RESUMO
Type I IFN receptor type 2 (IFNAR2) expression correlates significantly with clinical response to interferon (IFN)-alpha/5-fluorouracil (5-FU) combination therapy for hepatocellular carcinoma (HCC). However, some IFNAR2-positive patients show no response to the therapy. This result suggests the possibility of other factors, which would be responsible for resistance to IFN-alpha/5-FU therapy. The aim of this study was to examine the mechanism of anti-proliferative effects of IFN-alpha/5-FU therapy and search for a biological marker of chemoresistance to such therapy. Gene expression profiling and molecular network analysis were used in the analysis of non-responders and responders with IFNAR2-positive HCC. The Wnt/beta-catenin signalling pathway contributed to resistance to IFN-alpha/5-FU therapy. Immunohistochemical analysis showed positive epithelial cell adhesion molecule (Ep-CAM) expression, the target molecule of Wnt/beta-catenin signalling, only in non-responders. In vitro studies showed that activation of Wnt/beta-catenin signalling by glycogen synthesis kinase-3 inhibitor (6-bromoindirubin-3'-oxime (BIO)) induced chemoresistance to IFN-alpha/5-FU. BrdU-based cell proliferation ELISA and cell cycle analysis showed that concurrent addition of BIO and IFN-alpha/5-FU significantly to hepatoma cell cultures reduced the inhibitory effects of the latter two on DNA synthesis and accumulation of cells in the S-phase. The results indicate that activation of Wnt/beta-catenin signalling pathway induces chemoresistance to IFN-alpha/5-FU therapy and suggest that Ep-CAM is a potentially useful marker for resistance to such therapy, especially in IFNAR2-positive cases.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/fisiologia , Carcinoma Hepatocelular/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/fisiologia , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial , Feminino , Fluoruracila/administração & dosagem , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/administração & dosagem , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptor de Interferon alfa e beta/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
The aim of this study was to assess the value of alphafeto protein (AFP) mRNA-expressing cells detected in peripheral blood for predicting tumor recurrence after living donor liver transplantation (LDLT) in patients with hepatocellular carcinoma (HCC). The test group consisted of 25 patients who underwent LDLT for end-stage liver disease with HCC while the control group consisted of 37 living donors. Quantitative real-time reverse-transcriptase polymerase chain reaction was used for detection of AFP mRNA-expressing cells in peripheral blood. Nine (36%) of 25 patients developed tumor recurrences (four lung; one liver; one peritoneum; two bone; one adrenal gland) during the follow-up period. Perioperatively, AFP mRNA was positive in peripheral blood of eight patients (32.0%) but only in 1 (2.7%) of the control. Preoperative AFP mRNA was positive in three cases. Univariate analyses revealed that preoperative and perioperative AFP mRNA and microscopical vascular invasion were the significant predictors for HCC recurrence (P = .007, .037, and .005, respectively). In the patients with HCC exceeding Milan criteria (n = 15), the presence of AFP mRNA-positive cells in the peripheral blood correlated significantly with HCC recurrence (P = .033). We concluded that the presence of AFP mRNA-expressing cells could be a useful predictor of HCC recurrence in liver transplant patients.
Assuntos
Carcinoma Hepatocelular/cirurgia , Neoplasias Hepáticas/cirurgia , Transplante de Fígado/fisiologia , RNA Mensageiro/sangue , RNA Mensageiro/genética , alfa-Fetoproteínas/genética , Adulto , Carcinoma Hepatocelular/genética , Intervalo Livre de Doença , Humanos , Neoplasias Hepáticas/genética , Período Pós-Operatório , Valor Preditivo dos Testes , Recidiva , Estudos RetrospectivosRESUMO
The aim of this study was to investigate the risk factors for graft dysfunction after adult-to-adult living donor liver transplantation (LDLT). Thirty-nine adults with chronic cirrhosis underwent LDLT between 1999 and 2004. Their postoperative courses were uneventful with no vascular or bile duct complications early after LDLT, except one mild hepatic artery stenosis. The preoperative MELD scores were significantly higher in the failed graft group (n=5) than the functioning graft group (n=34; P=.004), while the graft liver weight/standard liver volume ratio was similar between these groups. We concluded that a high preoperative MELD score was associated with postoperative graft failure and that graft size had little impact on graft outcome. Although large grafts would seem intuitively more suitable for sick recipients, we did not show a benefit among this cohort; the MELD score was the best predictor, a finding that is also most consistent with donor safety.
Assuntos
Cirrose Hepática/cirurgia , Transplante de Fígado/efeitos adversos , Doadores Vivos , Adulto , Humanos , Transplante de Fígado/mortalidade , Pessoa de Meia-Idade , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/prevenção & controle , Estudos Retrospectivos , Fatores de Risco , Análise de Sobrevida , Falha de Tratamento , Resultado do TratamentoRESUMO
Several clinical trials have demonstrated the effectiveness of combination therapy with 5-fluorouracil (5-FU) and IFN-alpha in colon cancer, hepatocellular carcinoma (HCC), and other malignancies. In our preliminary clinical studies, we have observed outstanding effects with this combination therapy in patients with advanced HCC. However, the underlying mechanism by which IFN-alpha modulates the effects of 5-FU is unknown. We, therefore, conducted a mechanistic study using two HCC cell lines, PLC/PRF/5 and HuH7. IFN-alpha significantly enhanced the growth inhibitory effect of 5-FU in PLC/PRF/5 cells but not in HuH7 cells, and the isobolographic analysis indicated that this effect was synergistic. Flow cytometric analysis showed a delay in the progression of G0-G1 to S phase in PLC/PRF/5, and a sustained, induction of the cyclin-dependent kinase inhibitor p27-Kip1 and down-regulation of cyclin D1 was observed. Moreover, increased expression of p27Kip1 was associated with reduced CDK-2-associated kinase activity. Another difference in the two cell types was that PLC/PRF/5 expressed abundant IFN receptors, but HuH7 did not. Apoptosis assays were not helpful in explaining the mechanism. Our results suggest that the synergistic effects of 5-FU and IFN-alpha may in part be attributable to alterations in cell cycle progression via up-regulation of p27Kip1.
Assuntos
Apoptose/efeitos dos fármacos , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclo Celular/efeitos dos fármacos , Fluoruracila/toxicidade , Interferon-alfa/toxicidade , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Supressoras de Tumor , Carcinoma Hepatocelular , Divisão Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/análise , Quinases Ciclina-Dependentes/antagonistas & inibidores , Sinergismo Farmacológico , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Cinética , Neoplasias Hepáticas , Proteínas de Membrana , Proteínas Associadas aos Microtúbulos/análise , Proteínas Serina-Treonina Quinases/análise , Receptor de Interferon alfa e beta , Receptores de Interferon/análise , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Recent studies have shown increased levels of cyclooxygenase-2 (COX-2) in a variety of human malignancies including hepatocellular carcinoma (HCC), but little is known about the prognostic value of COX-2 in HCC or its associated nontumor liver tissue. We examined the expression of COX-2 protein by immunohistochemistry in 53 patients with HCCs whose corresponding nontumor tissues were hepatitis C virus-related chronic hepatitis (n = 21) and cirrhosis (n = 32). Samples of nine histologically normal livers and eight precancerous dysplasias were also analyzed. The level of COX-2 increased from normal liver to chronic hepatitis to cirrhosis. The majority of cirrhotic livers (81%) displayed marked COX-2 expression. In dysplasias, COX-2 expression was mainly moderate or strong (88%). In HCC, 17% of samples displayed a high COX-2 expression, and 37% of samples expressed COX-2 at a moderate level. Concordant results were obtained with reverse transcription-PCR and Western blot analyses. Clinicopathological survey indicated a significant correlation between COX-2 expression and differentiated carcinoma (P = 0.019). Although there was no correlation between COX-2 expression in HCC and prognosis, a striking difference was found between COX-2 expression in nontumor tissue and shorter disease-free survival (P = 0.0132). Moreover, high COX-2 expression in nontumor tissue was significantly correlated with the presence of active inflammation (P < 0.0001). The present findings suggest that COX-2 expression in nontumor tissue may play a positive role in relapse of HCC after surgery.
Assuntos
Carcinoma Hepatocelular/enzimologia , Isoenzimas/biossíntese , Neoplasias Hepáticas/enzimologia , Fígado/enzimologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Ciclo-Oxigenase 2 , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Fígado/patologia , Fígado/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Prostaglandina-Endoperóxido Sintases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The level of cyclooxygenase (COX)-2 has been investigated recently in various human carcinomas. In the present study, we examined the distribution and extent of COX-2 protein in human pancreatic tumors using immunohistochemistry. A strong expression of COX-2 protein was present in 23 of 52 (44%) pancreatic carcinomas, a moderate expression was present in 24 of 52 (46%) pancreatic carcinomas, and a weak expression was present in 5 of 52 (10%) pancreatic carcinomas. In contrast, benign tumors showed weak expression or no expression of COX-2, and only islet cells displayed COX-2 expression in normal pancreatic tissues. Overexpression of COX-2 in carcinoma tissues was also confirmed by Western blot analysis. Furthermore, consistent with the results at protein levels, reverse transcription-PCR analyses indicated that COX-2 mRNA was overexpressed in 7 of 13 (54%) carcinomas, but in none of 3 benign tumors. Our findings suggest that COX-2 inhibitors might be potentially effective against pancreatic carcinomas and that COX-2 may be involved in certain biological processes in pancreatic islets.
Assuntos
Adenocarcinoma/enzimologia , Carcinoma Adenoescamoso/enzimologia , Isoenzimas/biossíntese , Neoplasias Pancreáticas/enzimologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Western Blotting , Ciclo-Oxigenase 2 , Humanos , Imuno-Histoquímica , Isoenzimas/metabolismo , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The effect of donor-recipient strain combination and supplemental rapamycin (Rapa) on tolerance induction by intrathymic (IT) injection of donor splenocytes was examined in a mouse skin allograft model. In an MHC class I-mismatched C3H/He skin to (C57BL/6 x A)F1 (B6AF1) mouse combination, IT injection of 50 x 10(6) donor splenocytes with transient immunosuppression by rabbit anti-mouse lymphocyte serum (ALS) induced significant prolongation of skin allograft survival with a median survival time (MST) of 115 days versus an MST of 24.5 days in controls given ALS alone. With an additional short course of supplemental Rapa treatment at a dose of 1.5 mg/kg i.p. every other day from day 0 to 12, all C3H/He skin allografts survived indefinitely (> 350 days) in ALS-treated, donor splenocyte intrathymically injected B6AF1 recipient mice. Tolerance was antigen-specific, since the second donor-type skin allografts were accepted while third-party skin allografts were acutely rejected in these mice bearing long-term C3H/He skin allografts. In MHC class I- and II-disparate (DBA/2 to B6AF1) and fully MHC-incompatible (AKR to B6) strain combinations, IT injection of donor splenocytes and ALS treatment failed to prolong skin allograft survival over ALS controls. When supplemental Rapa was used, long-term skin allograft acceptance was observed with an MST of 127 days for the DBA/2 to B6AF1 combination and 70 days for the AKR to B6 combination. In contrast, supplemental treatment with cyclosporine was not effective in these combinations, which suggests that supplemental Rapa may have a unique effect in augmenting IT tolerance induction. Thymectomy within 7 days after IT injection significantly shortened the allograft survival, which suggests that interaction of the host thymus and the injected donor splenocytes, which takes place early after IT injection, plays an important role in the induction of allograft tolerance in this model.
Assuntos
Tolerância Imunológica , Terapia de Imunossupressão/métodos , Polienos/administração & dosagem , Transplante de Pele/imunologia , Baço/imunologia , Timo/imunologia , Animais , Soro Antilinfocitário/administração & dosagem , Ciclosporina/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Histocompatibilidade , Imunização Passiva , Imunossupressores/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Sirolimo , TimectomiaRESUMO
The immunosuppressive potentials of mAbs to lymphocyte function-associated antigen-1 (LFA-1) and CD2 molecules were examined in murine islet transplantation. Crude digested islets from BALB/c (H-2d) mice were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (H-2b) mice. The rat mAbs of KBA (anti-LFA-1) and RM2-1 (anti-CD2) were given intraperitoneally immediately after transplantation and on the first day after grafting at a dose of 0.1 mg/mouse/day. In nontreated animals, the islet allografts were acutely rejected with a mean survival time (MST) of 19.6 +/- 8.3 days. Control isotype-matched anti-CD18 treatment did not prolong the MST of 12.8 +/- 1.6 days. Anti-LFA-1 treatment alone produced indefinite survival in 5 of 10 recipients with MST of 72.2 +/- 33.4 days. Anti-CD2 treatment failed to do so, although MST was marginally prolonged to 32.8 +/- 20.5 days. When both mAbs were given together, additional benefit with anti-CD2 treatment was not observed (MST: 77.4 +/- 31.1 days). In spite of the unresponsiveness to islet allografts, the animals did not suffer from any severe infectious disease. Mice bearing long-term functioning islets rejected third-party skin grafts as well as islet donor strain skin grafts. The long-term surviving islet allografts were also rejected coincidentally. These results indicate that a perioperative short course of anti-LFA-1 mAb treatment can induce unresponsiveness to islet allografts, although it is not systemic, and that costimulatory signals through these adhesion molecules play a central role in inducing an immune response leading to rejection of the allografted islets.
Assuntos
Terapia de Imunossupressão/métodos , Transplante das Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos CD2 , Sobrevivência de Enxerto , Transplante das Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Receptores Imunológicos/imunologiaRESUMO
This study tried to improve the number of viable islets isolated from a pancreas because a sufficient number cannot be obtained when the organ is preserved in the manner used for pancreas transplantation. The mechanism involved in the decrease in islet yield during preservation was studied to try to develop a better method for islet preparation. First, the integrity of the ductal system was compared between fresh and 6-hr simply preserved (in Hanks' balanced salt solution) rat pancreases. The ductal pressure after ductal injection of HBSS reached a plateau earlier and was significantly lower for the preserved pancreases (0.073 +/- 0.026 min, 410 +/- 17 mmHg, n = 5) than for the fresh ones (0.176 +/- 0.086 min, 561 +/- 103 mmHg, n = 7, P less than 0.05). Second, the extent of pancreatic distention was examined following ductal injection of barium gelatin solution. Solution leakage occurred earlier and distention was less in the preserved pancreas. In addition, the gelatin was found in the capillaries within some islets of the preserved pancreas. These results indicated that the preservation led to a rapid loss of integrity of the ductal system before collagenase injection. We therefore tested the efficacy of ductal collagenase injection at the time of harvesting: 15 ml of 1.0 mg/ml collagenase HBSS was intraductally injected and the pancreas was preserved at 4 degrees C for 2, 4, 6, and 24 hr. The isolation procedure was similar to that used for the fresh pancreas. The yield was significantly better than that of the simply preserved pancreas at 4 hr (241 +/- 22, n = 3, vs. 140 +/- 58, n = 3, P less than 0.05) and at 6 hr (171 +/- 58, n = 14, vs. 32 +/- 33, n = 6, P less than 0.01). These isolated islets were spherical-oval and their viability was confirmed by the ability to reverse STZ-induced diabetes in mice. These results indicated that the integrity of the ductal system, which is necessary for distention of the whole pancreas, was lost during preservation. To solve this problem, ductal collagenase injection should be done at the time of pancreas harvesting and then followed by simple preservation. This method is recommended to obtain viable islets from a preserved pancreas.
Assuntos
Ilhotas Pancreáticas/citologia , Pâncreas/citologia , Animais , Separação Celular/métodos , Sobrevivência Celular , Temperatura Baixa , Isquemia , Masculino , Colagenase Microbiana , Preservação de Órgãos/métodos , Pâncreas/irrigação sanguínea , Ductos Pancreáticos/fisiologia , Pressão , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
We examined the efficacy of relatively low temperature collagenase digestion at 20 degrees C on the yield and viability of islets after long-term cold preservation. Wistar rat pancreases were distended with University of Wisconsin solution via a pancreatic duct at the time of harvesting to which collagenase and 2.5 mM calcium chloride were added. The pancreases were cold-preserved at 4 degrees C for 24 or 48 hr. After storage, they were incubated for collagenase digestion at 37 degrees C or 20 degrees C for various incubation periods to obtain the peak yield. At 20 degrees C, in vitro collagenase activity measured by the FALGPA method was one fourth of that at 37 degrees C, and pancreases were well digested with a prolonged digestion period (60-90 min vs. 15-20 min for the 37 degrees C group). In vitro insulin secretion of islets isolated from freshly removed pancreases was maintained at 20 degrees C for 120 min in University of Wisconsin solution as compared with 30 min at 37 degrees C. Therefore, the preserved pancreases used in this study were incubated either at 37 degrees C or 20 degrees C at various times in order to obtain peak islet yields. The islet yields from 24-hr cold-preserved pancreases at 37 degrees C and 20 degrees C digestion were 573 +/- 59/rat (n = 6) and 497 +/- 84/rat (n = 11), respectively, and those from 48-hr cold-preserved pancreases were 395 +/- 113/rat (n = 6) and 414 +/- 75/rat (n = 6), respectively. The yields from 24- and 48-hr cold-preserved pancreases were significantly low compared with 635 +/- 52/rat for fresh pancreases (n = 15), but there was no significant difference between the 2 methods. The viability of the isolated islets, which was examined by transplantation to streptozotocin-induced diabetic C57BL/6 mice, showed a significant difference in the capacity to ameliorate diabetes. The functional success rate of islet transplantation after 24-hr cold preservation was equally good (8/8 for 37 degrees C group vs. 9/10 for 20 degrees C group), but the rate for those from 48-hr cold-preserved pancreases was significantly better with digestion at 20 degrees C than at 37 degrees C (1/8 for 37 degrees C group vs. 7/8 for 20 degrees C group, P < 0.05). We concluded that viable islets can be isolated from 48-hr cold-preserved pancreases with the low temperature collagenase digestion method, which shows promise as a modality for successful clinical islet transplantation.
Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , Animais , Glicemia/metabolismo , Temperatura Baixa , Colagenases/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Preservação de Tecido/métodos , Transplante HeterólogoRESUMO
The effect of intrathymic (IT) injection of donor splenocytes and a short course of rapamycin (Rapa) treatment on rat to mouse skin xenograft survival was investigated. ACI rat skin xenografts were transplanted to (C57BL/6 x A)F1 mice treated with rabbit anti-mouse lymphocyte serum (ALS) on days -1, +2, and +4 relative to skin grafting on day 0. Fifty million donor-type splenocytes were injected intrathymically on day 7 after transplantation. Rapa was given intraperitoneally every other day from day 0 to day 12 at a dose of 3.0 mg/kg. Prolonged skin xenograft survival was observed in ALS- and Rapa-treated recipients (no IT injection) with a median survival time of 47 days. However, skin graft survival was markedly more prolonged in the group treated with ALS, Rapa, and IT injection of donor splenocytes did not have a beneficial effect on skin xenograft survival in ALS-treated recipients. An increased presence of donor-type cells was observed in the thymus of the ALS- and Rapa-treated recipients for 7 days after IT injection of donor splenocytes. In conclusion, a short course of Rapa markedly augments rat skin xenograft survival in ALS-treated mice injected intrathymically with donor-type splenocytes.
Assuntos
Soro Antilinfocitário/farmacologia , Imunossupressores/farmacologia , Polienos/farmacologia , Transplante de Pele/imunologia , Baço/citologia , Transplante Heterólogo/imunologia , Animais , Feminino , Sobrevivência de Enxerto/efeitos dos fármacos , Cobaias , Imunoterapia Adotiva , Injeções , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos Lew , Sirolimo , Timo , Fatores de Tempo , Quimeras de TransplanteRESUMO
BACKGROUND: Antiadhesion therapy using monoclonal antibodies (mAbs) to adhesion molecules in vivo has been shown to produce significant prolongation of graft survival in various transplantation models. However, it remains unclear whether antiadhesion therapy operates by merely blocking adhesion between antigen-presenting cells and T cells physically and/or by blocking costimulatory signals while preserving signals mediated through T-cell receptors in vivo. We examined antigen-specific T-cell responses during and after antiadhesion therapy. METHODS: BALB/c islets were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 mice given anti-lymphocyte function-associated antigen (LFA)-1 and/or anti-intercellular adhesion molecule-1 mAb treatment. The animals bearing surviving islet allografts were challenged with BALB/c or third-party islets on day 7 or more than 100 days after transplantation. RESULTS: Islet allografts were acutely rejected in untreated animals, with a mean survival time (MST) of 19+/-8 days. Administration of anti-LFA-1 mAb induced significant prolongation of graft survival with a mean survival time of 72+/-33 days, and half of the allografts showed indefinite survival. The animals given anti-LFA-1 mAb alone 7 days before transplantation showed acute rejection of BALB/c islets, whereas a significant number of animals given anti-LFA-1 mAb and the BALB/c islet allograft simultaneously accepted secondary BALB/c islets, but rejected third-party islets. Likewise, most of the animals bearing long-term functioning BALB/c allografts for more than 100 days accepted secondary BALB/c islets, but rejected C3H islets acutely. Interestingly, the spleen cells from these animals transferred unresponsiveness to BALB/c islets into the 2.5-Gy x-irradiated recipients, whereas those from naive animals induced acute rejection. CONCLUSIONS: These results indicate that anti-LFA-1 mAb treatment prevents T-cell activation leading to rejection, but results in a T-cell receptor engagement leading to antigen-specific unresponsiveness maintained by transferrable suppressor cells.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Sobrevivência de Enxerto/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Isoantígenos/análise , Antígeno-1 Associado à Função Linfocitária/imunologia , Animais , Terapia de Imunossupressão/métodos , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Reoperação , Fatores de Tempo , Transplante HomólogoRESUMO
BACKGROUND: Induction of unresponsiveness to graft is one of major interest in xenotransplantation. Two different modalities [direct graft treatment by mitomycin C (MMC) and blockage of the lymphocyte function-associated antigen-1/intracellular adhesion molecule-1 (LFA-1/ICAM-1) pathway in recipients by species-specific mAbs] were tested for their ability to produce unresponsiveness to secondary islet xenografts. METHODS: Collagenase-digested WS (RT1k) rat islets, purified by Ficoll density gradient, were incubated for 30 min with MMC 10 microg/ml, cultured for 20 hr, and transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (H-2b) mice. Recipient mice were divided into experimental groups according to anti-rat ICAM-1 and/or anti-mouse LFA-1 mAb treatment and transplantation of MMC-treated or nontreated islets. RESULTS: MMC pretreatment alone prolonged graft survival, with a mean survival time (MST) of 23.0+/-7.4 days, compared with that of cultured islets (12.4+/-2.7 days; P<0.01). MMC treatment of islets significantly augmented graft survival, compared with that of crude islet grafts under treatment with anti-donor ICAM-1 mAb (MST: >41.3+/-30 vs. 16.6+/-5.4 days, P<0.01), anti-recipient LFA-mAb (MST: >70.3+/-28.9 vs. 30.4+/-10.4 days, P<0.001), or both mAbs (MST: >88.1+/-24.1 vs. 23+/-7.4 days, P<0.0001). One of six, four of nine, and six of eight animals accepted MMC-treated islet xenografts over 100 days after treatment with anti-rat ICAM-1, anti-mouse LFA-1, or both mAbs treatments, respectively, whereas none of the animals accepted nontreated islets under the same treatment. When the mice bearing long-term functioning xenografts were challenged with the secondary graft from the original donor strain, the animals previously treated with anti-recipient LFA-1 and anti-donor ICAM-1 mAbs were more prone to accept it than animals given anti-recipient LFA-1 mAb alone (MST: 55.8+/-25.7 vs. 15+/-2.4 respectively; P<0.001), although they rejected the third-party xenograft and allograft acutely. CONCLUSIONS: In the xenogeneic islet transplantation model, MMC graft pretreatment and blockage of the ICAM-1/LFA-1 pathway constitute a potent protocol for inducing unresponsiveness to islet xenografts.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Molécula 1 de Adesão Intercelular/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Mitomicina/uso terapêutico , Transplante Heterólogo/imunologia , Animais , Diabetes Mellitus Experimental/cirurgia , Quimioterapia Combinada , Sobrevivência de Enxerto/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ratos , Ratos EndogâmicosRESUMO
BACKGROUND: There is a general agreement that a preferential accumulation of alloantigens within the liver could induce hyporesponsiveness to the inoculated antigens. Entrapment of antigens in the liver may evoke an unique immune response in the organ and play a key role in determination of the fate of the transplanted grafts. To understand the immune response in the liver after inoculation of allogeneic donor antigens, we examined the immune response to systemically inoculated alloantigen in rats whose sensitized liver was replaced with that of naive rats or in naive rats whose liver was replaced with that of sensitized rats. METHODS: Using implantation of syngeneic liver (alloantigen-accumulated/naive) in rats (naive/alloantigen-sensitized), we compared the immune responses to alloantigen between rats with hepatic/extrahepatic alloantigen at 24 hr after alloantigen inoculation. This was called sensitized-liver-grafted (SLG)/sensitized-liver-removed (SLR) treatment. The immune response to donor alloantigen in this model was evaluated by survival of skin or heart grafts, complement-dependent cytotoxicity (CDC) titer and delayed-type hypersensitivity (DTH) response. RESULTS: Compared with the mean survival time (MST) in donor spleen cell inoculated (DSI) rats (skin and heart, MST: 8.2+/-1.1 and 10.7+/-2.3 days), SLG rats rejected allografts in an accelerated fashion (skin and heart, MST: 5.5+/-0.5 and 4.2+/-0.8 days), associated with higher CDC titer and DTH response. In contrast, allograft survival was moderately prolonged in SLR (skin and heart, MST: 16.5+/-2.6 and 29.5+/-3.7 days) associated with suppressed CDC titer and DTH response. The survival of third-party allograft after SLG or SLR treatment (skin, MST: 9.3+/-1.5 or 9.7+/-0.6 days) indicated that immunological hyper/hyporesponsiveness was donor-specific. CONCLUSIONS: A strong anti-donor immune response was induced by the transfer of donor antigen-baring liver to naive rats 24 hr after alloantigen inoculation, whereas removal of the liver suppressed alloimmune response. Our results indicate that vigorous anti-alloimmune response occurred in the liver after systemic inoculation of donor spleen cells.
Assuntos
Rejeição de Enxerto/imunologia , Isoantígenos/imunologia , Transplante de Fígado/imunologia , Fígado/imunologia , Baço/citologia , Baço/transplante , Transferência Adotiva , Animais , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Hipersensibilidade Tardia/imunologia , Transfusão de Linfócitos , Masculino , Ratos , Ratos Wistar , Transplante de Pele/imunologia , Baço/imunologiaRESUMO
BACKGROUND: Prostaglandin E1 (PGE1) is known to inhibit ischemia-reperfusion injury of the liver. The calcium-dependent neutral proteinase, calpain-mu, is involved in oxidative stress-induced hepatocyte injury. We investigated the mechanisms of cytoprotection by PGE1, focusing on the elevation of intracellular calcium ([Ca2+]i), activation of calpain-mu, and calpain-mu-mediated activation of protein kinase C-alpha (PKC-alpha). METHODS: Cultured hepatocytes were treated with various amounts of PGE1 (0, 0.1, 1.0, 10, and 100 ng/ml) for 30 min and subsequently with 0.5 mM tert-butyl hydroperoxide (TBHP). Cell injury was evaluated by the release of lactate dehydrogenase. Plasma membrane bleb formation was examined by phase contrast microscopy. Activation of calpain-mu and limited degradation of PKC-alpha was evaluated by Western blotting using antibodies that specifically recognize the amino-terminal regions of calpain-mu and PKC-alpha. [Ca2+]i was measured by confocal microscopy using Fluo-3AM. RESULTS: LDH release from cells treated with 10 ng/ml PGE1 was significantly lower than from untreated cells (135 +/- 12 vs. 258 +/- 18 IU/L, respectively; P < 0.05). Morphologically, many blebs were observed in untreated cells, but very few were seen in those treated with 10 ng/ml PGE1. Western blotting revealed that the amount of activated calpain-mu and [Ca2+]i increased up to 1,300 nM at 35 min after the addition of TBHP (0.5 mmol/L) in control experiments (without PGE1). PGE1 (10 ng/ml) delayed the rise in [Ca2+]i for about 30 min, but did not suppress it completely. PKC-alpha decreased in experiments using PGE1 (10 ng/ml). CONCLUSION: PGE1 exerts its cytoprotective effect in TBHP-induced hepatocyte injury partly by inhibiting Ca2+-calpain-mu-mediated mechanisms.
Assuntos
Alprostadil/farmacologia , Calpaína/metabolismo , Fígado/metabolismo , Fígado/patologia , Estresse Oxidativo/fisiologia , Animais , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Membranas Intracelulares/metabolismo , Isoenzimas/metabolismo , Fígado/efeitos dos fármacos , Masculino , Concentração Osmolar , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Ratos , Ratos WistarRESUMO
Cellular interactions that lead to graft rejection were examined in a rat-to-mouse xenogeneic combination using species-specific monoclonal antibodies (mAbs) against donor and recipient intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) molecules, respectively. Although both mAbs displayed moderate blocking activity in an in vitro mixed lymphocyte response assay, strong suppression was observed when anti-donor (rat) ICAM-1 mAb was combined with anti-recipient (mouse) LFA-1 mAb. Likewise, significant prolongation of islet xenograft survival was observed with these mAbs. Thus, 0.05 mg of anti-mouse LFA-1 mAb and anti-rat ICAM-1 mAb given on days 0 and 1 produced significant prolongation of graft survival over the control (51+/-20 days vs. 10+/-3 days, P<0.0001), but not when anti-mouse ICAM-1 mAb was combined with anti-mouse LFA-1 mAb (13+/-3 days). In this species combination, mouse T cells were able to proliferate in the presence of rat antigen-presenting cells (APCs) in a cell number-dependent manner, but not in the presence of mouse APCs. The binding assay showed that LFA-1 molecules on mouse T cells can bind immobilized rat ICAM-1 molecules. These results suggest that rat ICAM-1 molecules on APCs can interact with mouse LFA-1 molecules on T cells across a species barrier and that this binding generates the consequent immune responses leading to rejection. mAb treatment against these adhesion molecules of recipient as well as donor is crucial for preventing rejection in a xenogeneic transplantation model.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Rejeição de Enxerto/imunologia , Molécula 1 de Adesão Intercelular/fisiologia , Transplante das Ilhotas Pancreáticas/imunologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Linfócitos T/imunologia , Transplante Heterólogo/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Sobrevivência de Enxerto/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/isolamento & purificação , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Antígeno-1 Associado à Função Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Fatores de TempoRESUMO
BACKGROUND: Intrathymic microchimerism (MC) is thought to be responsible for inducing allograft tolerance. However, the role of MC in the thymus gland after transplantation, particularly in the rejection response, is unknown. We investigated serial changes in intrathymic cytokine production associated with MC and allograft rejection. METHODS: Donor-specific cell injection (DSI) and heterotopic heart transplantation (HTx) were performed in the fully allogeneic combination using DA rats (RT1a) as donors and WS rats (RT1k)as recipients. MC was checked by polymerase chain reaction (PCR) using a donor RT1.Bbeta domain 1 region sequence-specific primers. Reverse transcription (RT)-PCR analysis of cytokine (interleukin [IL]-2, interferon-gamma, IL-4, and IL-10) profiles of the thymus was performed in animals given DSI, HTx, or DSI/HTx. RESULTS: DSI alone resulted in an immediate development of MC, detected by PCR, in various organs including the thymus, spleen, liver, and blood, of most rats, lasting for over 2 months. However, DSI-induced MC selectively disappeared in the thymus on day 7 after grafting, several days before the rejection of cardiac allograft. RT-PCR analysis of cytokine profiles showed that the levels of Th1 (IL-2 and interferon-gamma) cytokines transcribed in the thymus were higher than in the spleen. MC reappeared in the thymus on day 21 after grafting, but was not associated with elevation of Th1 cytokine transcription when allograft was replaced by fibrosis. CONCLUSIONS: Intrathymic MC does not always confer unresponsiveness to alloantigen, but can be eliminated after anti-donor response.
Assuntos
Quimera/fisiologia , Citocinas/genética , Rejeição de Enxerto/genética , Transplante de Coração , Linfócitos T Auxiliares-Indutores/fisiologia , Timo/fisiologia , Transcrição Gênica/fisiologia , Animais , Transplante de Células , Transfusão de Eritrócitos , Masculino , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Ratos Wistar , Baço/metabolismo , Baço/patologia , Células Th1/metabolismo , Células Th2/metabolismo , Timo/citologia , Timo/metabolismo , Timo/patologiaRESUMO
BACKGROUND: Rejection of pancreatic islet grafts is still a serious problem. We evaluated the effect of mitomycin C (MMC) on the survival of crude islets grafts after xenogeneic islet transplantation. METHODS: WS (RT1k) rat islets pretreated with various concentrations of MMC (0, 1, 3.2, 10, 32, 50, 100, 320, and 1,000 microg/ml) were transplanted into C57BL/6 mice with streptozotocin-induced diabetes. In vivo graft function was assessed by a daily measurement of nonfasting blood glucose concentration in each animal. We also examined the separate effect of MMC on purified islets and contaminants present in the crude islet preparation. RESULTS: MMC at doses of 10, 32, 50, and 100 microg/ml resulted in a significant prolongation of the mean graft survival time from a control of 12.4+/-2.5 days to 23+/-7.4, 17.5+/-5.4, 25.5+/-14.7, and 26.7+/-8.9 days, respectively. Deterioration of glucose metabolism was noted when the dose exceeded 32 microg/ml, whereas at 320 microg/ ml, MMC failed to restore normoglycemia. Prolongation of survival time of crude islets was the result of its effect on islets and contaminant components of the crude islet preparation. In vitro study showed that MMC treatment at a higher concentration than 10 microg/ml reduces the stimulatory as well as proliferative capacity of lymph node cells. CONCLUSIONS: Pretreatment of pancreatic islets with MMC at 10 microg/ml prolongs xenograft survival without deterioration of in vivo graft function. This novel treatment modality represents a new strategy for the modulation of immunity of islets and contaminants in crude islet preparations.
Assuntos
Ilhotas Pancreáticas/efeitos dos fármacos , Mitomicina/farmacologia , Transplante Heterólogo/imunologia , Animais , Divisão Celular , Glucose/metabolismo , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante das Ilhotas Pancreáticas/imunologia , Linfonodos/citologia , Masculino , Camundongos , Pré-Medicação , RatosRESUMO
To reveal the implication in gastric cancer pathogenesis of the novel human gene referred to as CA11, which was recently isolated by a differential display technique using normal gastric mucosa and gastric cancer tissue, we examined CA11 expression in 50 primary gastric cancers and also introduced the CA11 gene into gastric cancer cells. RNA dot blot analysis against various human organs and developmental stages demonstrated that CA11 was intensively expressed especially in normal stomach tissue. Northern blot analysis showed that expression of the CA11 gene in cancer tissue was down-regulated compared with normal tissue. Semi-quantitative RT-PCR also demonstrated that CA11 gene expression was decreased in 41 out of 50 (82%) of the gastric cancer tissues, when compared with normal stomach tissues, while no relationship was found between CA11 expression and various clinicopathological characteristics including histological type, depth of invasion, lymph node metastasis, and clinical stage. Immunohistochemical analysis with anti CA11 antibody showed that CA11-positive staining was observed in the surface regions of normal gastric epithelium, but was found faintly or not at all in cancer tissues. CA11 transfected MKN28 cells also displayed a marked decrease in the number of colony formations when compared to double normal controls. These findings suggest that the loss of CA11 expression in gastric tissues may play an important role in gastric carcinogenesis.
Assuntos
Adenocarcinoma/metabolismo , Carcinoma de Células em Anel de Sinete/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Northern Blotting , Carcinoma de Células em Anel de Sinete/patologia , Regulação para Baixo , Etiquetas de Sequências Expressas , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Técnicas Imunoenzimáticas , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Invasividade Neoplásica , Proteínas de Neoplasias/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/patologiaRESUMO
In order to detect a hematogenous spread of tumor cells in patients with hepatocellular carcinoma, reverse transcription-polymerase chain reaction assay has been used. In this study, we quantified alpha-fetoprotein (AFP) messenger RNA by real-time PCR approach using LightCyclertrade mark technique. AFP messenger RNA in the blood from 23 hepatocellular carcinoma patients undergoing hepatic resection, 31 healthy volunteers, 10 patients with liver cirrhosis and 5 patients underwent hepatectomy except for hepatocellular carcinoma was quantitated. In the real-time PCR, fluorescence was undetectable in any of the controls. On the contrary, fluorescent signals were detected in 10 out of 39 blood specimens collected from 23 HCC patients. AFP-positive status was significantly associated with the existence of multiple intrahepatic nodules. Out of 8 cases with AFP-positive status, intra- and/or extra-hepatic recurrence has been observed in 3 cases. The quantities of AFP messenger RNA in these 3 cases were relatively high among 8 cases with AFP-positive status. AFP messenger RNA was detectable by newly developed real-time PCR approach with LightCycler and it is suggested that this approach could be applicable in detection of small amounts of tumor cells in the blood of HCC patients.