Beige mucosa observable under narrow-band imaging indicates the active sites of eosinophilic esophagitis.

BACKGROUND AND AIM: The majority of patients with eosinophilic esophagitis (EoE) are likely to have observable features under narrow-band imaging, namely beige mucosa. However, the histological features and clinical implications of beige mucosa have not been investigated. The aim of this study was to determine whether beige mucosa could serve as an endoscopic marker for predicting active inflammatory sites of EoE. METHODS: We retrospectively analyzed both the narrow-band images and biopsied specimens of 77 esophageal lesions from 35 consecutive patients with EoE. We divided these specimens into two groups: target biopsied specimens from beige mucosa (beige group) and specimens biopsied from non-beige mucosa (non-beige group). The number of eosinophils per high-powered field, thickness of the superficial differentiated cell layer, and depth of the hemoglobin component from the surface layer were compared between the two groups. RESULTS: Forty-four out of the 45 specimens were diagnosed as histological active lesions in the beige group. The sensitivity, specificity, and overall accuracy of beige mucosa in predicting EoE activity were 97.8%, 96.9%, and 97.8%, respectively. Compared with the non-beige group, specimens in the beige group had a significantly thinner superficial differentiated cell layer. CONCLUSIONS: Beige mucosa is associated with thinning of the normal superficial differentiated cell layer, and these histological changes in the active inflammatory sites of EoE could be recognized endoscopically as color differences. Beige mucosa may serve as an endoscopic indicator for predicting the histological activity of EoE.

An Evaluation of Endoscopic Images from Over 15 Years Prior to the Diagnosis of Autoimmune Gastritis: A Report of Three Patients.

We herein describe three patients whose endoscopic images from over 15 years prior to their diagnosis of autoimmune gastritis (AIG) were available for review. All patients had corpus-dominant atrophic gastritis at the time of the diagnosis of AIG. Previous endoscopic images without severe atrophy showed erythema restricted to the fundic mucosa. These findings are suggestive of ongoing gastritis in patients with AIG. Initial endoscopy in Patient 2 showed multiple hyperplastic polyps that decreased in size and number over the course of 15 years. In this patient, circular wrinkle-like patterns and remnant oxyntic mucosa were visible after the atrophy had become quite prominent.

Discovery of 4-[4-({(3R)-1-butyl-3-[(R)-cyclohexyl(hydroxy)methyl]-2,5-dioxo-1,4,9-triazaspiro[5.5]undec-9-yl}methyl)phenoxy]benzoic acid hydrochloride: a highly potent orally available CCR5 selective antagonist.

Based on the original spirodiketopiperazine design framework, further optimization of an orally available CCR5 antagonist was undertaken. Structural hybridization of the hydroxylated analog 4 derived from one of the oxidative metabolites and the new orally available non-hydroxylated benzoic acid analog 5 resulted in another potent orally available CCR5 antagonist 6a as a clinical candidate. Full details of a structure-activity relationship (SAR) study and ADME properties are presented.

Prevalence of Autoimmune Disease in Patients with Eosinophilic Esophagitis: A Cross-sectional Study of Three Hospitals in Japan.

Objective Recent studies have found higher rates of autoimmune diseases, such as celiac disease, Crohn's disease, and ulcerative colitis, in patients with eosinophilic esophagitis (EoE) than in the general population. This study investigated the concomitant rate of autoimmune disease among Japanese patients with EoE and evaluated the clinicopathological characteristics of EoE patients with autoimmune disease. Methods The medical records of patients diagnosed with EoE between April 1, 2016, and June 30, 2020, were reviewed. We analyzed the concomitant rate of autoimmune diseases in patients with EoE and compared the clinical and histological differences between patients with and without autoimmune disease. Results Sixty-four patients with EoE were enrolled. Of them, 1 had ulcerative colitis (1.6%), 1 had autoimmune polyendocrine syndrome type 2 (1.6%), and 6 had endometriosis (28.5% of women, 44.4% of reproductive-aged women). No significant differences in the clinical course or histological findings were found between EoE patients with and without autoimmune diseases. No complications, including stenosis, were seen in EoE with autoimmune disease, and most patients with EoE and autoimmune diseases were responsive to proton pump inhibitors (PPIs). Conclusion In this study, 8 out of 64 patients with EoE had an autoimmune-related disease, including ulcerative colitis (n=1, 1.6%), autoimmune polyendocrine syndrome type 2 (n=1, 1.6%), or endometriosis (n=6, 44.4% of women of reproductive age), which is higher than that previously reported in the general population. Further investigations are required to clarify the relationship between EoE and autoimmune diseases.

Annexin A2 expression and phosphorylation are up-regulated in hepatocellular carcinoma.

Annexins (ANXs) constitute a family of Ca2+-dependent membrane-binding proteins; at least 20 of them have been described to date. Among these, Annexin A2 (ANXA2) has been revealed as a multi-functional protein in vitro. Its actual role in vivo, however, requires further investigation. We already reported that ANX-I (ANXA1) was up-regulated in hepatocellular carcinoma (HCC). The role of ANXA2 in various liver diseases including HCC remains obscure. In the present study, the protein and mRNA levels of ANXA2, as well as its localization, were determined for the normal human liver, chronic hepatitis liver, and non-tumorous and tumorous portions of HCC tissues. ANXA2 was rarely detected in either normal or chronic hepatitis liver tissues, whereas it was overexpressed at both the transcriptional and translational levels in tumorous and non-tumorous regions of HCC. In addition, in many cases, more ANXA2 was expressed in the tumorous portion than in the non-tumorous portion of HCC. The expression of ANXA2 was mainly localized in cancer cells, especially in poorly differentiated HCC. Furthermore, ANXA2 was tyrosine-phosphorylated in HCC. These data suggest that overexpression and tyrosine phosphorylation of ANXA2 play important roles in the malignant transformation process leading to HCC and are related to the histological grade of HCC.

Outcomes of gastrointestinal defect closure with an over-the-scope clip system in a multicenter experience: An analysis of a successful suction method.

AIM: To demonstrate the clinical outcomes of a multicenter experience and to suggest guidelines for choosing a suction method. METHODS: This retrospective study at 5 medical centers involved 58 consecutive patients undergoing over-the-scope clips (OTSCs) placement. The overall rates of technical success (TSR), clinical success (CSR), complications, and procedure time were analyzed as major outcomes. Subsequently, 56 patients, excluding two cases that used the Anchor device, were divided into two groups: 14 cases of simple suction (SS-group) and 42 cases using the Twin Grasper (TG-group). Secondary evaluation was performed to clarify the predictors of OTSC success. RESULTS: The TSR, CSR, complication rate, and median procedure time were 89.7%, 84.5%, 1.8%, and 8 (range 1-36) min, respectively, demonstrating good outcomes. However, significant differences were observed between the two groups in terms of the mean procedure time (5.9 min vs 14.1 min). The CSR of the SS- and TG-groups among cases with a maximum defect size ≤ 10 mm and immediate or acute refractory bleeding was 100%, which suggests that SS is a better method than TG in terms of time efficacy. The CSR in the SS-group (78.6%), despite the technical success of the SS method (TSR, 100%), tended to decrease due to delayed leakage compared to that in the TG-group (TSR, CSR; 88.1%), indicating that TG may be desirable for leaks and fistulae with defects of the entire layer. CONCLUSION: OTSC system is a safe and effective therapeutic option for gastrointestinal defects. Individualized selection of the suction method based on particular clinical conditions may contribute to the improvement of OTSC success.

Expression of cytokeratin 19 in human hepatocellular carcinoma cell lines.

It has been reported that the cytokeratin 19 (CK19) fragment, CYFRA 21-1, is increased in sera of some hepatocellular carcinoma patients. We hypothesized that CYFRA 21-1 might be released directly from hepatocellular carcinoma. To investigate this mechanism, we evaluated expression of CK19 in 8 human cell lines by immunoradiometric assay, Western blotting, immunohistochemistry, and reverse transcriptase-polymerase chain reaction in Chang liver cells, 2 cholangiocarcinoma cell lines, and 5 hepatocellular carcinoma cell lines. Three of 5 hepatocellular carcinoma cell lines released CYFRA 21-1, synthesized CK19 protein and expressed mRNA for CK19, as observed in 2 cholangiocarcinoma cell lines. In contrast, there was no expression of CK19 in Chang liver cells or 2 of 5 hepatocellular carcinoma cell lines. Analysis of genomic DNA for CK19 demonstrated that exon 1 was not amplified in the 3 cell lines not expressing CK19. However, there were no point mutations within exon 1 and the promoter region of CK19 in hepatocellular carcinoma cell lines. Our present study demonstrates that: i) the expression of CK19 was evident in some human hepatocellular carcinoma cell lines, and ii) the expression of mRNA for CK19 was related to the release of CYFRA 21-1. These results partially clarify the mechanism by which some hepatocellular carcinoma cell lines produce CK19 and others do not.

Combination therapy of percutaneous ethanol injection and radiofrequency ablation against hepatocellular carcinomas difficult to treat.

Radiofrequency ablation (RFA) is an effective modality for the treatment of hepatocellular carcinoma (HCC), because it can induce large coagulated necrosis in a few sessions. We have recently reported that the combination therapy of percutaneous ethanol injection (PEI) with RFA (PEI-RFA) created enhancement of coagulated necrosis compared with RFA alone. In the present study, we adopted PEI-RFA for the treatment of HCCs located in the regions that are difficult to treat with RFA alone. Five patients with biopsy-proven HCC and liver cirrhosis underwent PEI-RFA therapy. In these patients, HCCs were located beside the gallbladder, inferior vena cava or portal vein or kidney, or immediately under the diaphragm. Prior to RFA, 99.5% ethanol was injected into the region of HCC located in the regions where RFA energy appears to be difficult to reach. In all cases, HCC was totally coagulated by PEI-RFA. Injecting ethanol prior to RFA therapy caused no major side effects. These results indicate that PEI-RFA may be effective for the treatment of HCCs located in the regions that are difficult to treat with RFA alone as well as large-sized HCCs.

CYFRA 21-1 is released in TNF-alpha-induced apoptosis in the hepatocellular carcinoma cell line HuH-7.

Many types of cancer cells widely express cytokeratin 19 (CK19). Clinical investigations have suggested that serum CYFRA 21-1, a fragment of CK19, is one of the most useful tumor markers. In the present study, we hypothesized that released CYFRA 21-1 is closely associated with cellular apoptosis during tumor growth. Apoptosis was induced by tumor necrosis factor-alpha (TNF-alpha) in the HuH-7 hepatocellular carcinoma cell line. Both TNF-alpha-treated and non-treated cells of HuH-7 were simultaneously examined by immunoradiometric assay, annexin-V apoptosis analysis, immunohistochemical staining and colorimetric protease measurement. Levels of CYFRA 21-1 increased significantly in TNF-alpha-treated cells displaying a high percentage of apoptosis, granular-like aggregation of CK19, and elevated activity of caspase-3 in contrast to non-treated cells. Levels of CYFRA 21-1 decreased significantly after caspase-3 was inhibited in TNF-alpha-treated cells. Thus, the release of CYFRA 21-1 may suggest cellular apoptosis in the process of tumor growth.

Clinical significance of lens culinaris agglutinin-reactive fraction of serum alpha-fetoprotein in patients with hepatocellular carcinoma.

alpha-fetoprotein (AFP) is an important marker for the diagnosis of hepatocellular carcinoma (HCC) and has been widely used in clinical settings. Recently, the importance of lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) has been indicated. However, the clinical significance of the level of AFP-L3 protein in relation to the characteristics of HCC has not been fully evaluated. In the present study, both the ratio of AFP-L3 (AFP-L3%) and the absolute value of AFP-L3 (AFP-L3-AV) were examined in 80 patients with HCC, and evaluated with respect to characteristics of HCC such as grade of differentiation, size of tumor and morphological findings. Among HCC-specific tumor markers, AFP, AFP-L3% and protein induced in vitamin K absence (PIVKA-II), AFP showed the highest positive rate in patients with HCC, while AFP-L3% showed the lowest rate. AFP-L3% and AFP-L3-AV were, however, most significantly correlated with the grade of HCC differentiation, while AFP showed the least significant correlation. Furthermore, AFP-L3% was most significantly correlated with the size of HCC in patients with solitary HCC. Conversely, neither AFP-L3-AV nor PIVKA-II showed a significant correlation with the size of HCC. In relation to morphological differences of HCC, although AFP-L3%, AFP-L3-AV and PIVKA-II were significantly higher in the diffuse type of HCC than in the nodular type of HCC, AFP was most significantly correlated with the morphological differences of HCC. These results indicate that tumor markers for HCC, such as AFP, AFP-L3%, AFP-L3-AV and PIVKA-II, may play different roles in predicting the characteristics of HCC.

Aldehyde dehydrogenase-2 genotypes and HLA haplotypes in Japanese patients with esophageal cancer.

The aim of this study was to examine how aldehyde dehydrogenase-2 (ALDH2) genotypes and human leukocyte antigen (HLA) haplotypes contribute to the risk for esophageal cancer. We examined ALDH2 genotypes and HLA haplotypes in 29 Japanese patients with esophageal cancer. The ratio of patients who experienced current or former intense vasodilatation upon consuming alcohol (flushing type) was much higher in individuals with the inactive form of ALDH2 encoded by the ALDH2(2)/2(2) or ALDH2(1)/2(2) genotype than in those with the active form of ALDH2 encoded by the ALDH2(1)/2(1) genotype. The ratio of inactive ALDH2 was significantly higher in patients with esophageal cancer than in control normal subjects, suggesting that alcoholics with inactive ALDH2 were susceptible to esophageal cancer. HLA haplotypes A24, A26, B54, B61 and DR9 were prevalent in patients with esophageal cancer (82.8, 24.1, 34.5, 37.9 and 44.8%, respectively). HLA haplotype of A24 and inactive ALDH2 were simultaneously found in 58.6% of patients with esophageal cancer. Furthermore, we found other primary malignancies in 6 of 29 (20.7%) patients with esophageal cancer, and 4 of these 6 patients had both the inactive form of ALDH2 and the HLA A24 haplotype. The present study showed the high prevalence of the inactive form of ALDH2 and HLA haplotypes A24, A26, B54, B61 and DR9 in Japanese patients with esophageal cancer. Therefore, the examination of genotypes of ALDH2 loci and HLA haplotypes may allow the early detection of esophageal cancer in the Japanese population.

Autoreactive helper T cells specific for cytokeratin 19 in patients with autoimmune hepatitis.

Recently we have demonstrated the existence of anti-cytokeratin 19 (CK19) antibodies in sera of patients with autoimmune hepatitis (AIH). In the present study, we examined the existence of T lymphocytes specific for CK19 in patients with AIH. The frequency of responders having CK19-specific T lymphocytes was significantly higher in AIH patients than in chronic hepatitis C (CH-C) patients and normal subjects. Furthermore, the stimulation index of proliferative responses of peripheral blood mononuclear cells (PBMCs) was significantly higher in AIH patients than in CH-C patients and normal subjects. The phenotype of proliferating PBMCs specific for CK19 was shown to be predominantly CD4(+) T lymphocytes and these CD4(+) T lymphocytes had a Th1 subtype. The present findings demonstrate that there is some population of CD4(+) T lymphocytes with a Th1 subtype specific for CK19 in peripheral blood of patients with AIH. The Th1-predominat pattern of cytokines may induce cytotoxic T lymphocytes (CTLs) specific for the antigenic peptides derived from the autoantigen, including CK19. In addition, these CTLs may attack the hepatocytes and this may be one of the important etiologies of AIH.

Aberrant cytokeratin expression and high susceptibility to apoptosis in autoimmune hepatitis.

Immunopathological differences between autoimmune hepatitis (AIH) and chronic hepatitis C (CH-C) have not been well investigated. Therefore, we immunohistochemically examined the expression of various cytokeratins (CKs) not only in liver tissues of AIH but also in those of CH-C at the active stage. Furthermore, to evaluate the immune surveillance system and the susceptibility to apoptosis, immunohistochemical staining of human leukocyte antigen (HLA)-DRalpha, cathepsin D, B cell leukemia-2 (bcl-2), bcl-2-associated X protein (bax) and caspase 3 was also performed. Heterogeneous expression of CK 8 and CK 18 was observed in hepatocytes of AIH, while homogeneous expression was observed in hepatocytes of CH-C. Aberrant expression of CK 7 and CK 19 was observed in hepatocytes of AIH, while it was not in hepatocytes of CH-C. Expression of HLA-DRalpha was observed in hepatocytes of AIH but not in those of CH-C. Furthermore, expression of cathepsin D, bax and caspase 3 was much stronger in hepatocytes of AIH than in those of CH-C. These results indicate that cytoskeletal alterations of hepatocytes in AIH may increase the susceptibility to apoptosis and induce hepatocyte destruction.

Design and synthesis of inhibitors of inducible nitric oxide synthase. Discovery of a new chemical lead with potential for oral bioavailability.

A series of 2-iminopiperidines fused to small-membered rings (Tables 1 and 2) were synthesised and biologically evaluated using an in vitro human nitric oxide synthase (NOS) inhibition assay. Fused bicyclic compounds 5-9 exhibited nearly the same potency as compound 1 in the hiNOS inhibition assay. Among these, the 1-methyl analogues 8 and 9 showed better isoform selectivity than their corresponding unsubstituted analogues 7 and 6, respectively. Compounds 5 and 6 were also evaluated by an in vivo NO accumulation assay in a mouse model. The discovery process of new chemical leads for an orally bioavailable inhibitor of human inducible NOS (iNOS) is reported. The structure-activity relationship (SAR) study and chemistry of these compounds are also reported.

Clinical characteristics of patients with autoimmune hepatitis seropositive for anticentromere antibody.

AIM: Recent studies have revealed that primary biliary cirrhosis patients with anticentromere antibody (ACA) commonly develop portal hypertension. However, the clinical characteristics of autoimmune hepatitis (AIH) remain uncertain. We investigated the clinical features of patients with AIH seropositive for ACA (ACA-AIH), comparing them with those of patients with AIH seropositive for other immunofluorescent patterns of antinuclear antibodies (ANA) (other-AIH). METHODS: AIH was diagnosed on the basis of the scoring system proposed by the International Autoimmune Hepatitis Group. Seropositivity for ACA was determined by a discrete speckled pattern on HEp-2 cells by an immunofluorescent technique. The severity of histological grading and staging was evaluated by the histological activity index (HAI) score. RESULTS: Eight (17%) of 47 patients with AIH had ACA. No significant differences in age, sex, onset pattern of the disease, progression to hepatic failure and relapse rate were present between the ACA-AIH and other-AIH groups. The frequency of concurrent autoimmune diseases in ACA-AIH was significantly higher than that in other-AIH (75% vs 36%, P = 0.0406). Biochemical analysis revealed a significantly lower mean immunoglobulin G (IgG) level than that in other-AIH (2176 +/- 641 vs 3013 +/- 923 mg/dL, P = 0.0150). However, there were no differences in serum alanine aminotransferase levels, titers of ANA, HAI scores or the positive rate of human leukocyte antigen (HLA)-DR4 between the groups. CONCLUSION: These results suggest that the emergence of ACA is not a distinct entity of AIH, despite its clinical characteristics of a significantly higher frequency of concurrent autoimmune diseases and lower serum IgG levels.

Use of protein array to investigate receptor tyrosine kinases activated in gastric cancer.

Our study used protein array technology to analyze the expression status of various activated receptor tyrosine kinases (RTKs) in gastric carcinoma; then, we sought to discover an effective therapeutic receptor tyrosine kinase for this disease and investigated the anti-tumor mechanism of the therapeutic RTK. In addition to the expressions of activated RTKs in human gastric cancer and adjacent normal mucosa, the expression of activated RTKs in gastric cancer cell lines, MKN74, MKN45, MKN7 and MKN1, were also studied. The RTKs activated in gastric cancer tissue are EGFR, ErbB2, FGFR1, FGFR2alpha insulin R, and EphA4. Among the RTKs activated in gastric cancer tissues, EGFR and ErbB2 were also activated in all gastric cell lines examined in this study. A subsequent in vitro experiment using subcutaneous gastric cancer-bearing athymic nude mice demonstrated that the ErbB2-targeting drug trastuzumab markedly suppressed the growth of gastric cancer. Moreover, using an angiogenesis protein array, the expressions of Ang I, FGF-alpha, FGF-beta TGF-beta and IL-8 in MKN74 xenograft tumors were found to be significantly reduced by treatment with trastuzumab, indicating that trastuzumab may inhibit the expression of angiogenic molecules in MKN74 cells in vivo. These data suggest that ErbB2 is activated in gastric cancer, and the ErbB2-targeting drug trastuzumab may be related to the reduction of Ang 1, FGFalpha, FGFbeta, TGFalpha and IL-8.

Further optimization of sulfonamide analogs as EP1 receptor antagonists: synthesis and evaluation of bioisosteres for the carboxylic acid group.

4-{[2-[(2-Furylsulfonyl)(isobutyl)amino]-5-(trifluoromethyl)phenoxy]methyl}benzoic acid analogs 2a and b and a series of the acid analogs, in which the carboxylic acid residue of 2b was replaced with various kinds of carboxylic acid bioisosteres, were synthesized and evaluated as EP1 receptor antagonists. Compound 2b and its monocyclic acid analogs, in which the carboxylic acid residue of 2b was replaced with monocyclic acid bioisosteres, were found to show potent EP1 receptor antagonist activity. Optimization of the linker Y between the phenyl moiety and the carboxylic acid residue of 2b was also carried out (Table 5). Compounds 2b and 16 and 17 possessing conformationally restricted linker Y were found to show the most optimized potency among the tested compounds. Cytochrome P450 inhibition of optimized compounds was also investigated. Details of the structure-activity relationship study are presented.

Discovery of heteroaryl sulfonamides as new EP1 receptor selective antagonists.

4-({2-[Isobutyl(phenylsulfonyl)amino]-5-(trifluoromethyl)phenoxy}methyl)benzoic acid (1) is a functional PGE2 antagonist selective for EP1 receptor subtype. Analogs of 1, in which the phenyl-sulfonyl moiety has been replaced with more hydrophilic heteroarylsulfonyl moieties, exhibited more optimized antagonist activity, while some of them showed in vivo antagonist activity. Structure-activity relationship (SAR) studies are also presented.

Design and synthesis of orally bioavailable inhibitors of inducible nitric oxide synthase. Identification of 2-azabicyclo[4.1.0]heptan-3-imines.

Further chemical modification of 2-iminopiperidines fused to cyclopropane rings was performed. Optically active isomers 2 and 13 were synthesized and their biological activity was evaluated. Compound 2 exhibited greater potency and more isoform selectivity than enantiomer 13 in the iNOS inhibition assay. One of the gem-chlorines on the fused cyclopropane moiety of 2 was eliminated to produce 3, which showed reduced potency for iNOS inhibition, as well as 4 with an increased potency. The isoform selectivity of 4 was also much higher than that of 3. This was also true for the corresponding methyl derivatives 6-9. The structure-activity relationship (SAR) study and computer aided docking study of the most optimized structure 4 with human iNOS will also be reported.