Combination of tea catechins and ornithine effectively activates the urea cycle: an in vitro and human pilot study.

PURPOSE: Accumulation of ammonia causes central and peripheral fatigue. This study aimed to investigate the synergistic effect of tea catechins and low-dose ornithine in activating the urea cycle to reduce blood ammonia levels during exercise. METHODS: We used hepatocyte-like cells derived from human-induced pluripotent stem (iPS) cells to assess the effect of tea catechins combined with ornithine on urea cycle activity. The urea production and expression of key genes involved in the metabolism of urea were investigated. We then examined the synergistic improvement in ammonia metabolism by tea catechins in combination with ornithine in a human pilot study. RESULTS: Tea catechins combined with ornithine increased urea cycle activity in hepatocyte-like cells derived from human iPS cells. Intake of 538.6 mg of tea catechins with 1592 mg of ornithine for 2 consecutive days during exercise loading suppressed the exercise-induced increase in the blood ammonia concentration as well as stabilized blood glucose levels. CONCLUSION: Controlling the levels of ammonia, a toxic waste produced in the body, is important in a variety of situations, including exercise. The present study suggests that a heterogeneous combination of polyphenols and amino acids efficiently suppresses elevated ammonia during exercise in humans by a mechanism that includes urea cycle activation. TRIAL REGISTRATION: This study was registered in the University Hospital Medical Information Network Clinical Trial Registry (No. UMIN000035484, dated January 8, 2019).

Pentachlorophenol exposure induced neurotoxicity by disrupting citrulline metabolism in larvae and adult zebrafish.

Pentachlorophenol (PCP) is a ubiquitous environmental toxicant with various adverse effects. Although its neurotoxicity has been reported, the underlying mechanism and subsequent detoxification remain unclear. In this study, embryos and adult zebrafish were exposed to PCP to determine its potential neurotoxic mechanism and protective indicators. The survival rate, heart rate, mobility time, active status and moving distance were significantly decreased in larvae after 30 µg/L PCP exposure. Likewise, the mobile time, latency to the first movement, velocity and moving distance of adult zebrafish were significantly reduced by PCP exposure. Untargeted metabolomics analysis of larvae revealed that arginine and proline metabolism was the primary pathway affected by PCP exposure, reflected by increased proline and decreased citrulline (CIT) contents, which were confirmed by quantitative data. PCP exposure suppressed the conversion from arginine to CIT in larvae by downregulating the expression of nos1 and nos2a. Ornithine content was increased in the brains and intestines of adult zebrafish after PCP exposure, which inhibited ornithine catabolism to CIT by downregulating otc, resulting in reduced CIT. Intriguingly, CIT supplementation significantly restored the neurobehavioral defects induced by PCP in larvae and adult zebrafish. CIT supplementation upregulated the expression of ef1α and tuba1 in larvae and inhibited the downregulation of ef1α in the brains of adult zebrafish. Taken together, these results indicated that CIT supplementation could protect against PCP-induced neurotoxicity by upregulating the expression of genes involved in neuronal development and function.

An orally deliverable ornithine-based self-assembling polymer nanomedicine ameliorates hyperammonemia in acetaminophen-induced acute liver injury.

l-Ornithine (Orn) is a core amino acid responsible for ammonia detoxification in the body via the hepatic urea cycle. Clinical studies in Orn therapy have focused on interventions for hyperammonemia-associated diseases, such as hepatic encephalopathy (HE), a life-threatening neurological symptom affecting more than 80% of patients with liver cirrhosis. However, its low molecular weight (LMW) causes Orn to diffuse nonspecifically and be rapidly eliminated from the body after oral administration, resulting in unfavorable therapeutic efficacy. Hence, Orn is constantly supplied by intravenous infusion in many clinical settings; however, this treatment inevitably decreases patient compliance and limits its application in long-term management. To improve the performance of Orn, we designed self-assembling polyOrn-based nanoparticles for oral administration through ring-opening polymerization of Orn-N-carboxy anhydride initiated with amino-ended poly(ethylene glycol), followed by acylation of free amino groups in the main chain of the polyOrn segment. The obtained amphiphilic block copolymers, poly(ethylene glycol)-block-polyOrn(acyl) (PEG-block-POrn(acyl)), enabled the formation of stable nanoparticles (NanoOrn(acyl)) in aqueous media. We employed the isobutyryl (iBu) group for acyl derivatization in this study (NanoOrn(iBu)). In the healthy mice, daily oral administration of NanoOrn(iBu) for one week did not induce any abnormalities. In the mice exhibiting acetaminophen (APAP)-induced acute liver injury, oral pretreatment with NanoOrn(iBu) effectively reduced systemic ammonia and transaminases levels compared to the LMW Orn and untreated groups. The results suggest that the application of NanoOrn(iBu) is of significant clinical value with the feasibility of oral delivery and improvement in APAP-induced hepatic pathogenesis. STATEMENT OF SIGNIFICANCE: Liver injury is often accompanied by hyperammonemia, a life-threatening condition characterized by elevated blood ammonia levels. Current clinical treatments for reducing ammonia typically entail the invasive approach of intravenous infusion, involving the administration of l-ornithine (Orn) or a combination of Orn and L-aspartate. This method is employed due to the poor pharmacokinetics associated with these compounds. In our pursuit of enhancing therapy, we have developed an orally administrable nanomedicine based on Orn-based self-assembling nanoparticle (NanoOrn(iBu)), which provides sustained Orn supply to the injured liver. Oral administration of NanoOrn(iBu) to healthy mice did not cause any toxic effects. In a mouse model of acetaminophen-induced acute liver injury, oral administration of NanoOrn(iBu) surpassed Orn in reducing systemic ammonia levels and liver damage, thereby establishing NanoOrn(iBu) as a safe and effective therapeutic option.

13 C NMR quantification of polyamine syntheses in rat prostate.

Currently, many prostate cancer patients, detected through the prostate specific antigen test, harbor organ-confined indolent disease that cannot be differentiated from aggressive cancer according to clinically and pathologically known measures. Spermine has been considered as an endogenous inhibitor for prostate-confined cancer growth and its expression has shown correlation with prostate cancer growth rates. If established clinically, measurements of spermine bio-synthesis rates in prostates may predict prostate cancer growth and patient outcomes. Using rat models, we tested the feasibility of quantifying spermine bio-synthesis rates with 13 C NMR. Male Copenhagen rats (10 weeks, n = 6) were injected with uniformly 13 C-labeled L-ornithine HCl, and were sacrificed in pairs at 10, 30, and 60 min after injection. Another two rats were injected with saline and sacrificed at 30 min as controls. Prostates were harvested and extracted with perchloric acid and the neutralized solutions were examined by 13 C NMR at 600 MHz. 13 C NMR revealed measurable ornithine, as well as putrescine-spermidine-spermine syntheses in rat prostates, allowing polyamine bio-synthetic and ornithine bio-catabolic rates to be calculated. Our study demonstrated the feasibility of 13 C NMR for measuring bio-synthesis rates of ornithine to spermine enzymatic reactions in rat prostates. The current study established a foundation upon which future investigations of protocols that differentiate prostate cancer growth rates according to the measure of ornithine to spermine bio-synthetic rates may be developed.

Inducement of ER Stress by PAD Inhibitor BB-Cl-Amidine to Effectively Kill AML Cells.

OBJECTIVE: Acute myeloid leukemia (AML) is a highly heterogeneous and recurrent hematological malignancy. Despite the emergence of novel chemotherapy drugs, AML patients' complete remission (CR) remains unsatisfactory. Consequently, it is imperative to discover new therapeutic targets or medications to treat AML. Such epigenetic changes like DNA methylation and histone modification play vital roles in AML. Peptidylarginine deminase (PAD) is a protein family of histone demethylases, among which the PAD2 and PAD4 expression have been demonstrated to be elevated in AML patients, thus suggesting a potential role of PADs in the development or maintenance of AML and the potential for the identification of novel therapeutic targets. METHODS: AML cells were treated in vitro with the pan-PAD inhibitor BB-Cl-Amidine (BB-Cl-A). The AML cell lines were effectively induced into apoptosis by BB-Cl-A. However, the PAD4-specific inhibitor GSK484 did not. RESULTS: PAD2 played a significant role in AML. Furthermore, we found that BB-Cl-A could activate the endoplasmic reticulum (ER) stress response, as evidenced by an increase in phosphorylated PERK (p-PERK) and eIF2α (p-eIF2α). As a result of the ER stress activation, the BB-Cl-A effectively induced apoptosis in the AML cells. CONCLUSION: Our findings indicated that PAD2 plays a role in ER homeostasis maintenance and apoptosis prevention. Therefore, targeting PAD2 with BB-Cl-A could represent a novel therapeutic strategy for treating AML.

Compromised glycolysis contributes to foot process fusion of podocytes in diabetic kidney disease: Role of ornithine catabolism.

INTRODUCTION: Compromised glycolysis in podocytes contributes to the initiation of diabetic kidney disease (DKD). Podocyte injury is characterized by cytoskeletal remodeling and foot process fusion. Compromised glycolysis in diabetes likely leads to switch of energy supply in podocyte. However, the underlying mechanism by which disturbed energy supply in podocytes affects the cytoskeletal structure of podocytes remains unclear. METHODS: Metabolomic and transcriptomic analyses were performed on the glomeruli of db/db mice to examine the catabolism of glucose, fatty, and amino acids. Ornithine catabolism was targeted in db/db and podocyte-specific pyruvate kinase M2 knockout (PKM2-podoKO) mice. In vitro, expression of ornithine decarboxylase (ODC1) was modulated to investigate the effect of ornithine catabolism on mammalian target of rapamycin (mTOR) signaling and cytoskeletal remodeling in cultured podocytes. RESULTS: Multi-omic analyses of the glomeruli revealed that ornithine metabolism was enhanced in db/db mice compared with that in db/m mice under compromised glycolytic conditions. Additionally, ornithine catabolism was exaggerated in podocytes of diabetic PKM2-podoKO mice compared with that in diabetic PKM2flox/flox mice. In vivo, difluoromethylornithine (DFMO, inhibitor of ODC1) administration reduced urinary albumin excretion and alleviated podocyte foot process fusion in db/db mice. In vitro, 2-deoxy-d-glucose (2-DG) exposure induced mTOR signaling activation and cytoskeletal remodeling in podocytes, which was alleviated by ODC1-knockdown. Mechanistically, a small GTPase Ras homolog enriched in the brain (Rheb), a sensor of mTOR signaling, was activated by exposure to putrescine, a metabolic product of ornithine catabolism. CONCLUSION: These findings demonstrate that compromised glycolysis in podocytes under diabetic conditions enhances ornithine catabolism. The metabolites of ornithine catabolism contribute to mTOR signaling activation via Rheb and cytoskeletal remodeling in podocytes in DKD.

Blood-brain barrier dysfunction in L-ornithine induced acute pancreatitis in rats and the direct effect of L-ornithine on cultured brain endothelial cells.

BACKGROUND: In severe acute pancreatitis (AP) the CNS is affected manifesting in neurological symptoms. Earlier research from our laboratory showed blood-brain barrier (BBB) permeability elevation in a taurocholate-induced AP model. Here we aimed to further explore BBB changes in AP using a different, non-invasive in vivo model induced by L-ornithine. Our goal was also to identify whether L-ornithine, a cationic amino acid, has a direct effect on brain endothelial cells in vitro contributing to the observed BBB changes. METHODS: AP was induced in rats by the intraperitoneal administration of L-ornithine-HCl. Vessel permeability and the gene expression of the primary transporter of L-ornithine, cationic amino acid transporter-1 (Cat-1) in the brain cortex, pancreas, liver and lung were determined. Ultrastructural changes were followed by transmission electron microscopy. The direct effect of L-ornithine was tested on primary rat brain endothelial cells and a triple co-culture model of the BBB. Viability and barrier integrity, including permeability and TEER, nitrogen monoxide (NO) and reactive oxygen species (ROS) production and NF-κB translocation were measured. Fluorescent staining for claudin-5, occludin, ZO-1, ß-catenin, cell adhesion molecules Icam-1 and Vcam-1 and mitochondria was performed. Cell surface charge was measured by laser Doppler velocimetry. RESULTS: In the L-ornithine-induced AP model vessel permeability for fluorescein and Cat-1 expression levels were elevated in the brain cortex and pancreas. On the ultrastructural level surface glycocalyx and mitochondrial damage, tight junction and basal membrane alterations, and glial edema were observed. L-ornithine decreased cell impedance and elevated the BBB model permeability in vitro. Discontinuity in the surface glycocalyx labeling and immunostaining of junctional proteins, cytoplasmic redistribution of ZO-1 and ß-catenin, and elevation of Vcam-1 expression were measured. ROS production was increased and mitochondrial network was damaged without NF-κB, NO production or mitochondrial membrane potential alterations. Similar ultrastructural changes were seen in L-ornithine treated brain endothelial cells as in vivo. The basal negative zeta potential of brain endothelial cells became more positive after L-ornithine treatment. CONCLUSION: We demonstrated BBB damage in the L-ornithine-induced rat AP model suggesting a general, AP model independent effect. L-ornithine induced oxidative stress, decreased barrier integrity and altered BBB morphology in a culture BBB model. These data suggest a direct effect of the cationic L-ornithine on brain endothelium. Endothelial surface glycocalyx injury was revealed both in vivo and in vitro, as an additional novel component of the BBB-related pathological changes in AP.

Population Pharmacokinetic Analysis to Assist Dose Selection of the L-Ornithine Salt of Phenylacetic Acid.

BACKGROUND AND OBJECTIVE: L-Ornithine phenylacetate is an intravenous formulation of the L-ornithine salt of phenylacetic acid under development for the treatment of hepatic encephalopathy. Very limited clinical data in patients are available, with a phase II study in target patients not designed for dose finding, to support phase III dose selection in a global development program. The objective of the present population pharmacokinetic modeling and simulation was to evaluate dose selection for target patient populations with a low body weight, ethnicity, and hepatic impairment in a global clinical study. METHODS: A population pharmacokinetic model was developed based on plasma concentrations of L-ornithine, phenylacetic acid, and phenylacetylglutamine data from four clinical trials in healthy subjects and patients with stable cirrhosis or hospitalized adult patients with liver cirrhosis and hepatic encephalopathy. A covariate analysis was conducted to identify source of variability to support dose selection for global clinical development of L-ornithine phenylacetate. Phenylacetylglutamine formation in the pharmacokinetic model also quantified pharmacodynamic effects measured by ammonia removal. RESULTS: Body weight and hepatic function were significant covariates determining phenylacetic acid exposure. After accounting for body weight, there was no difference between tested Caucasian and Asian populations in phenylacetic acid exposure. Renal dysfunction significantly reduced phenylacetylglutamine excretion. However, renal impairment had no impact on plasma phenylacetic acid and free ammonia levels. Exploratory modeling suggested that L-ornithine might enhance the removal of ammonia. CONCLUSIONS: With a flat dosing algorithm, special consideration must be given to patients with a small body size (i.e., body weight ≤ 50 kg) and severe hepatic impairment.

Review of arginase as a promising biocatalyst: characteristics, preparation, applications and future challenges.

As a committed step in the urea cycle, arginase cleaves l-arginine to form l-ornithine and urea. l-Ornithine is essential to: cell proliferation, collagen formation and other physiological functions, while the urea cycle itself converts highly toxic ammonia to urea for excretion. Recently, arginase was exploited as an efficient catalyst for the environmentally friendly synthesis of l-ornithine, an abundant nonprotein amino acid that is widely employed as a food supplement and nutrition product. It was also proposed as an arginine-reducing agent in order to treat arginase deficiency and to be a means of depleting arginine to treat arginine auxotrophic tumors. Targeting arginase inhibitors of the arginase/ornithine pathway offers great promise as a therapy for: cardiovascular, central nervous system diseases and cancers with high arginase expression. In this review, recent advances in the characteristics, structure, catalytic mechanism and preparation of arginase were summarized, with a focus being placed on the biotechnical and medical applications of arginase. In particular, perspectives have been presented on the challenges and opportunities for the environmentally friendly utilization of arginase during l-ornithine production and in therapies.

Inhibition of PAD4-mediated NET formation by cl-amidine prevents diabetes development in nonobese diabetic mice.

Many evidences indicated that neutrophil extracellular traps (NETs) play pathogenic roles in type 1 diabetes (T1D). Peptidylarginine deiminases 4 (PAD4) has been proved to be indispensable for generation of NETs. In the current study, we investigated whether oral administration of cl-amidine, an effective inhibitor of PAD4, protects non-obese diabetic (NOD) mice from T1D development. Female NOD mice were orally administrated with cl-amidine (5 µg/g body weight) from the age of 8 weeks up to 16 weeks. It showed that cl-amidine inhibit NET formation in vitro and in vivo. The onset of T1D was delayed nearly 8 weeks and the incidence of disease was significantly decreased in cl-amidine treated mice compared with the control group. Moreover, cl-amidine decreased the serum levels of anti-citrullinated peptide antibody (ACPA) and anti-neutrophil cytoplasmic antibodies (ANCA) in NOD mice. Also, it decreased generation of T1D autoantibodies such as glutamic acid decarboxylase antibody (GADA), tyrosine phosphatase-related islet antigen-2 antibody (IA2A) and zinc transporter 8 antibody (ZnT8A), which were strongly correlated with the reduced serum PAD4 and MPO-DNA levels. Furthermore, cl-amidine administration inhibited pancreatic inflammation and increased frequency of regulatory T cells in pancreatic lymph nodes (PLNs). In addition, cl-amidine improved gut barrier dysfunction and decreased the serum level of lipopolysaccharide (LPS), which was positively correlated with the NETs markers (PAD4 and MPO-DNA) and T1D autoantibody IA2A. In conclusion, our data showed that orally delivery of cl-amidine effectively prevent T1D development and suggested inhibition of PAD4-dependent NET formation as a potential way of clinical treatment in T1D.

Systemic L-ornithine supplementation specifically increases ovarian putrescine levels during ovulation in mice†.

In all mammalian species examined thus far, the ovaries produce a burst of ornithine decarboxylase (ODC) and putrescine during ovulation or after application of human chorionic gonadotropin (hCG). Aged mice have significantly reduced levels of this periovulatory ODC and putrescine rise. Putrescine supplementation, in vitro during oocyte maturation or in mouse drinking water during the periovulatory period, reduces egg aneuploidies and embryo resorption, improving fertility of aged mice. These studies suggest that periovulatory putrescine supplementation may be a simple and effective therapy for reproductive aging for women. However, putrescine supplementation is expected to increase widespread tissue putrescine levels, raising concerns of nonspecific and unwanted side effects. Given that ODC is highly expressed in the ovaries during ovulation but otherwise exhibits low activity in most tissues, we hypothesized that periovulatory supplementation of L-ornithine, the substrate of ODC, might be suitable for delivering putrescine specifically to the ovaries. In this study, we have demonstrated that systemic application of L-ornithine via oral gavage or subcutaneous injection increased ovarian putrescine levels; the increase was restricted to animals that had been injected with hCG. Furthermore, L-ornithine specifically increased ovarian putrescine levels without affecting putrescine levels in any other tissues. However, our attempts to improve fertility of aged mice through L-ornithine supplementation in mouse drinking water produced either no effects (1% L-ornithine) or negative impact on fertility (4% ornithine). Our results suggest that it might not be feasible to achieve fertility-enhancing ovarian putrescine levels via L-ornithine supplementation in drinking water without encountering undesired consequences of high dose of exogenous L-ornithine.

Additive Effects of L-Ornithine on Preferences to Basic Taste Solutions in Mice.

In addition to the taste receptors corresponding to the six basic taste qualities-sweet, salty, sour, bitter, umami, and fatty-another type of taste receptor, calcium-sensing receptor (CaSR), is found in taste-bud cells. CaSR is called the 'kokumi' receptor because its agonists increase sweet, salty and umami tastes to induce 'koku', a Japanese word meaning the enhancement of flavor characters such as thickness, mouthfulness, and continuity. Koku is an important factor for enhancing food palatability. However, it is not well known whether other kokumi-receptors and substances exist. Here, we show that ornithine (L-ornithine but not D-ornithine) at low concentrations that do not elicit a taste of its own, enhances preferences to sweet, salty, umami, and fat taste solutions in mice. Increased preference to monosodium glutamate (MSG) was the most dominant effect. Antagonists of G-protein-coupled receptor family C group 6 subtype A (GPRC6A) abolished the additive effect of ornithine on MSG solutions. The additive effects of ornithine on taste stimuli are thought to occur in the oral cavity, and are not considered post-oral events because ornithine's effects were confirmed in a brief-exposure test. Moreover, the additive effects of ornithine and the action of the antagonist were verified in electrophysiological taste nerve responses. Immunohistochemical analysis implied that GPRC6A was expressed in subsets of type II and type III taste cells of mouse circumvallate papillae. These results are in good agreement with those reported for taste modulation involving CaSR and its agonists. The present study suggests that ornithine is a kokumi substance and GPRC6A is a newly identified kokumi receptor.

Protective Effects of Swertiamarin against Methylglyoxal-Induced Epithelial-Mesenchymal Transition by Improving Oxidative Stress in Rat Kidney Epithelial (NRK-52E) Cells.

Increased blood glucose in diabetic individuals results in the formation of advanced glycation end products (AGEs), causing various adverse effects on kidney cells, thereby leading to diabetic nephropathy (DN). In this study, the antiglycative potential of Swertiamarin (SM) isolated from the methanolic extract of E. littorale was explored. The effect of SM on protein glycation was studied by incubating bovine serum albumin with fructose at 60 °C in the presence and absence of different concentrations of swertiamarin for 24 h. For comparative analysis, metformin was also used at similar concentrations as SM. Further, to understand the role of SM in preventing DN, in vitro studies using NRK-52E cells were done by treating cells with methylglyoxal (MG) in the presence and absence of SM. SM showed better antiglycative potential as compared to metformin. In addition, SM could prevent the MG mediated pathogenesis in DN by reducing levels of argpyrimidine, oxidative stress and epithelial mesenchymal transition in kidney cells. SM also downregulated the expression of interleukin-6, tumor necrosis factor-α and interleukin-1ß. This study, for the first time, reports the antiglycative potential of SM and also provides novel insights into the molecular mechanisms by which SM prevents toxicity of MG on rat kidney cells.

Folate transporter dynamics and therapy with classic and tumor-targeted antifolates.

There are three major folate uptake systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). We studied the functional interrelationships among these systems for the novel tumor-targeted antifolates AGF94 (transported by PCFT and FRs but not RFC) and AGF102 (selective for FRs) versus the classic antifolates pemetrexed, methotrexate and PT523 (variously transported by FRs, PCFT and RFC). We engineered HeLa cell models to express FRα or RFC under control of a tetracycline-inducible promoter with or without constitutive PCFT. We showed that cellular accumulations of extracellular folates were determined by the type and levels of the major folate transporters, with PCFT and RFC prevailing over FRα, depending on expression levels and pH. Based on patterns of cell proliferation in the presence of the inhibitors, we established transport redundancy for RFC and PCFT in pemetrexed uptake, and for PCFT and FRα in AGF94 uptake; uptake by PCFT predominated for pemetrexed and FRα for AGF94. For methotrexate and PT523, uptake by RFC predominated even in the presence of PCFT or FRα. For both classic (methotrexate, PT523) and FRα-targeted (AGF102) antifolates, anti-proliferative activities were antagonized by PCFT, likely due to its robust activity in mediating folate accumulation. Collectively, our findings describe a previously unrecognized interplay among the major folate transport systems that depends on transporter levels and extracellular pH, and that determines their contributions to the uptake and anti-tumor efficacies of targeted and untargeted antifolates.

Neutrophil Extracellular Traps (NETs) and Vasculitis.

Background: Neutrophil extracellular traps (NETs) have been implicated in host immune responses. Attempts have been made to examine how NETs affect the pathogenesis of complications such as autoimmune and vascular disorders. Aim: This study aimed to explore the relationship between NETs and vasculitis. Material and Methods: The current study entailed the searching of PsycINFO, PubMed, Web of Science, and CINAHL for articles related to the research topic. The search terms and phrases included "vasculitis," "NETs," "neutrophil extracellular traps," "NETosis," and "pathogenesis." The search was limited to articles published between 2009 and 2019. Results: Researchers have shown that NETs contribute to the pathogenesis of vasculitis through different mechanisms and processes, including renal failure and vascular damage. The protective effects of NETs have also been highlighted. Discussion: Overall, some scholars have shown the effectiveness of using DNase I and the PAD4 inhibitor Cl-amidine to treat vasculitis by restricting NET formation. However, observations have been noted in only animal experimental models. Conclusion: Neutrophil hyperactivity and its role in vasculitis are not yet fully understood. More studies aiming to determine the accurate function of NETs in vasculitis pathogenesis, particularly in humans, should be undertaken. Intensive research on NETs and vasculitis can increase the knowledge of medical practitioners and contribute to the development of new treatment methods to enhance patient outcomes in the future.

Peptidylarginine Deiminase Inhibitor Application, Using Cl-Amidine, PAD2, PAD3 and PAD4 Isozyme-Specific Inhibitors in Pancreatic Cancer Cells, Reveals Roles for PAD2 and PAD3 in Cancer Invasion and Modulation of Extracellular Vesicle Signatures.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with limited survival rate. Roles for peptidylarginine deiminases (PADs) have been studied in relation to a range of cancers with roles in epigenetic regulation (including histone modification and microRNA regulation), cancer invasion, and extracellular vesicle (EV) release. Hitherto though, knowledge on PADs in PDAC is limited. In the current study, two PDAC cell lines (Panc-1 and MiaPaCa-2) were treated with pan-PAD inhibitor Cl-amidine as well as PAD2, PAD3, and PAD4 isozyme-specific inhibitors. Effects were assessed on changes in EV signatures, including EV microRNA cargo (miR-21, miR-126, and miR-221), on changes in cellular protein expression relevant for pancreatic cancer progression and invasion (moesin), for mitochondrial housekeeping (prohibitin, PHB), and gene regulation (deiminated histone H3, citH3). The two pancreatic cancer cell lines were found to predominantly express PAD2 and PAD3, which were furthermore expressed at higher levels in Panc-1, compared with MiaPaCa-2 cells. PAD2 isozyme-specific inhibitor had the strongest effects on reducing Panc-1 cell invasion capability, which was accompanied by an increase in moesin expression, which in pancreatic cancer is found to be reduced and associated with pancreatic cancer aggressiveness. Some reduction, but not significant, was also found on PHB levels while effects on histone H3 deimination were variable. EV signatures were modulated in response to PAD inhibitor treatment, with the strongest effects observed for PAD2 inhibitor, followed by PAD3 inhibitor, showing significant reduction in pro-oncogenic EV microRNA cargo (miR-21, miR-221) and increase in anti-oncogenic microRNA cargo (miR-126). While PAD2 inhibitor, followed by PAD3 inhibitor, had most effects on reducing cancer cell invasion, elevating moesin expression, and modulating EV signatures, PAD4 inhibitor had negligible effects and pan-PAD inhibitor Cl-amidine was also less effective. Compared with MiaPaCa-2 cells, stronger modulatory effects for the PAD inhibitors were observed in Panc-1 cells, which importantly also showed strong response to PAD3 inhibitor, correlating with previous observations that Panc-1 cells display neuronal/stem-like properties. Our findings report novel PAD isozyme regulatory roles in PDAC, highlighting roles for PAD isozyme-specific treatment, depending on cancer type and cancer subtypes, including in PDAC.

Extracellular DNA Correlates with Intestinal Inflammation in Chemically Induced Colitis in Mice.

Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.

Understanding the influence of trenbolone acetate and polyamines on proliferation of bovine satellite cells.

Approximately 90% of beef cattle on feed in the United States receive at least one anabolic implant, which results in increased growth, efficiency, and economic return to producers. However, the complete molecular mechanism through which anabolic implants function to improve skeletal muscle growth remains unknown. This study had 2 objectives: (1) determine the effect of polyamines and their precursors on proliferation rate in bovine satellite cells (BSC); and (2) understand whether trenbolone acetate (TBA), a testosterone analog, has an impact on the polyamine biosynthetic pathway. To address these, BSC were isolated from 3 finished steers and cultured. Once cultures reached 75% confluency, they were treated in 1% fetal bovine serum (FBS) and/or 10 nM TBA, 10 mM methionine (Met), 8 mM ornithine (Orn), 2 mM putrescine (Put), 1.5 mM spermidine (Spd), or 0.5 mM spermine (Spe). Initially, a range of physiologically relevant concentrations of Met, Orn, Put, Spd, and Spe were tested to determine experimental doses to implement the aforementioned experiments. One, 12, or 24 h after treatment, mRNA was isolated from cultures and abundance of paired box transcription factor 7 (Pax7), Sprouty 1 (Spry), mitogen-activated protein kinase-1 (Mapk), ornithine decarboxylase (Odc), and S adenosylmethionine (Amd1) were determined, and normalized to 18S. No treatment × time interactions were observed (P ≥ 0.05). Treatment with TBA, Met, Orn, Put, Spd, or Spe increased (P ≤ 0.05) BSC proliferation when compared with control cultures. Treatment of cultures with Orn or Met increased (P ≤ 0.01) expression of Odc 1 h after treatment when compared with control cultures. Abundance of Amd1 was increased (P < 0.01) 1 h after treatment in cultures treated with Spd or Spe when compared with 1% FBS controls. Cultures treated with TBA had increased (P < 0.01) abundance of Spry mRNA 12 h after treatment, as well as increased mRNA abundance of Mapk (P < 0.01) 12 h and 24 h after treatment when compared with 1% FBS control cultures. Treatment with Met increased (P < 0.01) mRNA abundance of Pax7 1 h after treatment as compared with 1% FBS controls. These results indicate that treatments of BSC cultures with polyamines and their precursors increase BSC proliferation rate, as well as abundance of mRNA involved in cell proliferation. In addition, treatment of BSC cultures with TBA, polyamines, or polyamine precursors impacts expression of genes related to the polyamine biosynthetic pathway and proliferation.

Peptidylarginine Deiminase Inhibition Prevents Diabetes Development in NOD Mice.

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice. This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated glucose-regulated protein 78, and reduced spontaneous neutrophil extracellular trap formation of bone marrow-derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate characterized by reduced frequencies of effector memory CD4+ T cells and a modest reduction in the frequency of interferon-γ-producing CD4+ and CD8+ T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes.

Neutrophil extracellular traps are induced in a psoriasis model of interleukin-36 receptor antagonist-deficient mice.

Loss-of-function mutations in the interleukin (IL)-36 gene IL36RN are associated with psoriasis. The importance of neutrophil extracellular traps (NETs), web-like structures composed of neutrophil DNA, in the pathogenesis of psoriasis has been unclear. Here, we aimed to clarify the role of NET signaling in the deficiency of IL36 receptor antagonist (DITRA). We evaluated the severity of psoriasis-like lesions induced by imiquimod cream treatment in Il36rn-/- mice. The mRNA levels of psoriasis-related cytokines were measured via real-time reverse transcription polymerase chain reaction, and the effects of Cl-amidine, a peptidyl arginine deiminase 4 (PAD4) inhibitor, on psoriasis-like lesions were evaluated. PAD4 is a histone-modifying enzyme that is involved in NET formation. Psoriasis area and severity index scores, epidermal thickness, and infiltrated neutrophil counts were significantly increased in Il36rn-/- mice; NET formation was confirmed pathologically. Several cytokines and chemokines were upregulated in the skin lesions of Il36rn-/- mice and Cl-amidine treatment improved these psoriasis-like lesions. These results suggest that NET formation plays an important role in the pathology of psoriasis-like lesions in these mice and might represent a promising therapeutic target for DITRA.