RESUMEN
A 21-yr-old man of blood type O receiving hemodialysis for IgA nephropathy underwent living-related ABO-incompatible (ABOI) renal transplantation from his mother, whose blood type is A. He was negative for flow cross-match, anti-human leukocyte antigen (HLA) antibody, and anti-MICA antibody. Pre-treatment anti-A IgG titer was 1:256. Desensitization consisted of tacrolimus, mycophenolate mofetil, methylprednisolone, rituximab, and plasmapheresis. He developed acute antibody rejection at day 2 post-transplant, which was successfully treated. After renal artery reconstruction surgery at day 91 for renovascular hypertension caused by renal artery stricture, the patient suffered from acute prostatitis, which subsequently induced type III acute antibody-mediated rejection. Even after recovery from the rejection after temporary hemodialysis, graft function progressively deteriorated and consecutive allograft biopsy showed progressive thrombotic microangiopathy (TMA) without any evidence of donor-specific antibody other than anti-A antibody. The tacrolimus dose was kept low for fear of tacrolimus-induced TMA. Despite these efforts, the patient resumed hemodialysis six months' post-transplant.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/inmunología , Incompatibilidad de Grupos Sanguíneos/inmunología , Isoanticuerpos/inmunología , Fallo Renal Crónico/cirugía , Trasplante de Riñón/efectos adversos , Microangiopatías Trombóticas/etiología , Adulto , Progresión de la Enfermedad , Rechazo de Injerto , Humanos , Inmunosupresores/administración & dosificación , Fallo Renal Crónico/inmunología , Donadores Vivos , Masculino , Pronóstico , Diálisis Renal , Microangiopatías Trombóticas/fisiopatología , Adulto JovenRESUMEN
Matrix metalloproteinase 9 (MMP-9) is a critical mediator of leukocyte migration in hepatic ischemia/reperfusion (I/R) injury. To test the relevance of inducible nitric oxide synthase (iNOS) expression on the regulation of MMP-9 activity in liver I/R injury, our experiments included both iNOS-deficient mice and mice treated with ONO-1714, a specific iNOS inhibitor. The inability of iNOS-deficient mice to generate iNOS-derived nitric oxide (NO) profoundly inhibited MMP-9 activity and depressed leukocyte migration in livers after I/R injury. While macrophages expressed both iNOS and MMP-9 in damaged wild-type livers, neutrophils expressed MMP-9 and were virtually negative for iNOS; however, exposure of isolated murine neutrophils and macrophages to exogenous NO increased MMP-9 activity in both cell types, suggesting that NO may activate MMP-9 in leukocytes by either autocrine or paracrine mechanisms. Furthermore, macrophage NO production through the induction of iNOS was capable of promoting neutrophil transmigration across fibronectin in a MMP-9-dependent manner. iNOS expression in liver I/R injury was also linked to liver apoptosis, which was reduced in the absence of MMP-9. These results suggest that MMP-9 activity induced by iNOS-derived NO may also lead to detachment of hepatocytes from the extracellular matrix and cell death, in addition to regulating leukocyte migration across extracellular matrix barriers. These data provide evidence for a novel mechanism by which MMP-9 can mediate iNOS-induced liver I/R injury.
Asunto(s)
Quimiotaxis de Leucocito/fisiología , Hígado/inmunología , Metaloproteinasa 9 de la Matriz/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Daño por Reperfusión/inmunología , Animales , Apoptosis/fisiología , Western Blotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Hígado/lesiones , Hígado/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Óxido Nítrico/inmunología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/deficiencia , Daño por Reperfusión/metabolismoRESUMEN
Activation of the complement cascade represents an important event during ischemia/reperfusion injury (IRI). This work was designed to investigate the role of the membrane attack complex (MAC; C5b-9) in the pathogenesis of hepatic IRI. Livers from B&W/Stahl/rC6(+) and C6(-) rats were harvested, stored for 24 hours at 4 degrees C, and then transplanted [orthotopic liver transplantation (OLT)] to syngeneic recipients. There were 4 experimental groups: (1) C6(+)-->C6(+), (2) C6(+)-->C6(-), (3) C6(-)-->C6(+), and (4) C6(-)-->C6(-). At day +1, C6(-) OLTs showed decreased vascular congestion/necrosis, contrasting with extensive necrosis in C6(+) livers, that was independent of the recipient C6 status (Suzuki score: 7.2 +/- 0.9, 7.3 +/- 1.3, 4.5 +/- 0.6, and 4.8 +/- 0.4 for groups 1-4, respectively, P < 0.05). The liver function improved in recipients of C6(-) grafts (serum glutamic oxaloacetic transaminase: 2573 +/- 488, 1808 +/- 302, 1170 +/- 111, and 1188 +/- 184 in groups 1-4, respectively, P < 0.05). Intragraft macrophage infiltration (ED-1 immunostaining) and neutrophil infiltration (myeloperoxidase activity) were reduced in C6(-) grafts versus C6(+) grafts (P = 0.001); these data were confirmed by esterase staining (naphthol). The expression of proinflammatory interferon-gamma, interleukin-1beta, and tumor necrosis factor messenger RNA/protein was also reduced in C6(-) OLTs in comparison with C6(+) OLTs. Western blot-assisted expression of proapoptotic caspase-3 was decreased in C6(-) OLTs versus C6(+) OLTs (P = 0.006), whereas antiapoptotic Bcl-2/Bag-1 was enhanced in C6(-) OLTs compared with C6(+) OLTs (P = 0.001). Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining of apoptotic cells was enhanced (P < 0.05) in C6(+) OLTs compared with C6(-) OLTs. Thus, the terminal products of the complement system are essential in the mechanism of hepatic IRI. This is the first report using a clinically relevant liver cold ischemia model to show that local MAC inhibition attenuates IRI cascade in OLT recipients.
Asunto(s)
Complemento C6/deficiencia , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Hepatopatías/metabolismo , Trasplante de Hígado , Daño por Reperfusión/metabolismo , Animales , Apoptosis , Isquemia Fría , Complemento C6/genética , Citocinas/metabolismo , Femenino , Hígado/patología , Hepatopatías/inmunología , Hepatopatías/patología , Trasplante de Hígado/inmunología , Trasplante de Hígado/patología , Macrófagos/fisiología , Infiltración Neutrófila , Ratas , Daño por Reperfusión/inmunología , Daño por Reperfusión/patologíaRESUMEN
A 61-year-old male presented with advanced gastric cancer with lymph node swelling around the root of superior mesenteric artery lymph nodes and invasion of pancreas. We thought a complete resection would be difficult, so he was given neo-adjuvant chemotherapy in combination with S-11 20 mg/body/day (3 weeks administration and 1 week rest) and paclitaxel (PTX) 80 mg/m(2) (day 1, 8, 15). After 2 courses of this neo-adjuvant chemotherapy, the tumor and lymph node swelling decreased in size. Total gastrectomy, Roux-en Y and D1+beta type nodal dissection were performed. Intraoperative findings included tumor exposure on the serous membrane and enlarged lymph nodes on the lesser curvature; however, no marked pancreatic invasion was observed and the lymph nodes had become scarred. The changes with neo-adjuvant chemotherapy were judged to be grade 2. After the operation, there was no side effect, though he received the same chemotherapy as an outpatient in three courses. Five years passed from the first medical examination. The patient remains alive, and the neo-adjuvant chemotherapy proved effective.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Terapia Neoadyuvante , Ácido Oxónico/uso terapéutico , Paclitaxel/uso terapéutico , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patología , Tegafur/uso terapéutico , Combinación de Medicamentos , Gastroscopía , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Gástricas/diagnóstico por imagen , Neoplasias Gástricas/cirugía , Factores de Tiempo , Tomografía Computarizada por Rayos XRESUMEN
Ischemia-reperfusion injury (IRI) contributes to early and late dysfunction of liver transplants. We have shown that sentinel Toll-like receptor-4 (TLR4) plays a key role in the activation of T cell immune responses during hepatic IRI. We have also documented that overexpression of heme oxygenase-1 (HO-1) exerts potent cytoprotective effects. This study analyzes how adenovirus (Ad)-based viral interleukin-10 (vIL-10) gene transfer affects TLR4 and HO-1 signaling in host innate and adaptive immunity during liver IRI. Using a partial lobar warm IRI model, groups of wild-type and HO-1(+/-) knockout (KO) mice were assessed for severity of hepatocellular damage after 90 min of warm ischemia followed by 6 hr of reperfusion. Both wild-type and HO-1 (+/-) KO mice treated with Ad-vIL-10 have shown improved hepatic function (serum glutamic-oxaloacetic transaminase levels), ameliorated histological signs of IRI (Suzuki's score), decreased neutrophil accumulation (myeloperoxidase activity), and depressed tumor necrosis factor-alpha/IL-1beta, IL-2/interferon-gamma, E-selectin, and macrophage inflammatory protein-2 expression. These effects were IL-10 dependent as treatment with neutralizing antibody re-created liver IRI. In contrast, untreated wild-type and HO-1 (+/-) KO mice, as well as wild-type and HO-1 (+/-) KO mice treated with Ad-beta-Gal, showed severe hepatocellular damage due to IRI. Unlike in controls, wild-type and HO-1 (+/-) KO mice treated with Ad-vIL-10 revealed markedly depressed TLR4 and NF-kappaB expression, along with increased HO-1 and Bcl-2/Bcl-x(L) expression, as compared with respective controls. Thus, vIL-10 gene transfer prevents hepatic IRI in association with depressed expression of innate TLR4, and adaptive Th1 cytokine/chemokine programs. The induction of antioxidant HO-1 and anti-apoptotic Bcl-2/Bcl-x(L) by vIL-10 exerts synergistic cytoprotective function against antigen-independent hepatic inflammatory response triggered by IRI.
Asunto(s)
Terapia Genética , Hemo-Oxigenasa 1/fisiología , Interleucina-10/genética , Hígado/irrigación sanguínea , Daño por Reperfusión/prevención & control , Receptor Toll-Like 4/metabolismo , Animales , Citocinas/metabolismo , Citoprotección/genética , Citoprotección/inmunología , Regulación hacia Abajo , Selectina E/metabolismo , Técnicas de Transferencia de Gen , Hemo-Oxigenasa 1/genética , Inmunidad Innata/genética , Interleucina-10/análisis , Hígado/inmunología , Hígado/patología , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Daño por Reperfusión/inmunología , Daño por Reperfusión/patología , Transducción de SeñalRESUMEN
BACKGROUND: Toll-like receptor (TLR)-4 signaling plays a key role in initiating exogenous antigen-independent innate immunity-dominated liver ischemia/reperfusion injury (IRI). Heme oxygenase (HO)-1, a heat-shock protein 32, exerts potent adaptive anti-oxidant and anti-inflammatory functions. Signal transducers and activator of transcription (STAT)-1 activation triggers interferon (IFN)-inducible protein 10 (CXCL-10), one of major products of type-1 IFN pathway downstream of TLR4. This study focuses on the role of type-1 IFN pathway in the mechanism of HO-1 cytoprotection during liver IRI. METHODS AND RESULTS: Cobalt protoporphyrin (CoPP)-induced HO-1 overexpression ameliorated liver damage in a well-defined mouse model of liver warm IRI, as evidenced by improved hepatic function (serum alanine aminotransferase levels) and liver histology (Suzuki's scores). HO-1 downregulated phospho-STAT-1 and its key product, CXCL-10. In contrast, TLR4 expression remained elevated regardless of the IRI status. To dissect the mechanism of HO-1 upon CXCL-10, we cultured RW 264.7 (macrophage) cells with exogenous rIFN-beta to stimulate CXCL-10 production via TLR4 pathway in vitro. Indeed, CoPP-induced HO-1 suppressed otherwise highly upregulated rIFN-beta-triggered CXCL-10. Moreover, consistent with our in vitro data, CoPP pretreatment diminished rIFN-beta-induced CXCL-10 production in normal mouse livers. CONCLUSION: Hepatic IRI activates TLR4 signaling in vivo to elaborate CXCL-10. HO-1 overexpression downregulates activation of STAT1 via type-1 IFN pathway downstream of TLR4, which in turn decreases CXCL-10 production. This study provides evidence for a novel mechanism by which HO-1 exerts adaptive cytoprotective and anti-inflammatory functions in the context of innate TLR4 activation.
Asunto(s)
Hemo-Oxigenasa 1/biosíntesis , Interferón Tipo I/antagonistas & inhibidores , Isquemia/prevención & control , Hepatopatías/prevención & control , Hígado/enzimología , Daño por Reperfusión/prevención & control , Animales , Aspartato Aminotransferasas/sangre , Línea Celular , Quimiocina CXCL10 , Quimiocinas CXC/genética , Inducción Enzimática/efectos de los fármacos , Hemo-Oxigenasa 1/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Hipoxantina Fosforribosiltransferasa/genética , Interferón Tipo I/fisiología , Isquemia/patología , Hepatopatías/patología , Macrófagos/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Protoporfirinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Receptor Toll-Like 4/metabolismoRESUMEN
BACKGROUND: Ischemia-reperfusion (I/R) injury is a prime antigen-independent inflammatory factor in the dysfunction of liver transplants. The precise contribution of T cells in the mechanism of I/R injury remains to be elucidated. As the CD154-CD40 co-stimulation pathway provides essential second signal in the initiation and maintenance of T-cell-dependent immune responses, this study was designed to assess the role of CD154 signaling in the pathophysiology of liver I/R injury. METHODS: A mouse model of partial 90-min warm hepatic ischemia followed by 6 hr of reperfusion was used. Three animal groups were studied: (1) wild-type (WT) mice treated with Ad-(-gal versus Ad-CD40 immunoglobulin; (2) untreated WT versus CD154 (MR1) monoclonal antibody-treated WT mice; and (3) untreated WT versus CD154 knockout mice. RESULTS: The disruption of CD154 signaling in all three animal groups ameliorated otherwise fulminant liver injury, as evidenced by depressed serum glutamic oxaloacetic transaminase levels, compared with controls. These beneficial effects were accompanied by depressed hepatic T-cell sequestration, local decrease of vascular endothelial growth factor expression, inhibition of tumor necrosis factor-(and T-helper type 1 cytokine production, and induction of antiapoptotic (Bcl-2/Bcl-xl) but depression of proapoptotic (caspase-3) proteins. CONCLUSIONS: By using in parallel a gene therapy approach, pharmacologic blockade, and genetically targeted mice, these findings document the benefits of disrupting CD154 to selectively modulate inflammatory responses in liver I/R injury. This study reinforces the key role of CD154-CD40 T-cell co-stimulation in the pathophysiology of liver I/R injury.
Asunto(s)
Antígenos CD40/inmunología , Ligando de CD40/genética , Ligando de CD40/inmunología , Circulación Hepática/inmunología , Hígado/irrigación sanguínea , Daño por Reperfusión/inmunología , Linfocitos T/inmunología , Animales , Apoptosis/inmunología , Citocinas/inmunología , Isquemia , Hígado/inmunología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , ARN/aislamiento & purificación , Daño por Reperfusión/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Células TH1/inmunologíaRESUMEN
BACKGROUND: Pyrrolidine dithiocarbamate (PDTC) represents a class of antioxidants and is a potent inducer of the heme oxygenase-1 (HO-1) gene and an inhibitor of nuclear factor-kappa B (NF-kappa B). We examined the impact of PDTC preconditioning against cold ischemia and reperfusion injury in the rat liver. METHODS: Lewis rats were treated subcutaneously with saline or PDTC solution 24 hours before harvesting. Some animals pretreated with PDTC were also given zinc protoporphyrin IX intravenously immediately after reperfusion. HO-1 expression and enzyme activity in liver tissues were analyzed at different time points after each treatment. After transplantation of 24-hour preserved livers, serum levels of transaminases and gene expression of tumor necrosis factor-alpha, interleukin-1 beta, and NF-kappa B were measured. Animal survival and cellular viability were monitored. RESULTS: HO-1 gene expression and protein synthesis were enhanced in PDTC-treated livers, leading to increased enzyme activity (P <.05). The PDTC treatment group showed lower transaminase levels (P <.05), lower cytokine and NF-kappa B messenger RNA expression (P <.05), and fewer nonviable cells (P <.05) than did the control group, whereas these PDTC effects were abolished with zinc protoporphyrin injection after reperfusion (P <.05). The best animal survival rate was observed in the PDTC group (P <.05). CONCLUSION: PDTC preconditioning reduces inflammatory responses during reperfusion. PDTC appears to exert this protective effect by induction of an antioxidative stress protein and inhibition of proinflammatory cytokines.
Asunto(s)
Antioxidantes/farmacología , Criopreservación , Trasplante de Hígado/efectos adversos , Preservación de Órganos/efectos adversos , Prolina/análogos & derivados , Prolina/farmacología , Daño por Reperfusión/etiología , Daño por Reperfusión/prevención & control , Tiocarbamatos/farmacología , Animales , Supervivencia Celular , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Interleucina-1/genética , Hígado/enzimología , Trasplante de Hígado/mortalidad , Masculino , FN-kappa B/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Transaminasas/sangre , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Ischemia/reperfusion injury (IRI) is a multifactorial antigen-independent process that affects both early and late graft function after transplantation. The complex mechanism of IRI can be attributed to neutrophil accumulation at the site of tissue injury, release of pro-inflammatory mediators such as oxygen free radicals (OFRs), and cytokines, which lead to cellular injury that culminates in the ultimate graft failure. The heme oxygenase-1 (HO-1) system is among the most critical of cytoprotective mechanisms activated during the cellular stress. The cytoprotection often seen in the transplanted organ following local HO-1 overexpression may include several factors, such as: a) antioxidant function, b) maintenance of microcirculation, c) anti-apoptotic function, and d) anti-inflammatory function. The role of enhanced endogenous HO-1 overexpression, and HO-1 downstream mediators (bilirubin, ferritin, CO), which protect against the IRI sequel, remain currently one of the most active areas of investigation. Indeed, HO-1, which functions to amplify multiple intracellular cytoprotective pathways, may serve as a novel therapeutic concept in transplantation to maximize organ donor pool through their safer use despite prolonged periods of cold ischemia.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Daño por Reperfusión/enzimología , Animales , Bilirrubina/metabolismo , Biliverdina/metabolismo , Monóxido de Carbono/metabolismo , Inducción Enzimática , Ferritinas/metabolismo , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemo-Oxigenasa 1 , Humanos , Hierro/metabolismo , Proteínas de la Membrana , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & controlRESUMEN
Data of 36 months were accumulated regarding the effects of lanthanum carbonate (LA) on serum phosphate concentrations in dialysis patients. Fifty-three patients (average age and dialysis history 58.4 years and 9.1 years) were included in this study who have been receiving outpatient treatment since March 2009, and who have been unable to maintain serum phosphate concentrations of ≤6.0 mg/dL via traditional therapeutic agents used for hyperphosphatemia. Patients were given dosage of LA in addition to, or instead of, co-hyperphosphatemia treatments already being received. Mean dosages of calcium carbonate (CC) and sevelamer hydrochloride (SH) before starting LA administration were 1301.9 mg and 2462.3 mg, respectively. Dosage of LA for all cases was 750 mg at initial dose; 1528.3 mg at 5 months; and 1416.7 mg at 30 months. Dosage of other phosphate binders were 905.7 mg of CC and 820.8 mg of SH at 5 months; and 687.5 mg of CC and 1031.3 mg of SH at 30 months. Serum phosphorus levels (P levels) were significantly decreased at 1 month of LA administration, and continued until 30 months of La treatment. These results suggest that LA successfully controlled serum P and Ca concentrations simultaneously within target ranges without affecting serum intact parathyroid hormone concentration, although further long-term prospective cohort study on LA would be required.
Asunto(s)
Hiperfosfatemia/tratamiento farmacológico , Fallo Renal Crónico/terapia , Lantano/uso terapéutico , Diálisis Renal/métodos , Anciano , Calcio/sangre , Carbonato de Calcio/administración & dosificación , Carbonato de Calcio/uso terapéutico , Estudios de Cohortes , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Humanos , Lantano/administración & dosificación , Masculino , Persona de Mediana Edad , Fosfatos/sangre , Poliaminas/administración & dosificación , Poliaminas/uso terapéutico , Estudios Prospectivos , Sevelamer , Factores de Tiempo , Resultado del TratamientoRESUMEN
Cyclooxygenase-2 (COX-2) is a prostanoid-synthesizing enzyme that is critically implicated in a variety of pathophysiological processes. Using a COX-2-deficient mouse model, we present data that suggest that COX-2 has an active role in liver ischemia/reperfusion (I/R) injury. We demonstrate that COX-2-deficient mice had a significant reduction in liver damage after I/R insult. The inability of COX-2(-/-) to elaborate COX-2 products favored a Th2-type response in these mice. COX-2(-/-) livers after I/R injury showed significantly decreased levels of IL-2, as well as IL-12, a cytokine known to have a central role in Th1 effector cell differentiation. Moreover, such livers expressed enhanced levels of the anti-inflammatory cytokine IL-10, shifting the balance in favor of a Th2 response in COX-2-deficient mice. The lack of COX-2 expression resulted in decreased levels of CXCL2, a neutrophil-activating chemokine, reduced infiltration of MMP-9-positive neutrophils, and impaired late macrophage activation in livers after I/R injury. Additionally, Bcl-2 and Bcl-x(L) were normally expressed in COX-2(-/-) livers after injury, whereas respective wild-type controls were almost depleted of these two inhibitors of cell death. In contrast, caspase-3 activation and TUNEL-positive cells were depressed in COX-2(-/-) livers. Therefore, our data support the concept that COX-2 is involved in the pathogenic events occurring in liver I/R injury. The data also suggest that potential valuable therapeutic approaches in liver I/R injury may result from further studies aimed at identifying specific COX-2-derived prostanoid pathways.
Asunto(s)
Ciclooxigenasa 2/deficiencia , Hígado/irrigación sanguínea , Hígado/inmunología , Infiltración Neutrófila , Daño por Reperfusión/inmunología , Células Th2/inmunología , Animales , Celecoxib , Quimiocina CXCL2/metabolismo , Ciclooxigenasa 2/efectos de los fármacos , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Citocinas/genética , Citocinas/metabolismo , Regulación hacia Abajo , Hígado/enzimología , Activación de Macrófagos , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Noqueados , Infiltración Neutrófila/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirazoles/administración & dosificación , Daño por Reperfusión/enzimología , Daño por Reperfusión/prevención & control , Sulfonamidas/administración & dosificación , Células TH1/inmunología , Proteína bcl-X/metabolismoRESUMEN
This study analyzes how toll-like receptor 4 (TLR4) signaling in the donor organ affects the ischemia and reperfusion injury (IRI) sequel following liver transplantation. Isogenic orthotopic liver transplantations (OLTs) with rearterialization were performed in groups of wild-type (WT) and TLR4 knockout (KO) mice after donor liver preservation in University of Wisconsin solution at 4 degrees C for 24 hours. Unlike WT OLTs, TLR4-deficient OLTs transplanted to either WT or TLR4 KO recipients suffered significantly less hepatocellular damage, as evidenced by serum alanine aminotransferase levels, and histological Suzuki's grading of liver IRI. Disruption of TLR4 signaling in OLTs decreased local neutrophil sequestration, CD4+ T cell infiltration, interferon (IFN)-gamma-inducible protein 10 (CXCL10) and an intercellular adhesion molecule (ICAM-1), as well as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-2, and IFN-gamma, yet increased IL-4 and IL-10 expression. The well-functioning OLTs from TLR4 KO donors revealed attenuated activity of capase-3, and enhanced heme oygenase-1 (HO-1) expression, along with decreased levels of apoptotic endothelial cells/hepatocytes, as compared with WT OLTs with intact TLR4 signaling. Thus, the functional sentinel TLR4 complex in the donor organ plays a key role in the mechanism of hepatic IRI after OLT. Disruption of TLR4 pathway downregulated the early proinflammatory responses and ameliorated hepatic IRI. These results provide the rationale to locally modify innate TLR4 signaling in the donor organ to more efficiently control the adaptive posttransplantation IRI-dependent responses.
Asunto(s)
Trasplante de Hígado/efectos adversos , Hígado/patología , Daño por Reperfusión/prevención & control , Transducción de Señal/fisiología , Receptor Toll-Like 4/metabolismo , Animales , Apoptosis , Western Blotting , Caspasa 3/genética , Caspasa 3/metabolismo , ADN/genética , Modelos Animales de Enfermedad , Expresión Génica , Inmunohistoquímica , Interleucinas/genética , Interleucinas/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Peroxidasa/metabolismo , Daño por Reperfusión/etiología , Daño por Reperfusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Although hypoxia stimulates the expression of vascular endothelial growth factor (VEGF), little is known of the role or mechanism by which VEGF functions after ischemia and reperfusion (I/R) injury. In this report, we first evaluated the expression of VEGF in a mouse model of liver warm ischemia. We found that the expression of VEGF increased after ischemia but peaked between 2 and 6 hours after reperfusion. Mice were treated with a neutralizing anti-mouse VEGF antiserum (anti-VEGF) or control serum daily from day -1 (1 day before the initiation of ischemia). Treatment with anti-VEGF significantly reduced serum glutaminic pyruvic transaminase levels and reduced histological evidence of hepatocellular damage compared with controls. Anti-VEGF also markedly decreased T-cell, macrophage, and neutrophil accumulation within livers and reduced the frequency of intrahepatic apoptotic terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells. Moreover, there was a reduction in the expression of pro-inflammatory cytokines (tumor necrosis factor-alpha and interferon-gamma), chemokines (interferon-inducible protein-10 and monocyte chemoattractant protein-1) and adhesion molecules (E-selectin) in parallel with enhanced expression of anti-apoptotic genes (Bcl-2/Bcl-xl and heme oxygenase-1) in anti-VEGF-treated animals. In conclusion, hypoxia-inducible VEGF expression by hepatocytes modulates leukocyte trafficking and leukocyte-induced injury in a mouse liver model of warm I/R injury, demonstrating the importance of endogenous VEGF production in the pathophysiology of hepatic I/R injury.
Asunto(s)
Quimiotaxis de Leucocito , Hepatopatías/prevención & control , Daño por Reperfusión/prevención & control , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Selectina E/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Hemo-Oxigenasa 1/metabolismo , Humanos , Inmunoglobulina G/inmunología , Etiquetado Corte-Fin in Situ , Hepatopatías/enzimología , Hepatopatías/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Conejos , Daño por Reperfusión/metabolismo , Linfocitos T/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Heme oxygenase-1 (HO-1) overexpression protects against tissue injury in many inflammatory processes, including ischemia/reperfusion injury (IRI). This study evaluated whether genetically decreased HO-1 levels affected susceptibility to liver IRI. Partial warm ischemia was produced in hepatic lobes for 90 min followed by 6 h of reperfusion in heterozygous HO-1 knockout (HO-1(+/-)) and HO-1(+/+) wild-type (WT) mice. HO-1(+/-) mice demonstrated reduced HO-1 mRNA/protein levels at baseline and postreperfusion. This corresponded with increased hepatocellular damage in HO-1(+/-) mice, compared with WT. HO-1(+/-) mice revealed enhanced neutrophil infiltration and proinflammatory cytokine (TNF-alpha, IL-6, and IFN-gamma) induction, as well as an increase of intrahepatic apoptotic TUNEL(+) cells with enhanced expression of proapoptotic genes (Bax/cleaved caspase-3). We used cobalt protoporphyrin (CoPP) treatment to evaluate the effect of increased baseline HO-1 levels in both WT and HO-1(+/-) mice. CoPP treatment increased HO-1 expression in both animal groups, which correlated with a lower degree of hepatic damage. However, HO-1 mRNA/protein levels were still lower in HO-1(+/-) mice, which failed to achieve the degree of antioxidant hepatoprotection seen in CoPP-treated WT. Although the baseline and postreperfusion HO-1 levels correlated with the degree of protection, the HO-1 fold induction correlated instead with the degree of damage. Thus, basal HO-1 levels are more critical than the ability to up-regulate HO-1 in response to the IRI and may also predict the success of pharmacologically induced cytoprotection. This model provides an opportunity to further our understanding of HO-1 in stress defense mechanisms and design new regimens to prevent IRI.
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Antioxidantes/metabolismo , Citoprotección/fisiología , Hemo-Oxigenasa 1/metabolismo , Hígado/enzimología , Daño por Reperfusión/enzimología , Animales , Apoptosis/fisiología , Western Blotting , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Hemo-Oxigenasa 1/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Hígado/patología , Ratones , Ratones Noqueados , Infiltración Neutrófila/efectos de los fármacos , Protoporfirinas/farmacología , ARN Mensajero/análisis , Daño por Reperfusión/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
P-selectin glycoprotein ligand-1 (PSGL-1) mediates the initial tethering of leukocytes to activated platelets and endothelium. We report molecular cloning and characterization of the rat PSGL-1 gene. A neutralizing Ab was generated, and its binding epitope was mapped to the N-terminal binding region of rat PSGL-1. We examined the effects of early PSGL-1 blockade in rat liver models of cold ischemia, followed by ex vivo reperfusion or transplantation (orthotopic liver transplantation (OLT)) using an anti-PSGL-1 Ab with diminished Fc-mediated effector function. In the ex vivo hepatic cold ischemia and reperfusion model, pretreatment with anti-PSGL-1 Ab improved portal venous flow, increased bile production, and decreased hepatocellular damage. Rat pretreatment with anti-PSGL-1 Ab prevented hepatic insult in a model of cold ischemia, followed by OLT, as assessed by 1) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic-pyruvic transaminase levels), and ameliorated histological features of ischemia/reperfusion injury, consistent with extended OLT survival; 2) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin, ED-1, CD3, and OX-62 cells; 3) inhibited expression of proinflammatory cytokine genes (TNF-alpha, IL-1beta, IL-6, IFN-gamma, and IL-2); and 4) prevented hepatic apoptosis accompanied by up-regulation of antiapoptotic Bcl-2/Bcl-xL protective genes. Thus, targeting PSGL-1 with a blocking Ab that has diminished Fc-mediated effector function is a simple and effective strategy that provides the rationale for novel therapeutic approaches to maximize the organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.
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Anticuerpos Bloqueadores/farmacología , Leucocitos/inmunología , Trasplante de Hígado/inmunología , Daño por Reperfusión/prevención & control , Secuencia de Aminoácidos , Animales , Apoptosis/inmunología , Secuencia de Bases , Sitios de Unión de Anticuerpos/genética , Western Blotting , Biblioteca Genómica , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Humanos , Etiquetado Corte-Fin in Situ , Leucocitos/metabolismo , Trasplante de Hígado/patología , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas Lew , Daño por Reperfusión/inmunología , Daño por Reperfusión/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
The current models of liver ischemia/reperfusion injury (IRI) in mice are largely limited to a warm ischemic component. To investigate the mechanism of hepatic "cold" IRI, we developed and validated a new mouse model of prolonged cold preservation followed by syngeneic orthotopic liver transplantation (OLT). Two hundred and forty-three OLTs with or without rearterialization and preservation in University of Wisconsin solution at 4 degrees C were performed in Balb/c mice. The 14-day survivals in the nonarterialized OLT groups were 92% (11/12), 82% (9/11), and 8% (1/12) after 1-hour, 6-hour and 24-hour preservation, respectively. In contrast, hepatic artery reconstruction after 1-hour, 6-hour, and 24-hour preservation improved the outcome as evidenced by 2-week survival of 100% (12/12), 100% (10/10), and 33% (4/12), respectively, and diminished hepatocellular damage (serum alanine aminotransferase /histology). Moreover, 24-hour (but not 1-h) cold preservation of rearterialized OLTs increased hepatic CD4+ T-cell infiltration and proinflammatory cytokine (tumor necrosis factor-alpha, interleukin 2, interferon-gamma) production, as well as enhanced local apoptosis, and Toll-like receptor 4/caspase 3 expression. These cardinal features of hepatic IRI validate the model. In conclusion, we have developed and validated a new mouse model of IRI in which hepatic artery reconstruction was mandatory for long-term animal survival after prolonged (24-h) OLT preservation. With the availability of genetically manipulated mouse strains, this model should provide important insights into the mechanism of antigen-independent hepatic IRI and help design much needed refined therapeutic means to combat hepatic IRI in the clinics.
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Inflamación/etiología , Isquemia , Trasplante de Hígado/métodos , Hígado/irrigación sanguínea , Adenosina , Alopurinol , Animales , Apoptosis , Recuento de Linfocito CD4 , Modelos Animales de Enfermedad , Glutatión , Inmunohistoquímica , Insulina , Trasplante de Hígado/inmunología , Trasplante de Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Soluciones Preservantes de Órganos , Complicaciones Posoperatorias/etiología , Rafinosa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Linfocitos T/patología , Resultado del TratamientoRESUMEN
We report our experience of cytapheresis using a nonwoven polyester fiber filter to treat critical states of immune diseases. In 7 critical states of ulcerative colitis (UC), cytapheresis was effective in improving symptoms of UC. Administration of steroids was important in some cases. In 3 cases of renal transplantation, cytapheresis was also effective in controlling rejection. IgA nephropathy of transplanted cases was well controlled. Furthermore, an original disease such as focal segmental glomerulosclerosis (FCGS) in a transplant patient was well treated by extracorporeal immune modulation of the cytapheresis.
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Colitis Ulcerosa/terapia , Cuidados Críticos/métodos , Citaféresis , Adulto , Anciano , Colitis Ulcerosa/inmunología , Citaféresis/métodos , Femenino , Glomeruloesclerosis Focal y Segmentaria/inmunología , Glomeruloesclerosis Focal y Segmentaria/terapia , Humanos , Inmunidad Celular , Trasplante de Riñón , Masculino , Persona de Mediana EdadRESUMEN
Heme oxygenase-1 (HO-1) has been shown to increase cellular resistance against oxidative injury, but the functional significance of this is currently obscure. We investigated the protective role of HO-1, induced by tin-protoporphyrin IX (SnPP), in attenuating liver transplantation injury. Lewis rats were intraperitoneally treated with saline as control, 50 micro mol/kg of SnPP, or 2 mg/kg of cycloheximide (CHX) before SnPP injection. Gene expression of HO-1 was induced after either treatment with SnPP- or CHX + SnPP instead of saline, whereas HO-1 protein synthesis was enhanced in Kupffer-like dendritic cells of the SnPP-treated group. Following reperfusion of liver grafts preserved for 30 h, there were fewer intercellular adhesion molecule-1-positive cells in SnPP-treated livers, significantly reduced numbers of dead cells, and enhanced graft viability. The present data suggest that increased synthesis of HO-1 protein by SnPP pre-conditioning is linked to the improved liver graft viability through inhibition of inflammatory adhesion molecules.
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Criopreservación , Hemo Oxigenasa (Desciclizante)/metabolismo , Trasplante de Hígado , Hígado/fisiopatología , Daño por Reperfusión/prevención & control , Estrés Fisiológico/enzimología , Animales , Supervivencia Celular , Cicloheximida/farmacología , Susceptibilidad a Enfermedades , Sinergismo Farmacológico , Inducción Enzimática/efectos de los fármacos , Expresión Génica , Hemo Oxigenasa (Desciclizante)/genética , Hemo-Oxigenasa 1 , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/metabolismo , Hígado/enzimología , Hígado/patología , Trasplante de Hígado/mortalidad , Masculino , Metaloporfirinas/farmacología , Protoporfirinas/farmacología , Ratas , Ratas Endogámicas Lew , Reperfusión , Daño por Reperfusión/fisiopatología , Factores de Tiempo , Acondicionamiento Pretrasplante , Trasplante HomólogoRESUMEN
BACKGROUND: Heme oxygenase (HO)-1 is induced as a unique stress response and leads to a transient resistance against oxidative damage, including ischemia and reperfusion (I/R) injury. In the present study, we examined whether HO-1 induction may confer a protection against I/R injury in the rat kidney. METHODS: Lewis rats were divided into four groups as follows: (1) vehicle group; (2) group treated with ferri-protoporphyrin IX (hemin), an inducer of HO; (3) group treated with low-dose tin-protoporphyrin IX (SnPP), an inhibitor of HO; and (4) group treated with high-dose SnPP. Renal warm ischemia for 60 minutes was performed 24 hours after each treatment. RESULTS: At 24 hours after treatment, hemin induced a significant increase in renal HO activity, but failed to induce HO-1 protein synthesis. Although both low- and high-dose SnPP reduced HO activity, a marked HO-1 expression was observed only in the high-dose SnPP-treated kidney. Hemin exacerbated the renal function after reperfusion, while high-dose SnPP significantly suppressed the intercellular adhesion molecule (ICAM)-1 expression, the infiltration of ED-1-positive macrophages and the expression of activated caspase-3, which resulted in attenuation of apoptotic cell death and ameliorated I/R injury. CONCLUSION: These results suggest that prior induction of HO-1 protein by high-dose SnPP may lead to anti-inflammatory and antiapoptotic effects on warm renal I/R injury independently of its enzyme activity, and that HO enzyme activation may not always act as an antioxidant, especially under I/R-induced oxidative stress.