Treatment strategy changes for inflammatory bowel diseases in biologic era: results from a multicenter cohort in Japan, Far East 1000.

Many molecular targeted agents, including biologics, have emerged for inflammatory bowel diseases (IBD), but their high prices have prevented their widespread use. This study aimed to reveal the changes in patient characteristics and the therapeutic strategies of IBD before and after the implementation of biologics in Japan, where the unique health insurance system allows patients with IBD and physicians to select drugs with minimum patient expenses. The analysis was performed using a prospective cohort, including IBD expert and nonexpert hospitals in Japan. In this study, patients were classified into two groups according to the year of diagnosis based on infliximab implementation as the prebiologic and biologic era groups. The characteristics of therapeutic strategies in both groups were evaluated using association analysis. This study analyzed 542 ulcerative colitis (UC) and 186 Crohn's disease (CD). The biologic era included 53.3% of patients with UC and 76.2% with CD, respectively. The age of UC (33.9 years vs. 38.8 years, P < 0.001) or CD diagnosis (24.3 years vs. 31.9 years, P < 0.001) was significantly higher in the biologic era group. The association analysis of patients with multiple drug usage histories revealed that patients in the prebiologic era group selected anti-tumor necrosis factor (TNF)-α agents, whereas those in the biologic era group preferred biologic agents with different mechanisms other than anti-TNF-α. In conclusion, this study demonstrated that both patient characteristics and treatment preferences in IBD have changed before and after biologic implementation.

Pulmonary and Disseminated Mycobacterium avium Complex Cases Confirmed by Tissue-Direct Polymerase Chain Reaction-Based Nucleic Acid Lateral Flow Immunoassay of Formalin-Fixed Paraffin-Embedded Tissues.

Background: Detection of Mycobacterium avium complex (MAC) in tissue is essential for the diagnosis of MAC infections when the Mycobacterium is not isolated from sputum. However, detection of MAC in paraffin-embedded sections has not been established. Methods: We encountered two patients with suspected MAC infections after surgery: patient 1 had a pulmonary nodule that was initially suspected to be lung cancer and was excised under video-assisted thoracoscopic surgery (VATS). Patient 2, who was under treatment with steroids and anti-IL-6 inhibitors for rheumatoid arthritis, was suspected to have disseminated ileocecal cancer with metastasis to the lung and skin. In both cases, we postoperatively detected MAC genes in paraffin-embedded tissue sections using the novel mycobacterial nucleic acid identification test, ie tissue-direct polymerase chain reaction (tdPCR)-based nucleic acid lateral flow immunoassay (NALFIA). Both patients showed granulomatous lesions with hematoxylin-eosin staining, and mycobacteria by Ziehl-Neelsen staining in tissue sections from the lung and skin, respectively, although MAC were not isolated from the sections. MAC genes were finally detected by tdPCR-NALFIA in both cases. Conclusion: Although Ziehl-Neelsen staining and culture tests are the gold standard in identifying causative mycobacteria, the rapid results of tdPCR-NALFIA performed simultaneously with sputum and/or tissue culture may make it an important auxiliary diagnostic tool for identifying mycobacterial infection, leading to improvement in the management of MAC patients.

Clinical evaluation of cell-direct polymerase chain reaction-based nucleic acid lateral flow immunoassay for rapid detection of bacterial pathogens in clinically suspected sepsis: A multi-center study in Japan.

Blood culture, a method for identifying causative agents of bacterial sepsis, requires several days. The combination of cell-direct polymerase chain reaction and nucleic acid lateral flow immunoassay (cdPCR-NALFIA) is a simple and sensitive detection method for identifying pathogenic bacteria. Furthermore, this assay, when applied directly to blood samples yields results within 4.5 h, without requiring culture. This study was performed at five hospitals in Japan between 2013 and 2016. Blood samples from 73 patients with clinically suspected sepsis yielded 18 positive blood cultures, and the isolated bacterial species were detectable using cdPCR-NALFIA in nine samples. Thirteen samples were positive on cdPCR-NALFIA. In total, 17 samples confirmed to have bacterial species were detectable using cdPCR-NALFIA and/or blood culture with a true positive rate of 76.5% and 64.7%, respectively. The combination of blood culture and cdPCR-NALFIA could improve the rate of detection of bacterial sepsis.

Emergence of fibrocytes showing morphological changes in the inflamed colonic mucosa.

Fibrocytes contribute to wound healing and are uniquely defined by coexpression of hematopoietic and mesenchymal cell markers. In this study, trafficking of fibrocytes was determined in a murine model of colitis induced by administering 3% dextran sodium sulfate (DSS) for seven days. Colonic tissues were immunostained for CD45, collagen type I (Col I), and alpha-SMA. On day 0, there were no CD45(+)Col I(+) cells in colonic tissues. However, on day 7 when inflammatory cells showed remarkable accumulation, oval-shaped CD45(+)Col I(+) fibrocytes were obvious in the submucosal layer. On day 14 when colonic tissues were in the healing phase, numerous spindle-shaped CD45(+)Col I(+) fibrocytes were observed. Emergence of CD45(+)Col I(+) fibrocytes preceded the appearance of alpha-SMA(+) myofibroblasts. Oval-shaped fibrocytes recruited as early as the inflammatory phase of colitis are likely to differentiate into spindle-shaped fibrocytes in the healing phase, suggesting that fibrocytes may promote wound healing in inflamed colonic tissues.

Molecular basis for the selectivity of the mammalian bombesin peptide, neuromedin B, for its receptor.

The mammalian bombesin (Bn) peptides, neuromedin B (NMB) and gastrin-releasing peptide (GRP), have widespread actions in many tissues, and their effects are mediated by two closely related G-protein-coupled receptors, the NMBR and GRPR. Little is known about the structural determinants of NMBR selectivity for NMB, in contrast to GRP selectivity for the GRPR, which has been extensively studied. To provide insight, chimeric NMBR-GRPR loss-of-affinity and gain-of-affinity mutants were made, as well as NH(2)-terminally truncated NMBR and point mutants using site-directed mutagenesis. Receptors were expressed in Balb-3T3-cells or CHOP cells, and affinities were determined. NMB had 115-fold greater affinity for NMBR than GRPR. Receptor-chimeric studies showed that NMBR selectivity for NMB was primarily determined by differences in the third extracellular (EC3) regions of GRPR-NMBR and adjacent upper-transmembrane-5 (TM5) region. In this region, 24 NMB gain-of-affinity GRPR mutants or NMBR loss-of-affinity point/combination mutants were made. Three gain-of-affinity mutant GRPRs [[A198I] (EC3), [H202Q] (EC3), [S215I] (upper TM5)] had increased NMB affinity (2.4-21-fold), and these results were confirmed with NMBR loss-of-affinity mutants [I199A,Q203H,I215S-NMBR]. The combination mutant [A198I,S215]GRPR had the greatest effect causing a complete NMB gain-of-affinity. The importance of differences at position 199NMBR or 203NMBR was studied by substituting amino acids with various properties. Our results show that NMBR selectivity for NMB is due to differences in the EC3 of NMBR-GRPR and the adjacent upper-TM5 region. Within these regions, isoleucines in NMBR [position 199 (EC3)] (instead of A198GRPR) and in 215NMBR (TM5) (instead of S214GRPR), as well as Q203NMBR (instead of H202GRPR) are responsible for high NMB-affinity/selectivity of NMBR. The effect at position 199 is primarily due to differences in hydrophobicity of the substitution, whereas steric factors and charge of the substitution at position 203 were important determinants of NMB selectivity.

[Barostat procedures].

Barostat is equipment for studying smooth muscle tone and sensory thresholds in the gastrointestinal (GI) tract. The principle of it is to maintain a constant pressure within an air-filled bag positioned in the lumen of the GI tract to be studied. Patients with irritable bowel syndrome (IBS) have been shown to have lower pain thresholds by barostat. Barostat would be one of the key procedures for studying pathophysiology in patients with IBS, although this method should be further standardized.

Pharmacotherapy of Zollinger-Ellison syndrome.

INTRODUCTION: The role of pharmacotherapy in the management of patients with Zollinger-Ellison syndrome (ZES) is often equated with the medical management of acid hypersecretion. However, pharmacotherapy is also increasingly involved in the other management areas of these patients. AREAS COVERED: This paper reviews the role of pharmacotherapy in all aspects of the management of patients with ZES. Newer aspects are emphasized. This includes the difficulty of diagnosing ZES in patients taking proton pump inhibitors. Also covered is the role of pharmacotherapy in controlling acid hypersecretion and other hormonal hypersecretory states these patients may develop, including hyperparathyroidism in patients with multiple endocrine neoplasia type 1 and ZES; tumor localization; and the treatment of advanced metastatic disease. The last includes chemotherapy, liver-directed therapies, biotherapy (somatostatin/interferon), peptide radio-receptor therapy and molecular-targeted therapies including the use of mTor inhibitors (everolimus) and tyrosine kinase inhibitors (sunitinib). EXPERT OPINION: Pharmacotherapy is now involved in all aspects of the management of patients with ZES, with the result that ZES has progressed from being considered an entirely surgical disease initially to the present where medical treatment plays a major role in almost all aspects of the management of these patients.

Causes of death and prognostic factors in multiple endocrine neoplasia type 1: a prospective study: comparison of 106 MEN1/Zollinger-Ellison syndrome patients with 1613 literature MEN1 patients with or without pancreatic endocrine tumors.

Multiple endocrine neoplasia type 1 (MEN1) is classically characterized by the development of functional or nonfunctional hyperplasia or tumors in endocrine tissues (parathyroid, pancreas, pituitary, adrenal). Because effective treatments have been developed for the hormone excess state, which was a major cause of death in these patients in the past, coupled with the recognition that nonendocrine tumors increasingly develop late in the disease course, the natural history of the disease has changed. An understanding of the current causes of death is important to tailor treatment for these patients and to help identify prognostic factors; however, it is generally lacking.To add to our understanding, we conducted a detailed analysis of the causes of death and prognostic factors from a prospective long-term National Institutes of Health (NIH) study of 106 MEN1 patients with pancreatic endocrine tumors with Zollinger-Ellison syndrome (MEN1/ZES patients) and compared our results to those from the pooled literature data of 227 patients with MEN1 with pancreatic endocrine tumors (MEN1/PET patients) reported in case reports or small series, and to 1386 patients reported in large MEN1 literature series. In the NIH series over a mean follow-up of 24.5 years, 24 (23%) patients died (14 MEN1-related and 10 non-MEN1-related deaths). Comparing the causes of death with the results from the 227 patients in the pooled literature series, we found that no patients died of acute complications due to acid hypersecretion, and 8%-14% died of other hormone excess causes, which is similar to the results in 10 large MEN1 literature series published since 1995. In the 2 series (the NIH and pooled literature series), two-thirds of patients died from an MEN1-related cause and one-third from a non-MEN1-related cause, which agrees with the mean values reported in 10 large MEN1 series in the literature, although in the literature the causes of death varied widely. In the NIH and pooled literature series, the main causes of MEN1-related deaths were due to the malignant nature of the PETs, followed by the malignant nature of thymic carcinoid tumors. These results differ from the results of a number of the literature series, especially those reported before the 1990s. The causes of non-MEN1-related death for the 2 series, in decreasing frequency, were cardiovascular disease, other nonendocrine tumors > lung diseases, cerebrovascular diseases. The most frequent non-MEN1-related tumor deaths were colorectal, renal > lung > breast, oropharyngeal. Although both overall and disease-related survival are better than in the past (30-yr survival of NIH series: 82% overall, 88% disease-related), the mean age at death was 55 years, which is younger than expected for the general population.Detailed analysis of causes of death correlated with clinical, laboratory, and tumor characteristics of patients in the 2 series allowed identification of a number of prognostic factors. Poor prognostic factors included higher fasting gastrin levels, presence of other functional hormonal syndromes, need for >3 parathyroidectomies, presence of liver metastases or distant metastases, aggressive PET growth, large PETs, or the development of new lesions.The results of this study have helped define the causes of death of MEN1 patients at present, and have enabled us to identify a number of prognostic factors that should be helpful in tailoring treatment for these patients for both short- and long-term management, as well as in directing research efforts to better define the natural history of the disease and the most important factors determining long-term survival at present.

The molecular basis for high affinity of a universal ligand for human bombesin receptor (BnR) family members.

There is increased interest in the Bn-receptor family because they are frequently over/ectopically expressed by tumors and thus useful as targets for imaging or receptor-targeted-cytotoxicity. The synthetic Bn-analog, [D-Tyr(6), ß-Ala(11), Phe(13), Nle(14)]Bn(6-14) [Univ.Lig] has the unique property of having high affinity for all three human BNRs (GRPR, NMBR, BRS-3), and thus could be especially useful for this approach. However, the molecular basis of this property is unclear and is the subject of this study. To accomplish this, site-directed mutagenesis was used after identifying potentially important amino acids using sequence homology analysis of all BnRs with high affinity for Univ.Lig compared to the Cholecystokinin-receptor (CCK(A)R), which has low affinity. Using various criteria 74 amino acids were identified and 101 mutations made in GRPR by changing each to those of CCK(A)R or to alanine. 22 GRPR mutations showed a significant decrease in affinity for Univ.Lig (>2-fold) with 2 in EC2[D97N, G112V], 1 in UTM6[Y284A], 2 in EC4[R287N, H300S] showing >10-fold decrease in Univ.Lig affinity. Additional mutations were made to explore the molecular basis for these changes. Our results show that high affinity for Univ.Lig by human Bn-receptors requires positively charged amino acids in extracellular (EC)-domain 4 and to a lesser extent EC2 and EC3 suggesting charge-charge interactions may be particularly important for determining the general high affinity of this ligand. Furthermore, transmembrane amino acids particularly in UTM6 are important contributing both charge-charge interactions as well as interaction with a tyrosine residue in close proximity suggesting possible receptor-peptide cation-π or H-bonding interactions are also important for determining its high affinity.

Pharmacology and selectivity of various natural and synthetic bombesin related peptide agonists for human and rat bombesin receptors differs.

The mammalian bombesin (Bn)-receptor family [gastrin-releasing peptide-receptor (GRPR-receptor), neuromedin B-receptor (NMB receptor)], their natural ligands, GRP/NMB, as well as the related orphan receptor, BRS-3, are widely distributed, and frequently overexpressed by tumors. There is increased interest in agonists for this receptor family to explore their roles in physiological/pathophysiological processes, and for receptor-imaging/cytotoxicity in tumors. However, there is minimal data on human pharmacology of Bn receptor agonists and most results are based on nonhuman receptor studies, particular rodent-receptors, which with other receptors frequently differ from human-receptors. To address this issue we compared hNMB-/GRP-receptor affinities and potencies/efficacies of cell activation (assessing phospholipase C activity) for 24 putative Bn-agonists (12 natural, 12 synthetic) in four different cells with these receptors, containing native receptors or receptors expressed at physiological densities, and compared the results to native rat GRP-receptor containing cells (AR42J-cells) or rat NMB receptor cells (C6-glioblastoma cells). There were close correlations (r=0.92-99, p<0.0001) between their affinities/potencies for the two hGRP- or hNMB-receptor cells. Twelve analogs had high affinities (≤ 1 nM) for hGRP receptor with 15 selective for it (greatest=GRP, NMC), eight had high affinity/potencies for hNMB receptors and four were selective for it. Only synthetic Bn analogs containing ß-alanine(11) had high affinity for hBRS-3, but also had high affinities/potencies for all GRP-/hNMB-receptor cells. There was no correlation between affinities for human GRP receptors and rat GRP receptors (r=0.131, p=0.54), but hNMB receptor results correlated with rat NMB receptor (r=0.71, p<0.0001). These results elucidate the human and rat GRP-receptor pharmacophore for agonists differs markedly, whereas they do not for NMB receptors, therefore potential GRP-receptor agonists for human studies (such as Bn receptor-imaging/cytotoxicity) must be assessed on human Bn receptors. The current study provides affinities/potencies on a large number of potential agonists that might be useful for human studies.

Pharmacology of putative selective hBRS-3 receptor agonists for human bombesin receptors (BnR): affinities, potencies and selectivity in multiple native and BnR transfected cells.

The orphan receptor, bombesin receptor subtype-3(BRS-3) is a G-protein-coupled receptor classified in the bombesin (Bn) receptor family because of its high homology (47-51%) with other members of this family [gastrin-releasing peptide receptor [GRPR] and neuromedin B receptor [NMBR]]. There is increasing interest in BRS-3, because primarily from receptor knockout studies, it seems important in energy metabolism, glucose control, insulin secretion, motility and tumor growth. Pharmacological tools to study the role of BRS-3 in physiology/pathophysiology are limited because the natural ligand is unknown and BRS-3 has low affinity for all naturally occurring Bn-related peptides. However, a few years ago a synthetic high-affinity agonist [dTyr(6),betaAla(11),Phe(13),Nle(14)]Bn-(6-14) was described but was nonselective for BRS-3 over other Bn receptors. Based on this peptide, in various studies a number of putative selective, high-potency hBRS-3 agonists were described, however the results on their selectivity are conflicting in a number of cases. The purpose of the present study was to thoroughly study the pharmacology of four of the most select/potent putative hBRS-3 agonists (#2-4, 16a). Each was studied in multiple well-characterized Bn receptor-transfected cells and native Bn receptor bearing cells, using binding studies, alterations in cellular signaling (PLC, PKD) and changes in cellular function(growth). Two peptides (#2, #3) had nM affinities/potencies for hBRS-3, peptide #4 had low affinity/potency, and peptide #16a very low (>3000 nM). Peptide#3 had the highest selectivity for hBRS-3 (100-fold), whereas #2, 4 had lower selectivity. Peptide #16a's selectivity could not be determined because of its low affinity/potencies for all hBn receptors. These results show that peptide #3 is the preferred hBRS-3 agonist for studies at present, although its selectivity of only 100-fold may limit its utility in some cases. This study underscores the importance of full pharmacological characterization of newly reported selective agonists.

Urocortin I is present in the enteric nervous system and exerts an excitatory effect via cholinergic and serotonergic pathways in the rat colon.

Corticotropin-releasing factor (CRF) and urocortin I (UcnI) have been shown to accelerate colonic transit after central nervous system (CNS) or peripheral administration, but the mechanism of their peripheral effect on colonic motor function has not been fully investigated. Furthermore, the localization of UcnI in the enteric nervous system (ENS) of the colon is unknown. We investigated the effect of CRF and UcnI on colonic motor function and examined the localization of CRF, UcnI, CRF receptors, choline acetyltransferase (ChAT), and 5-HT. Isometric tension of rat colonic muscle strips was measured. The effect of CRF, UcnI on phasic contractions, and electrical field stimulation (EFS)-induced off-contractions were examined. The effects of UcnI on both types of contraction were also studied in the presence of antalarmin, astressin2-B, tetrodotoxin (TTX), atropine, and 5-HT antagonists. The localizations of CRF, UcnI, CRF receptors, ChAT, and 5-HT in the colon were investigated by immunohistochemistry. CRF and UcnI increased both contractions dose dependently. UcnI exerted a more potent effect than CRF. Antalarmin, TTX, atropine, and 5-HT antagonists abolished the contractile effects of UcnI. CRF and UcnI were observed in the neuronal cells of the myenteric plexus. UcnI and ChAT, as well as UcnI and 5-HT, were colocalized in some of the neuronal cells of the myenteric plexus. This study demonstrated that CRF and UcnI act on the ENS and increase colonic contractility by enhancing cholinergic and serotonergic neurotransmission. These peptides are present in myenteric neurons. CRF and, perhaps, to a greater extent, UcnI appear to act as neuromodulators in the ENS of the rat colon.