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1.
Thorac Cardiovasc Surg ; 57(5): 276-80, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19629889

RESUMO

BACKGROUND: Atrial fibrillation (AF) is a common complication after coronary artery bypass grafting operation (CABG). Experimental data have shown antiarrhythmic effects of n-3 polyunsaturated fatty acids (PUFA) on myocardial cells. Orally administered PUFA could significantly reduce the rate of postoperative AF. We assessed the efficacy of PUFA for the prevention of AF after CABG. PUFA were given intravenously to prevent variation in bioavailability. METHODS AND RESULTS: 52 patients were randomized to the interventional group, 50 served as controls. In the control group free fatty acids (100 mg soya oil/kg body weight/day) were infused via perfusion pump, starting on admission to hospital and ending at discharge from intensive care. In the interventional group PUFA were given at a dosage of 100 mg fish oil/kg body weight/day. Primary end point was the postoperative development of AF, documented by surface ECG. Secondary end point was the length of stay in the ICU. The demographic, clinical and surgical characteristics of the patients in the two groups were similar. Postoperative AF occurred in 15 patients (30.6 %) in the control and in 9 (17.3 %) in the PUFA group ( P < 0.05). After CABG, the PUFA patients had to be treated in the ICU for a shorter time than the control patients. No adverse effects were observed. CONCLUSIONS: Perioperative intravenous infusion of PUFA reduces the incidence of AF after CABG and leads to a shorter stay in the ICU and in hospital. Our data suggest that perioperative intravenous infusion of PUFA should be recommended for patients undergoing CABG.


Assuntos
Antiarrítmicos/administração & dosagem , Fibrilação Atrial/prevenção & controle , Ponte de Artéria Coronária/efeitos adversos , Ácidos Graxos Ômega-3/administração & dosagem , Óleos de Peixe/administração & dosagem , Óleo de Soja/administração & dosagem , Idoso , Fibrilação Atrial/etiologia , Fibrilação Atrial/fisiopatologia , Cuidados Críticos , Método Duplo-Cego , Eletrocardiografia , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Infusões Intravenosas , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Tempo , Resultado do Tratamento
2.
Transplant Proc ; 40(4): 974-7, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18555093

RESUMO

Acute cardiac allograft rejection remains a major cause of morbidity and mortality after heart transplantation and predisposes for the development of graft vasculopathy. The aim of this study was to investigate the immunomodulatory effect of preconditioning of the donor and recipient with medical ozone (O(3)/O(2)) on acute allograft rejection. Minimizing the initial ischemia-reperfusion injury may result in a reduction of graft vasculopathy and ameliorate long-term outcomes after cardiac transplantation. Lewis rats were challenged with Wistar-Furth cardiac allograft. In donor and recipient animals a medical ozone (O(3)/O(2))-pneumoperitoneum was induced by single (1x) or repetitive (5x) insufflation (concentration: 50 microg/mL, 80 mL/kg body weight) of medical ozone intraperitoneally. Without immunomodulatory therapy (n = 11) cardiac allograft survival was 5.9 +/- 0.9 days. Preconditioning with medical ozone alone (single bolus as well as repetitive administration, n = 7) of the donor and recipient animals prolonged cardiac allograft survival significantly to 7.6 +/- 1.4 days (P < .05), without any adjunctive immunosuppressive therapy. In this pilot study, the intraperitoneal administration of ozone in donor and recipient animals protected from ischemia-reperfusion injury, reduced the immunogenicity of the graft, and prolonged cardiac allograft survival. Further studies are warranted to elucidate the underlying mechanisms and--more important--to investigate the effect on the development of graft vasculopathy, the major obstacle to long-term graft and patient survivals.


Assuntos
Rejeição de Enxerto/prevenção & controle , Transplante de Coração/fisiologia , Imunossupressores/uso terapêutico , Ozônio/uso terapêutico , Transplante Homólogo/fisiologia , Animais , Masculino , Modelos Animais , Ratos , Ratos Endogâmicos WF , Condicionamento Pré-Transplante/métodos
3.
Cancer Res ; 52(1): 108-16, 1992 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-1727369

RESUMO

A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta-actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100-fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations.


Assuntos
Amplificação de Genes/genética , NAD(P)H Desidrogenase (Quinona)/genética , Neoplasias/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Actinas/genética , Sequência de Bases , DNA de Neoplasias/genética , Fluoruracila/uso terapêutico , Humanos , Leucovorina/uso terapêutico , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Neoplásico/genética , Reprodutibilidade dos Testes , Transcrição Gênica
4.
Cancer Res ; 52(4): 797-802, 1992 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-1737339

RESUMO

NAD(P)H:quinone oxidoreductase (DT-diaphorase; DTD) is an obligate two-electron reductase which may play a role in the bioactivation of antitumor quinones such as mitomycin C (MMC). We studied 10 colon carcinoma cell lines showing different levels of DTD activity (range, 0-3447 nmol/min/mg protein), as measured by the reduction of dichlorophenolindophenol. Expression of the NAD(P)H:quinone reductase gene (NQO1), which codes for the DTD enzyme, as measured by a polymerase chain reaction amplification technique was then correlated with enzymatic activity in all cell lines. HT-29 cells, which have intermediate DTD activity (769 +/- 144 nmol/min/mg protein, mean +/- SD) and are sensitive to MMC, showed high NQO1 expression relative to beta-actin (taken as 100% here for comparative purposes). BE cells which have no detectable DTD activity and are resistant to MMC showed moderate NQO1 expression (91% of HT-29). RNA single-strand conformational polymorphism analysis and subsequent sequencing of BE complementary DNA revealed a C to T mutation in the NQO1 complementary DNA. This confers a proline to serine substitution in the amino acid sequence of the protein. Additionally, HCT-116 cells showed both moderate DTD activity (390 +/- 41 nmol/min/mg protein) and NQO1 expression (41% of HT-29), while resistant subclones of these cells, exposed to MMC during 11 and 44 weeks, showed low gene expression (5 and 9% of HT-29 respectively) and enzymatic activity (11 +/- 6 and 36 +/- 16 nmol/min/mg protein). These results support the ideas that reductive activation of MMC by DTD may be important in the cytotoxicity of MMC and that polymerase chain reaction may be a useful technique for quantitating the relative expression of genes in human tumors.


Assuntos
Neoplasias do Colo/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Mitomicina/farmacologia , NAD(P)H Desidrogenase (Quinona)/genética , Actinas/genética , Sequência de Bases , Neoplasias do Colo/enzimologia , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Humanos , Cinética , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Transcrição Gênica , Células Tumorais Cultivadas
5.
J Cardiovasc Surg (Torino) ; 56(6): 827-36, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26381216

RESUMO

AIM: In Germany, every surgical or endovascular procedure on the extracranial carotid artery is documented in a mandatory quality assurance registry. The purpose of this study is to describe the patient characteristics, the indications for treatment, and the short-term outcomes as well as to analyse the corresponding trends from 2003 to 2014. METHODS: Data on demographics, peri-procedural measures, and outcomes were extracted from the annual quality reports published by the Federal Agency for Quality Assurance and the Institute for Applied Quality Improvement and Research in Health Care. Data were available from 2003 to 2014 for carotid endarterectomy (CEA) and from 2012 to 2014 for carotid artery stenting (CAS). The primary outcome event of this study is any stroke or death until discharge from hospital. Temporal trends of categorical variables were statistically analysed using the Cochran-Armitage test for trend. RESULTS: Between 2003 and 2014, 309,405 CEAs and 18,047 CAS procedures were documented in the database; 68.1% of all patients were male. The mean age of patients treated with CEA increased from 68.9 years in 2003 to 70.9 years in 2014. The proportion of patients with ASA stages III to V increased from 65% to 71% in CEA, whereas it decreased from 44% to 41% in CAS patients. 53.1% of all CEAs were performed for asymptomatic patients (group A), 34.4% for symptomatic patients treated electively (group B), and 11.2% a in a collective group including other indications for CEA or CAS (such as recurrent stenosis, carotid aneurysms, emergency treatment due to stroke-in-evolution). The corresponding data for CAS are 49.3%, 26.1% and 26.3% respectively. In group B, the interval between the neurological index event and CEA decreased from 28 to 8 days (P<0.001). In patients treated with CAS, this interval was 9 days in 2012 (no further data available). On average, 67.1% and 48.2% of surgically treated patients as well as 77.8% and 69.8% of CAS patients were neurologically assessed before and after the procedure, respectively. From 2003 to 2014, CEA procedures were performed more frequently in locoregional anesthesia (10.1% to 29.1%, P<0.001). The same trend was observed for the application of the eversion technique (37.0% to 41.6%, P<0.001), the neurophysiological monitoring (49.8% to 61.8%, P<0.001), and the intra-procedural assessment of the treated artery (44.5% to 69.7%, P<0.001). In contrast, shunting was used less frequently (48.1% to 43.0%, P<0.001). Averagely 95.7% of all endovascular procedures were performed using stent-angioplasty. In 54.2% a protection device was used. Nitinol and bare metal stents were used in 74.1% and 21.4% of cases, respectively. The in-hospital rate of any stroke or death decreased from 2.0% to 1.1% in asymptomatic patients treated with CEA without a contralateral stenosis ≥75% or occlusion, P<0.001). In patients treated with CAS this rate did not increase (1.7% to 1.8%, p=0.909). The corresponding rates in CEA and CAS patients with severe contralateral stenosis or occlusion varied between 1.9%-3.1% and 2.2%-2.6%, respectively. In symptomatic patients (group B) with a stenosis of 50 percent or more, the rate of any stroke or death decreased significantly after CEA from 4.2% to 2.4% (P<0.001) and remained stable after CAS (3.9% to 3.5%, P=0.577). CONCLUSION: This report on 327,452 carotid procedures analysed one of the largest quality registries on CEA and CAS worldwide. Data indicate that treated patients became older and sicker, whereas in contrast, the in-hospital rates of stroke or death are decreasing over time.


Assuntos
Angioplastia/tendências , Doenças das Artérias Carótidas/terapia , Endarterectomia das Carótidas/tendências , Fatores Etários , Idoso , Angioplastia/efeitos adversos , Angioplastia/instrumentação , Angioplastia/mortalidade , Doenças das Artérias Carótidas/diagnóstico , Doenças das Artérias Carótidas/mortalidade , Dispositivos de Proteção Embólica , Endarterectomia das Carótidas/efeitos adversos , Endarterectomia das Carótidas/mortalidade , Feminino , Alemanha , Mortalidade Hospitalar , Humanos , Masculino , Garantia da Qualidade dos Cuidados de Saúde/tendências , Indicadores de Qualidade em Assistência à Saúde/tendências , Sistema de Registros , Medição de Risco , Fatores de Risco , Stents , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/mortalidade , Fatores de Tempo , Resultado do Tratamento
6.
Transplantation ; 64(12): 1816-22, 1997 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-9422425

RESUMO

BACKGROUND: Peripheral and central immune mechanisms contribute to the induction of tolerance in acute rejection rodent transplant models after systemic administration of CTLA4Ig and intrathymic infusion of donor alloantigen, respectively. We have investigated the effects of CTLA4Ig-induced blockade of CD28-B7 T-cell co-stimulation in conjunction with intrathymic immunomodulation on cellular and humoral immune responses leading to accelerated rejection of cardiac allografts in presensitized rats. METHODS AND RESULTS: Lewis rats were challenged with Wistar-Furth (WF) skin transplants, followed 7 days later by transplantation of WF hearts. These cardiac allografts were rejected in a fulminant manner in <24 hr. A single infusion of human CTLA4Ig (0.5 mg/rat i.v.) at the time of cardiac engraftment (day 0) did not affect accelerated rejection. Intrathymic injection of WF spleen cells (2x10[7]) at the time of skin transplantation (day -7) abrogated <24-hr rejection and extended cardiac allograft survival to 6.6+/-0.6 days. Moreover, intrathymic host immunomodulation combined with administration of human CTLA4Ig (days 0-14, every other day) extended cardiac allograft survival synergistically to 27.7+/-7.5 days, and immunomodulation combined with murine CTLA4Ig extended survival to >42.5+/-4.8 days. The prolongation of allograft survival required the blockade of both B7-1 and B7-2 ligands and was accompanied by reduction of host proliferative responses (mixed lymphocyte response) and depression of anti-donor cytotoxic T-cell generation/function (lymphocyte-mediated cytotoxicity). CTLA4Ig therapy did not affect the strong systemic IgM and IgG alloantibody response seen otherwise after intrathymic immunomodulation. CONCLUSION: CTLA4Ig enhances the effects of intrathymic donor-type cell infusion in sensitized rat recipients of cardiac allografts, indicating that "peripheral" blockade of CD28-B7 T-cell co-stimulation synergizes with the "central" immunosuppressive effects of intrathymic immunomodulation.


Assuntos
Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Rejeição de Enxerto/imunologia , Imunoconjugados , Linfócitos T/imunologia , Abatacepte , Adjuvantes Imunológicos , Animais , Antígenos CD , Antígenos de Diferenciação/imunologia , Antígeno CTLA-4 , Citotoxicidade Imunológica , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Isoanticorpos/imunologia , Teste de Cultura Mista de Linfócitos , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Ratos Endogâmicos WF , Transplante de Pele/imunologia , Análise de Sobrevida , Timo/imunologia
7.
J Neuroimmunol ; 96(2): 158-66, 1999 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-10337914

RESUMO

Blockade of the CD28-B7 or CD40L-CD40 T cell costimulatory signals prevents induction of experimental autoimmune encephalomyelitis (EAE). However, the effect of simultaneous blockade of these signals in EAE is unknown. We show that administration of either MR1 (to block CD40L) or CTLA4Ig (to block B7) after immunization or after the first attack protects from EAE. Treatment with a combination of CTLA4Ig and MR1 provides additive protection, and is associated with complete absence of mononuclear cell infiltrates in the central nervous system, and marked suppression of proliferation of primed T cells in the periphery. Selective B7-1 blockade did not protect from EAE. These observations have implications for therapy of autoimmune diseases.


Assuntos
Antígeno B7-1/farmacologia , Antígenos CD28/farmacologia , Encefalomielite Autoimune Experimental/prevenção & controle , Imunoconjugados , Glicoproteínas de Membrana/farmacologia , Transdução de Sinais/efeitos dos fármacos , Abatacepte , Animais , Antígenos CD , Antígenos de Diferenciação/farmacologia , Ligante de CD40 , Antígeno CTLA-4 , Relação Dose-Resposta a Droga , Esquema de Medicação , Sinergismo Farmacológico , Encefalomielite Autoimune Experimental/patologia , Feminino , Imunossupressores/farmacologia , Glicoproteínas de Membrana/administração & dosagem , Camundongos , Proteína Básica da Mielina/farmacologia , Recidiva , Linfócitos T/efeitos dos fármacos , Fatores de Tempo
8.
Transplantation ; 63(10): 1495-500, 1997 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-9175816

RESUMO

The CD26 antigen, one of the major costimulatory molecules in T cell activation, was shown to possess dipeptidyl peptidase IV (DPP IV) activity. Previously, we demonstrated that immunosuppressed kidney transplant patients exhibit lower DPP IV serum activity as compared with healthy individuals. In the present study, we analyzed the role of CD26/DPP IV in the immune cascade triggered by organ transplantation and leading to acute rejection of cardiac allografts in rat recipients. Transplantation of hearts from (Lewis x Brown Norway)F1 donors into Lewis hosts resulted in an early (24 hr) increase in cellular CD26 expression, followed by a rise in DPP IV serum activity, which peaked at day 6, i.e., before the time of actual graft loss. Specific targeting of DPP IV activity with a novel, low-molecular-weight inhibitor of the diphenyl-phosphonate group (prodipine) abrogated acute rejection and prolonged cardiac allograft survival to 14.0+/-0.9 days (P<0.0001). Prodipine treatment prevented the early peak of cellular CD26 expression and thoroughly suppressed systemic DPP IV activity. The inhibition of DPP IV was associated with severely impaired host cytotoxic T lymphocyte responses in vitro. These results demonstrate the role of CD26/DPP IV in alloantigen-mediated immune regulation in vivo and provide the first direct evidence that CD26/DPP IV plays an important role in the mechanism of allograft rejection. The model of targeting CD26/DPP IV may reveal essential interactions on the level of costimulatory alternate T cell activation pathways, allowing a more subtle approach for more selective immunosuppression in transplant recipients.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Transplante de Coração/imunologia , Inibidores de Proteases/farmacologia , Animais , Dipeptidil Peptidase 4/análise , Dipeptidil Peptidase 4/sangue , Sobrevivência de Enxerto/efeitos dos fármacos , Imunossupressores/farmacologia , Linfócitos/imunologia , Masculino , Piperidinas/farmacologia , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/fisiologia , Transplante Homólogo/imunologia
9.
J Heart Lung Transplant ; 19(6): 566-75, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10867337

RESUMO

BACKGROUND: Intrathymic injection of alloantigen in the form of donor cells, soluble major histocompatibility complex (MHC) molecules, or MHC allopeptides induces donor-specific tolerance in a variety of acute allograft rejection models. We have previously shown that a single intrathymic injection of donor spleen cells into pre-sensitized rats abrogates accelerated (circa 24-hour) rejection and prolongs the survival of cardiac allografts to about 7 days. The present study was designed to investigate the mechanisms by which intrathymic administration of donor cells modifies the course of accelerated rejection. METHODS: Lewis RT1(1) (LEW) rats sensitized by transplantation with Wistar-Furth RT1(u) (WF) skin grafts received WF cardiac allografts 7 days later-a classic model of accelerated rejection. At the time of skin challenge, however, certain animals received intrathymic cell suspensions (either allogeneic or syngeneic) or donor-derived class I and/or class II MHC peptides. RESULTS: Control animals (sensitized by skin grafts but receiving no other treatment) rejected cardiac allografts within 24 hours. Intrathymic injection of WF splenocytes at the time of skin transplantation abrogated rejection at 24 hours and prolonged cardiac allograft survival to 6.6+/-0.6 days (p<0.001), whereas intrathymic administration of syngeneic (LEW) or allogeneic third party Brown Norway RT1(n) cells was ineffective in this regard. Intrathymic injection of gamma-irradiated donor cells marginally extended cardiac allograft survival to 3.0+/-0.9 days (p< 0.001), but the grafts were still rejected in an accelerated fashion. Intrathymic injection of donor-derived class I and/or class II MHC allopeptides at the same time period also failed to prolong cardiac allograft survival beyond 3 days. In the group receiving unmodified donor cells, elevated immunoglobulin M (IgM) and immunoglobulin G (IgG) allo-antibodies were found at the time of cardiac transplantation; this pattern was not observed with any other treatment. CONCLUSION: The superiority of non-modified donor spleen cells over gamma-irradiated donor cells or donor specific allopeptides in modifying the course of accelerated cardiac rejection suggests that direct allorecognition is the dominant pathway initiating rejection in sensitized transplant recipients. Marked alterations in the antidonor IgM and IgG responses are associated with successful abrogation of accelerated rejection by thymic immunomodulatory mechanisms.


Assuntos
Transplante de Células , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/imunologia , Imunização/métodos , Isoantígenos/administração & dosagem , Baço/citologia , Timo/imunologia , Animais , Biomarcadores/sangue , Genes MHC Classe I/imunologia , Genes MHC da Classe II/imunologia , Rejeição de Enxerto/sangue , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Injeções , Isoanticorpos/sangue , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Ratos Endogâmicos WF , Baço/imunologia , Transplante Homólogo
10.
Ann Thorac Surg ; 58(6): 1614-6, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7979724

RESUMO

This study describes the placement of a newly designed implantable cardioverter defibrillator in a subpectoral device pocket using the incision for venous access in 16 patients undergoing implantation of an implantable cardioverter defibrillator with a nonthoracotomy lead system. The endocardial lead system consisted of a right atrial/superior vena cava defibrillation spring electrode and a right ventricular bipolar sensing/defibrillation electrode, inserted by cephalic venotomy or by puncturing of the subclavian vein. As a result of intraoperative testing using biphasic shocks the defibrillation threshold (DFT) had to be less than 24 J, otherwise an additional subcutaneous patch electrode was placed in the lateral chest wall near the cardiac apex through another incision. All patients received a nonthoracotomy lead system in combination with a subpectoral device placement. In 11 of 16 patients the endocardial leads alone were sufficient (DFT, 13.4 +/- 7.0 J), 5 of 16 patients (31%) required an additional subcutaneous patch electrode to achieve proper device function (DFT, 14.6 +/- 9.0 J). The operation lasted 93 +/- 20 minutes. This was a significant (p < 0.05) lower time consumption than standard nonthoracotomy approach combined with abdominal device placement (120 +/- 50 minutes). There were no postoperative complications. During follow-up period (average, 4 months), none of the patients reported major local symptoms, especially no device migration occurred. This approach, in contrast to an abdominal device placement, avoids another incision and subcutaneous tunneling of leads. In 11 of 16 patients defibrillator implantation by a single incision in the deltoideopectoral groove was possible.


Assuntos
Desfibriladores Implantáveis , Idoso , Arritmias Cardíacas/cirurgia , Procedimentos Cirúrgicos Cardíacos/métodos , Cardiomiopatia Dilatada/cirurgia , Eletrodos , Desenho de Equipamento , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Toracotomia , Disfunção Ventricular Direita/cirurgia
11.
Methods Mol Biol ; 15: 177-88, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-21400275

RESUMO

Quantitative measurement of specific mRNA species is of major importance for approaching many fundamental questions in biology. Until now, quantitation of gene expression has usually been done by Northern blotting, but this procedure is relatively insensitive, requiring microgram amounts of RNA. Furthermore, unless linear ranges of RNA concentration are determined, the procedure is semiquantitative at best. Because of the limitations of Northern blotting, various strategies have been developed for quantitation of cDNA by polymerase chain reaction (PCR)-based methods (1-3), most of them utilizing the principle of competitive PCR in which a synthetic segment is coamplified along the target DNA segment. We were interested in comparing the expression of drug target genes in very small samples of human tumor material, such as would be obtained from biopsies or even paraffin blocks. The competitive PCR was not entirely suitable for this purpose because: (1) the "input" RNA or DNA concentration must be known with precision in order to provide a normalization factor, and (2) our results suggested that the competitor and target were not necessarily amplified with the same efficiency even when the segments to be amplified were the same size (4). To overcome these problems, we developed an alternate method of PCR quantitation with the following key features:

18.
Transplant Proc ; 41(6): 2616-20, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19715986

RESUMO

p38MAP kinase plays a crucial role in intracellular signal transduction of inflammation. The inhibitor of p38 MAP kinase, FR167653, has been proven to be effective to suppress proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in various animal models. The aim of our study was to investigate p38MAP kinase inhibition by FR167653 on the inflammatory profile of cells involved in vascular injury. HUVEC incubated with FR167653 in concentrations of 0.1 to 20 mumol for 24 hours were stimulated with TNF-alpha (20 ng/mL). Human monocytes were incubated with equal concentrations of FR167653 and stimulated with lipopolysaccharides (LPS; 10 microg/mL). In monocytes, p38 MAP kinase could be inhibited by FR167653 (Western blot). The cytokines IL-6 and IL-8 were dose dependently downregulated by FR167653 (enzyme-linked immunosorbent assay) [ELISA]. These results were confirmed at a transcriptional level by real-time polymerase chain reaction (PCR). Gene expression of IL-6 and IL-8 was dose dependently downregulated. The expression pattern of ICAM-1 and VCAM-1 was not altered by FR167653 (ELISA). In HUVEC, the cytokines IL-6 and IL-8 were dose dependently downregulated by FR167653 (ELISA). These results were confirmed on a transcriptional level by real-time PCR. Gene expression of IL-6 and IL-8 were also dose dependently suppressed by FR167653. In addition FR167653 downregulated the expression of the adhesion molecules ICAM-1 and VCAM-1 (ELISA). FR167653 suppressed the development of a proinflamatory profile of HUVEC and human monocytes after stimulation with TNF-alpha or LPS, respectively. These results indicated anti-inflammatory properties of FR167653 on endothelial and inflammatory cells, which may be therapeutically useful to ameliorate vascular injury.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Endotélio Vascular/fisiologia , Inflamação/prevenção & controle , Monócitos/fisiologia , Pirazóis/farmacologia , Piridinas/farmacologia , Veias Umbilicais/fisiologia , Técnicas de Cultura de Células , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/genética , Cinética , Lipopolissacarídeos/farmacologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
19.
Transplant Proc ; 41(6): 2621-4, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19715987

RESUMO

Accelerated rejection due to host sensitization to major histocompatibility complex antigens is a critical problem in clinical organ transplantation in patients who have previously received an organ transplant, experienced acute rejection episodes, received blood transfusions, or been pregnant. The precise pathologic mechanisms underlying accelerated rejection have not been characterized. Herein, we describe apoptosis during T- and B-cell-driven accelerated rejection of cardiac allografts in presensitized recipients. In an established accelerated rejection model, Lewis rats were sensitized to skin grafts from Wistar-Furth rats; after 7 days, they received Wistar-Furth hearts. These grafts were rejected within 24 hours posttransplantation compared with 10 days in nonsensitized recipients (acute rejection, n = 5). Apoptosis was observed during accelerated rejection of cardiac allografts but not in naïve recipients of hearts, as demonstrated at DNA laddering and TUNEL (terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling) assay. Apoptosis was discovered as a thus far unknown effector mechanism in accelerated cardiac transplant rejection that accompanies combined cellular and humoral immune alloreactivity. Apoptotic cell death in accelerated rejection and the cascade of upstream and downstream events leading to or resulting from this process should be considered critical steps in the pathogenesis of accelerated rejection.


Assuntos
Transplante de Coração/imunologia , Transplante de Coração/patologia , Animais , Apoptose , Morte Celular , Feminino , Rejeição de Enxerto/epidemiologia , Imunização/métodos , Marcação In Situ das Extremidades Cortadas , Masculino , Gravidez , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos WF , Transplante de Pele/imunologia , Transplante Homólogo/imunologia , Transplante Homólogo/patologia
20.
Transplant Proc ; 41(6): 2625-7, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19715988

RESUMO

Graft vasculopathy (GVP) is one of the major obstacles to long-term graft and patient survival after cardiac transplantation and a major reason for morbidity and mortality. Antigen-dependent and antigen-independent factors play causal roles in the development of GVP. The aim of this study was to evaluate antigen-dependent and -independent factors in the development of GVR in a clinically relevant fully allogeneic rat cardiac model under immunosuppression with cyclosporine (CyA). Lewis rats were challenged with Wistar-Furth cardiac allografts. Acute rejection occurred within 10 days after engraftment (n = 6). Daily SC administration of CyA (2.5 mg/kg body weight, n = 12) led to long-term graft survival (>100 days) but did not prevent GVP (Adams Score: 1.7 +/- 1.9, n = 4). Isografts did not develop GVP. In allografts, the dose modification of CyA to 5 mg or 1.25 mg/kg body weight as well as the prolongation of ischemia from 45 minutes to 4 hours did not increase the development of GVP. In isografts, the prolongation of ischemic time from 45 minutes to 4 hours significantly increased the development of GVP (Adams score, 0.3 +/- 0.8 [n = 7] vs 1.2 +/- 1.9 [n = 6]; P < .05). In this fully allogeneic cardiac allograft model with clinically relevant immunosuppressive therapy, GVP was induced independent of the applied CyA dose. In addition, the prolongation of ischemic time did not increase the development of GVP. Isografts only developed significant GVP with long ischemia times. Therefore, an initial injury, either prolonged ischemia time or an allogeneic immune response, predispressed to the development of GVP.


Assuntos
Ciclosporina/uso terapêutico , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Transplante de Coração/patologia , Doenças Vasculares/patologia , Animais , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/efeitos dos fármacos , Imunossupressores/uso terapêutico , Modelos Animais , Isquemia Miocárdica/fisiopatologia , Complicações Pós-Operatórias/patologia , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos WF , Transplante Homólogo/imunologia , Transplante Homólogo/patologia , Doenças Vasculares/etiologia
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