RESUMEN
The clinical success of new treatment strategies aiming on inducing permanent graft acceptance will rely on the ability to determine whether specific unresponsiveness to donor alloantigens has developed and for how long it is maintained. To identify markers for such posttransplant monitoring, genes differentially expressed by graft infiltrating leukocytes during tolerance induction or rejection after kidney transplantation in rats were compared. A subsequently performed full kinetic analysis in two different transplant models, kidney and heart, in two species, rat and mouse identified two markers (TOAG-1, alpha-1,2-mannosidase) with high specificity and reproducibility, which are highly expressed during induction and maintenance of acceptance, and downregulated during rejection. Expression level of these markers showed a strong positive correlation with graft function. In addition, expression of both genes was downregulated in the peripheral blood and the graft prior to rejection, suggesting that these markers may be useful for monitoring in clinical transplantation where peripheral blood is the most easily accessible patient sample. Interestingly, downregulation of TOAG-1 and alpha-1,2-mannosidase expression occurred in graft infiltrating cells and expression of both genes was also downregulated after T-cell activation in vitro.
Asunto(s)
Marcadores Genéticos/fisiología , Rechazo de Injerto/genética , Supervivencia de Injerto/genética , Trasplante de Corazón/fisiología , Trasplante de Riñón/fisiología , Manosidasas/genética , Animales , Complejo CD3/genética , Complejo CD3/fisiología , Regulación de la Expresión Génica/fisiología , Marcadores Genéticos/genética , Rechazo de Injerto/inmunología , Rechazo de Injerto/fisiopatología , Supervivencia de Injerto/inmunología , Supervivencia de Injerto/fisiología , Leucocitos/metabolismo , Masculino , Manosidasas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Perforina , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/fisiología , Valor Predictivo de las Pruebas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas , Trasplante HomólogoRESUMEN
Angiotensin-II (Ang-II) type 1 (AT(1)) receptor blockers may delay the progression of chronic allograft nephropathy (CAN). However, neither the optimal time for initiating AT(1) receptor blockade in order to delay CAN potentially nor the role of Ang-II type 2 (AT(2)) receptors under AT(1) receptor blockade is known. Both AT receptors can regulate p53 expression and apoptosis. We investigated what time of initiation with AT(1) blockers most effectively delayed CAN as well as the role of the AT(2) receptor, and how angiotensin receptor blockade affected apoptosis and its regulating factors in this context in a rat model. Kidneys of Fisher (F344) rats were transplanted into Lewis rats. Animals were treated with AT(1) (candesartan) and/or AT(2) (PD123319) receptor antagonists, a calcium channel blocker, or vehicle (treatment periods: day -7 before to week 24 after transplantation (long term), week 12 to week 24 (late), day -7 to day +5 (early)) and observed the animals for 24 weeks. Reduction of proteinuria, grade of CAN, and number of apoptotic cells was most pronounced in animals receiving long-term AT(1) receptor blockade. A combined AT(1)/AT(2) blocker treatment reduced CAN similarly to AT(1) blocker treatment alone. The number of apoptotic cells and the level of p53 mRNA were significantly lower in long-term AT(1) blocker-treated animals. In summary, AT(1) receptor blockade delayed the progression of CAN, particularly in animals treated long term. Reduction of apoptosis could be related to these beneficial effects. The AT(2) receptor does not appear to play an important role in CAN.
Asunto(s)
Antagonistas de Receptores de Angiotensina , Bencimidazoles/farmacología , Imidazoles/farmacología , Trasplante de Riñón/efectos adversos , Síndrome Nefrótico/prevención & control , Piridinas/farmacología , Tetrazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo , Enfermedad Crónica , Interacciones Farmacológicas , Estudios de Seguimiento , Masculino , Síndrome Nefrótico/etiología , Proteinuria/tratamiento farmacológico , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Factores de Tiempo , Trasplante Homólogo , Proteína p53 Supresora de Tumor/metabolismoAsunto(s)
Fumaratos/uso terapéutico , Rechazo de Injerto/prevención & control , Trasplante de Riñón/fisiología , Enfermedad Aguda , Animales , Enfermedad Crónica , Creatinina/sangre , Evaluación Preclínica de Medicamentos , Rechazo de Injerto/sangre , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas WF , Factores de Tiempo , Trasplante HomólogoAsunto(s)
Trasplante de Riñón/inmunología , Linfocitos T/inmunología , Traslado Adoptivo/métodos , Animales , Movimiento Celular , Proteínas Fluorescentes Verdes , Indicadores y Reactivos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Activación de Linfocitos , Ratas , Ratas Endogámicas LewRESUMEN
BACKGROUND: E1-deleted adenoviral vectors are frequently used for in vivo gene therapy. However, gene expression after adenovirus-(ad) mediated gene transfer is known to be transient due to the generation of an immune response against virus-infected cells. In this study, we asked whether an anti-CD4 mAb (RIB 5/2) treatment may improve the gene transfer into rat cardiac grafts. METHODS: We injected recombinant ad-constructs encoding for Escherichia coli beta-gal into syngeneic rat heart transplants via the proximal aorta. One-half of the recipients of genetically modified grafts received the anti-CD4 mAb RIB 5/2, whereas the other half received no monoclonal antibody treatment. Genetically unmodified isografts without any treatment of the recipients were used as additional controls. At different time points hearts were harvested and analyzed for reporter gene expression, intragraft cellular infiltration, and cytokine gene expression (quantitative "real time" reverse transcriptase polymerase chain reaction). Serum samples were analyzed for anti-ad-Ig using enzyme-linked-immunosorbent-assay. RESULTS: In control animals the beta-gal reporter gene expression slowly increased until day 7 and then declined. The immunohistological and reverse transcriptase polymerase chain reaction intragraft analyses revealed a strong inflammatory response (cellular infiltration, cytokine expression) in ad-transfected grafts that may explain the delayed expression and fast down-regulation of the transgene. Treatment with RIB 5/2 mAb resulted in a faster and prolonged reporter gene expression, reduced graft infiltration, reduced anti-ad-Ig titers and less interferon-gamma up-regulation. CONCLUSIONS: Our results indicate that modulation of the anti-ad immune response using a nondepleting anti-CD4 mAb may increase the efficiency of ad-vectors for gene therapy in the transplant setting.
Asunto(s)
Adenoviridae/genética , Adenoviridae/inmunología , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/inmunología , Técnicas de Transferencia de Gen , Trasplante de Corazón , Animales , Anticuerpos Antivirales/sangre , Interferón gamma/genética , Masculino , Ratones , ARN Mensajero/análisis , Ratas , Ratas Endogámicas Lew , TransgenesRESUMEN
E1-deleted adenoviral vectors are efficient vectors for somatic gene therapy. Recently, we have shown that intratracheal application of an adenoviral reporter construct leads to significant reporter gene expression in rat lungs within 24 h. In contrast, reporter gene expression in syngeneic rat heart transplants after adenovirus-mediated gene transfer was delayed. Since the adenovirus cannot replicate, down-regulation of the hCMV-IE promoter controlled reporter gene expression in initially infected cells by cytokines, which are released as a result of ischemia/reperfusion injury, might be involved. In order to investigate the role of proinflammatory cytokines, eg TNF-alpha in affecting hCMV-IE promoter-driven reporter gene expression, transient blockade of TNF-alpha was achieved by local co-application of an Ad-construct encoding for a soluble TNFRp55-Ig chimeric molecule in a syngeneic rat heart transplantation model. Co-application of the reporter construct together with the TNFRp55-Ig chimeric molecule significantly increased the early reporter gene expression after transplantation. Moreover, infiltration of inflammatory cells (T cells, macrophages, NK cells) and production of TNF-alpha in the transplant was markedly reduced. Our results indicate that: (1) proinflammatory cytokines are involved in down-regulation of reporter gene expression in ischemia/reperfusion injured tissues; and (2) inhibition of TNF-alpha might be a useful tool to increase early gene expression in gene therapy protocols, particularly in transplantation. Gene Therapy (2000) 7, 1238-1243.
Asunto(s)
Adenoviridae/genética , Antígenos CD/uso terapéutico , Trasplante de Corazón , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Daño por Reperfusión/terapia , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Antígenos CD/genética , Regulación hacia Abajo , Genes Reporteros , Supervivencia de Injerto , Regiones Promotoras Genéticas/genética , Ratas , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Daño por Reperfusión/metabolismoRESUMEN
BACKGROUND: Recently we have demonstrated that the nondepleting anti-CD4 monoclonal antibody (mAb) RIB5/2 induces long-term acceptance of kidney and heart allografts in all rat strain combinations tested. Cytokine gene expression studies by reverse transcriptase-polymerase chain reaction revealed a reversed intragraft interleukin (IL)-4/interferon-gamma ratio. Whether IL-4 mediated immune deviation contributes to transplantation tolerance is not clear so far. METHODS: To learn more about the functional relevance of the relative IL-4 up-regulation, IL-4 was overexpressed in rat heart allografts by using ex vivo adenoviral gene transfer. The efficiency of gene transfer was analyzed by reporter gene assays as well by reverse transcriptase-polymerase chain reaction analysis of IL-4 mRNA expression. RESULTS: The intragraft overexpression of IL-4 did not prolong the allograft survival compared with controls. Moreover, neutralization of IL-4 by OX81 mAb did not prevent tolerance induction by RIB5/2 treatment. CONCLUSIONS: Anti-CD4 mAb-induced tolerance is associated with an intragraft type1/type2 shift, however, the up-regulation of IL-4 alone is neither sufficient nor essential to induce tolerance to cardiac allografts in a high-responder strain combination.
Asunto(s)
Trasplante de Corazón/inmunología , Tolerancia Inmunológica , Interleucina-4/fisiología , Animales , Anticuerpos Monoclonales/uso terapéutico , Técnicas de Transferencia de Gen , Supervivencia de Injerto , Interleucina-4/genética , Interleucina-4/inmunología , Ratas , Ratas Endogámicas Lew , Trasplante HomólogoAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Supervivencia de Injerto/fisiología , Terapia de Inmunosupresión/métodos , Interleucina-4/fisiología , Trasplante de Riñón/inmunología , Animales , Antígenos CD4/inmunología , Creatinina/sangre , Interleucina-4/inmunología , Trasplante de Riñón/fisiología , Masculino , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas , Trasplante HomólogoAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/inmunología , Supervivencia de Injerto/inmunología , Terapia de Inmunosupresión/métodos , Interleucina-2/farmacología , Trasplante de Riñón/inmunología , Animales , Humanos , Ratas , Ratas Endogámicas ACI , Ratas Endogámicas Lew , Ratas Endogámicas , Proteínas Recombinantes/farmacología , Trasplante Homólogo , Trasplante IsogénicoRESUMEN
Although CD4-targeted therapy abrogates acute rejection and may induce permanent graft acceptance in rodents, little is known about the mechanisms of long-term graft survival in these models. Recently, we have shown that treatment with a nondepleting anti-CD4 monoclonal antibody (mAb) (RIB-5/2) induces long-term survival of renal, heart, and skin allografts in strong major histocompatibility complex I/II incompatible rat strains. Here, we demonstrate that the development of major histocompatibility complex-specific and tissue-nonspecific tolerance rather than graft adaptation is responsible for long-term anti-CD4 mAb-induced transplant survival. Donor-specific but not third-party heart and pancreatic islet grafts were accepted permanently without adjunctive therapy in long-term kidney allograft recipients, and infusion of naive or alloimmune splenocytes failed to break the tolerant state. Interestingly, alloreactive T cells were not depleted in these long-term survivors, as ex vivo donor-specific mixed lymphocyte reaction was largely unaffected. The reverse transcriptase-polymerase chain reaction analyses of long-term renal allografts before and after donor-specific antigen challenge revealed no changes in CD3 mRNA level, but showed up-regulation of CD25, interleukin (IL) 2, interferon (IFN) gamma, IL-4, and IL-10 mRNA in the early phase, suggesting the presence of alloreactive T cells in tolerant rats. At later time points, the expression of IFN-gamma declined rapidly, whereas IL-4 persisted, resulting in a reversal of IFN-gamma/IL-4 ratio. Our data demonstrate the stability of anti-CD4 mAb-induced tolerance despite persistence of alloreactive T cells, suggesting the role of active tolerance-maintaining mechanisms. The T helper (Th) 1/Th2 shift may be involved in this regulatory process, as anti-CD4 mAb prevents acute graft-deteriorating rejection by effectively blocking Th1 responses, and well-functioning grafts may tolerize themselves by inducing regulatory cells.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Linfocitos T CD4-Positivos/inmunología , Trasplante de Riñón/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Citocinas/genética , Expresión Génica , Supervivencia de Injerto/efectos de los fármacos , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/inmunología , Terapia de Inmunosupresión , Depleción Linfocítica , Masculino , Reacción en Cadena de la Polimerasa/métodos , ADN Polimerasa Dirigida por ARN , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WF , Células TH1/fisiología , Células Th2/fisiología , Trasplante Homólogo/inmunologíaAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/inmunología , Rechazo de Injerto/prevención & control , Trasplante de Riñón/inmunología , Enfermedad Aguda , Animales , Enfermedad Crónica , Glomeruloesclerosis Focal y Segmentaria/patología , Glomeruloesclerosis Focal y Segmentaria/prevención & control , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Terapia de Inmunosupresión , Trasplante de Riñón/patología , Ratas , Ratas Endogámicas WF , Ratas Sprague-Dawley , Receptores de Interleucina-2/inmunología , Trasplante HomólogoAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos de Diferenciación/uso terapéutico , Antígenos CD4/inmunología , Supervivencia de Injerto , Inmunoconjugados , Inmunosupresores/uso terapéutico , Trasplante de Riñón/inmunología , Abatacept , Animales , Antígenos CD , Antígenos de Diferenciación/análisis , Antígeno CTLA-4 , Creatinina/sangre , Citocinas/análisis , Sinergismo Farmacológico , Trasplante de Riñón/patología , Trasplante de Riñón/fisiología , Ratas , Ratas Endogámicas , Ratas Endogámicas WF , Trasplante HomólogoRESUMEN
In rats, transient prophylactic anti-CD4 therapy with the nondepleting mAB RIB5/2 prevents acute rejection of MHC-mismatched allografted kidneys and induces long-lasting unresponsiveness. However, little is known about long-term benefits of this prophylactic anti-CD4 regimen. Here we report experimental results of permanently accepted rat renal allografts after prophylactic anti-CD4 treatment in regard to signs of chronic rejection. Kidneys from Wistar Furth donors were orthotopically grafted into bilateral nephrectomized BDIX recipients under the cover of anti-CD4 treatment (20 mg/kg b.w). Kidney function was serially monitored by measurement of serum creatinine and urine protein excretion. After 100 or 300 days respectively renal allografts were harvested, histologically and immunohistologically assessed and intragraft cytokine gene expression determined. Serum creatinine increased in few allografted rats. 30% of the 300-day-old grafts had an increased proteinuria and higher degrees of glomerular sclerosis. In these grafts cellular infiltration was more pronounced. However, no activated leukocytes (IL-2 receptor positive) were detected. Correspondingly, intragraft gene expression of CD3, IL-10 and IFN gamma was low. The results of our study indicate that a prophylactic anti-CD4 regimen diminishes chronic rejection to a level comparable to isografted or naive mass-reduced or ischemic kidneys. Thus, the signs of chronic rejection observed seem to be mainly caused by alloantigen-independent processes.