Relapse rate and predictors of relapse after cessation of glucocorticoid maintenance therapy in type 1 autoimmune pancreatitis: a multicenter retrospective study.

BACKGROUND: Type 1 autoimmune pancreatitis responds well to glucocorticoid therapy with a high remission rate. Moreover, glucocorticoid maintenance therapy can help prevent relapse. However, the relapse rate following cessation of long-term glucocorticoid therapy is unknown. The aim of this study was to clarify the relapse rate and predictors of relapse following long-term glucocorticoid therapy cessation. METHODS: We analyzed 94 patients who achieved remission after undergoing glucocorticoid therapy, discontinued treatment after at least 6 months of maintenance therapy, and were subsequently followed up for at least 6 months. The patients were divided into three groups based on treatment duration (< 18, 18-36, and ≥ 36 months), and their relapse rates were compared. Univariate and multivariate analyses of clinical factors were conducted to identify relapse predictors. RESULTS: After discontinuing glucocorticoid therapy, relapse was observed in 43 (45.7%) patients, with cumulative relapse rates of 28.2% at 1 year, 42.1% at 3 years, 47.0% at 5 years, and a plateau of 77.6% at 9 years. Of the 43 patients who relapsed, 25 (58.1%) relapsed within 1 year after after cessation of glucocorticoid therapy. Relapse and cumulative relapse rates did not differ significantly according to treatment duration. In the multivariate analysis, an elevated serum IgG4 level at the time of glucocorticoid cessation was found to be an independent predictor of relapse (hazard ratio, 4.511; p < 0.001). CONCLUSIONS: A high relapse rate occurred after cessation of glucocorticoid maintenance therapy, regardless of the duration of maintenance therapy, especially within the first year after cessation. However, the normalization of long-term serum IgG4 levels may be a factor in considering cessation.

Distribution of three isoforms of antimicrobial peptide, chrysophsin-1, -2 and -3, in the red sea bream, Pagrus (Chrysophrys) major.

We report here a liquid chromatography/electrospray ionization-tandem mass spectrometry assay for the quantification of three isoforms of antimicrobial peptide (AMP), chrysophsin-1, -2 and -3, in the red sea bream, Pagrus (Chrysophrys) major. Chrysophsin-1 was mainly distributed in the pyloric caeca and gills, followed by intestine and stomach. Chrysophsin-2 was detected in the gills and stomach, but chrysophsin-3 was only in the gills. The present procedure is valuable as a general method for simultaneous determination of the level of multiple AMP isoforms in fish tissues, and the data give important information for understanding the significance of each AMP isoform in host defense.

Utility of Covered Self-Expanding Metal Stents for Biliary Drainage during Neoadjuvant Chemotherapy in Patients with Borderline Resectable Pancreatic Cancer.

OBJECTIVES: We aimed to compare the utility of covered self-expanding metal stents (CSEMSs) with that of plastic stents (PSs) for biliary drainage during neoadjuvant chemotherapy in patients with borderline resectable pancreatic cancer. METHODS: Forty patients with borderline resectable pancreatic cancer underwent biliary stenting during neoadjuvant chemotherapy at Hiroshima University Hospital. PSs and CSEMSs were placed in 19 and 21 patients, respectively. Two gemcitabine-based regimens for chemotherapy were used. Treatment outcomes and postoperative complications were compared between both groups. RESULTS: The incidence of recurrent biliary obstruction was significantly lower in the CSEMS group (0% vs. 47.4%, p < 0.001), and the median time to recurrent biliary obstruction in the PS group was 47 days. There was no difference in the incidence of other complications such as non-occlusive cholangitis, pancreatitis, and cholecystitis between the two groups. Delays in the chemotherapy schedule due to stent-related complications were significantly frequent in the PS group (52.6% vs. 4.8%, p = 0.001). There was no significant difference in the incidence of postoperative complications between the two groups. CONCLUSIONS: CSEMSs may be the best choice for safely performing neoadjuvant chemotherapy for several months in patients with borderline resectable pancreatic cancer with bile duct stricture.

The Significance of Histopathological Findings on Clinical Outcomes in Endoscopic Papillectomy with Endocut.

This study aimed to evaluate primary clinical outcomes in patients who underwent endoscopic papillectomy (EP) using the Endocut mode while examining the pathological characteristics of the margin of the resected specimen. To this end, 70 patients who underwent Endocut EP were included. Resection margins were classified according to pathological findings as "negative", "positive", or "uncertain (difficult pathological evaluation)". The effect of pathological resection margins on residual tumor recurrence rates was evaluated. The median follow-up was 47 months (range, 22-84). Eleven patients (15.7%) were diagnosed with residual tumors, ten of whom were diagnosed within 6 months after EP. The resection margins were pathologically negative in 27 patients, positive in 15, and uncertain in 28; residual tumors occurred in 5 patients (33.3%) in the positive group, 5 (17.9%) in the uncertain group, and 1 (3.7%) in the negative group. The patient in the negative group had familial adenomatous polyposis (FAP). Female sex, FAP, and uncertain or positive resection margins were significantly more common in residual patients (p = 0.009, 0.044, and 0.041, respectively). Pathological resection margins can be used to infer the residual tumor incidence, leading to early post-treatment of residual tumors.

Ontogeny of gene expression of group IB phospholipase A2 isoforms in the red sea bream, Pagrus (Chrysophrys) major.

The red sea bream (Pagrus major) was previously found to express mRNAs for two group IB phospholipase A(2) (PLA(2)) isoforms, DE-1 and DE-2, in the digestive organs, including the hepatopancreas, pyloric caeca, and intestine. To characterize the ontogeny of the digestive function of these PLA(2)s, the present study investigated the localization and expression of DE-1 and DE-2 PLA(2) genes in red sea bream larvae/juveniles and immature adults, by in situ hybridization. In the adults, DE-1 PLA(2) mRNA was expressed in pancreatic acinar cells. By contrast, DE-2 PLA(2) mRNA was detected not only in digestive tissues, such as pancreatic acinar cells, gastric glands of the stomach, epithelial cells of the pyloric caeca, and intestinal epithelial cells, but also in non-digestive ones, including cardiac and lateral muscle fibers and the cytoplasm of the oocytes. In the larvae, both DE-1 and DE-2 PLA(2) mRNAs first appeared in pancreatic tissues at 3 days post-hatching (dph) and in intestinal tissue at 1 dph, and expression levels for both gradually increased after this point. In the juvenile stage at 32 dph, DE-1 PLA(2) mRNA was highly expressed in pancreatic tissue, and DE-2 PLA(2) mRNA was detected in almost all digestive tissues, including pancreatic tissue, gastric glands, pyloric caeca, and intestine, including the myomere of the lateral muscles. In conclusion, both DE-1 and DE-2 PLA(2) mRNAs are already expressed in the digestive organs of red sea bream larvae before first feeding, and larvae will synthesize both DE-1 and DE-2 PLA(2) proteins.

Partial purification and characterization of pro-phospholipase A2 activating proteases from gill membranes of the red sea bream, Chrysophrys major.

We previously reported that gill group IB secretory phospholipase A(2) (sPLA(2)) exists as an inactive pro-sPLA(2) with the dipeptide Ala-Arg, at the N-terminus of mature sPLA(2) in mucous cells. Pro-sPLA(2) should be activated after being secreted to the surface of gill epithelia by trypsin-like protease. To clarify the above hypothesis, we investigated the existence of pro-sPLA(2) activating protease (PAP) in the gills of the red sea bream, using gill pro-sPLA(2) as a substrate. PAP was solubilized from the membrane fraction of the gills with 2% sodium cholate and partially purified by benzamidine-Sepharose chromatography and reversed-phase HPLC. Partially purified proteases, PAP1 and PAP2 showed a high molecular mass of about 200 kDa by gelatin zymography. PAP1 and PAP2 had optimal pH from 7 to 9 and were inhibited by trypsin inhibitors. These properties of PAP1 and PAP2 suggest that both enzymes belong to the membrane-associated trypsin-like serine protease family, such as enteropeptidase and corin. This is the first report verifying the existence of the activating protease of group IB pro-sPLA(2) isoforms in a non-digestive tissue.

Partial cloning and expression of mRNA coding choline acetyltransferase in the spinal cord of the goldfish, Carassius auratus.

Choline acetyltransferase (ChAT, EC 2.3.1.6) synthesizes a neurotransmitter, acetylcholine in cholinergic neurons. ChAT is considered to be the most specific marker for cholinergic neurons. To obtain a better marker of the neurons, as the first step, we isolated a partial ChAT cDNA from the goldfish (Carassius auratus) brain by RT-PCR methods. The partial cDNA of the goldfish ChAT was composed of 718 nucleotides. The amino acid sequence of the goldfish ChAT is approximately 70% identical to those of mammalian and chicken ChAT. Northern blot analysis demonstrated that ChAT mRNA was expressed in the brain and the spinal cord of the goldfish, and much abundant in the spinal cord. In the spinal cord of the goldfish, ChAT-positive neurons were detected mainly in the ventral horn by in situ hybridization. In addition, fluorescence in situ hybridization combined with a retrograde labeling by using True Blue demonstrated ChAT mRNA positive neurons were exactly motoneurons. In the cord, putative presynaptic sympathetic neurons were also labeled.

Molecular cloning, expression, and characterization of secretory phospholipase A2 in tobacco.

Phospholipase A2 (PLA2) activity was investigated in various tissues of tobacco (Nicotiana tabacum). PLA2 activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA2 activity in the flower appears to have originated from both Ca2+-dependent and -independent PLA2. A cDNA clone for protein with homology to animal secretory PLA2 (sPLA2), denoted as Nt PLA2, was isolated from the tobacco flower. The cDNA of Nt PLA2 encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence for mature Nt PLA2 contains 12 cysteines, a Ca2+ binding loop, and a catalytic domain that are commonly conserved in animal sPLA2. The Nt PLA2 mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA2 was expressed as a fusion protein with thioredoxin in Escherichia coli. From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then the recombinant mature Nt PLA2 was purified by ion exchange chromatography. It was discovered that the purified Nt PLA2 essentially requires Ca2+, for the enzyme activity when the activity was determined using mixed-micellar phospholipid substrates with sodium cholate. The optimal activity of Nt PLA2 was at pH 8-10 when PC was used as a substrate.

Characterization of secretory phospholipase A2 with phospholipase A1 activity in tobacco, Nicotiana tabacum (L.).

A cDNA encoding protein with homology to plant secretory phospholipase A2 (sPLA2), denoted as Nt1 PLA2, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA2 has 12 cysteines, Ca²âº binding loop and catalytic site domain that are commonly conserved in plant sPLA2s. The recombinant Nt1 PLA2 was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA2 could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²âº essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA2 mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.

Molecular cloning and effect of c-fos mRNA on pharmacological stimuli in the goldfish brain.

c-fos is an immediate early gene, and is rapidly and transiently induced in neurons of the central nervous system according to their activities. To investigate neuronal activities in the brain of the goldfish (Carassius auratus), we considered that expression of c-fos mRNA would be an available marker for the neuronal activities. Therefore, we firstly isolated a cDNA clone encoding c-Fos from the goldfish brain by RT-PCR and RACE methods. A full length cDNA of the goldfish c-fos was composed of 1044 bp open reading frame. The amino acid sequence of the goldfish c-Fos was approximately 56-90% identical to those of other teleostean fish c-Fos. Northern blot analysis showed that the expression of c-fos mRNA was rapidly and transiently induced in the brain of the goldfish by the intraperitoneal administration of kainic acid. We also showed that the identification of the c-fos mRNA expression site by in situ hybridization will be able to be used as an anatomical marker for the identification of the activated neuronal region in the goldfish brain.

Purification and characterization of three isoforms of chrysophsin, a novel antimicrobial peptide in the gills of the red sea bream, Chrysophrys major.

We report here the isolation of three isoforms of a novel C-terminally amidated peptide from the gills of red sea bream, Chrysophrys (Pagrus) major. Peptide sequences were determined by a combination of Edman degradation, MS and HPLC analysis of native and synthetic peptides. Three peptides, named chrysophsin-1, chrysophsin-2, and chrysophsin-3, consist of 25, 25, and 20 amino acids, respectively, and are highly cationic, containing an unusual C-terminal RRRH sequence. The alpha-helical structures of the three chrysophsin peptides were predicted from their secondary structures and were confirmed by CD spectroscopy. The synthetic peptides displayed broad-spectrum bactericidal activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Bacillus subtilis, and fish and crustacean pathogens. The three peptides were also hemolytic. Immunohistochemical analysis showed that chrysophsins were localized in certain epithelial cells lining the surface of secondary lamellae and eosinophilic granule cell-like cells at the base of the secondary lamellae in red sea bream gills. Their broad ranging bactericidal activities, combined with their localization in certain cells and eosinophilic granule cell-like cells in the gills, suggest that chrysophsins play a significant role in the innate defense system of red sea bream gills.