Mapping of the binding sites of human diamine oxidase (DAO) monoclonal antibodies.

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.

Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies.

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.

Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies.

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.

Histamine transport and metabolism are deranged in salivary glands in Sjogren's syndrome.

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 ∼100 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.

Characterization of diamine oxidase from human seminal plasma.

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.

New tools for studying old questions: antibodies for human diamine oxidase.

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.

Histamine can be Formed and Degraded in the Human and Mouse Heart.

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.

Autonomous histamine metabolism in human melanoma cells.

Melanoma cells constitutively produce various cytokines as well as growth factors and express their corresponding receptors. Exogenous histamine is known to be a growth factor for some tumours while in other cases histamine inhibits tumour growth, and acts on G protein-coupled H1 and H2 histamine receptors. In previous studies we have detected the expression of the l-histidine decarboxylase (HDC) gene and the presence of HDC protein in human melanoma cell lines. In the present study, the activities of the histamine-forming enzyme HDC and of the degrading enzymes diamine oxidase (DAO) and histamine N-methyltransferase (HNMT) were measured in primary (WM35 and WM983) and metastatic (M1 and HT168) human melanoma cell lines. HDC activity was found in WM35 and WM983 cell lines, while detectable HNMT activity was measured in WM983, M1 and HT168 lines. In contrast, DAO showed very low activity in melanoma cell lines. Melanoma cells release a detectable amount of histamine into the medium without external stimuli. These findings support the possibility of autonomous histamine metabolism in melanoma cells. Our results suggest that not only exogenous histamine but also histamine produced and released by the melanoma cells and acting as an autocrine and paracrine factor may influence cell proliferation and modulate the in situ immune response of the host.

Continuous administration of heparin in patients with deep vein thrombosis can increase plasma levels of diamine oxidase.

Besides its anticoagulant activity, the sulfated polysaccharide heparin has numerous other biological effects. Especially the antiinflammatory and immunoregulatory properties of heparin may be associated with its ability to release the histamine-degrading enzyme diamine oxidase (DAO) from tissue-bound sites into the circulation. Whereas DAO activity is at the limits of detection in normal human plasma, the application of heparin leads to a significant increase of plasma DAO activity. However, previously, only the effect of bolus injection of unfractionated heparin (UFH) had been studied. To investigate DAO release during continuous heparin infusion, 28 patients with deep vein thrombosis (DVT) undergoing heparin therapy were analyzed. Whereas continuous heparin infusion did not lead to any increase of plasma DAO activity in 12 patients (43%), 6 patients (21%) showed a single elevated and 10 patients (36%) permanently elevated plasma DAO activity. The groups of patients exhibiting different DAO release responses did not differ in age, sex, body weight, concomitant diseases, heparin infusion rates, coagulation indices, location and extension of thrombosis, or clinical outcome. However, the rate of idiopathic DVT was significantly higher in the group of patients releasing DAO. This study shows, for the first time, that continuous heparin infusion can lead to DAO release and that individuals exhibit considerable differences in their release response. Although the significance of heparin-induced DAO release needs further clarification, our results indicate that postheparin plasma DAO activity could be an interesting parameter correlated with idiopathic DVT.

Diets high in heat-treated soybean meal reduce the histamine-induced epithelial response in the colon of weaned piglets and increase epithelial catabolism of histamine.

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.

Influence of magnetic resonance contrast media on the activity of histamine inactivating enzymes.

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.

Decreased histamine catabolism in the colonic mucosa of patients with colonic adenoma.

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.

Functional characteristics of the porcine colonic epithelium following transportation stress and Salmonella infection.

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.

Histamine inactivation in the colon of pigs in relationship to abundance of catabolic enzymes.

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.

Histamine-degrading enzymes as cellular markers of acute small bowel allograft rejection.

Intestinal histamine-degrading enzymes diamine oxidase (DAO) and histamine N-methyltransferase (HNMT) activities are relatively constant per individual and bowel segment, and they reflect the functional integrity of the intestinal mucosa. It was, therefore, hypothesised that a decrease in these enzymes could be indicative of acute rejection of an intestinal allograft. Enzymatic activities of DAO and HNMT were determined in mucosal biopsies of isogeneic (Lewis-to-Lewis, n=48) and allogeneic (Brown Norway-to-Lewis, n=48) heterotopic small bowel transplants in a rat model at various time periods. Allograft recipients were not given any immunosuppression. While no changes in enzyme activities were observed in isografts up to day 8 following transplantation, significantly reduced activities of both enzymes were found in all allografts 6-8 days after transplantation. Activities of both DAO and HNMT exhibited a strong negative correlation with the histological rejection score ( P<0.01). We can conclude that DAO and HNMT activities in gut mucosa are reliable quantitative markers of acute intestinal allograft rejection in the rat that support histopathological analysis.