Nonsteroidal anti-inflammatory drugs (NSAIDs) induce cytokines, including tumor necrosis factor-α and interleukins (ILs), in the small intestine via a Toll-like receptor 4 (TLR4)-dependent pathway, ...leading to intestinal ulceration. Activation of the inflammasome promotes pro-caspase-1 cleavage, leading to pro-IL-1β maturation. We examined the role of NLRP3 inflammasome in NSAID-induced enteropathy. Small intestinal damage developed 3 h after indomethacin administration, accompanied by increases in IL-1β and NLRP3 mRNA expression and mature caspase-1 and IL-1β levels. In vivo blocking of IL-1β using neutralizing antibodies attenuated indomethacin-induced damage, whereas exogenous IL-1β aggravated it. NLRP3(-/-) and caspase-1(-/-) mice exhibited resistance to the damage with reduction of mature IL-1β production. This resistance was abolished by exogenous IL-1β. TLR4 deficiency prevented intestinal damage and inhibited upregulation of NLRP3 and IL-1β mRNAs and maturation of pro-caspase-1 and pro-IL-1β, whereas TLR4 activation by its agonists exerted opposite effects. Apyrase, an adenosine triphosphate (ATP) scavenger, or Brilliant Blue G, a purinergic P2X7 receptor antagonist, inhibited the damage as well as caspase-1 activation and IL-1β processing, despite there being sufficient amounts of pro-IL-1β and NLRP3. These results suggest that NLRP3 inflammasome-derived IL-1β plays a crucial role in NSAID-induced enteropathy and that both TLR4- and P2X7-dependent pathways are required for NLRP3 inflammasome activation.
Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is ...customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5–2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein‐A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.
Amino acids are widely used in biotechnology applications. Since amino acids are natural compounds, they can be safely used in pharmaceutical applications, e.g., as a solvent additive for protein ...purification and as an excipient for protein formulations. At high concentrations, certain amino acids are found to raise intra-cellular osmotic pressure and adjust to the high salt concentrations of the surrounding medium. They are called “compatible solutes”, since they do not affect macromolecular function. Not only are they needed to increase the osmotic pressure, they are known to increase the stability of the proteins. Sucrose, glycerol and certain amino acids were used to enhance the stability of unstable proteins after isolation from natural environments. The mechanism of the action of these protein-stabilizing amino acids is relatively well understood. On the contrary, arginine was accidentally discovered as a useful reagent for assisting in the refolding of recombinant proteins. This effect of arginine was ascribed to its ability to suppress aggregation of the proteins during refolding, thereby increasing refolding efficiency. By the same mechanism, arginine now finds much wider applications than previously anticipated in the research and development of proteins, in particular in pharmaceutical applications. For example, arginine solubilizes proteins from loose inclusion bodies, resulting in efficient production of active proteins. Arginine suppresses protein-protein interactions in solution and also non-specific adsorption to gel permeation chromatography columns. Arginine facilitates elution of bound proteins from various column resins, including Protein-A or dye affinity columns and hydrophobic interaction columns. This review covers various biotechnology applications of amino acids, in particular arginine.
Summary
Background
The features of proton pump inhibitor‐responsive oesophageal eosinophilia (PPI‐REE) are similar to those of eosinophilic oesophagitis (EoE), but PPI‐REE demonstrates symptomatic ...and histological responses to PPI therapy. Several studies have shown that basophils play a crucial role in the pathogenesis of allergic diseases.
Aim
To identify and compare basophil infiltration in the oesophageal epithelium in patients with EoE, PPI‐REE, gastroesophageal reflux disease (GERD) and normal oesophagus (controls).
Methods
Biopsy specimens from 43 patients, including 12 with EoE, 11 with PPI‐REE, 10 with GERD and 10 normal oesophagus, were analysed. Immunohistochemistry was performed to quantify the number of basophils and mast cells in the oesophageal epithelium. Double immunofluorescence staining for thymic stromal lymphopoietin (TSLP) and basophils was performed. Patients with EoE were treated with swallowed fluticasone.
Results
There were no differences in clinical, endoscopic or histological features between patients with EoE and PPI‐REE. There were more basophils and mast cells in patients with EoE and PPI‐REE than in patients with GERD and control subjects. Basophil infiltration of the oesophageal epithelium in patients with EoE was higher than that in patients with PPI‐REE (3.6 ± 2.8 per high power field vs. 1.2 ± 0.9 per high power field respectively; P = 0.02); however, there was no significant difference in mast cell infiltration between the two groups. TSLP was highly expressed in the oesophageal epithelium in areas infiltrated by basophils. Steroid therapy significantly decreased intraepithelial basophils in patients with EoE.
Conclusion
Basophils may play an important role in the pathogenesis of eosinophilic oesophagitis.
Background
Functional dyspepsia (FD), a heterogeneous disorder, involves multiple pathogenetic mechanisms. Developing treatments for FD has been challenging. We performed a randomized, ...placebo‐controlled, double‐blind clinical trial to determine the efficacy of rikkunshito, a Japanese herbal medicine, in FD patients.
Methods
FD patients (n = 192) who met the Rome III criteria without Helicobacter pylori infection, predominant heartburn, and depression were enrolled at 56 hospitals in Japan. After 2 weeks of single‐blind placebo treatment, 128 patients with continuous symptoms were randomly assigned to 8 weeks of rikkunshito (n = 64) or placebo (n = 61). The primary efficacy endpoint was global assessment of overall treatment efficacy (OTE). The secondary efficacy endpoints were improvements in upper gastrointestinal symptoms evaluated by the Patient Assessment of Upper Gastrointestinal Disorders‐Symptom Severity Index (PAGI‐SYM), the Global Overall Symptom scale (GOS), and the modified Frequency Scale for the Symptoms of Gastroesophageal Reflux Disease (m‐FSSG), and psychological symptoms evaluated by the Hospital Anxiety and Depression Scale (HADS).
Key Results
Rikkunshito increased OTE compared to placebo at 8 weeks (P = .019). Rikkunshito improved upper gastrointestinal symptoms (PAGI‐SYM, GOS, and m‐FSSG) at 8 weeks, especially postprandial fullness/early satiety (P = .015 and P = .001) and bloating (P = .007 and P = .002) of the PAGI‐SYM subscales at 4 weeks and 8 weeks. Improvement of HADS at 8 weeks (P = .027) correlated with those of PAGI‐SYM (r = .302, P = .001), GOS (r = .186, P = .044), and m‐FSSG (r = .462, P < .001), postprandial fullness/early satiety (r = .226, P = .014), dyspepsia (r = .215, P = .019), and PDS (r = .221, P = .016).
Conclusion & inferences
Rikkunshito may be beneficial for FD patients to simultaneously treat gastrointestinal and psychological symptoms.
In the RCT conducted for Helicobacter pylori‐negative FD patients, rikkunshito significantly relieved dyspeptic symptoms compared to placebo possibly via improvement for physiological dysfunction based on the changes in PAGI‐SYM scores. Attenuation of anxiety correlated with improvement of dyspeptic symptoms such as postprandial fullness/early satiety. Rikkunshito exhibits relevant pharmacological actions throughout the brain‐gut axis and improves dyspeptic symptoms mediated by modulating both the visceral and central factors in FD patients.
Enterobacteria and cytokines both play roles in the pathophysiology of NSAID-induced enteropathy. Toll-like receptor (TLR) 4 recognises lipopolysaccharide (LPS), resulting in activation of an ...inflammatory cascade via the accessory protein MyD88.
To investigate role of TLR4 in inflammatory responses in indomethacin-induced enteropathy.
Indomethacin was administered p.o. to non-fasting rats and mice to induce small intestinal damage. The extent of such damage was evaluated by measuring the injured area stained dark blue with Evans blue. Rats were given antibiotics (ampicillin, aztreonam or vancomycin) p.o., or intraperitoneal LPS (a TLR4 ligand) or neutralising antibodies against neutrophils, tumour necrosis factor (TNF)-alpha, or monocyte chemotactic protein (MCP)-1. Furthermore, the intestinal ulcerogenicity of indomethacin was examined in TLR4-mutant, TLR4(-/-), and MyD88(-/-) mice.
Indomethacin induced small intestinal damage with an increase in expression of TNF-alpha and MCP-1 in both rats and mice. Antibodies against neutrophils, TNF-alpha and MCP-1 inhibited the damage by 83%, 67% and 63%, respectively, in rats. Ampicillin and aztreonam also inhibited this damage, and decreased the number of Gram-negative bacteria in the small intestinal contents of the rat. However, vancomycin, which exhibited no activity against Gram-negative bacteria, had no preventive effect against this damage. Administration of LPS 1 h after indomethacin aggravated the damage, whereas LPS pretreatment inhibited it with reduction of expression of TLR4 and cytokines. In TLR4-mutant mice, the damage and cytokine expression were markedly inhibited. TLR4(-/-) and MyD88(-/-) mice were also resistant to the damage.
Indomethacin may injure the small intestine through a TLR4/MyD88-dependent pathway.
This prospective study aimed to evaluate the feasibility and safety of locoregional mitomycin C (MMC) injection to treat refractory esophageal strictures after endoscopic submucosal dissection (ESD) ...for superficial esophageal carcinoma. Patients with dysphagia and strictures that were refractory to repeated endoscopic balloon dilation (EBD) were eligible. After EBD, MMC was injected into the dilated site. Between June 2009 and August 2010, five patients were recruited. The treatment was performed once in two patients and twice in three patients with recurrent dysphagia or restenosis. In all patients, passing a standard endoscope through the site was easy and the dysphagia grade improved (grade 3→1 in 3 patients, grade 4→2 in 2 patients). No serious complications were noted. During the observation period of 4.8 months, neither recurrent dysphagia nor re-stricture appeared in any of the patients. The combination of locoregional MMC injections and EBD is feasible and safe for the treatment of esophageal strictures after ESD.Recently, endoscopic submucosal dissection (ESD) has been developed and accepted as a new endoscopic treatment for gastrointestinal tumors. ESD is a promising treatment for superficial esophageal carcinoma (SEC), and it has a reliable en bloc resection rate. However, the application of ESD for widespread lesions is challenging because of the high risk of the development of severe strictures, which lead to a low quality of life after ESD. Although endoscopic balloon dilation (EBD) is effective for benign strictures, it needs to be performed frequently until the dysphagia disappears 1. Mitomycin C (MMC), which is a chemotherapeutic agent derived from some Streptomyces species 2, reduces scar formation when topically applied to a surgical lesion. MMC has been applied to treat strictures in a variety of anatomical locations, including a variety of organs 3. The aim of this study was to prospectively evaluate both the feasibility and the safety of locoregional MMC injection therapy in patients with refractory esophageal strictures after ESD for SEC.
Background
Enteric glial cells (EGCs) play important roles in enteric integrity and regulation of gastrointestinal function. However, whether EGCs undergo pathophysiological changes in ...stress‐associated gastrointestinal disorders is unknown. We investigated structural and functional alterations in colonic EGCs and their roles in colonic contraction in an irritable bowel syndrome (IBS) model.
Methods
As a chronic stress, male Wistar rats underwent 3‐h maternal separation during postnatal days 2–14. As an acute stress, we used water‐immersion stress (4 h) in adulthood (at 8 weeks). We quantitatively and morphologically evaluated enteric neurons and EGCs using whole‐mount longitudinal muscle‐myenteric plexus preparations. Colonic contraction was analyzed with electrical field stimulation (EFS).
Key Results
Glial fibrillary acidic protein (GFAP) expression and the number of total, cholinergic, and nitrergic neurons were unchanged in maternally separated rats with acute stress (combined stress: an IBS model) compared with controls. However, the density of GFAP‐positive EGC processes that apparently overlapped with the neurons and the extent of bulbous swelling of terminals increased according to the stress intensity: control, acute stress, maternal separation, and combined stress. EFS‐induced colonic contractions were significantly greater in the combined stress rats than in controls. Higher dose of fluorocitrate, a selective inhibitor of EGC metabolism, was required to inhibit both EFS‐induced contraction and EGCs activation in the combined stress rats than in controls.
Conclusions & Inferences
Colonic EGCs exhibited structural alterations according to the stress intensity. EGCs were associated with stress‐induced colonic hyper‐contraction in the combined stress rats, which may underlie the pathogenesis of IBS.
Enteric glial cells (EGCs) play important roles in the regulation of gastrointestinal function, but manifestations of colonic EGCs in irritable bowel syndrome (IBS) patients remain unknown. EGCs exhibited hyperplasia of their processes that apparently overlapped with the neurons and changes in bulbous swelling of terminals, and were also associated with stress‐induced hyper colonic contraction in the maternally separated rats. Such changes in EGCs may underlie the pathogenesis of IBS.
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