Vascular adhesion protein-1 blockade in primary sclerosing cholangitis: Open-label, multicenter, single-arm, phase II trial.

BACKGROUND: Primary sclerosing cholangitis is a progressive inflammatory liver disease characterized by biliary and liver fibrosis. Vascular adhesion protein-1 (VAP-1) is important in the inflammatory process driving liver fibrosis. We evaluated the safety and efficacy of VAP-1 blockade with a monoclonal antibody (timolumab, BTT1023) in patients with primary sclerosing cholangitis. METHODS: BUTEO was a prospective, single-arm, open-label, multicenter, phase II trial, conducted in 6 centers in the United Kingdom. Patients with primary sclerosing cholangitis aged 18-75 years had an alkaline phosphatase value of >1.5 times the upper limit of normal. The dose-confirmatory stage aimed to confirm the safety of timolumab through the incidence of dose-limiting toxicity and sufficient trough levels of circulating antibody to block VAP-1 function. The primary outcome of the dose-expansion portion of the trial was patient's response to timolumab at day 99, as measured by a reduction in serum alkaline phosphatase by 25% or more from baseline to day 99. RESULTS: Twenty-three patients were recruited: 7 into the initial dose-confirmatory stage and a further 16 into an expansion stage. Timolumab (8 mg/kg) was confirmed to be safe for the duration of administration with sufficient circulating levels. Only 2 of the 18 evaluable patients (11.1%) achieved a reduction in alkaline phosphatase levels of 25% or more, and both the proportion of circulating inflammatory cell populations and biomarkers of fibrosis remained unchanged from baseline. CONCLUSIONS: The BUTEO trial confirmed 8 mg/kg timolumab had no short-term safety signals and resulted in sufficient circulating levels of VAP-1 blocking timolumab. However, the trial was stopped after an interim assessment due to a lack of efficacy as determined by no significant change in serum liver tests.

Pilot Study on the Prevalence of Diamine Oxidase Gene Variants in Patients with Symptoms of Histamine Intolerance.

A retrospective pilot study was carried out to investigate the prevalence of four variants of the diamine oxidase (DAO) encoding gene (AOC1) in Caucasian adults with symptoms of histamine intolerance. In a cohort of 100 patients and 100 healthy individuals, DAO-encoding gene non-synonymous Single Nucleotide Variations (SNVs) were genotyped by multiplex single-nucleotide primer extension (SNPE) and capillary electrophoresis, and serum DAO activity was analyzed with a radio-extraction assay. The study found that 79% of individuals with symptoms of histamine intolerance harbored one or more of the four SNVs associated with reduced DAO activity. No significant differences were found in the prevalence of any variant between the group of patients and healthy controls. However, when considering the status of the alleles associated with DAO deficiency, more homozygous alleles were observed in histamine-intolerant patients. Moreover, a slightly but statistically higher percentage of patients had a high genetic risk score, reflecting the cumulative effect of carrying multiple DAO deficiency-associated gene variants and a high load of risk alleles (homozygous). A relationship between serum DAO activity and the genetic load of one specific SNV was observed, with DAO activity being significantly lower in patients homozygous for rs2052129. These results potentially support that carrying multiple DAO deficiency-associated gene variants and a high load of risk alleles (homozygous) is more relevant than the mere presence of one or more SNVs. Further studies are needed to determine the predictive value of these DAO-encoding gene variants.

Bifidobacterium longum K5 Prevents Enterohaemorrhagic Escherichia coli O157:H7 Infection in Mice through the Modulation of the Gut Microbiota.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a commonly encountered foodborne pathogen that can cause hemorrhagic enteritis and lead to hemolytic uremic syndrome (HUS) in severe cases. Bifidobacterium is a beneficial bacterium that naturally exists in the human gut and plays a vital role in maintaining a healthy balance in the gut microbiota. This study investigated the protective effects of B. longum K5 in a mouse model of EHEC O157:H7 infection. The results indicated that pretreatment with B. longum K5 mitigated the clinical symptoms of EHEC O157:H7 infection and attenuated the increase in myeloperoxidase (MPO) activity in the colon of the mice. In comparison to the model group, elevated serum D-lactic acid concentrations and diamine oxidase (DAO) levels were prevented in the K5-EHEC group of mice. The reduced mRNA expression of tight junction proteins (ZO-1, Occludin, and Claudin-1) and mucin MUC2, as well as the elevated expression of virulence factors Stx1A and Stx2A, was alleviated in the colon of both the K5-PBS and K5-EHEC groups. Additionally, the increase in the inflammatory cytokine levels of TNF-α and IL-1ß was inhibited and the production of IL-4 and IL-10 was promoted in the K5-EHEC group compared with the model group. B. longum K5 significantly prevented the reduction in the abundance and diversity of mouse gut microorganisms induced by EHEC O157:H7 infection, including blocking the decrease in the relative abundance of Roseburia, Lactobacillus, and Oscillibacter. Meanwhile, the intervention with B. longum K5 promoted the production of acetic acid and butyric acid in the gut. This study provides insights into the use of B. longum K5 for developing probiotic formulations to prevent intestinal diseases caused by pathogenic bacterial infections.

Crosstalk of cell death pathways unveils an autophagy-related gene AOC3 as a critical prognostic marker in colorectal cancer.

The intricate crosstalk of various cell death forms was recently implicated in cancers, laying a foundation for exploring the association between cell death and cancers. Recent evidence has demonstrated that biological networks outperform snapshot gene expression profiles at discovering promising biomarkers or heterogenous molecular subtypes across different cancer types. In order to investigate the behavioral patterns of cell death-related interaction perturbation in colorectal cancer (CRC), this study constructed the interaction-perturbation network with 11 cell death pathways and delineated four cell death network (CDN) derived heterogeneous subtypes (CDN1-4) with distinct molecular characteristics and clinical outcomes. Specifically, we identified a subtype (CDN4) endowed with high autophagy activity and the worst prognosis. Furthermore, AOC3 was identified as a potential autophagy-related biomarker, which demonstrated exceptional predictive performance for CDN4 and significant prognostic value. Overall, this study sheds light on the complex interplay of various cell death forms and reveals an autophagy-related gene AOC3 as a critical prognostic marker in CRC.

Effects of Lonicerae flos and Turmeric extracts on growth performance and intestinal health of yellow-feathered broilers.

This experiment aimed to investigate the effect of Lonicerae flos and Turmeric extracts (LTE) added to diets on growth performance and intestinal health of broilers. A total of 720 healthy 21-day-old yellow-feathered broilers were randomly divided into 3 treatment groups, with 6 replicates and 40 broilers per replicate. These 3 dietary treatments included a basal diet + 0 g/t LTE (CON), a basal diet + 300 g/t LTE (LTE300), and a basal diet + 500 g/t LTE (LTE500). The results showed that dietary supplementation of LTE linearly increased (P < 0.05) average daily gain (d 21-38) and average daily feed intake (d 21-60). At d 60, LTE300 had the highest serum total antioxidant capacity and total superoxide dismutase (P < 0.05), and LTE500 had the lowest malondialdehyde level (P < 0.05) among the three groups. Moreover, compared to CON, LTE300 significantly (P < 0.05) reduced endotoxin (d 38 and d 60) and diamine oxidase activity (d 38); LTE500 significantly (P < 0.05) reduced endotoxin (d 38 and d 60) and diamine oxidase levels (d 60) in the serum. LTE groups significantly (P < 0.05) increased ileal the ratio of villus height to crypt depth and serum immunoglobulin G. Furthermore, dietary supplementation of LTE also improved the intestinal epithelial barrier by the up-regulated mRNA expression of Claudin-1, Occludin and zonula occludens-1, and decreased the mRNA expression of interleukin-2, interleukin-8, tumor necrosis factor-α, nuclear factor κB, myeloid differentiation factor 88 and toll-like receptor 4. Compared to CON, 16S rRNA sequencing analysis showed that LTE300 had a better effect on the microbial diversity and composition in the ileum, and Bacillus and Lactobacillus_agilis were significantly enriched in LTE300. PICRUSt results showed that LTE300 was significantly (P < 0.05) enriched in four pathway pathways at KEGG level 2. In conclusion, dietary supplementation with LTE improved growth performance and intestinal health by enhancing antioxidant capacity, intestinal barrier and immune function, and regulating intestinal flora of yellow-feathered broilers.

ω-(5-Phenyl-2H-tetrazol-2-yl)alkyl-substituted glycine amides and related compounds as inhibitors of the amine oxidase vascular adhesion protein-1 (VAP-1).

Vascular adhesion protein-1 (VAP-1), also known as plasma amine oxidase or semicarbazide-sensitive amine oxidase, is an enzyme that degrades primary amines to aldehydes with the formation of hydrogen peroxide and ammonia. Among others, it plays a role in inflammatory processes as it can mediate the migration of leukocytes from the blood to the inflamed tissue. We prepared a series of ω-(5-phenyl-2H-tetrazol-2-yl)alkyl-substituted glycine amides and related compounds and tested them for inhibition of purified bovine plasma VAP-1. Compounds with submicromolar activity were obtained. Studies on the mechanism of action revealed that the glycine amides are substrate inhibitors, i.e., they are also converted to an aldehyde derivative. However, the reaction proceeds much more slowly than that of the substrate used in the assay, whose conversion is thus blocked. Examination of the selectivity of the synthesized glycine amides with respect to other amine oxidases showed that they inhibited diamine oxidase, which is structurally related to VAP-1, but only to a much lesser extent. In contrast, the activity of monoamine oxidase A and B was not affected. Selected compounds also inhibited VAP-1 in human plasma. The IC50 values measured were higher than those determined with the bovine enzyme. However, the structure-activity relationships obtained with the glycine amides were similar for both enzymes.

Genome-Wide Identification, Characterization, and Expression Analysis of the Copper-Containing Amine Oxidase Gene Family in Mangrove Kandelia obovata.

Copper-containing amine oxidases (CuAOs) are known to have significant involvement in the process of polyamine catabolism, as well as serving crucial functions in plant development and response to abiotic stress. A genome-wide investigation of the CuAO protein family was previously carried out in sweet orange (Citrus sinensis) and sweet cherry (Prunus avium L.). Six CuAO (KoCuAO1-KoCuAO6) genes were discovered for the first time in the Kandelia obovata (Ko) genome through a genome-wide analysis conducted to better understand the key roles of the CuAO gene family in Kandelia obovata. This study encompassed an investigation into various aspects of gene analysis, including gene characterization and identification, subcellular localization, chromosomal distributions, phylogenetic tree analysis, gene structure analysis, motif analysis, duplication analysis, cis-regulatory element identification, domain and 3D structural variation analysis, as well as expression profiling in leaves under five different treatments of copper (CuCl2). Phylogenetic analysis suggests that these KoCuAOs, like sweet cherry, may be subdivided into three subgroups. Examining the chromosomal location revealed an unequal distribution of the KoCuAO genes across four out of the 18 chromosomes in Kandelia obovata. Six KoCuAO genes have coding regions with 106 and 159 amino acids and exons with 4 and 12 amino acids. Additionally, we discovered that the 2.5 kb upstream promoter region of the KoCuAOs predicted many cis elements linked to phytohormones and stress responses. According to the expression investigations, CuCl2 treatments caused up- and downregulation of all six genes. In conclusion, our work provides a comprehensive overview of the expression pattern and functional variety of the Kandelia obovata CuAO gene family, which will facilitate future functional characterization of each KoCuAO gene.

LncRNA AOC4P recruits TRAF6 to regulate EZH2 ubiquitination and participates in trophoblast glycolysis and M2 macrophage polarization which is associated with recurrent spontaneous abortion.

During embryo implantation, trophoblast cells rely on large amounts of energy produced by glycolysis for their rapid growth and invasion. The disorder of trophoblast metabolism may lead to recurrent spontaneous abortion (RSA). Lactate, which is produced by the glycolysis of trophoblast cells during early pregnancy, can promote the polarization of M2 macrophages and maintain an anti-inflammatory environment at the maternal-fetal interface. Our study found that amine oxidase copper-containing 4 pseudogene (AOC4P) was abnormally increased in villi from RSA patients. It inhibited the glycolysis of trophoblast cells and thus hindered the polarization of M2 macrophages. Further studies showed that AOC4P combines with tumor necrosis factor receptor-associated factor 6 (TRAF6) to upregulate TRAF6 expression. TRAF6 acted as an E3 ubiquitin ligase to promote ubiquitination and degradation of zeste homolog 2 (EZH2). These results provided new insights into the important role played by AOC4P at the maternal-fetal interface.

A randomised Phase IIa trial of amine oxidase copper-containing 3 (AOC3) inhibitor BI 1467335 in adults with non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a progressive, inflammatory liver disease with no approved pharmacological treatment. This Phase IIa, double-blind, placebo-controlled, multicentre trial (ClinicalTrials.gov: NCT03166735) investigated pharmacodynamics and safety of BI 1467335, an amine oxidase copper-containing 3 (AOC3) inhibitor, in adults with NASH from Europe and North America. Participants from 44 centres across the US, Germany, Spain, Belgium, the UK, Netherlands, Canada, France and Ireland were randomised (2:1:1:1:2; 27 July 2017 to 14 June 2019) to daily oral BI 1467335 1 mg (n = 16), 3 mg (n = 16), 6 mg (n = 17), 10 mg (n = 32) or placebo (n = 32) for 12 weeks, with follow-up to Week 16. Primary endpoint was AOC3 activity relative to baseline (%), 24 hours post-dose after 12 weeks' treatment. Secondary biomarker endpoints included changes from baseline at Week 12 in alanine aminotransferase (ALT) and caspase-cleaved cytokeratin 18 (CK-18 caspase). Mean AOC3 activities relative to baseline at Week 12: 90.4% (placebo; n = 32), 26.5% (1 mg; n = 16), 10.4% (3 mg; n = 16), 5.0% (6 mg; n = 16), 3.3% (10 mg; n = 32). These changes indicated that BI 1467335 dose-dependently inhibited AOC3 activity; ≥3 mg doses achieved >80% inhibition ( < 20% activity) at Week 4. At Week 12 following doses of BI 1467335 ≥ 3 mg, ALT and CK-18 caspase decreased dose-dependently. All tested BI 1467335 doses were well tolerated, with no clinically relevant treatment-emergent safety signals. BI 1467335 strongly inhibited AOC3 in participants with NASH, with doses ≥3 mg dose-dependently reducing the levels of liver injury biomarkers, ALT and CK-18. This trial was registered with ClinicalTrials.gov (NCT03166735) and the European Union Drug Regulating Authorities Clinical Trials Database (EudraCT 2016-000499-83).

Recombinant human diamine oxidase prevents hemodynamic effects of continuous histamine infusion in guinea pigs.

OBJECTIVE: To test whether recombinant human diamine oxidase (rhDAO) with a mutated heparin-binding motif (mHBM), which shows an increased alpha-distribution half-life, prevents histamine-induced hemodynamic effects. MATERIAL: Thirty-eight female guinea pigs were either pretreated with rhDOA_mHBM or buffer. TREATMENT AND METHODS: Guinea pigs received a continuous infusion of histamine. Heart rate (HR), body core temperature and mean arterial pressure (MAP) were measured and blood was collected. RESULTS: Continuous intravenous infusion of 8 µg/kg/min histamine increased mean peak plasma histamine levels from 5 (± 0.3 SEM) to 28 ng/mL (± 4.9 SEM) after 30 min but had no effect on oxygen saturation. Guinea pigs pretreated with 4 mg/kg rhDAO_mHBM showed lower mean HR (p = 0.008), histamine plasma concentrations (p = 0.002), and higher body core temperatures at the end of the histamine challenge (p = 0.02) compared to controls. Cessation of histamine infusion led to a rebound increase in MAP, but this hemodynamic instability was prevented by rhDAO_mHBM. Pretreatment with 4 mg/kg rhDAO_mHBM reduced urinary histamine (p = 0.004) and 1-Methylhistamine (p < 0.0001) concentrations compared to controls. CONCLUSIONS: Prophylactic infusion of rhDAO_mHBM prevents hemodynamic effects in a guinea pig model of continuous histamine infusion. These findings might help in the translation from animals to humans and in the selection of the optimal dosing of rhDAO_mHBM during human histamine challenge studies.

Evaluation of Serum Diamine Oxidase as a Diagnostic Test for Histamine Intolerance.

Histamine intolerance (HIT) is a clinical condition caused by decreased intestinal degradation of ingested histamine, primarily due to reduced enzyme diamine oxidase (DAO) activity, leading to histamine accumulation and causing various clinical manifestations. The measurement of serum DAO is commonly used as the main diagnostic test for HIT, although its diagnostic use is still uncertain. In this retrospective study, we aimed to assess the validity of DAO determination in patients with clinically suspected HIT. We measured DAO levels in 249 patients with suspected HIT and 50 healthy adult controls without HIT-related problems. Based on five clinical criteria, we divided patients into two groups: high (all five inclusion criteria; 41 patients) and low probability of HIT (≤4 inclusion criteria; 208 patients). Patients with a "high probability of HIT" had the lowest DAO (median: 8 U/mL, IQR: 6-10) in comparison to patients with a "low probability of HIT (median: 10 U/mL, IQR: 7-16, p = 0.0006) and healthy controls (median: 18 U/mL, IQR: 14-22, p < 0.0001). The specificity and sensitivity for DAO levels < 3/< 10 U/mL (manufacturer's set cut-off) to discriminate between patients with ''high probability of HIT'' and healthy controls were 100%/92% and 2%/71%. On the other hand, the specificity and sensitivity to discriminate between patients with ''high probability of HIT'' and ''low probability of HIT'' were 97%/61% and 2%/71%, respectively. Serum DAO determination represents an additional asset to the diagnosis of HIT based on clinical evaluation and assessment, but the diagnosis should not solely rely on DAO measurements.

LOXL2 in Cancer: A Two-Decade Perspective.

Lysyl Oxidase Like 2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises five lysine tyrosylquinone (LTQ)-dependent copper amine oxidases in humans. In 2003, LOXL2 was first identified as a promoter of tumour progression and, over the course of two decades, numerous studies have firmly established its involvement in multiple cancers. Extensive research with large cohorts of human tumour samples has demonstrated that dysregulated LOXL2 expression is strongly associated with poor prognosis in patients. Moreover, investigations have revealed the association of LOXL2 with various targets affecting diverse aspects of tumour progression. Additionally, the discovery of a complex network of signalling factors acting at the transcriptional, post-transcriptional, and post-translational levels has provided insights into the mechanisms underlying the aberrant expression of LOXL2 in tumours. Furthermore, the development of genetically modified mouse models with silenced or overexpressed LOXL2 has enabled in-depth exploration of its in vivo role in various cancer models. Given the significant role of LOXL2 in numerous cancers, extensive efforts are underway to identify specific inhibitors that could potentially improve patient prognosis. In this review, we aim to provide a comprehensive overview of two decades of research on the role of LOXL2 in cancer.

Placebo-Controlled Histamine Challenge Disproves Suspicion of Histamine Intolerance.

BACKGROUND: Histamine intolerance (HIT) is frequently diagnosed in patients with polysymptomatic otherwise unexplained symptoms. OBJECTIVES: To exclude HIT by a single-blind placebo-controlled histamine challenge (SBPCHC), to study clinical features of patients with positive challenge, and to examine the predictability of HIT by biomarkers. METHODS: SBPCHC was performed in 59 patients with suspected HIT. History and clinical data, including serum diamine oxidase (DAO) and histamine skin test wheal size of patients with positive versus negative SBPCHC, were compared. RESULTS: Patients were predominantly middle-aged women (84.7%). Three-quarters reported improvement but never resolution of symptoms during a histamine-low diet. Histamine provocation was safe; only 1 patient was treated with antihistamines. Thirty-seven patients (62.7%) displayed symptoms to placebo. HIT was excluded in 50 patients (84.7%). Objective symptoms occurred in 4 of 59 cases (6.8%) after histamine but not after placebo challenge. These were diagnosed with "plausible HIT" because reactions occurring by chance could not be excluded. Another 5 patients (8.5%) were diagnosed with "possible HIT" after case-dependent detailed analysis. Patients with plausible/possible HIT had reported more gastrointestinal symptoms (P = .01), but comparable diet response and equal histamine skin prick test wheal sizes to those without HIT. Serum DAO activity tended to be lower in patients with HIT (P = .08), but was highly variable in those without, limiting its value as a biomarker. CONCLUSIONS: SBPCHC disproves HIT in the majority of patients. Placebo-controlled challenges are needed as placebo reactions were frequent. Gastrointestinal symptoms after food intake and reduced DAO levels are markers for HIT; however, specificity is not sufficient enough for making the diagnosis.

The Use of DAO as a Marker for Histamine Intolerance: Measurements and Determinants in a Large Random Population-Based Survey.

Histamine intolerance (HIT) is a common adverse reaction to food where elimination and reintroduction of histamine-rich food is part of the investigation. Analysis of the enzyme diamine oxidase (DAO) is sometimes used as an additional tool for diagnosis. This study aimed to describe the distribution of DAO in a large representative cohort of adults and to determine the association between DAO activity and possible associated factors. The study is based on the population-based West Sweden Asthma Study and includes 1051 subjects. Subjects underwent structured interviews including questions on demography, asthma, allergy symptoms, and lifestyle factors. Subjects were assessed for specific-IgE-antibodies and measurement of DAO activity in serum. Previously suggested cut-off levels for low values (<3 U/mL), normal values (>10 U/mL), and median levels of DAO were used. In the group of 1051 subjects, only a few presented reactions upon histamine intake, whereas 44% presented DAO levels below the suggested normal cut-off levels. BMI and age were shown to have an impact on DAO activity among women with increasing activity of DAO with increasing BMI and age. Among men, only increasing age was seen to have an impact on DAO levels. There was no difference in DAO levels with different sensitization status to common foods or airborne allergens. No association between DAO levels and reported symptoms to histamine-rich foods could be found. In conclusion, the determination of the DAO enzyme needs to be re-evaluated and may not be used as a valuable tool for histamine intolerance using current cut-off values. Further studies are needed to improve the use of DAO as a biomarker for histamine intolerance.

P19-derived neuronal cells express H1, H2, and H3 histamine receptors: a biopharmaceutical approach to evaluate antihistamine agents.

Histamine is a biogenic amine implicated in various biological and pathological processes. Convenient cellular models are needed to screen and develop new antihistamine agents. This report aimed to characterize the response of neurons differentiated from mouse P19 embryonal carcinoma cells to histamine treatment, and to investigate the modulation of this response by antihistamine drugs, vegetal diamine oxidase, and catalase. The exposure of P19 neurons to histamine reduced cell viability to 65% maximally. This effect involves specific histamine receptors, since it was prevented by treatment with desloratadine and cimetidine, respectively, H1 and H2 antagonists, but not by the H3 antagonist ciproxifan. RT-PCR analysis showed that P19 neurons express H1 and H2 receptors, and the H3 receptor, although it seemed not involved in the histamine effect on these cells. The H4 receptor was not expressed. H1 and H2 antagonists as well as vegetal diamine oxidase diminished the intracellular Ca2+ mobilization triggered by histamine. The treatment with vegetal diamine oxidase or catalase protected against mortality and a significant reduction of H2O2 level, generated from the cells under the histamine action, was found upon treatments with desloratadine, cimetidine, vegetal diamine oxidase, or catalase. Overall, the results indicate the expression of functional histamine receptors and open the possibility of using P19 neurons as model system to study the roles of histamine and related drugs in neuronal pathogenesis. This model is less expensive to operate and can be easily implemented by current laboratories of analysis and by Contract Research Organizations.

Pathogenetic Mechanisms of Mental Disorders: Endogenous Intoxication.

The review describes the syndrome of endogenous intoxication in patients with mental disorders. Oxidative stress, middle-mass endotoxic molecules, impaired functional properties of serum albumin and albumin thiol groups, neurotrophic factors, and enzymes, including monoamine oxidase and semicarbazide-sensitive amine oxidase contribute to the development of endogenous intoxication. Possible pathogenetic mechanisms of the endogenous intoxication development in mental disorders and approaches to its treatment are discussed.

Evaluation of tributyrin supplementation in milk replacer on diarrhoea occurrence in preweaning Holstein calves.

Neonatal calf diarrhoea is one of the most important health challenges in cattle herds causing substantial economic losses and antimicrobial use. Due to the raising problem of antimicrobial resistance, effective alternatives are urgently required, in line with European policies. The aim of this study was to evaluate the effect of tributyrin supplementation in milk replacer on diarrhoea, performance and metabolic status in preweaning Holstein calves. Twelve newborn calves, after colostrum administration, were randomly allotted in two experimental groups for 42 days: control (CTRL) fed milk replacer, tributyrin (TRIB) fed milk replacer supplemented with 0.3% of liquid tributyrin on milk powder weight. Calves BW was recorded on a weekly basis from day 7 to day 42, and feed intake was recorded daily to calculate zootechnical performance. Faecal consistency was assessed daily through the faecal score (0-3 scale; considering diarrhoea moderate = 2 and severe = 3). Faecal samples were collected weekly from rectal ampulla for microbiological analysis by plate counting method evaluating the number of total bacteria, lactic acid bacteria and coliform bacteria. On day 0 and day 42, individual blood samples were collected from jugular vein for metabolic profile analysis. Serum samples of day 42 were also evaluated for the antioxidant barrier using a colorimetric test, while glucagon-like peptide 2 and diamine oxidase concentrations were measured through immunoenzymatic assays. Tributyrin supplementation did not influence the zootechnical performance of calves over 42 days of trial. Diarrhoea frequency was significantly lower in TRIB compared to CTRL group (27.91 and 38.37%; P < 0.01) considering the whole experimental period. In particular, the major effect was observed for moderate diarrhoea in TRIB group that showed a significantly reduced frequency compared to CTRL (P < 0.01) thus suggesting a preventive effect of tributyrin. Faecal total bacterial, lactic acid and coliform bacteria counts did not show differences between groups. Urea serum concentrations tended to be lower in TRIB compared to CTRL, indicating an efficient utilisation of dietary protein. Antioxidant barrier and glucagon-like peptide 2 were comparable between CTRL and TRIB on day 42. Diamine oxidase concentrations were significantly decreased in TRIB compared to CTRL group after 42 days of trial (P < 0.01), suggesting a higher gut epithelial integrity probably due to lower diarrhoea frequency and the nourish effect of tributyrin on enterocytes. In conclusion, tributyrin could be considered as a valuable bioactive feed additive to decrease the neonatal diarrhoea occurrence and support intestinal integrity in preweaning calves.

Strikingly conserved gene expression changes of polyamine regulating enzymes among various forms of acute and chronic kidney injury.

The polyamines spermidine and spermine and their common precursor molecule putrescine are involved in tissue injury and repair. Here, we test the hypothesis that impaired polyamine homeostasis contributes to various kidney pathologies in mice during experimental models of ischemia-reperfusion, transplantation, rhabdomyolysis, cyclosporine treatment, arterial hypertension, diabetes, unilateral ureteral obstruction, high oxalate feeding, and adenine-induced injuries. We found a remarkably similar pattern in most kidney pathologies with reduced expression of enzymes involved in polyamine synthesis together with increased expression of polyamine degrading enzymes. Transcript levels of amine oxidase copper-containing 1 (Aoc1), an enzyme which catalyzes the breakdown of putrescine, were barely detectable by in situ mRNA hybridization in healthy kidneys. Aoc1 was highly expressed upon various experimental kidney injuries resulting in a significant reduction of kidney putrescine content. Kidney levels of spermine were also significantly reduced, whereas spermidine was increased in response to ischemia-reperfusion injury. Increased Aoc1 expression in injured kidneys was mainly accounted for by an Aoc1 isoform that harbors 22 additional amino acids at its N-terminus and shows increased secretion. Mice with germline deletion of Aoc1 and injured kidneys showed no decrease of kidney putrescine content; although they displayed no overt phenotype, they had fewer tubular casts upon ischemia-reperfusion injury. Hyperosmotic stress stimulated AOC1 expression at the transcriptional and post-transcription levels in metanephric explants and kidney cell lines. AOC1 expression was also significantly enhanced after kidney transplantation in humans. These data demonstrate that the kidneys respond to various forms of injury with down-regulation of polyamine synthesis and activation of the polyamine breakdown pathway. Thus, an imbalance in kidney polyamines may contribute to various etiologies of kidney injury.

Diamine Oxidase as a Therapeutic Enzyme: Study of Germination from Vegetal Sources and Investigation of the Presence of ß-N-Oxalyl-L-α,ß-diaminopropionic Acid (ß-ODAP) Using LC-MS/MS.

Vegetal diamine oxidase (vDAO), an enzyme proposed to relieve symptoms of histaminosis, shows better reactivity with histamine and aliphatic diamines, as well as higher enzymatic activity than DAO of animal origin. The objective of this study was to evaluate the enzyme activity of vDAO from germinating grains from Lathyrus sativus (grass pea) and Pisum sativum (pea), and to verify the presence of a neurotoxin, ß-N-Oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), in the crude extract obtained from their seedlings. A targeted liquid chromatography-multiple-reaction monitoring mass spectrometry method was developed and used to quantify ß-ODAP in the analysed extracts. An optimized sample preparation procedure, involving protein precipitation with acetonitrile followed by mixed-anion exchange solid-phase extraction, allowed for high sensitivity and good peak shape for ß-ODAP detection. The Lathyrus sativus extract exhibited the highest vDAO enzyme activity of the extracts, followed by the extract from pea cultivar Amarillo from the Crop Development Centre (CDC). The results have also shown that even though ß-ODAP was present in the crude extract from L. sativus, its content was far below the toxicity threshold (300 mg of ß-ODAP/kg body/day). CDC Amarillo showed 5000-fold less ß-ODAP than the undialysed L. sativus extract. It was concluded that both species can be considered as convenient sources of vDAO for potential therapeutic use.

The Impact of Semicarbazide Sensitive Amine Oxidase Activity on Rat Aortic Vascular Smooth Muscle Cells.

Semicarbazide-sensitive amine oxidase (SSAO) is both a soluble- and membrane-bound transmembrane protein expressed in the vascular endothelial and in smooth muscle cells. In vascular endothelial cells, SSAO contributes to the development of atherosclerosis by mediating a leukocyte adhesion cascade; however, its contributory role in the development of atherosclerosis in VSMCs has not yet been fully explored. This study investigates SSAO enzymatic activity in VSMCs using methylamine and aminoacetone as model substrates. The study also addresses the mechanism by which SSAO catalytic activity causes vascular damage, and further evaluates the contribution of SSAO in oxidative stress formation in the vascular wall. SSAO demonstrated higher affinity for aminoacetone when compared to methylamine (Km = 12.08 µM vs. 65.35 µM). Aminoacetone- and methylamine-induced VSMCs death at concentrations of 50 & 1000 µM, and their cytotoxic effect, was reversed with 100 µM of the irreversible SSAO inhibitor MDL72527, which completely abolished cell death. Cytotoxic effects were also observed after 24 h of exposure to formaldehyde, methylglyoxal and H2O2. Enhanced cytotoxicity was detected after the simultaneous addition of formaldehyde and H2O2, as well as methylglyoxal and H2O2. The highest ROS production was observed in aminoacetone- and benzylamine-treated cells. MDL72527 abolished ROS in benzylamine-, methylamine- and aminoacetone-treated cells (**** p < 0.0001), while ßAPN demonstrated inhibitory potential only in benzylamine-treated cells (* p < 0.05). Treatment with benzylamine, methylamine and aminoacetone reduced the total GSH levels (**** p < 0.0001); the addition of MDL72527 and ßAPN failed to reverse this effect. Overall, a cytotoxic consequence of SSAO catalytic activity was observed in cultured VSMCs where SSAO was identified as a key mediator in ROS formation. These findings could potentially associate SSAO activity with the early developing stages of atherosclerosis through oxidative stress formation and vascular damage.