Homólogo/patologia
18.
Bone Marrow Transplant ; 32(10): 1027-30, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14595391

RESUMO

An X-linked severe combined immunodeficient (SCID) patient received a nonirradiated erythrocyte transfusion and developed transfusion-associated graft-versus-host disease (TAGVHD), which was controllable with high-dose corticosteroids. Haplo-identical SCT was performed, after a myeloablative conditioning regimen. At day +26, he developed GVHD. Chimerism studies revealed DNA of the erythrocyte transfusion donor (ETD) and recipient only. Because of early nonengraftment and the presence of alloreactive T cells of ETD origin, the patient was treated with an immunosuppressive conditioning regimen followed by a second SCT from the same donor. While tapering immunosuppression, he again developed mild GVHD, and DNA of ETD and bone marrow donor origin were both present. On cyclosporin, the ETD-DNA signal finally disappeared. High-resolution HLA typing revealed haplo-identity between BMD, ETD and the patient, which might have contributed to the relative mild course of the TAGVHD.


Assuntos
Transfusão de Eritrócitos/efeitos adversos , Doença Enxerto-Hospedeiro/etiologia , Imunodeficiência Combinada Severa/terapia , Corticosteroides/uso terapêutico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Masculino , Imunodeficiência Combinada Severa/complicações , Quimeras de Transplante/genética , Resultado do Tratamento
20.
J Neuroimmunol ; 133(1-2): 198-204, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12446023

RESUMO

Vbeta usage of muscle-infiltrating T lymphocytes in polymyositis (PM) and sporadic inclusion body myositis (s-IBM) was correlated with clinical and histopathological features. Immunohistochemical analysis was combined with complementarity-determining region 3 (CDR3) length analysis in nine muscle biopsies of eight PM patients and six biopsies of five s-IBM patients. Vbeta usage was heterogeneous in seven patients. Four of these patients had definite PM with endomysial located T cell infiltrates, but T cells specifically surrounding and invading individual non-necrotic fibers were not found. In two s-IBM patients, Vbeta 2 usage was increased. In one of them, a repeat biopsy showed a heterogeneous Vbeta usage. We conclude that clonal expansion of muscle-infiltrating T cells could only be detected in part of the patients. Explanations may be that clonal expansion does not take place in all disease phases and that PM is a heterogeneous disease with respect to pathogenesis.


Assuntos
Quimiotaxia de Leucócito/imunologia , Miosite de Corpos de Inclusão/imunologia , Polimiosite/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Antígenos de Superfície/imunologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/imunologia , Músculo Esquelético/patologia , Miosite de Corpos de Inclusão/patologia , Polimiosite/patologia
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