RESUMEN
Tolerogenic dendritic cells (tolDC) are a new immunotherapeutic tool for the treatment of rheumatoid arthritis (RA) and other autoimmune disorders. We have established a method to generate stable tolDC by pharmacological modulation of human monocyte-derived DC. These tolDC exert potent pro-tolerogenic actions on CD4+ T cells. Lack of interleukin (IL)-12p70 production is a key immunoregulatory attribute of tolDC but does not explain their action fully. Here we show that tolDC express transforming growth factor (TGF)-ß1 at both mRNA and protein levels, and that expression of this immunoregulatory cytokine is significantly higher in tolDC than in mature monocyte-derived DC. By inhibiting TGF-ß1 signalling we demonstrate that tolDC regulate CD4+ T cell responses in a manner that is at least partly dependent upon this cytokine. Crucially, we also show that while there is no significant difference in expression of TGF-ßRII on CD4+ T cells from RA patients and healthy controls, RA patient CD4+ T cells are measurably less responsive to TGF-ß1 than healthy control CD4+ T cells [reduced TGF-ß-induced mothers against decapentaplegic homologue (Smad)2/3 phosphorylation, forkhead box protein 3 (FoxP3) expression and suppression of (IFN)-γ secretion]. However, CD4+ T cells from RA patients can, nonetheless, be regulated efficiently by tolDC in a TGF-ß1-dependent manner. This work is important for the design and development of future studies investigating the potential use of tolDC as a novel immunotherapy for the treatment of RA.
Asunto(s)
Artritis Reumatoide/terapia , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica , Inmunoterapia/métodos , Factor de Crecimiento Transformador beta1/metabolismo , Artritis Reumatoide/inmunología , Células Cultivadas , Colecalciferol/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/trasplante , Dexametasona/farmacología , Factores de Transcripción Forkhead/metabolismo , Humanos , Inmunomodulación , Interleucina-12/genética , Interleucina-12/metabolismo , Activación de Linfocitos , Proteína Smad2/metabolismoRESUMEN
Biliary epithelial cells (BEC) are important targets in some liver diseases, including acute allograft rejection. Although some injured BEC die, many can survive in function compromised states of senescence or phenotypic de-differentiation. This study was performed to examine changes in the phenotype of BEC during acute liver allograft rejection and the mechanism driving these changes. Liver allograft sections showed a positive correlation (p < 0.0013) between increasing T cell mediated acute rejection and the number of BEC expressing the senescence marker p21(WAF1/Cip) or the mesenchymal marker S100A4. This was modeled in vitro by examination of primary or immortalized BEC after acute oxidative stress. During the first 48 h, the expression of p21(WAF1/Cip) was increased transiently before returning to baseline. After this time BEC showed increased expression of mesenchymal proteins with a decrease in epithelial markers. Analysis of TGF-ß expression at mRNA and protein levels also showed a rapid increase in TGF-ß2 (p < 0.006) following oxidative stress. The epithelial de-differentiation observed in vitro was abrogated by pharmacological blockade of the ALK-5 component of the TGF-ß receptor. These data suggest that stress induced production of TGF-ß2 by BEC can modify liver allograft function by enhancing the de-differentiation of local epithelial cells.
Asunto(s)
Conductos Biliares Intrahepáticos/patología , Senescencia Celular , Células Epiteliales/patología , Rechazo de Injerto/patología , Trasplante de Hígado/patología , Enfermedad Aguda , Conductos Biliares Intrahepáticos/metabolismo , Biopsia , Western Blotting , Células Cultivadas , Densitometría , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Rechazo de Injerto/genética , Rechazo de Injerto/metabolismo , Humanos , Inmunohistoquímica , Estrés Oxidativo/genética , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Crecimiento Transformador beta2/biosíntesis , Factor de Crecimiento Transformador beta2/genética , Trasplante HomólogoRESUMEN
BACKGROUND: Oesophagogastric cancer surgery is immunosuppressive. This may be modulated by omega-3 fatty acids (O-3FAs). The aim of this study was to assess the effect of perioperative O-3FAs on clinical outcome and immune function after oesophagogastric cancer surgery. METHODS: Patients undergoing subtotal oesophagectomy and total gastrectomy were recruited and allocated randomly to an O-3FA enteral immunoenhancing diet (IED) or standard enteral nutrition (SEN) for 7 days before and after surgery, or to postoperative supplementation alone (control group). Clinical outcome, fatty acid concentrations, and HLA-DR expression on monocytes and activated T lymphocytes were determined before and after operation. RESULTS: Of 221 patients recruited, 26 were excluded. Groups (IED, 66; SEN, 63; control, 66) were matched for age, malnutrition and co-morbidity. There were no differences in morbidity (P = 0·646), mortality (P = 1·000) or hospital stay (P = 0·701) between the groups. O-3FA concentrations were higher in the IED group after supplementation (P < 0·001). The ratio of omega-6 fatty acid to O-3FA was 1·9:1, 4·1:1 and 4·8:1 on the day before surgery in the IED, SEN and control groups (P < 0·001). There were no differences between the groups in HLA-DR expression in either monocytes (P = 0·538) or activated T lymphocytes (P = 0·204). CONCLUSION: Despite a significant increase in plasma concentrations of O-3FA, immunonutrition with O-3FA did not affect overall HLA-DR expression on leucocytes or clinical outcome following oesophagogastric cancer surgery. REGISTRATION NUMBER: ISRCTN43730758 (http://www.controlled-trials.com).
Asunto(s)
Nutrición Enteral/métodos , Neoplasias Esofágicas/cirugía , Ácidos Grasos Omega-3/administración & dosificación , Neoplasias Gástricas/cirugía , Adulto , Anciano , Análisis de Varianza , Proteína C-Reactiva/metabolismo , Suplementos Dietéticos , Neoplasias Esofágicas/sangre , Neoplasias Esofágicas/inmunología , Esofagectomía/métodos , Ácidos Grasos/metabolismo , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-6/metabolismo , Femenino , Gastrectomía/métodos , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Cuidados Posoperatorios/métodos , Complicaciones Posoperatorias/etiología , Cuidados Preoperatorios/métodos , Estudios Prospectivos , Neoplasias Gástricas/sangre , Neoplasias Gástricas/inmunología , Linfocitos T/metabolismoRESUMEN
More than 45 members of the family of chemotactic cytokines have been described. These chemokines control the migration of leukocytes throughout the whole alloimmune response from initial ischemic damage to acute inflammation and eventual resolution. Several chemokines have been strongly linked to allograft rejection. Recent studies have described powerful endogenous mechanisms that regulate chemokine biology. This review will describe a new class of chemokine receptor that bind ligands with high affinity but lack the capacity for signaling. Atypical receptors represent a new paradigm in chemokine biology and may hold the key to our eventual manipulation of chemokine-driven allograft inflammation.
Asunto(s)
Quimiocinas/inmunología , Trasplante de Órganos , Receptores de Quimiocina/inmunología , Animales , Antiinflamatorios/inmunología , Rechazo de Injerto/inmunología , Humanos , Leucocitos/inmunología , Transducción de Señal , Trasplante Homólogo/inmunologíaRESUMEN
Epithelial to mesenchymal transition (EMT) has been implicated in the pathogenesis of obliterative bronchiolitis (OB) after lung transplant. Although TNF-alpha accentuates TGF-beta1 driven EMT in primary human bronchial epithelial cells (PBECs), we hypothesized that other acute pro-inflammatory cytokines elevated in the airways of patients with OB may also accentuate EMT and contribute to dysregulated epithelial wound repair. PBECs from lung transplant recipients were stimulated with TGF-beta1+/-IL-1beta, IL-8, TNF-alpha or activated macrophages in co-culture and EMT assessed. The quality and rate of wound closure in a standardized model of lung epithelial injury was assessed in response to above stimuli. Co-treatment with TGF-beta1+TNF-alpha or IL-1beta significantly accentuates phenotypic and some functional features of EMT compared to TGF-beta1 alone. Co-treatment with TGF-beta1+TNF-alpha or IL-1beta accelerates epithelial wound closure however the quality of repair is highly dysregulated. Co-treatment with TGF-beta1+IL-8 has no significant effect on EMT or the speed or quality of wound healing. Activated macrophages dramatically accentuate TGF-beta1-driven EMT and cause dysregulated wound repair. Crosstalk between macrophage-derived acute inflammation in the airway and elevated TGF-beta1 may favor dysregulated airway epithelial repair and fibrosis in the lung allograft via EMT.
Asunto(s)
Epitelio/patología , Inflamación , Trasplante de Pulmón/métodos , Mesodermo/citología , Cicatrización de Heridas , Línea Celular Tumoral , Técnicas de Cocultivo , Fibrosis/patología , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Macrófagos/metabolismo , Modelos Biológicos , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Chemokines are immobilized by binding to glycosaminoglycans (GAGs). A non-GAG-binding mutant CCL7 (mtCCL7) was developed that retained its affinity for chemokine receptors. This mtCCL7 induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration (p<0.01). Unlike wild-type CCL7, mtCCL7 persisted in the circulation of BALB/c mice for more than 6 h and prevented leukocyte infiltration of skin isografts (p<0.05). Treatment with mtCCL7 marginally increased the survival of C57BL/6 to BALB/c skin allografts and reduced graft infiltration by CD3+ cells (p<0.05). Importantly, mtCCL7 promoted long-term (>40 day) graft survival following minor histocompatibility (HY) antigen mismatched C57BL/6 skin transplantation; control grafts were rejected by day 24. Treatment with mtCCL7 produced a significant decrease in the frequency of IFN-gamma producing donor-reactive splenic T cells, reduced CCR2 expression by circulating leukocytes for 6 h (p<0.01) and blocked the normal increase in affinity of alpha4beta1 integrins for VCAM-1 following transient chemokine stimulation. These data suggest that mtCCL7 persists in the circulation and reduces both specific T-cell priming and the capacity of circulating immune cells to respond to GAG-bound chemokine at sites of developing inflammation.
Asunto(s)
Quimiocina CCL7/genética , Quimiocina CCL7/uso terapéutico , Trasplante de Piel/inmunología , Trasplante de Piel/patología , Animales , Sitios de Unión/genética , Quimiocina CCL7/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/inmunología , Femenino , Glicosaminoglicanos/metabolismo , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Antígeno H-Y/administración & dosificación , Técnicas In Vitro , Inflamación/inmunología , Inflamación/patología , Inflamación/prevención & control , Integrinas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Receptores de Quimiocina/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patología , Trasplante HomólogoRESUMEN
BACKGROUND/AIMS: At present, there is no simple test for predicting severity in acute pancreatitis. We investigated the use of an assay of soluble E-cadherin (sE-cadherin). METHODS: Concentrations of sE-cadherin, from 19 patients with mild acute pancreatitis, 7 patients with severe acute pancreatitis, 11 patients with other acute gastrointestinal pathologies, and 12 healthy subjects were measured using a commercially available sandwich ELISA kit based on two monoclonal antibodies specific to the extracellular fragment of human E-cadherin. Measurements were made at 12 hours or less from onset of pain and also at 24 and 48 hours after onset of pain. RESULTS: Mean (standard deviation) concentration of sE-cadherin in patients with severe acute pancreatitis at <12 hours was 17780 ng/mL (7853), significantly higher than that of healthy volunteers 5180 ng/mL (1350), P = .0039, patients with other gastrointestinal pathologies 7358 ng/mL (6655), P = .0073, and also significantly higher than that of patients with mild pancreatitis, 7332 ng/mL (2843), P = .0019. DISCUSSION: Serum sE-cadherin could be an early (within 12 hours) objective marker of severity in acute pancreatitis. This molecule warrants further investigation in the form of a large multicentre trial.
Asunto(s)
Cadherinas/sangre , Pancreatitis/sangre , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
BACKGROUND: Aberrant epithelial repair is a key event in the airway remodelling which characterises obliterative bronchiolitis (OB) in the transplanted lung. The potential for airway epithelium from lung transplant recipients to undergo epithelial to mesenchymal cell transition (EMT) was assessed in culture and in vivo in lung allograft tissue. METHODS: Change in epithelial and mesenchymal marker expression was assessed after stimulation with transforming growth factor beta(1) (TGF-beta(1)) alone or in combination with tumour necrosis factor alpha (TNFalpha) and compared with untreated controls. The ability of cells to deposit extracellular matrix, secrete matrix metalloproteinases (MMPs) and invade collagen was investigated. Immunolocalisation of epithelial and mesenchymal markers was compared in airway tissue from stable recipients and those with OB. RESULTS: Untreated cells maintained epithelial morphology and phenotype. TGF-beta(1) reduced expression of epithelial markers, increased expression of vimentin and fibronectin, promoted collagen I and fibronectin deposition and increased MMP-9 production. Co-treatment with TNFalpha dramatically accentuated phenotypic and some functional features of EMT. Airway epithelial biopsies from recipients with OB demonstrated significantly increased staining for mesenchymal markers and significantly reduced E-cadherin staining compared with stable recipients. CONCLUSIONS: These observations demonstrate the ability of human airway epithelium to undergo EMT and suggest this phenomenon may be a potential link between inflammatory injury and TGF-beta(1)-driven airway remodelling in the development of OB.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/fisiología , Bronquiolos/patología , Bronquiolitis Obliterante/patología , Células Epiteliales/patología , Trasplante de Pulmón/patología , Mesodermo/patología , Biomarcadores/metabolismo , Bronquiolitis Obliterante/etiología , Cadherinas/metabolismo , Transdiferenciación Celular/fisiología , Células Epiteliales/metabolismo , Femenino , Fibronectinas/metabolismo , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Mesodermo/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Vimentina/metabolismoRESUMEN
Expression of the chemokine receptor CXCR4 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express CXCL12. In this study, we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides. Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I(125) CXCL12. Heparin dodecasaccharides were found to be the minimal chain length required to efficiently bind CXCL12 (71% inhibition; P<0.001). These oligosaccharides also significantly inhibited CXCL12-induced migration of CXCR4-expressing LMD MDA-MB 231 breast cancer cells. In addition, heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product, Tinzaparin. When given subcutaneously in a SCID mouse model of human breast cancer, heparin dodecasaccharides had no effect on the number of lung metastases, but did however inhibit (P<0.05) tumour growth (lesion area) compared to control groups. In contrast, polymeric heparin significantly inhibited both the number (P<0.001) and area of metastases, suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Quimiocinas CXC/metabolismo , Neoplasias Pulmonares/prevención & control , Oligosacáridos/farmacología , Receptores CXCR4/metabolismo , Animales , Neoplasias de la Mama/patología , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12 , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Heparina de Bajo-Peso-Molecular/farmacología , Heparitina Sulfato/metabolismo , Humanos , Inmunohistoquímica , Radioisótopos de Yodo , Neoplasias Pulmonares/secundario , Ratones , Ratones SCID , Polímeros/metabolismo , Ensayo de Unión Radioligante , Receptores CXCR4/efectos de los fármacos , TinzaparinaRESUMEN
The effectiveness of lung transplantation is marred by the relatively high incidence of rejection. The lung normally contains a large population of lymphocytes in contact with the airway epithelium, a proportion of which expresses the mucosal integrin, alpha(E)(CD103)beta(7). This integrin is not a homing receptor, but is thought to retain lymphocytes at the epithelial surface. Following transplantation, a population of 'tissue-restricted' cytotoxic T cells (CTL) have been identified which have the ability to lyse epithelial cells, but not major histocompatibility complex (MHC)-identical splenic cells. We tested the hypothesis that expression of the mucosal integrin confers the ability of CTL to target and destroy e-cadherin expressing targets. Immunohistochemical and flow cytometric analyses were used to demonstrate the relevance of this model to human lung. Allo-activated CTL were generated in mixed leucocyte reactions and CD103 expression up-regulated by the addition of transforming growth factor (TGF)-beta. The functional effect of CD103 expression was investigated in (51)Cr-release assays using e-cadherin-expressing transfectant targets. Human lung epithelial cells express e-cadherin and one-third of intraepithelial lymphocytes (IEL) expressed CD103. Allo-activated and bronchoalveolar lavage (BAL) lymphocytes express more CD103 than those in blood. Transfection of e-cadherin into murine fibroblasts conferred susceptibility to lysis by alpha(E)beta(7)-expressing CTL which could be blocked by specific monoclonal antibodies to CD103 and e-cadherin. CD103 functions to conjugate CTL effectors to e-cadherin-expressing targets and thereby facilitates cellular cytotoxicity. E-cadherin is expressed prominently by epithelial cells in the lung, enabling CTL to target them for destruction.
Asunto(s)
Antígenos CD/inmunología , Cadenas alfa de Integrinas/inmunología , Trasplante de Pulmón/inmunología , Linfocitos T Citotóxicos/inmunología , Anciano , Animales , Antígenos CD/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular/inmunología , Línea Celular , Proliferación Celular , Células Epiteliales/inmunología , Humanos , Inmunidad Mucosa/inmunología , Inmunofenotipificación , Cadenas alfa de Integrinas/metabolismo , Pulmón/inmunología , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Persona de Mediana Edad , TransfecciónRESUMEN
Sympathetic discharge and hypertensive crisis often accompany brain death, causing neurogenic pulmonary edema. Progressive systemic inflammatory response develops, which can injure the lung further. We investigated whether (a) early hemodynamic injury during donor brain death increases reperfusion injury after lung transplantation and (b) delaying lung recovery would augment reperfusion injury further, because of the progressive systemic inflammatory response in the donor. Brain death was induced by intracranial balloon inflation in rats, with or without alpha-adrenergic blockade pretreatment to prevent the hypertensive crisis. Another group of rats had a sham procedure. Lungs were retrieved 15 min after brain death or sham procedure and reperfused using recipient rats. In a fourth group, brain death was induced and the lungs were retrieved 5 h after brain death and reperfused. Postreperfusion, lungs retrieved early from untreated brain-dead donors developed more severe reperfusion injury, as assessed by functional parameters and inflammatory markers, than those from sham or alpha-blockade-treated donors. Lungs retrieved late from brain-dead donors had similar inflammatory markers after reperfusion to those retrieved early, but significantly lower pulmonary vascular resistance. Early hemodynamic damage during donor brain death increases reperfusion injury after lung transplantation. Delaying retrieval may allow the lung to recover from the hemodynamic injury.
Asunto(s)
Muerte Encefálica/patología , Funcionamiento Retardado del Injerto/etiología , Trasplante de Pulmón/efectos adversos , Donantes de Tejidos , Enfermedades Vasculares/patología , Animales , Hipertensión , Inflamación , Pulmón/patología , Masculino , Modelos Animales , Ratas , Ratas Wistar , Daño por Reperfusión/patología , Enfermedades Vasculares/etiologíaRESUMEN
BACKGROUND AND OBJECTIVE: Gingival overgrowth (GO) is a side-effect of cyclosporin A (CsA) therapy and is characterised by enlargement of the gingiva with epithelial thickening and overproduction of extracellular matrix components. The pathogenesis of the epithelial thickening in GO remains obscure. The objective of the present study was to investigate the effects of CsA on the growth of oral epithelial cells in vitro and to test the hypothesis that CsA influences apoptosis in these cells. MATERIAL AND METHODS: Cyclosporin was cocultured with an immortalized normal human oral keratinocyte cell line (HOK-16B), an epitheloid cervical carcinoma cell line (HeLa) and primary oral keratinocytes. Cell division was quantified using a CyQUANT kit. Apoptosis was induced using tumour necrosis factor-alpha (TNF-alpha) and assayed by analysis of caspase-3 activity. Expression of the anti-apoptotic protein, Bcl-2, was measured by western blotting. RESULTS: CsA exhibited a dose- and time-dependent inhibition of cell division in all three keratinocyte cell cultures. Significantly, HOK-16B cells treated with high doses of CsA (10 alphag/ml) did not recover their proliferative capacity 3 d after withdrawal of CsA, indicating that CsA-induced inhibition of growth is not temporary. Concentrations of CsA that inhibited cell division (1 microg/ml) did not have any effect on constitutive or TNF-alpha -induced apoptosis or Bcl-2 expression in HOK-16B cells. CONCLUSION: CsA inhibits oral epithelial cell division and this effect is not associated with changes in apoptosis in these cells. The action of CsA on oral epithelial cells may be associated with a long-lasting stress signal, which might account for some of the pathological effects of this drug.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclosporina/farmacología , Encía/efectos de los fármacos , Inmunosupresores/farmacología , Queratinocitos/efectos de los fármacos , Caspasa 3 , Caspasas/metabolismo , Línea Celular Transformada , Proliferación Celular/efectos de los fármacos , Encía/citología , Encía/metabolismo , Células HeLa , Humanos , Queratinocitos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesisRESUMEN
It has been suggested that TGFbeta can cause chronic allograft nephropathy by inducing epithelial to mesenchymal transition (EMT); some studies show a reverse transition can be produced by bone morphogenetic protein-7 (BMP7). The current study assessed the relative contribution of signals generated within tubular epithelial cells by TGFbeta and BMP7 in normal kidney and after transplantation. Epithelial cells in normal human kidneys expressed phosphorylated forms of both Smad2/3 and Smad1/5/8 within their nuclei; cell culture experiments showed that these signaling molecules were generated in response to TGFbeta and BMP7, respectively. Phospho(p)-Smad2/3 was expressed at increased levels by tubular epithelial cells during acute renal allograft rejection and chronic allograft nephropathy but pSmad1/5/8 was expressed at very low levels; this staining profile was associated with induction of the EMT marker, S100A4. Further in vitro experiments demonstrated that this pattern of Smad signaling was a consequence of the stimulation of tubular epithelial cells with TGFbeta in the absence of BMP7. Importantly, addition of BMP7 to TGFbeta-stimulated cells enhanced the expression of pSmad1/5/8 and reduced expression of S100A4. These results suggest that exogenous BMP7 could restore the homeostatic balance of pSmad signaling found in normal kidneys, thereby preventing or reversing the development of chronic allograft nephropathy.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Células Epiteliales/citología , Corteza Renal/citología , Trasplante de Riñón/patología , Factor de Crecimiento Transformador beta/fisiología , Proteína Morfogenética Ósea 7 , Técnicas de Cultivo de Célula/métodos , Células Epiteliales/fisiología , Rechazo de Injerto/patología , Humanos , Transducción de Señal , Donantes de Tejidos , Trasplante HomólogoRESUMEN
BACKGROUND: Obliterative bronchiolitis in chronic rejection of lung allografts is characterised by airway epithelial damage and fibrosis. The process whereby normal epithelium is lost and replaced by fibroblastic scar tissue is poorly understood, but recent findings suggest that epithelial cells can become fibroblasts through epithelial-mesenchymal transition (EMT). It is hypothesised that EMT occurs in lung allografts and plays a potential role in airway remodelling. METHODS: Sixteen stable lung transplant recipients underwent bronchoscopy with bronchoalveolar lavage (BAL), endobronchial biopsies, and bronchial brushings. Biopsy sections were stained for the fibroblast marker S100A4. Brushings were cultured on collagen, stained with anti-S100A4, and examined for further EMT markers including matrix metalloproteinase (MMP) zymographic activity and epithelial invasion through collagen coated filters. RESULTS: A median 15% (0-48%) of the biopsy epithelium stained for S100A4 in stable lung transplant recipients and MMP-7 co-localisation was observed. In non-stimulated epithelial cultures from lung allografts, S100A4 staining was identified with MMP-2 and MMP-9 production and zymographic activity. MMP total protein and activity was increased following stimulation with transforming growth factor (TGF)-beta1. Non-stimulated transplant epithelial cells were invasive and penetration of collagen coated filters increased following TGF-beta1 stimulation. CONCLUSIONS: This study provides evidence of EMT markers in lung allografts of patients without loss of lung function. The EMT process may represent a final common pathway following injury in more common diseases characterised by airway remodelling.
Asunto(s)
Células Epiteliales/patología , Trasplante de Pulmón , Mesodermo/patología , Adulto , Biopsia/métodos , Bronquiolitis Obliterante , Líquido del Lavado Bronquioalveolar/citología , Femenino , Fibroblastos/patología , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasas de la Matriz/análisis , Persona de Mediana Edad , Fenotipo , Coloración y EtiquetadoRESUMEN
PURPOSE: Intravesical bacillus Calmette-Guerin (BCG) therapy is the principal treatment for high risk, noninvasive urothelial carcinoma and carcinoma in situ of the bladder. However, up to 40% of patients fail to respond to this treatment. In this study the potential for inhibition of PGE2 production by BCG treated dendritic cells (DCs) was studied in the context of preferential polarization of the immune response toward a cancer clearing T-helper type 1 immune response. MATERIALS AND METHODS: Murine bone marrow derived DCs were cultured with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. After 7 days the cells were stimulated with BCG. Cell surface expression of co-stimulatory molecules and phagocytic ability were measured by flow cytometry analysis to verify cell activation. The production of IL-10 and IL-12 was measured after DC stimulation with BCG in the presence of IL-10, prostaglandin E2(Cayman Chemical, Ann Arbor, Michigan), antiIL-10 antibody (Insight Biotechnology, Wembley, United Kingdom), NS-398 and indomethacin (Sigma, Poole, United Kingdom). RESULTS: Prostaglandin E2 stimulated a dose dependent increase in the levels of IL-10 produced by BCG activated DCs (p <0.01). IL-10 significantly decreased IL-12 production (p <0.001), while IL-10 blockade significantly increased IL-12 levels (p <0.05). The COX-2 selective inhibitor NS-398 caused a dose dependent increase in the concentration of IL-12 produced by BCG activated DCs (p <0.01). This effect was also seen with the partially selective COX-1 inhibitor indomethacin (p <0.05). CONCLUSIONS: The inhibition of PGE2 synthesis by COX inhibition favored the production of IL-12 by BCG activated DC. This potentially will result in the generation of a T-helper type 1, polarized T-cell response that may improve the efficacy of BCG therapy.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Vacuna BCG/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Dinoprostona/antagonistas & inhibidores , Dinoprostona/biosíntesis , Adyuvantes Inmunológicos/uso terapéutico , Animales , Vacuna BCG/uso terapéutico , Inhibidores de la Ciclooxigenasa/uso terapéutico , Sinergismo Farmacológico , Femenino , Inmunoterapia , Interleucinas/biosíntesis , Ratones , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
Renal, hepatic, and lung allografts are compromised by aggressively deteriorating function. This chronic process is produced by an overall burden of organ damage, but the pathophysiology remains poorly understood. Rates of chronic rejection in the lung, for example, have not substantially improved over the last decade, despite new immunosuppressive drugs and improvements in surgical procedure. We present a hypothesis that epithelial-to-mesenchymal transition is a common cause of chronic allograft failure. Research in this area may provide insights into chronic rejection of kidney, liver, and lung allografts that impact on future therapeutic strategies.
Asunto(s)
Células Epiteliales/patología , Trasplante de Corazón/patología , Trasplante de Riñón/patología , Trasplante de Pulmón/patología , Mesodermo/patología , Diferenciación Celular , Humanos , Trasplante Homólogo/patología , Insuficiencia del TratamientoRESUMEN
BACKGROUND AND AIMS: Induction of the pro-fibrotic growth factor TGF-beta1 has been suggested as a possible mechanism through which immunosuppressant drugs may induce gingival overgrowth. This study aims to investigate plasma levels of TGF-beta1 and relate them to the development and severity of gingival overgrowth in immunosuppressed transplant patients. MATERIALS AND METHODS: One hundred and thirty-two ciclosporin-treated and 13 tacrolimus-treated transplant patients and 24 drug-free control subjects underwent a full periodontal examination including a determination of the presence and severity of gingival overgrowth. RESULTS: Plasma TGF-beta1 concentrations were determined by ELISA, and were found to be significantly elevated in samples from the transplant patients (mean=29.1 ng/ml) as compared with controls (mean=6.1 ng/ml, p<0.0001). There was no significant difference between the levels of plasma TGF-beta1 in the ciclosporin- and tacrolimus-treated patient groups. CONCLUSIONS: Furthermore, concomitant treatment with calcium channel blockers did not influence the levels of plasma TGF-beta1 in the patients group. The relationship between gingival overgrowth, independent periodontal variables and TGF-beta1 plasma concentrations was examined using univariate and multivariate regression analyses; low TGF-beta1 plasma concentrations were found to be a risk factor for gingival overgrowth in immunosuppressed patients concomitantly receiving a calcium channel blocker.
Asunto(s)
Ciclosporina/uso terapéutico , Sobrecrecimiento Gingival/inmunología , Inmunosupresores/uso terapéutico , Tacrolimus/uso terapéutico , Factor de Crecimiento Transformador beta/sangre , Factores de Edad , Análisis de Varianza , Bloqueadores de los Canales de Calcio/uso terapéutico , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Huésped Inmunocomprometido , Masculino , Análisis de Regresión , Factor de Crecimiento Transformador beta1 , TrasplanteRESUMEN
A number of antigens implicated in the pathogenesis of autoimmune diseases including Sjogren's syndrome and systemic lupus erythematosus (SLE) are expressed aberrantly by apoptotic cells. It is also known that apoptogenic proteins are released from the mitochondrial intermembrane space at an early stage during the induction and development of apoptosis. Combination of this evidence led us to test the hypothesis that apoptotic mechanisms provide an explanation for the abnormal expression of the inner mitochondrial enzyme, pyruvate dehydrogenase complex (PDC), observed on the surface of some cells in patients with the autoimmune liver disease primary biliary cirrhosis (PBC). Using one murine and two human cell lines it was found that the induction of apoptosis led to early detection of PDC within the cytoplasm. However, cytochrome c oxidase subunit 4 (COX 4), which is also present on the inner surface of the inner mitochondrial membrane, remained within the mitochondria. Immunoreactive PDC was also detected on the outer surface of the intact plasma membrane of cells sampled after the induction of apoptosis. Serial release of PDC to the cytoplasm and then onto the external surface of the plasma membrane provides direct evidence that the antigen on the cell surface is of mitochondrial origin. Immunoreactivity specific for PDC is strongly implicated in the pathogenesis of PBC, but this autoantigen is normally concealed from the immune system by three membrane systems. Release of PDC onto the cell surface during apoptosis provides a possible route for recognition of this antigen by the immune system which could contribute to both afferent and efferent phases of the disease process.
Asunto(s)
Cirrosis Hepática Biliar/inmunología , Complejo Piruvato Deshidrogenasa/análisis , Linfocitos T/inmunología , Animales , Apoptosis , Caspasas/análisis , Línea Celular , Membrana Celular/química , Citometría de Flujo , Humanos , Immunoblotting , Células Jurkat , Cirrosis Hepática Biliar/patología , Ratones , Microscopía ConfocalRESUMEN
Intravesical bacillus Calmette-Guerin (BCG) is a treatment for transitional cell carcinoma (TCC) and carcinoma in situ (cis) of the urinary bladder, but some patients remain refractory. The mechanism of cancer clearance is not known, but T cells are thought to play a contributory role. Tissue dendritic cells (DCs) are known to initiate antigen-specific immune responses following activation of receptors, which recognise molecular patterns on the surface of microorganisms. A family of these receptors, the toll-like receptors (TLRs), are also crucial for activating DC to produce cytokines that polarise the T-cell response towards a T helper (Th)1 or Th2 phenotype. This study compared the potential of intact BCG to activate DC with that of the defined TLR4 ligand lipopolysaccharide (LPS) and the TLR9 ligand CpG-oligonucleotide. It was found that all three stimuli efficiently activated normal DC, but cells expressing a mutant TLR4 responded poorly to stimulation with LPS. Importantly, stimulation with BCG induced both IL-12 and IL-10, suggesting subsequent development of a poorly focused T-cell immune response containing both Th1 and Th2 immune function. By contrast, LPS- and CpG-oligonucleotides induced only IL-12, indicating the potential to produce a Th1 response, which is likely to clear cancer most efficiently. Given the toxicity of LPS, our data suggest that CpG-oligonucleotides may be beneficial for intravesical therapy of bladder cancer.