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Static cold storage is the cheapest and easiest method and current gold standard to store and preserve donor organs. This study aimed to compare the preservative capacity of gluconate-lactobionate-dextran (Unisol) solutions to histidine-tryptophan-ketoglutarate (HTK) solution. Murine syngeneic heterotopic heart transplantations (Balb/c-Balb/c) were carried out after 18 h of static cold storage. Cardiac grafts were either flushed and stored with Unisol-based solutions with high-(UHK) and low-potassium (ULK) ± glutathione, or HTK. Cardiac grafts were assessed for rebeating and functionality, histomorphologic alterations, and cytokine expression. Unisol-based solutions demonstrated a faster rebeating time (UHK 56 s, UHK + Glut 44 s, ULK 45 s, ULK + Glut 47 s) compared to HTK (119.5 s) along with a better contractility early after reperfusion and at the endpoint on POD 3. Ischemic injury led to a significantly increased leukocyte recruitment, with similar degrees of tissue damage and inflammatory infiltrate in all groups, yet the number of apoptotic cells tended to be lower in ULK compared to HTK. In UHK- and ULK-treated animals, a trend toward decreased expression of proinflammatory markers was seen when compared to HTK. Unisol-based solutions showed an improved preservative capacity compared with the gold standard HTK early after cardiac transplantation. Supplemented glutathione did not further improve tissue-protective properties.
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Trasplante de Corazón , Soluciones Preservantes de Órganos , Animales , Dextranos , Disacáridos , Gluconatos/farmacología , Glutatión , Trasplante de Corazón/métodos , Humanos , Isquemia , Ratones , Preservación de Órganos/métodos , Soluciones Preservantes de Órganos/farmacología , Perfusión/métodos , Donantes de TejidosRESUMEN
In search for novel biomarkers to assess graft quality, we investigated whether defined candidate genes are predictive for outcome after liver transplantation (LT). Zero-hour liver biopsies were obtained from 88 livers. Gene expression of selected candidate markers was analyzed and correlated with clinical parameters as well as short and long-term outcomes post LT. Whereas both, the calculated Eurotransplant Donor-Risk-Index and the donor body mass index, had either a poor or no predictive value concerning serum levels indicative for liver function (ALT, AST, GGT, bilirubin) after 6 months, chronological donor age was weakly predictive for serum bilirubin (AUC=0.67). In contrast, the major histcompatibility complex class I related chain A (MICA) mRNA expression demonstrated a high predictive value for serum liver function parameters revealing an inverse correlation (e.g. for ALT: 3 months p=0.0332; 6 months p=0.007, 12 months 0.0256, 24 months p=0.0098, 36 months, p=0.0153) and proved significant also in a multivariate regression model. Importantly, high expression of MICA mRNA revealed to be associated with prolonged graft survival (p=0.024; log rank test) after 10 years of observation, whereas low expression was associated with the occurrence of death in patients with transplant related mortality (p=0.031). Given the observed correlation with short and long-term graft function, we suggest MICA as a biomarker for pre-transplant graft evaluation.
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Biomarcadores/metabolismo , Rechazo de Injerto/diagnóstico , Antígenos de Histocompatibilidad Clase I/metabolismo , Trasplante de Hígado , Hígado/metabolismo , Factores de Edad , Bilirrubina/sangre , Femenino , Rechazo de Injerto/mortalidad , Supervivencia de Injerto , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Hígado/patología , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Análisis de SupervivenciaRESUMEN
Histamine is metabolized by several enzymes in vitro and in vivo. The relevance of this metabolism in the mammalian heart in vivo is unclear. However, histamine can exert positive inotropic effects (PIE) and positive chronotropic effects (PCE) in humans via H2-histamine receptors. In transgenic mice (H2-TG) that overexpress the human H2 receptor in cardiomyocytes but not in wild-type littermate mice (WT), histamine induced PIE and PCE in isolated left or right atrial preparations. These H2-TG were used to investigate the putative relevance of histamine degrading enzymes in the mammalian heart. Histidine, the precursor of histamine, increased force of contraction (FOC) in human atrial preparations. Moreover, histamine increased the phosphorylation state of phospholamban in human atrium. Here, we could detect histidine decarboxylase (HDC) and histamine itself in cardiomyocytes of mouse hearts. Moreover, our data indicate that histamine is subject to degradation in the mammalian heart. Inhibition of the histamine metabolizing enzymes diamine oxidase (DAO) and monoamine oxidase (MAO) shifted the concentration response curves for the PIE in H2-TG atria to the left. Moreover, activity of histamine metabolizing enzymes was present in mouse cardiac samples as well as in human atrial samples. Thus, drugs used for other indication (e.g. antidepressants) can alter histamine levels in the heart. Our results deepen our understanding of the physiological role of histamine in the mouse and human heart. Our findings might be clinically relevant because we show enzyme targets for drugs to modify the beating rate and force of the human heart.
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BACKGROUND: In an experimental murine liver clamping model, we aimed to investigate the efficacy of real-time confocal microscopy (RCM) in assessing viability of steatotic livers in comparison to standard assessment tools, including histopathological evaluation. METHODS: C57Bl/6 mice were subjected to a methionine-choline-deficient diet causing nonalcoholic fatty liver disease or to Lieber DeCarli diet causing ethanol-induced liver injury. Untreated animals served as controls. Liver biopsies were analyzed following challenge with 45 min of warm ischemia time and either 4 h of reperfusion or 24 h of cold storage. Organ quality assessment was performed at defined time points by RCM, histological staining, measurement of serum alanine aminotransferase activity, and expression analyses of proinflammatory cytokines. Additionally, survival analysis was performed. RESULTS: Cold as well as warm ischemia time resulted in a significant decrease in cell viability when compared with naive livers as well as nonischemic-challenged steatotic livers (P < 0.05) as assessed by RCM. Furthermore, RCM revealed the actual cellular damage at early time points, while established methods including H&E-staining and serum transaminase profile failed. CONCLUSIONS: In a translational attempt, we demonstrate that RCM is a suitable diagnostic tool to obtain information about functional damage of the liver apart from standard approaches.
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Hígado Graso Alcohólico/patología , Hígado/patología , Microscopía Confocal , Enfermedad del Hígado Graso no Alcohólico/patología , Animales , Biopsia , Deficiencia de Colina/complicaciones , Isquemia Fría/efectos adversos , Modelos Animales de Enfermedad , Hígado Graso Alcohólico/etiología , Hígado Graso Alcohólico/genética , Hígado Graso Alcohólico/metabolismo , Hígado/metabolismo , Masculino , Metionina/deficiencia , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Valor Predictivo de las Pruebas , Reperfusión/efectos adversos , Daño por Reperfusión/etiología , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Factores de Tiempo , Supervivencia Tisular , Isquemia Tibia/efectos adversosRESUMEN
OBJECTIVE: Recently we characterized five mouse monoclonal antibodies that allow the specific and sensitive detection of human diamine oxidase (DAO). To understand differences in binding characteristics and recognition of enzyme variants, we mapped the antibody binding sites. METHODS: Fragments of human DAO were expressed as glutathione-S-transferase fusion proteins that were used for testing antibody binding on immunoblots. Combined information from species cross-reactivity, sequence comparison and binding site-prediction software were used to localize the epitope recognized by each antibody. RESULTS: All five monoclonal DAO antibodies bound to linear epitopes between the N3 and enzymatic domains of the 732 amino acid protein. The binding sites could be mapped onto amino acid regions V262-E278 and P279-R288, respectively, which exhibit considerable sequence variation in mammals explaining the fact that the human DAO antibodies do not cross-react with DAO from other species. The antibodies efficiently bind only denatured human DAO but not the native protein. CONCLUSIONS: Characterization of the binding sites of the DAO antibodies revealed that the antibodies bind two adjacent epitopes and exhibit similar binding characteristics and species cross-reactivity. As the epitopes do not overlap any of the amino acid substitutions described for clinically significant DAO gene polymorphisms, our antibodies will also be useful for analyses of the mutant DAO proteins.
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Amina Oxidasa (conteniendo Cobre)/metabolismo , Anticuerpos Monoclonales/farmacología , Sitios de Unión , Amina Oxidasa (conteniendo Cobre)/genética , Amina Oxidasa (conteniendo Cobre)/inmunología , ADN Complementario/genética , Epítopos , Escherichia coli/genética , Humanos , Plásmidos , Unión ProteicaRESUMEN
One factor that significantly contributes to renal allograft loss is chronic calcineurin inhibitor (CNI) nephrotoxicity (CIN). Among other factors, the complement (C-) system has been proposed to be involved CIN development. Hence, we investigated the impact of CNIs on intracellular signalling and the effects on the C-system in human renal tubule cells. In a qPCR array, CNI treatment upregulated C-factors and downregulated SOCS-3 and the complement inhibitors CD46 and CD55. Additionally, ERK1/-2 was required for these regulations. Following knock-down and overexpression of SOCS-3, we found that SOCS-3 inhibits ERK1/-2 signalling. Finally, we assessed terminal complement complex formation, cell viability and apoptosis. Terminal complement complex formation was induced by CNIs. Cell viability was significantly decreased, whereas apoptosis was increased. Both effects were reversed under complement component-depleted conditions. In vivo, increased ERK1/-2 phosphorylation and SOCS-3 downregulation were observed at the time of transplantation in renal allograft patients who developed a progressive decline of renal function in the follow-up compared to stable patients. The progressive cohort also had lower total C3 levels, suggesting higher complement activity at baseline. In conclusion, our data suggest that SOCS-3 inhibits CNI-induced ERK1/-2 signalling, thereby blunting the negative control of C-system activation.
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Inhibidores de la Calcineurina/efectos adversos , Proteínas del Sistema Complemento/metabolismo , Ciclosporina/efectos adversos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Rechazo de Injerto/metabolismo , Enfermedades Renales/metabolismo , Trasplante de Riñón , Túbulos Renales/efectos de los fármacos , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Tacrolimus/efectos adversos , Anciano , Anciano de 80 o más Años , Apoptosis , Antígenos CD55/metabolismo , Inhibidores de la Calcineurina/uso terapéutico , Línea Celular , Supervivencia Celular , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Ciclosporina/uso terapéutico , Femenino , Regulación de la Expresión Génica , Humanos , Enfermedades Renales/terapia , Túbulos Renales/patología , Sistema de Señalización de MAP Quinasas , Masculino , Proteína Cofactora de Membrana/metabolismo , Persona de Mediana Edad , Fosforilación , ARN Interferente Pequeño/genética , Proteína 3 Supresora de la Señalización de Citocinas/genética , Tacrolimus/uso terapéuticoRESUMEN
OBJECTIVE: Recently, we characterized mouse monoclonal antibodies that allow the specific and sensitive detection of human histamine N-methyltransferase (HNMT). To understand differences in binding characteristics and recognition of enzyme variants we mapped the antibody binding sites. METHODS: Fragments of human HNMT were expressed as glutathione S-transferase fusion proteins that were used for testing antibody binding on immunoblots. Combined information from species cross-reactivity, sequence comparison, protein structure, and binding site prediction software were used to localize the epitope recognized by each antibody. RESULTS: All eight monoclonal HNMT antibodies bound to linear epitopes in the C-terminal domain of the 292 amino acid protein. Of the five antibodies cross-reacting with HNMT from other species, one bound region L182-T223, three region M224-E261, and one region L262-A292. All three antibodies recognising only human HNMT bound the C-terminal region L262-A292 that contains residues present only in the human protein. CONCLUSIONS: Our HNMT monoclonal antibodies bind in three different regions of the protein and those binding the same putative epitope exhibit similar binding characteristics and species cross-reactivity. Antibodies binding non-overlapping epitopes will facilitate analyses of all clinically relevant variants described for HNMT.
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Anticuerpos Monoclonales/metabolismo , Histamina N-Metiltransferasa/metabolismo , Anticuerpos Monoclonales/química , Sitios de Unión , Epítopos/química , Epítopos/metabolismo , Histamina N-Metiltransferasa/química , Histamina N-Metiltransferasa/genética , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The definition of biological donor organ age rather than chronological age seems obvious for the establishment of a valid pre-transplant risk assessment. Therefore, we studied gene expression for candidate markers in 60 zero-hour kidney biopsies. Compared with 29 younger donors under age 55, 31 elderly donors age 55 and older had significant mRNA expression for immunoproteasome subunits (PSMB8, PSMB9 and PSMB10), HLA-DRB, and transcripts of the activating cytotoxicity receptor NKG2D. Gene expression was validated in an independent donor cohort consisting of 37 kidneys from donors 30 years and under (Group I), 75 kidneys from donors age 31-54 years (Group II) and 75 kidneys from donors age 55 and older (Group III). Significant gene induction was confirmed in kidneys from Group III for PSMB9 and PSMB10. Strikingly, transcripts of NKG2D had the significantly highest gene induction in Group III versus Group II and Group I. Similar results were obtained for CDKN2A, but not for telomere length. Both NKG2D and CDKN2A mRNA expression were significantly correlated with creatinine levels at 24 months after transplantation. Univariate regression analysis showed significant predictive power regarding graft function at 6 and 12 months for NKG2D and CDKN2A. However, only NKG2D remained significantly predictive in the multivariate model at 12 months. Thus, our results reveal novel candidate markers in aged renal allografts, which could be helpful in the assessment of organ quality.
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Senescencia Celular , Selección de Donante , Trasplante de Riñón/métodos , Riñón/química , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Donantes de Tejidos , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Austria , Biopsia , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Cisteína Endopeptidasas/genética , Funcionamiento Retardado del Injerto/genética , Femenino , Regulación de la Expresión Génica , Marcadores Genéticos , Alemania , Rechazo de Injerto/genética , Supervivencia de Injerto , Cadenas beta de HLA-DR/genética , Humanos , Riñón/patología , Trasplante de Riñón/efectos adversos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Valor Predictivo de las Pruebas , Complejo de la Endopetidasa Proteasomal/genética , ARN Mensajero/genética , Factores de Riesgo , Homeostasis del Telómero , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
OBJECTIVE: The lack of suitable antibodies for the histamine inactivating enzyme histamine N-methyltransferase (HMT) has so far prevented the direct analysis of HMT proteins in man and other mammals. METHODS: A series of monoclonal antibodies was produced by immunizing mice with human and porcine HMT expressed in vitro. Antibodies were characterized by immunoblotting and immunohistochemical staining. RESULTS: Six different monoclonal antibodies specific for human HMT and four different monoclonal antibodies specific for porcine HMT were obtained that can detect HMT with up to tenfold greater sensitivity than the most sensitive enzymatic assays currently available. Using these antibodies allowed us to confirm the expression and cellular localization of HMT in various human and porcine tissues, where the presence of the enzyme had previously been deduced from activity measurement and HMT mRNA analysis. Immunohistochemical staining of human and porcine tissue sections clearly showed that HMT is a cytosolic protein, which is localized in specific cells of most mammalian tissues. CONCLUSIONS: The new monoclonal antibodies not only allow a comprehensive quantitative evaluation of the expression of HMT at the cellular level in man and other mammals but will also facilitate sensitive analyses of disease-associated alterations of this protein.
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Anticuerpos Monoclonales/farmacología , Histamina N-Metiltransferasa/inmunología , Histamina N-Metiltransferasa/metabolismo , Adulto , Animales , Femenino , Glutatión Transferasa/genética , Histamina N-Metiltransferasa/genética , Humanos , Riñón/metabolismo , Masculino , Ratones Endogámicos BALB C , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/inmunología , Porcinos , Adulto JovenRESUMEN
BACKGROUND: Many diseases and pathological conditions are characterized by transient or constitutive overproduction of reactive oxygen species (ROS). ROS are causal for ischemia/reperfusion (IR)-associated tissue injury (IRI), a major contributor to organ dysfunction or failure. Preventing IRI with antioxidants failed in the clinic, most likely due to the difficulty to timely and efficiently target them to the site of ROS production and action. IR is also characterized by changes in the activity of intracellular signaling molecules including the stress kinase p38MAPK. While ROS can cause the activation of p38MAPK, we recently obtained in vitro evidence that p38MAPK activation is responsible for elevated mitochondrial ROS levels, thus suggesting a role for p38MAPK upstream of ROS and their damaging effects. RESULTS: Here we identified p38MAPKα as the predominantly expressed isoform in HL-1 cardiomyocytes and siRNA-mediated knockdown demonstrated the pro-oxidant role of p38MAPKα signaling. Moreover, the knockout of the p38MAPK effector MAPKAP kinase 2 (MK2) reproduced the effect of inhibiting or knocking down p38MAPK. To translate these findings into a setting closer to the clinic a stringent kidney clamping model was used. p38MAPK activity increased upon reperfusion and p38MAPK inhibition by the inhibitor BIRB796 almost completely prevented severe functional impairment caused by IR. Histological and molecular analyses showed that protection resulted from decreased redox stress and apoptotic cell death. CONCLUSIONS: These data highlight a novel and important mechanism for p38MAPK to cause IRI and suggest it as a potential therapeutic target for prevention of tissue injury.
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Apoptosis , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Estrés Oxidativo , Animales , Línea Celular , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Proteína Quinasa 14 Activada por Mitógenos/genética , Miocitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Endogámicas LewRESUMEN
We examined the influence of dietary fermentable protein (fCP) and fermentable carbohydrates (fCHO) on the colonic epithelial response to histamine in pigs. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO, low fCP/high fCHO, high fCP/low fCHO and high fCP/high fCHO. After 21-23 days, the pigs were killed and tissue from the proximal colon was stimulated with carbachol, histamine, PGE2 or sodium hydrogen sulphide in Ussing chambers. Changes in short-circuit current and tissue conductance were measured. Diamine oxidase, histamine N-methyltransferase, stem cell growth factor receptor, Fc-epsilon receptor I and cystic fibrosis transmembrane conductance regulator gene expression was determined. Activities of diamine oxidase and histamine N-methyltransferase and numbers of colonic mast cells were measured. The change in the short-circuit current in response to histamine was lower (P = 0.002) and tended to be lower for PGE2 (P = 0.053) in high fCP groups compared to low fCP groups, irrespective of fCHO. Additionally, the change in tissue conductance after the application of histamine was lower (P = 0.005) in the high fCP groups. The expression of histamine N-methyltransferase mRNA (P = 0.033) and the activities of diamine oxidase (P = 0.001) and histamine N-methyltransferase (P = 0.006) were higher with high fCP in comparison with low fCP. The expression of mast cell markers, stem cell growth factor receptor (P = 0.005) and Fc-epsilon receptor I (P = 0.049) was higher with high fCP diets compared to diets low in fCP, whereas the mast cell count did not differ between groups. The expression of the cystic fibrosis transmembrane conductance regulator was reduced (P = 0.001) with high fCP diets compared to low fCP diets. The lower epithelial response to histamine and PGE2 and elevated epithelial histamine inactivation suggests an adaptation to high fCP diets.
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Colon/metabolismo , Dieta , Glycine max/química , Histamina/metabolismo , Mucosa Intestinal/metabolismo , Alimentación Animal , Animales , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , D-Aminoácido Oxidasa/genética , D-Aminoácido Oxidasa/metabolismo , Carbohidratos de la Dieta/metabolismo , Proteínas en la Dieta/metabolismo , Activación Enzimática , Femenino , Expresión Génica , Masculino , Comidas , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de IgE/genética , Receptores de IgE/metabolismo , Porcinos , DesteteRESUMEN
Lipocalin-2 (Lcn2) expression contributes to ischemia and reperfusion injury (IRI) by enhancing pro-inflammatory responses. The aim of this work was to elucidate the regulation of Lcn2 during hypoxia and its effects on the expression of key chemokines and adhesion molecules. Lcn2 wt and Lcn2(-/-) mice were used in a heterotopic heart transplantation model. Quantitative RT-PCR was applied for chemokine gene expression analysis. Reporter gene studies were used to elucidate the regulation of the Lcn2 promoter by hypoxia. HIF-1ß expression led to a 2.4-fold induction of the Lcn2 promoter. Apart from an earlier onset of granulocyte infiltration in the Lcn2 wt setting after 2 h of reperfusion compared with the Lcn2(-/-) setting (P < 0.013), exogenous application of recombinant Lcn2 revealed a trend toward increase of granulocyte infiltration. Analyzed chemokines were expressed significantly higher in the Lcn2 wt setting at 2 h of reperfusion (P ≤ 0.05). The number of apoptotic cells observed in Lcn2(-/-) grafts was significantly higher than in the Lcn2 wt setting. Our results indicate that Lcn2 affects granulocyte infiltration in the reperfused graft by modulating the expression of chemokines, their receptors and the apoptotic rate.
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Proteínas de Fase Aguda/fisiología , Quimiocinas/genética , Trasplante de Corazón , Lipocalinas/fisiología , Daño por Reperfusión Miocárdica/inmunología , Infiltración Neutrófila , Proteínas Oncogénicas/fisiología , Proteínas de Fase Aguda/genética , Animales , Apoptosis , Células COS , Moléculas de Adhesión Celular/genética , Chlorocebus aethiops , Lipocalina 2 , Lipocalinas/genética , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/patología , Proteínas Oncogénicas/genética , Regiones Promotoras Genéticas , Receptores de Quimiocina/genéticaRESUMEN
OBJECTIVE: To study histamine transport and metabolism of salivary gland (SG) epithelial cells in healthy controls and SS patients. METHODS: Enzymes and transporters involved in histamine metabolism were analysed in cultured human submandibular salivary gland (HSG) epithelial cells and tissue sections using quantitative real-time PCR and immunostaining. HSG cells were used to study [(3)H]histamine uptake [(±1-methyl-4-phenylpyridinium (MPP)] and efflux by liquid scintillation counting. RESULTS: mRNA levels of l-histidine decarboxylase (HDC) and histamine-N-methyltransferase (HNMT) were similar in the control and SS glands, but diamine oxidase was not expressed at all. Organic cation transporter 3 (OCT3) in healthy SG was localized in the acinar and ductal cells, whereas OCT2 was restricted to the myoepithelial cells. Both transporters were significantly decreased in SS at mRNA and protein levels. OCT3-mRNA levels in HSG cells were significantly higher than those of the other studied transporters. Uptake of [(3)H]histamine was inhibited by MPP in a time-dependent manner, whereas [(3)H]histamine-preloaded HSG cells released it. CONCLUSION: Ductal epithelial cells are non-professional histamine-producing cells able to release histamine via OCTs at the resting state up to â¼100 nM, enough to excite H3R/H4R(+) epithelial cells, but not H1R, which requires burst release from mast cells. At the stimulated phase, 50-60 µM histamine passes from the interstitial fluid through the acinar cells to saliva, whereas uptake by ductal cells leads to intracellular degradation by HNMT. OCT3/histamine/H4R-mediated cell maintenance and down-regulation of high histamine levels fail in SS SGs.
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Transporte Biológico/fisiología , Células Epiteliales/metabolismo , Histamina/metabolismo , Síndrome de Sjögren/metabolismo , Glándula Submandibular/metabolismo , Células Cultivadas , Regulación hacia Abajo , Histamina N-Metiltransferasa/genética , Histamina N-Metiltransferasa/metabolismo , Histidina Descarboxilasa/genética , Histidina Descarboxilasa/metabolismo , Humanos , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Transportador 2 de Cátion OrgánicoRESUMEN
Diamine oxidase (DAO) was purified to homogeneity from human seminal plasma by consecutive chromatographic fractionation on heparin-sepharose, phenyl-sepharose, CIM-QA, and Superdex 200. Human seminal plasma DAO behaves electrophoretically similar to DAO proteins from other human tissues and has very similar enzymatic properties with histamine and aliphatic diamines being the preferred substrates as well as significant conversion of polyamines. The cellular source and functional importance of DAO in human semen remain to be determined.
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Amina Oxidasa (conteniendo Cobre)/metabolismo , Semen/enzimología , Adulto , Humanos , Masculino , Persona de Mediana Edad , Poliaminas , Sefarosa/análogos & derivadosRESUMEN
Diamine oxidase (DAO) oxidatively deaminates histamine and other diamines. Due to the lack of antibodies for human DAO, many findings on this enzyme had not been confirmed in man. Therefore, we produced a series of monoclonal antibodies by immunizing mice with human DAO protein fragments expressed in vitro. Five different monoclonal antibodies specific for human DAO were obtained that do not recognize any other human protein and can detect DAO with 100-fold greater sensitivity than the most sensitive enzymatic assays currently available. Using these antibodies allowed confirming the expression and cellular localization of DAO in various human tissues such as kidney, intestine and placenta where the presence of the enzyme had previously been deduced from activity measurement and DAO mRNA analysis. Due to the high sensitivity of the novel monoclonal antibodies, DAO was also detected at sites that previously evaded unequivocal proof of DAO enzymatic activity such as the urine. On the other hand, with these antibodies it was possible to show that DAO is normally not present in human liver and blood serum. The new monoclonal antibodies not only allow a comprehensive quantitative evaluation of the expression of DAO at the cellular level in man but will also facilitate sensitive analyses of disease-associated alterations of this enzyme.
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Amina Oxidasa (conteniendo Cobre)/inmunología , Amina Oxidasa (conteniendo Cobre)/metabolismo , Anticuerpos Monoclonales/metabolismo , Amina Oxidasa (conteniendo Cobre)/química , Amina Oxidasa (conteniendo Cobre)/genética , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Intestinos/enzimología , Riñón/enzimología , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/inmunología , Placenta/enzimología , Embarazo , Suero/enzimología , PorcinosRESUMEN
RATIONALE AND OBJECTIVES: The observation that the intravenous application of gadolinium-based contrast media can lead to nephrogenic systemic fibrosis (NSF) has raised interest in their interactions with pathways. In this context, histamine is a focus because of its stimulating effect on fibrogenesis. In humans, histamine can be inactivated either by diamine oxidase (DAO) or by histamine N-methyltransferase (HMT), and numerous drugs are known to inhibit these enzymes. Therefore, it was the aim of this study to investigate whether magnetic resonance imaging contrast agents have an inhibitory effect on the enzymatic activities of DAO and HMT. MATERIALS AND METHODS: Seven gadolinium-based (gadoterate meglumine, gadoteridol, gadobutrol, gadobenate dimeglumine, gadopentetate dimeglumine, gadoxetate disodium, gadodiamide) and one manganese-containing (mangafodipir) contrast agents were tested in vitro. Following the preincubation of purified DAO and HMT with 0.1 to 10 mmol/L of the respective contrast medium, enzyme activities were determined using radiometric microassays. Enzyme activities measured in the absence of contrast agents and after preincubation with specific inhibitors of DAO and HMT, respectively, served as controls. RESULTS: The gadolinium-containing and manganese-containing contrast media tested did not show significant inhibition of the activities of DAO and HMT. No significant difference was observed between ionic and nonionic or between cyclic and linear gadolinium compounds. Preincubation of the enzymes with specific inhibitors led to complete inhibition of the respective enzymatic activity. CONCLUSION: The gadolinium-containing and manganese-based magnetic resonance imaging contrast media tested did not exhibit significant inhibition of histamine-inactivating enzymes at physiologically relevant concentrations.
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Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Medios de Contraste/química , Gadolinio/química , Histamina N-Metiltransferasa/antagonistas & inhibidores , Histamina/química , Imagen por Resonancia Magnética/métodos , Activación EnzimáticaRESUMEN
INTRODUCTION: Alterations in mucosal histamine degradation play an important role in various gastrotinestinal diseases including colonic adenoma. In humans, histamine can be catabolized either by oxidative deamination by diamine oxidase (DAO) or by ring methylation by histamine N-methyltransferase (HNMT). The significance of HNMT in this context was investigated for the first time in this project. METHODS: About 94 colonic biopsies were endoscopically obtained from 23 patients suffering from colonic adenoma and 26 biopsies from six healthy individuals. Each sample was mechanically homogenized, homogenates were cleared by centrifugation and used for determination of protein and histamine concentrations and enzyme activities of DAO and HNMT by radiometric assay. RESULTS: In adenoma patients DAO activities were slightly and HNMT activities were significantly decreased in normal mucosa compared to controls. Activities of both enzymes were significantly lower in adenoma tissue than in healthy mucosa in the same patients. A significant correlation was found between HNMT and DAO in all investigated samples. Histamine concentrations were elevated in adenoma patients. CONCLUSIONS: Histamine catabolism is decreased in the colonic mucosa of patients with colonic adenoma.
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Adenoma/metabolismo , Neoplasias del Colon/metabolismo , Histamina/metabolismo , Mucosa Intestinal/metabolismo , Adenoma Velloso/metabolismo , Adulto , Anciano , Amina Oxidasa (conteniendo Cobre)/metabolismo , Femenino , Histamina N-Metiltransferasa/metabolismo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: Stressful life events and infections contribute to gut disorders such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). We used a pig model to analyse whether this could be linked to altered mediator sensitivity of the epithelial lining. MATERIAL AND METHODS: Uninfected control pigs or pigs with subclinical Salmonella (S.) typhimurium DT 104 infection were killed either without (ConRest, InfRest) or with prior 8-h transportation (ConTrans, InfTrans). Short-circuit current (I(sc)), tissue conductance (G(t)) and release of mast cell mediators were monitored in isolated colonic epithelia mounted in Ussing chambers. Epithelia were exposed to histamine (100 microM, mucosally), substance P (SP; 1 microM, serosally), calcimycin A23187 (1 microM, serosally) and theophylline (10 mM, bilaterally). Transepithelial flux of histamine and colonic activities of histamine N-methyltransferase (HMT) and diamine oxidase (DAO) were determined. RESULTS: S. infection decreased baseline I(sc), G(t) and histamine fluxes, while transportation had no effect on these values. Mucosal histamine increased I(sc) only in ConTrans pigs. This was not associated with increased mucosal-to-serosal flux of histamine but with a 2-fold increased DAO activity. Serosal SP increased I(sc) only in transported animals, but the increase was six times higher in ConTrans versus InfTrans pigs. Effectiveness of SP was not dependent on the release of histamine or prostaglandin D2. A23187 and theophylline elicited increases in I(sc) that were not different between treatments. CONCLUSIONS: Transportation stress facilitates secretory responses of the colonic epithelium to SP and luminal histamine. This is suppressed by subclinical S. infection. Effects of S. infection on porcine colon resemble, in part, the known effects of an oral S. endotoxin application.
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Colon/fisiopatología , Mucosa Intestinal/fisiopatología , Infecciones por Salmonella/fisiopatología , Estrés Fisiológico/fisiopatología , Animales , Colon/metabolismo , Liberación de Histamina , Mucosa Intestinal/metabolismo , Masculino , Prostaglandina D2/metabolismo , Infecciones por Salmonella/metabolismo , Porcinos , TransportesRESUMEN
OBJECTIVE: Catabolism of histamine plays a crucial role in the intestine in preventing intoxication by luminal histamine. Two enzymes are involved, namely histamine N-methyltransferase (HMT) and diamine oxidase (DAO). The purpose of this study was to find a link between histamine catabolism and the activities of HMT and DAO. MATERIAL AND METHODS: Epithelia of porcine proximal colon were mounted in Ussing chambers. After mucosal addition of (3)H-histamine (100 micromol x l(-1)) and (14)C-mannitol, the appearance of non-catabolized histamine, (3)H-histamine label (hist-rad) and (14)C-mannitol label were measured in parallel on the serosal side. Activities of HMT and DAO were determined in the proximal colon and proximal jejunum. RESULTS: Differences between the fluxes of histamine and hist-rad indicated catabolic conversion of 81.4+/-1.6% histamine during epithelial transit. Fluxes of hist-rad and histamine increased linearly with increasing mannitol fluxes but the percentage of catabolized histamine was not related to either mannitol or hist-rad fluxes. However, the percentage of catabolized histamine rose with increasing DAO activity. Given a negative correlation between DAO and HMT activities, the fraction of catabolized histamine decreased with increasing HMT activity. HMT activity was comparable in the colon and jejunum, but DAO activity was approximately nine times higher in the jejunum. CONCLUSIONS: Permeation, but not the relative efficiency of catabolism, of histamine depends on epithelial/paracellular tightness. While previous studies have shown that colonic HMT essentially catabolizes the bulk of histamine during permeation, DAO activity seems to be more variable and limiting for the overall efficiency of the catabolic process.
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Amina Oxidasa (conteniendo Cobre)/metabolismo , Colon/metabolismo , Histamina N-Metiltransferasa/metabolismo , Histamina/metabolismo , Mucosa Intestinal/enzimología , Amina Oxidasa (conteniendo Cobre)/análisis , Animales , Radioisótopos de Carbono , Activación Enzimática , Histamina N-Metiltransferasa/análisis , Mucosa Intestinal/metabolismo , Masculino , Manitol/farmacocinética , Orquiectomía , Permeabilidad , Sus scrofaRESUMEN
BACKGROUND: Increased activity of the immuno-modulatory enzyme indoleamine-2,3-dioxygenase (IDO) during immune activation, results in tryptophan depletion. Tryptophan metabolic changes reduce serotonin production and cause mood disturbances, depression, and impaired satiety, ultimately leading to increased food intake and obesity. Bariatric surgery significantly diminishes immune mediators by substantial weight reduction. We examined IDO-mediated tryptophan-catabolism in morbidly obese patients compared to lean individuals. METHODS: Serum concentrations of kynurenine and tryptophan, calculated kynurenine to tryptophan ratios (kyn trp-1) as an indirect estimate of IDO activity, and neopterin levels reflecting IFN-gamma mediated immune activation, were assessed before and after bariatric surgery. The study population included 22 morbidly obese individuals and 20 normal-weight volunteers. RESULTS: Median weight loss after 24.4+/-5.1 months was 40.6 kg resulting in a reduction of BMI from 44.1 kg/m(2) to 29.9 kg/m(2) (P<0.001). Preoperative kyn trp-1 in morbidly obese patients was significantly increased compared to the control group (41.6+/-20.1 mmol/mol vs 26.5+/-5.1 mmol/mol; P<0.001). Postoperative weight reduction did not lead to normalization of kyn trp-1 (37.9+/-14.0 mmol/mol). As a consequence, tryptophan levels were significantly lower in morbidly obese patients (pre-: 51.5+/-9.2 micromol L(-1) and postoperatively: 46.9+/-7.6 micromol L(-1)) when compared with those of normal-weight controls (64.8+/-9.5 micromol L(-1); P<0.001). In addition, neopterin levels were elevated in the study population pre- and postoperatively compared to normal-weight volunteers (both P<0.001). CONCLUSIONS: Tryptophan depletion in morbidly obese patients is due to chronic immune activation and persists in spite of significant weight reduction following bariatric surgery. This might thereby be responsible for diminished serotonin functions, leading to unchanged satiety dysregulation and a reward-deficiency-syndrome.