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Intestinal stem cells (ISCs) are located at the crypt base and fine-tune the balance of their self-renewal and differentiation 1,2 , but the physiological mechanism involved in regulating that balance remains unknown. Here we describe a transcriptional regulator that preserves the stemness of ISCs by restricting their differentiation into secretory-cell lineages. Interferon regulatory factor 2 (IRF2) negatively regulates interferon signalling 3 , and mice completely lacking Irf2 4 or with a selective Irf2 deletion in their intestinal epithelial cells have significantly fewer crypt Lgr5 hi ISCs than control mice. Although the integrity of intestinal epithelial cells was unimpaired at steady state in Irf2-deficient mice, regeneration of their intestinal epithelia after 5-fluorouracil-induced damage was severely impaired. Similarly, extended treatment with low-dose poly(I:C) or chronic infection of lymphocytic choriomeningitis virus clone 13 (LCMV C13) 5 caused a functional decline of ISCs in wild-type mice. In contrast, massive accumulations of immature Paneth cells were found at the crypt base of Irf2 -/- as well as LCMV C13-infected wild-type mice, indicating that excess interferon signalling directs ISCs towards a secretory-cell fate. Collectively, our findings indicate that regulated interferon signalling preserves ISC stemness by restricting secretory-cell differentiation.

Background: Depressive symptoms cannot be ignored when exercise intervention is performed. The purpose of this study was to clarify the association between depressive symptoms and exercise capacity in community-dwelling older adults., Methods: In this cross-sectional study, we analyzed 110 community-dwelling older adults (mean age [standard deviation] = 70.7 [4.0] years old; women: 55 %). Depressive symptoms were measured using a Japanese version of the Geriatric Depression Scale. We evaluated exercise capacity by measuring distance (2MWD) during a 2-minute walk test. Linear regression models were applied to analyze the association between 2MWD and depressive symptoms., Results: The results suggest that depressive symptoms are associated with low 2MWD (beta = -5.87, 95 % confidence interval = -11.18 to -0.57, p <  0.05) even after adjusting for age, gender, Body Mass Index, cigarette smoking, alcohol consumption, pain severity, and the number of comorbidities., Conclusions: The results indicated that depressive symptoms are associated with decreased exercise capacity in older adults., Competing Interests: Declaration of Competing Interest The authors have no financial conflicts of interest to disclose concerning the study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Study Design: Retrospective study., Objective: To investigate the association between gestational weight gain (GWG) during pregnancy and persistent low back and pelvic pain (LBPP) after delivery., Summary of Background Data: Persistent LBPP after delivery is a risk factor for developing depression and chronic pain as well as incurring sick leave. Women experience weight gain during pregnancy. Excessive weight gain places a greater burden on the musculoskeletal system. However, little is known about how GWG is associated with LBPP after delivery., Methods: After Ethics Committee approval, we analyzed 330 women at 4 months after delivery who had LBPP during pregnancy. The exclusion criteria were as follows: specific low back pain, multiple birth, and incomplete data. Four months after delivery, LBPP was assessed using a self-report questionnaire. Persistent LBPP was defined as pain at 4 months after delivery with an onset during pregnancy or within 3 weeks after delivery. GWG was calculated as the difference between the pregnancy weight and the prepregnancy weight, which we categorized into three groups: <10, 10 to <15, and ≥15 kg. Other confounding factors including age, height, weight at 4 months after delivery, parity, gestational week, mode of delivery, weight of the fetus, and prepregnancy LBPP were assessed. We used logistic regression analysis to calculate LBPP odds ratios (ORs) according to GWG., Results: The prevalence of persistent LBPP was 34.1% (n = 113). Compared with women with a GWG of <10 kg, women with a GWG of ≥15 kg had a higher prevalence of persistent LBPP (OR = 2.77, 95% confidence interval (95% CI) = 1.28-5.96, adjusted OR = 2.35, 95% CI = 1.06-5.21); however, no significant difference was found for women with a GWG of 10 to <15 kg (OR = 1.18, 95% CI = 0.72-1.92, adjusted OR = 1.02, 95% CI = 0.61-1.72)., Conclusions: Our study showed that excessive weight gain during pregnancy is one of the risk factors of persistent LBPP. Appropriate weight control during pregnancy could help prevent persistent LBPP after delivery., Level of Evidence: 3.

Background: In breast cancer survivors, multiple risk factors for health-related quality of life (HRQoL) and chronic pain, including cancer treatment-related factors, psychosocial factors, and central sensitization (CS), have been suggested; however, there has been no comparative study between breast cancer survivors with and without pain. This study aimed to compare the demographic characteristics, psychological factors, and CS-related symptoms between breast cancer survivors with pain, those without pain, and healthy controls, and to investigate the relationships of these factors with HRQoL., Methods: We conducted a cross-sectional survey of 218 women, including patients who underwent breast cancer surgery and adjuvant therapy and healthy women., Results: Patients were divided into the pain group (n = 42), without-pain group (n = 51), and healthy group (n = 47); thus, among breast cancer survivors, 45% reported chronic pain. The proportion of participants who received breast cancer treatments, such as axillary lymph node dissection and chemotherapy, was higher in the pain group than in the without-pain group (p < 0.05). The Central Sensitization Inventory (CSI) and psychosocial factors in the pain group were higher than those in the without-pain group and healthy group (p < 0.01). The CSI and PCS showed larger effect sizes than treatment-related factors. Moreover, HRQoL was significantly correlated with CSI, PCS, Patient Health Questionnaire-2, and Generalized Anxiety Disorder-2 scale (all, p < 0.01). On multiple linear regression analysis, CSI accounted for 43% of the variance in HRQoL., Conclusions: CS and pain catastrophizing may be more associated with the development and/or maintenance of persistent pain than treatment-related factors.

Background: Cation transport regulator 1 (CHAC1), a newly discovered enzyme that degrades glutathione, is induced in Helicobacter pylori (H. pylori)-infected gastric epithelial cells in culture. The CHAC1-induced decrease in glutathione leads to an accumulation of reactive oxygen species and somatic mutations in TP53. We evaluated the possible correlation between H. pylori infection and CHAC1 expression in human gastric mucosa., Materials and Methods: Both fresh-frozen and formalin-fixed paraffin-embedded tissue samples of gastric mucosa with or without H. pylori infection were obtained from 41 esophageal cancer patients that underwent esophago-gastrectomy. Fresh samples were used for real-time polymerase chain reaction for H. pylori DNA and CHAC1 mRNA, and formalin-fixed samples were used for immunohistochemistry with anti-CHAC1 and anti-H. pylori monoclonal antibodies. Double-enzyme or fluorescence immunohistochemistry and immuno-electron microscopy were used for further analysis., Results: Significant CHAC1 overexpression was detected in H. pylori-infected parietal cells that expressed the human proton pump/H,K-ATPase α subunit, whereas a constitutively low level of CHAC1 mRNA expression was observed in the other samples regardless of the H. pylori infection status, reflecting the weak CHAC1 expression detected by immunohistochemistry in the fundic-gland areas. Immuno-electron microscopy revealed intact H. pylori cells in the secretory canaliculi of infected parietal cells. Some parietal cells exhibited positive nuclear signals for Ki67 in the neck zone of the gastric fundic-gland mucosa with H. pylori infection., Conclusion: Cation transport regulator 1 overexpression in H. pylori-infected parietal cells may cause the H. pylori-induced somatic mutations that contribute to the development of gastric cancer., (© 2019 The Authors. Helicobacter Published by John Wiley & Sons Ltd.)

Background: Cardiac resynchronization therapy (CRT) is an effective treatment of heart failure (HF) with ventricular dyssynchrony, but not all patients respond to a similar extent. We investigated the efficacy and safety of exercise training (ET) in patients without response to CRT. Methods and Results: Thirty-four patients who participated in a 3-month ET program and underwent cardiopulmonary exercise testing at baseline and after the program were divided into 17 responders and 17 non-responders based on echocardiographic response criteria: either an increase in ejection fraction (EF) ≥10% or a reduction in left ventricular (LV) end-systolic volume ≥10%. Baseline characteristics including peak oxygen uptake (V̇O 2 ) and isometric knee extensor muscle strength (IKEMS) were similar in both groups, but non-responders had lower EF and larger LV. During the ET program, neither group had exercise-related adverse event including life-threatening ventricular arrhythmia. Peak V̇O 2 and IKEMS were significantly improved in both groups and there was no significant difference in change in peak V̇O 2 or IKEMS between responders and non-responders. On multiple regression analysis, change in IKEMS was an independent predictor of change in peak V̇O 2 , whereas the response to CRT was not. Conclusions: In HF patients undergoing CRT implantation, ET safely improved exercise capacity regardless of response to CRT, suggesting that even advanced HF patients without response to CRT can possibly benefit from ET., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2019, THE JAPANESE CIRCULATION SOCIETY.)

This study aimed to elucidate the predictors of improvements in exercise capacity during cardiac rehabilitation (CR) in the recovery phase after coronary artery bypass graft surgery (CABG) versus acute myocardial infarction (AMI). We studied 152 patients (91 after AMI and 61 after CABG) who participated in a 3-month CR program. All patients underwent a cardiopulmonary exercise test, blood tests, maximal quadriceps isometric strength (QIS) measurement, and bioelectrical impedance body composition measurement at the beginning and end of the 3-month CR program. At baseline, the percentage of predicted peak oxygen uptake (%pred-PVO 2 ), maximal QIS, and hemoglobin (Hb) were significantly lower, while C-reactive protein (CRP) was significantly higher, in the CABG than the AMI group. After the 3-month CR, %change in PVO 2 (%ΔPVO 2 ) was significantly greater in the CABG than the AMI group (18 ± 15% vs 11 ± 12%, P < 0.01). At univariate analysis, baseline plasma brain natriuretic peptide (BNP), %change in maximal QIS after CR (%Δ maximal QIS), and change in plasma hemoglobin (ΔHb) significantly correlated with %ΔPVO 2 in the CABG group, whereas only baseline %pred-PVO 2 did so in the AMI group. Multiple regression analysis revealed that the same factors were independent and significant predictors of %ΔPVO 2 in the CABG and AMI groups. The predictors of improvements in exercise capacity after CR differed between patients after CABG or AMI. Specifically, in CABG patients both enhancing QIS and correcting anemia may contribute to greater improvements in exercise capacity after CR, while a more effective CR program should be designed for CABG patients with high baseline BNP.

Infection with Helicobacter pylori is known to decrease the level of glutathione in gastric epithelial cells and increase the production of reactive oxygen species (ROS), which can lead to DNA damage and the development of gastric cancer. Cation transport regulator 1 (CHAC1) has γ-glutamylcyclotransferase activity that degrades glutathione. We found that cagA -positive H. pylori infection triggered CHAC1 overexpression in human gastric epithelial (AGS) cells leading to glutathione degradation and the accumulation of ROS. Nucleotide alterations in the TP53 tumour suppressor gene were induced in AGS cells overexpressing CHAC1, whereas no mutations were detected in cells overexpressing a catalytically inactive mutant of CHAC1. A high frequency of TP53 mutations occurred in H. pylori -infected AGS cells, but this was prevented in cells transfected with CHAC1 siRNA. These findings indicate that H. pylori -mediated CHAC1 overexpression degrades intracellular glutathione, allowing the accumulation of ROS which subsequently causes mutations that could contribute to the development of gastric cancer.

Purpose: Although cardiac rehabilitation is recommended for patients early after heart transplantation (HTx), adequate exercise effect cannot always be obtained, partly because in patients with chronic heart failure, exercise capacity is reduced due to malnutrition while waiting for HTx. This study aimed to investigate the relationships between exercise capacity and clinical variables, including nutritional indicators, early after HTx., Patients and Methods: Forty-three HTx recipients were studied. The mean age at HTx was 38 ± 14 years, and 86% were male. We assessed the relationships between peak oxygen uptake (VO 2 ) and clinical variables, including plasma B-type natriuretic peptide (BNP), isometric knee extensor muscle strength (KEMS), and nutritional indicators within 1 week of their respective discharges., Results: Peak VO 2 correlated positively with isometric KEMS (r = .63, P < .0001) and negatively with BNP level (r = -.37, P = .015). Of the nutritional indicators, only cholinesterase levels had a significant relationship with peak VO 2 (r = .34, P = .028), whereas the Geriatric Nutritional Risk Index and the Controlling Nutritional Status scores did not. In multiple linear regression analysis, cholinesterase levels and isometric KEMS were independent predictors of peak VO 2 ., Conclusion: Cholinesterase levels predicted exercise capacity early after HTx., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Mucosal barrier dysfunction is considered a critical component of Crohn's disease (CD) pathogenesis after the identification of susceptibility genes. However, the precise mechanism underlying mucosal barrier dysfunction has not yet been elucidated. We therefore aimed to elucidate the molecular mechanism underlying the expression of human α-defensin 6 (HD6) in patients with CD., Methods: HD6 expression was induced by the transfection of an atonal homolog 1 (Atoh1) transgene and was assessed by reverse transcription polymerase chain reaction. The HD6 promoter region targeted by Atoh1 and β-catenin was determined by reporter analysis and chromatin immunoprecipitation assay. HD5/HD6/Atoh1/β-catenin expression in noninflamed jejunal samples collected by balloon endoscopy from 15 patients with CD and 9 non-inflammatory bowel disease patients were assessed by immunofluorescence., Results: Both promoter activity and gene expression of HD6 was significantly upregulated by the Atoh1 transgene in human colonic cancer cell line. We identified a TCF4 binding site and an E-box site, critical for the regulation of HD6 transcriptional activity by directly binding of Atoh1 in the 200-bp HD6 promoter region. The treatment with β-catenin inhibitor also decreases HD6 promoter activity and gene expression. Moreover, HD6 expression, but not HD5 expression, was found to be decreased in noninflamed jejunal regions from patients with CD. In HD6-negative crypts, nuclear accumulation of β-catenin was impaired., Conclusions: HD6 expression was found to be regulated by cooperation between Atoh1 and β-catenin within the HD6 promoter region. Downregulation of HD6 in noninflamed mucosa may contribute to mucosal barrier dysfunction of patients with CD.

Some acts such as the Basic Environment Act are aimed at managing environmental health for a productive living environment in Japan. School is not only a place where lessons for a better future are taught but also an environment in which children spend many hours of their day. Therefore, activities involving regular checks are important to maintain and improve the school environment. Article 5 of the School Health and Safety Act states that schools must make plans and carry out regular checks on school environmental health. Article 6 prescribes that the Minister of Education, Culture, Sports, Science and Technology establish a "school environmental health standard". This standard involves metrics on the classroom environment (quality of air, illumination, and noise levels), quality of drinking/pool water and so on, and their standard values and evaluation methods. Article 23 prescribes that each school except colleges/universities have a school pharmacist. The school pharmacist plays an important role in maintaining and improving the school's environmental health. However, the current actions taken are not adequate. Therefore, prospects for future activities will be discussed based on the current situations and problems.

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colitis-associated colorectal cancer (CAC). CAC cells often develop chemoresistance, resulting in a poorer prognosis than that of sporadic colorectal cancer (CRC). The mechanism by which CAC enhances malignant potential remains unknown. We have previously reported that the proteasomal degradation of the transcription factor Atonal homolog 1 (Atoh1) protein results in the non-mucinous form of CRC. It also remains unknown whether Atoh1 protein is expressed in CAC. Therefore, in the present study, we investigated whether Atoh1 protein stabilizes in CAC. Consequently, the treatment with TNF-α stabilized Atoh1 protein through the inactivation of GSK-3β via Akt, resulting in the mucinous form of CRC cell lines. Atoh1 protein also enriched cancer stem cells with upregulated Lgr5 expression and cells in G0/G1 cell cycle phase, resulting in both the chemoresistance to 5-fluorouracil and oxaliplatin and the promotion of cell migration. Immunofluorescence of the human mucinous CAC specimens showed the accumulation of NF-κB p65 at nuclei with the expression of Atoh1 in mucinous cancer. In conclusion, the inflammation associated with carcinogenesis may preserve the differentiation system of intestinal epithelial cell (IEC), resulting in the acquisition of both the mucinous phenotype and high malignant potential associated with the enrichment of cancer stem cell., (© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Conclusion: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) is considered to be a distinct entity in Japan. The combination of both HPV-DNA sequencing analysis and immunohistochemistry (IHC) for p16(INK4A) is useful to discriminate the OPSCC patients with a better prognosis from other cases, especially in the advanced stage. Surgical treatment is recommended for HPV-negative advanced cancer., Objective: The number of HPV-related OPSCCs has been increasing worldwide. However, the incidence and prognostic significance of this cancer in Japan have not yet been fully elucidated., Methods: Seventy-seven Japanese patients with OPSCC were enrolled in this study. The prevalence of HPV-DNA was assessed by PCR and sequencing. The expression of p16(INK4A) and p53 was examined by IHC. The clinicopathological parameters and disease-specific survival were analyzed for HPV-positive and -negative patients., Results: HPV-DNA was detected in 32 patients. Thirty-four patients were p16(INK4A)-positive by IHC. The patients who were positive for HPV infection were significantly younger. Furthermore, in the stage III or IV cases, the 3-year disease-specific survival of the HPV infection-positive group was significantly better than that of the HPV-negative group. Surgical treatment was demonstrated to lead to a good prognosis for the patients with HPV-negative advanced cancer.

Recently, overexpression of γ-glutamylcyclotransferase (GGCT) has been reported in various cancer tissues suggesting that it has significant potential as a diagnostic marker. The aim of this study was to examine the suitability of GGCT for the detection of high-risk lesions at an early stage of esophageal squamous cell carcinoma (ESCC). A total of 200 lesions, including 120 invasive ESCC, 80 esophageal squamous intraepithelial neoplasia consisting of 40 low-grade intraepithelial neoplasia (LGIEN) and 40 high-grade intraepithelial neoplasia (HGIEN), as well as 20 confounding lesions, were examined by immunohistochemical (IHC) staining for GGCT. IHC staining for Ki-67 and p53 was also performed in esophageal squamous intraepithelial neoplasia to compare the diagnostic power of GGCT. Increased expression of GGCT was common in invasive ESCC and HGIEN (87.5% and 85.0%, respectively), but was much less frequently observed in LGIEN (17.5%). GGCT expression significantly correlated with the presence of lymph node metastasis and the degree of differentiation. In the differential diagnosis of LGIEN and HGIEN, GGCT possessed both high sensitivity and high specificity, while Ki-67 and p53 only possessed either high sensitivity or high specificity. Additionally, GGCT expression was higher in 7 out of 8 ESCC cell lines (KYSE series) than in a normal esophageal squamous cell line (Het-1A). The expression levels strongly correlated with enzymatic activity (r=.92; P<.001). These results indicate that overexpressed GGCT retains its enzymatic activity and can become a valuable biomarker for the diagnosis of HGIEN that is likely to progress to subepithelial invasion., (© 2014.)

We introduced an attempt at cancer rehabilitation at Osaka Medical College Hospital. We also reported trends in the clinical department that ordered the cancer rehabilitation, and the days needed to consult the rehabilitation department after hospitalization for 1,028 patients who needed rehabilitation from January to June 2012. The number of rehabilitation orders for cancer patients has increased in comparison with the same period during 2009, and the percentage of cancer rehabilitation orders has also increased, both in total and in each clinical department consulted. In addition, clinical departments that introduced a rehabilitation schedule along with their treatments ordered cancer rehabilitations much earlier than those departments without such a schedule. In future, to start cancer rehabilitation at an earlier stage, we should endeavor to create awareness of the importance of cancer rehabilitation and the introduction of a rehabilitation schedule along with cancer treatments.

Background: Gastric "crawling-type" adenocarcinoma (CTAC) is a neoplasm histologically comprising irregularly fused glands with low-grade cellular atypia that tends to spread laterally in the mucosa. It is necessary to elucidate the clinicopathological characteristics of CTAC., Methods: We evaluated 25 CTACs-16 intramucosal (M-) and 9 submucosal invasive (SM-) cancers-clinicopathologically and immunohistochemically., Results: CTAC was most frequently located in the lesser curvature of the middle-third of the stomach. Macroscopically, 21 lesions were superficial-depressed and 4 were superficial-flat type. Histologically, all CTACs had cystic dilated glands and 16 lesions had focal signet-ring cells. All invasive areas of the SM-CTACs were occupied by poorly differentiated adenocarcinoma with an infiltrative growth pattern and abundant stroma. Fifteen CTACs were surrounded by mucosa with partial or no intestinal metaplasia. In the intramucosal area, 24 lesions were mixed phenotype with mucin and brush border immunoexpression. SM-CTAC was frequent in lesions with an intramucosal poorly differentiated component (PDC) greater than 10 mm in size (P = 0.041), and lymph node metastasis (LNM) was frequent in lesions with a PDC greater than 20 mm (P = 0.039). The frequency of an expanded pattern (Ki-67-positive cells occupying > 50 % of the mucosa) was higher in SM-CTAC than in M-CTAC (P = 0.027). p53 overexpression was not detected in the intramucosal areas of any of the lesions., Conclusion: CTAC is a distinct subgroup of gastric adenocarcinoma in the early phase. A larger PDC and a Ki-67 expanded pattern were predictive of submucosal invasion or LNM.

World Health Organization (WHO) indicated that recognition of the responsibility of individuals for their own health and awareness that professional care for minor ailments is often unnecessary were important points of view for "Self-Medication" in "Guidelines for the Regulatory Assessment of Medicinal Products for Use in Self-Medication" published in 2000. In Japan, educational curriculum in each school is formulated by "the Courses of Study". Education for health at junior and senior high school is mainly carried out in "Health and Physical Education" class. The objectives are to enable students to develop qualities and abilities to appropriately manage and improve their health throughout their lives through understanding of health and safety in personal and social life. This idea is common with self-medication at a basic concept level. The previous Courses of Study were revised in 2008 and 2009. They described about medicine for senior but not junior high school. The interim report of "The Central Council for Education" in 2005 pointed out that understanding about effects/side effects by medicines, and abilities to use them appropriately are one of minimal qualities that all children must acquire. Due to revision of the Pharmaceutical Affairs Law in 2006, the necessity of endeavoring to widespread knowledge and enlighten about proper use of medicines in school education was considered. Since the new Courses of Study in the junior high school shows content that medicines should be used properly, it is thought that educational frame of medicines for children is going into new era in Japan.

For patients chronically infected with hepatitis C virus (HCV), mutations in the non-structural 5A (NS5A) gene are important predictive factors for the response to interferon (IFN) therapy. In the present study, factor analysis of the therapeutic response of patients following pegylated IFN and ribavirin combination therapy was assessed in a multicenter study. Chronic HCV-infected patients with genotype 1b and high viral load (n=96, mean age 56.5 years; 59 males, 68 females) treated with pegylated IFN-α-2b and ribavirin combination therapy were enrolled. This study was conducted at Kobe University Hospital and 25 affiliated hospitals in Hyogo prefecture. Sixty-five patients (68%) completed treatment with both pegylated IFN and ribavirin at >80% of the weight-based scheduled dosages. Patients who reduced or terminated therapy were frequently aged women (mean age 60.8 years; 11 males, 17 females). Overall, a sustained viral response (SVR) was achieved in 42 (44%) patients out of 96. Based on per-protocol-based (PPB) analysis, the SVR rate in patients with ≥6 amino acid (aa) mutations in the IFN resistance-determining region (IRRDR) (75%) or ≥1 aa mutation in the IFN sensitivity-determining region (ISDR) (61%) was significantly higher than that in patients with <5 aa mutations in IRRDR (30%) or no mutation in ISDR (29%). Multivariate analysis revealed that rapid viral response (RVR) (odds ratio, 18.1) and mutations of ≥6 in IRRDR (odds ratio, 15.5) were significantly associated with SVR. In conclusion, mutations in the NS5A region, particularly in patients with ≥6 aa mutations in IRRDR were strongly associated with a therapeutic response to pegylated IFN and ribavirin combination therapy.

Histologic evaluation of low-grade or high-grade intraepithelial neoplasia (LG-IN or HG-IN) of the esophagus is important for estimating the risk of progression to invasive carcinoma. Discrimination between LG-IN and HG-IN, or neoplasia and non-neoplastic lesion (NNL), however, is occasionally difficult. This study was designed to evaluate whether cytokeratin expression can be used for discrimination of these lesions. Esophageal Iodine-unstained lesions (n=154), less than 10 mm, were classified into HG-IN, LG-IN, and NNL. These lesions together with 154 foci of normal esophageal epithelium (NEE) were examined by immunohistochemistry for cytokeratins (CK4 and CK13), p53 overexpression, and the MIB-1 labeling index. The ratios of CK4- and CK13-positive staining were scored from 1 to 3. The CK4- and CK13-positive staining ratios were decreased in NNL (73% and 78%), LG-IN (55% and 69%), and HG-IN (33% and 48%), compared to NEE (91% and 95%). The differences between LG-IN and HG-IN, neoplasia and NNL, and among these three lesions and NEE were statistically significant (p < 0.005). The cytokeratin scores correlated with the MIB-1 labeling index (both: p < 0.0001), but not with p53 overexpression. CK4 and CK13 immunohistochemistry could be an objective method for evaluating the risk for progression to invasive carcinoma.

Background: Double balloon endoscopy (DBE) enables the observation and collection of viable specimens from the entire intestine, thereby allowing more detailed investigation of how the structure and function of the human small intestine are regulated. The present study aimed to elucidate the regulation of cell formation in the human small intestine using biopsy specimens collected from an entire individual small intestine by DBE., Methods: The expression and the localization of representative genes for the differentiation program were analyzed in the entire small intestine of 10 patients. The functional correlation between Hath1 and Klf4 was analyzed in an intestinal cell line by using a Tet-On system., Results: In longitudinal cell formation in the small intestine, it was shown that goblet cells, but not Paneth cells, increased toward the ileum in each individual small intestine. Immunohistochemistry showed that Hath1-expressing cells migrated from the base of the crypt to the top of the villi in the terminal ileum, while Klf4-expressing cells migrated from the top of the villus, resulting in the colocalization of Hath1 and Klf4 in the terminal ileum. Coexpression of Hath1 and Klf4 upregulated the expression of phenotypic genes for goblet cells following the downregulation of those for Paneth cells., Conclusions: Using mapping biopsy by DBE, we have demonstrated, for the first time, the molecular basis of the villus structure in the entire human small intestine in vivo. The present study showed that longitudinal cell formation was regulated by the colocalization of Hath1 and Klf4 that converted Paneth cell differentiation into goblet cell differentiation.

Context: Although clinical data for beneficial effects of Betaferon, human recombinant-interferon (r-IFN) β-1b, are accumulating, what is less evident is how and why it works., Objective: The present study was carried out to examine whether Betaferon suppresses progression of experimental autoimmune encephalomyelitis (EAE)., Materials and Methods: The EAE model was employed in guinea pigs in vivo, and mononuclear cell proliferation and 2',5'-oligoadenylate synthetase activity were assessed in vitro., Results: Betaferon was more reactive in two assays of guinea pigs, mitogen-induced proliferation of peripheral blood mononuclear cells and 2',5'-oligoadenylate synthetase activity of blood, than in rats and rabbits. Guinea pigs were immunized actively by antigen, porcine myelin basic protein. The neurological deficits were assessed by clinical signs scored daily. Guinea pig Betaferon, replaced with guinea pig albumin (GPA), at 1.2 and 12.0 MIU/kg/day or vehicle was administered subcutaneously daily for 20 days in the immunized guinea pigs. GPA-Betaferon suppressed the manifestation of ataxia or more progression of chronic neurological deficits significantly at 1.2 MIU/kg (p <0.05). Two out of 10 animals manifested no clinical signs in the GPA-Betaferon-treated group with the higher dose, while all animals were worsened with typical clinical signs of EAE in the vehicle group where mononuclear cell infiltrates around blood vessels were seen in the spinal cord of vehicle-treated animals., Discussion and Conclusion: Human r-IFN β-1b attenuates progression of neurological deficits in the EAE model of guinea pigs with evidence for higher susceptibility of animal cells/tissues to the human cytokine, in contrast with rodents and rabbits.

Underdeveloped nations are relatively protected from the worldwide asthma epidemic; the hygiene hypothesis suggests this is due to suppression of Th2-mediated inflammation by increased exposure to pathogens and their products. Although microbial exposures can promote Th2-suppressing Th1 responses, even Th2-skewing infections, such as helminths, appear to suppress atopy, suggesting an alternate explanation for these observations. To investigate whether induction of regulatory responses by helminths may counter allergic inflammation, we examined the effects of helminth infection in a murine model of atopic asthma. We chose Heligosomoides polygyrus, a gastrointestinal nematode, as the experimental helminth; this worm does not enter the lung in its life cycle. We found that H. polygyrus infection suppressed allergen-induced airway eosinophilia, bronchial hyperreactivity, and in vitro allergen-recall Th2 responses in an IL-10-dependent manner; total and OVA-specific IgE, however, were increased by worm infection. Finally, helminth-infected mice were protected against eosinophilic inflammation induced by adoptive transfer of OVA-stimulated CD4(+) cells, and transfer of cells from helminth-infected/OVA-exposed mice suppressed OVA-induced eosinophilic inflammation, suggesting a role for regulatory cells. Increased CD4(+)CD25(+)Foxp3(+) cells were found in thoracic lymph nodes of helminth-infected/OVA-exposed mice. Helminthic colonization appears to protect against asthma and atopic disorders; the regulatory cytokine, IL-10, may be a critical player.

Murine models of acute atopic asthma may be inadequate to study the effects of recurrent exposure to inhaled allergens, such as the epithelial changes seen in asthmatic patients. We developed a murine model in which chronic airway inflammation is maintained by repeated allergen [ovalbumin (OVA)] inhalation; using this model, we examined the response to mucosal administration of CpG DNA (oligonucleotides) and specific antigen immunotherapy. Mice repeatedly exposed to OVA developed significantly greater airway hyperresponsiveness and goblet cell hyperplasia, but not airway eosinophilia, compared with those exposed only twice. CpG-based immunotherapy significantly reversed both acute and chronic markers of inflammation as well as airway hyperresponsiveness. We further examined the effect of mucosal immunotherapy on the response to a second, unrelated antigen. Mice sensitized to both OVA and schistosome eggs, challenged with inhaled OVA, and then treated with OVA-directed immunotherapy demonstrated significant reduction of airway hyperresponsiveness and a moderate reduction in eosinophilia, after inhalation challenge with schistosome egg antigens. In this model, immunotherapy treatment reduced bronchoalveolar lavage (BAL) levels of Th2 cytokines (IL-4, IL-5, IL-13, and IL-10) without changing BAL IFN-gamma. Antigen recall responses of splenocytes from these mice demonstrated an antigen-specific (OVA) enhanced release of IL-10 from splenocytes of treated mice. These results suggest that CpG DNA may provide the basis for a novel form of immunotherapy of allergic asthma. Both antigen-specific and, to a lesser extent, antigen-nonspecific responses to mucosal administration of CpG DNA are seen.

Background: We have previously demonstrated that CpG oligodeoxynucleotides (CpG-ODNs) protect against eosinophilia and airway hyperresponsiveness in murine models of allergen-induced asthma. Acute inflammation is hypothesized to induce chronic airway responses, but no previous studies have evaluated the effects of CpG-ODNs on allergen-induced airway remodeling. Because remodeling is thought to be responsible for many of the long-term adverse effects on asthmatic patients, we evaluated whether CpG-ODNs might similarly prevent these changes using a murine model of recurrent allergen exposure., Objective: The purpose of this study was to evaluate the effect of CpG-ODNs on chronic inflammatory changes and airway remodeling by using a murine model of chronic allergen-induced asthma., Methods: C57BL/6 mice were sensitized to ovalbumin (OVA) and subsequently exposed to nebulized OVA by means of inhalation 3 times weekly for 6 weeks. Some mice received CpG-ODNs by means of intraperitoneal injection at the time of sensitization. At the end of the exposure period, mice were evaluated for the development of airway inflammation, airway hyperresponsiveness, and airway remodeling., Results: OVA-sensitized mice exposed to recurrent airway challenge with OVA have chronic inflammation, persistent airway hyperresponsiveness, and evidence of airway remodeling, including subepithelial collagen deposition and goblet cell hyperplasia-metaplasia. These changes are significantly reduced in mice treated with CpG-ODNs. Interestingly, mice treated with CpG-ODNs exhibit increased levels of bronchoalveolar lavage transforming growth factor beta, suggesting that regulatory T cells might be responsible for some of these protective effects., Conclusion: CpG-ODNs are effective not only in preventing acute inflammation but also appear to reduce markers of airway remodeling that develop after chronic allergen exposure.

CpG oligodeoxynucleotides (CpG ODNs) are known to induce type 1 T-helper-cell (Th1) responses. We have previously demonstrated that CpG ODNs administered during sensitization prevent Th2-mediated eosinophilic airway inflammation in vivo. We also reported that key Th1 cytokines, gamma interferon (IFN-gamma) and interleukin 12 (IL-12), are not necessary for this protection. Recent in vivo data suggest that CpG ODNs might also reverse established pulmonary eosinophilia. In order to clarify how CpG ODNs can inhibit established Th2 responses, we evaluated the cytokine production from splenocytes from antigen- and alum-immunized mice. Restimulation with antigen induced IL-5, which was clearly inhibited by coculture with CpG ODNs in a concentration-dependent manner. CpG ODNs also induced IFN-gamma, but in a concentration-independent manner. The inhibition of IL-5 production was not mediated through natural killer cells or via CD8(+) T lymphocytes. Although IFN-gamma plays an important role in inhibition of antigen-induced IL-5 production by CpG ODNs, IFN-gamma was not the sole factor in IL-5 inhibition. CpG ODNs also induced IL-10, and this induction correlated well with IL-5 inhibition. Elimination of IL-10 reduced the anti-IL-5 effect of CpG ODNs, although incompletely. This may be because IFN-gamma, induced by CpG ODNs, is also inhibited by IL-10, serving as a homeostatic mechanism for the Th1-Th2 balance. Overproduction of IFN-gamma was downregulated by CpG ODN-induced IL-10 via modulation of IL-12 production. These data suggest that CpG ODNs may inhibit established Th2 immune responses through IFN-gamma and IL-10 production, the latter serving to regulate excessive Th1 bias. These properties of CpG ODNs might be a useful feature in the development of immunotherapy adjuvants against allergic diseases such as asthma.

Background: Allergic rhinosinusitis is characterized by eosinophilic inflammation of the upper airway, which is induced by TH-2 cytokines. CpG oligodeoxynucleotides (ODN) are known to induce TH-1 and to suppress TH-2 cytokines in a variety of settings, including murine models of asthma., Objective: To examine the effect of CpG ODN in a murine model of upper airway allergic inflammation and to correlate with reduction of its manifestations of sneezing and nasal scratching., Methods: BALB/c mice were sensitized using Ovalbumin (Ova) intraperitoneally and challenged with aerosolized Ova. CpG ODN were administered at the time of Ova sensitization. Outcomes measured included nasal symptoms, submucosal eosinophilia in the areas lined by respiratory or olfactory epithelium, and bone marrow eosinophilia. To delineate the mechanism of CpG ODN-induced suppression of eosinophilic inflammation, in vitro experiments were carried out to examine the effect of stimulation with Ova on splenocytes obtained from mice that were treated with CpG or control ODN (or no ODN) in vivo. Supernatant was collected after 72 hours of incubation and cytokines were measured by enzyme linked immunosorbent assay., Results: CpG ODN administered at the time of Ova sensitization effectively abrogated nasal symptoms and eosinophilic upper airway inflammation compared with mice treated with control ODN or with no ODN. Cytokine data revealed that Ova sensitization suppressed IFN-gamma and induced IL-4 and IL-5 compared with non-sensitized mice. CpG ODN treatment reversed these effects., Conclusion: CpG ODN prevents the development of TH-2-mediated eosinophilic inflammation and symptoms in a murine model of allergic rhinosinusitis.

Allergen immunotherapy is an effective but underutilized treatment for atopic asthma. We have previously demonstrated that CpG oligodeoxynucleotides (CpG ODN) can prevent the development of a murine model of asthma. In the current study, we evaluated the role of CpG ODN in the treatment of established eosinophilic airway inflammation and bronchial hyperreactivity in a murine model of asthma. In this model, mice with established ovalbumin (OVA)-induced airway disease were given a course of immunotherapy (using low doses of OVA) in the presence or absence of CpG ODN. All mice then were rechallenged with experimental allergen. Untreated mice developed marked airway eosinophilia and bronchial hyperresponsiveness, which were significantly reduced by treatment with OVA and CpG. CpG ODN leads to induction of antigen-induced Th1 cytokine responses; successful therapy was associated with induction of the chemokines interferon-gamma-inducible protein-10 and RANTES and suppression of eotaxin. Unlike previous studies, these data demonstrate that the combination of CpG ODN and allergen can effectively reverse established atopic eosinophilic airway disease, at least partially through redirecting a Th2 to a Th1 response.

The effect of a novel thiazole derivative, SM-8849, on experimental arthritis in mice was studied and compared to that of prednisolone. SM-8849 and prednisolone reduced the incidence and severity of type II collagen-induced arthritis in mice, as assayed by clinical observation and histopathological studies. Although both agents inhibited type II collagen-induced delayed type hypersensitivity (DTH) in arthritic mice, SM-8849 did not affect the production of humoral antibodies to type II collagen. To examine the inhibitory mechanism of SM-8849, the effects on T cell-dependent allergic inflammation were studied. SM-8849 clearly inhibited T cell-dependent reactions including staphylococcal enterotoxin B (SEB)-induced arthritis, SEB-induced CD25 expression on T cells and sheep red blood cell (SRBC)-induced DTH reaction. SM-8849, however, had no effect on the production of humoral antibody forming cells in the spleen of mice immunized with SRBC. These results indicate that inhibition of type II collagen-induced arthritis by SM-8849 is mainly due to the inactivation of T cells that are related to DTH reaction.

The effect of cyclosporin A on apoptosis in eosinophils was examined to clarify the inhibitory mechanisms of cyclosporin A on allergen-induced eosinophilia in the airway. Eosinophils in bronchoalveolar lavage fluid from sensitized rats after inhaling an allergen were used. More than 50% of the eosinophils that died spontaneously by apoptosis within 24 hour incubation in RPMI 1640 medium contained 10% fetal calf serum. The addition of cyclosporin A or dexamethasone significantly enhanced the eosinophils apoptosis at concentrations of more than 0.1 microM. Apoptosis in eosinophils was considerably suppressed in the presence of culture supernatant of activated splenocytes as a source of various cytokines. Even in the presence of culture supernatant of activated splenocytes, cyclosporin A or dexamethasone facilitated apoptosis in eosinophils. These results suggest that apoptotic death of activated eosinophils is augmented with cyclosporin A and that accelerated apoptosis in eosinophils of the airway may account for the inhibitory effect of cyclosporin A on eosinophilia.

Effects of R[+]-8-([1-[3,4-dimethoxyphenyl]-2-hydroxyethyl]amino) -3,7-dihydro-7-[2-methoxyethyl]-1,3-dimethyl-1H-purine-2,6-dione (MKS-492), a reported type III isozyme inhibitor of cyclic nucleotide phosphodiesterase, on antigen- or platelet activating factor (PAF)-induced bronchoconstriction and allergic reactions in guinea pigs and rats were investigated. 1) MKS-492 inhibited antigen-induced bronchoconstriction in guinea pigs. Aminophylline also inhibited the reaction. 2) MKS-492 inhibited PAF-induced bronchoconstriction and inhibited the increase in airway responsiveness to histamine in guinea pigs, although aminophylline failed to affect these reactions. 3) MKS-492 relaxed guinea pig tracheal muscle in vitro more potently than aminophylline. 4) MKS-492 inhibited leukotriene B4 (LTB4)-induced airway eosinophilia in guinea pigs. 5) MKS-492 inhibited passive cutaneous anaphylaxis and mediator-induced skin reactions in rats more potently than aminophylline. Both drugs inhibited antigen- and phospholipase A2-induced histamine release from guinea pig lung tissue. 6) MKS-492 inhibited PAF-induced O2- generation from guinea pig alveolar macrophages. These results indicate that MKS-492 is a more potent inhibitor of allergic bronchoconstriction and PAF- or LTB4-induced inflammatory reactions in guinea pigs and the allergic cutaneous reactions in rats when compared to aminophylline.

The effects of pretreatment with murine recombinant interleukin 5 (mrIL-5) on platelet activating factor (PAF)-induced bronchoconstriction and airway hyperreactivity were investigated in guinea pigs. The intratracheal administration of mrIL-5 (2.5-10 micrograms) augmented platelet activating factor (PAF; 50 ng/kg)-induced bronchoconstriction in guinea pigs. When IL-5 (2.5 micrograms) was injected intratracheally, PAF (25 ng/kg)-induced bronchoconstriction was not affected, but PAF-induced airway hyperresponsiveness to histamine was exacerbated. Airway inflammation, in terms of increased capillary permeability and the accumulation of leukocytes in bronchoalveolar lavage fluid, was not produced by pretreatment with PAF (25 ng/kg), mrIL-5 (2.5 micrograms), or by a combination of these agents. This mrIL-5-induced augmentation of airway hyperreactivity by PAF was clearly inhibited by the phosphodiesterase-type III inhibitors, SDZ-MKS-492 and AH 21-132, but not by aminophylline.

Anti-inflammatory properties of zinc protoporphyrin disodium (Zn-PP-2Na) were studied. Zn-PP-2Na exhibits anti-allergic action against type III and IV reactions (passive Arthus reaction in rats and tuberculin-induced footpad reaction in mice), but does not affect type I and II reactions (homologous passive cutaneous anaphylaxis in mice and reversed cutaneous anaphylaxis in rats). Zn-PP-2Na also clearly inhibits type II collagen-induced arthritis in mice. The agent inhibits general arthritis symptoms, anti-type-II collagen antibody production and type II collagen-induced delayed type hypersensitivity (DTH) in arthritic mice. Zn-PP-2Na, however, did not affect carrageenin-induced paw edema and histamine- and serotonin-induced skin reactions in rats. Zn-PP-2Na inhibits IL-1-induced mouse lymphocyte proliferation, but does not affect PMA-induced O2- generation from guinea-pig neutrophil. These results indicate that Zn-PP-2Na inhibits type II collagen-induced arthritis in mice due to the antagonism of IL-1 activity and the inhibition of DTH against type II collagen.

Tranilast, an anti-asthmatic drug with anti-allergic properties, inhibited endothelin (ET)-induced asthmatic like respiratory obstruction in guinea pigs when administered orally at 200 mg/kg 1 h prior to the intravenous injection of ET. ET also caused a bronchoconstriction detected as an increase in inflation pressure in Konzett-Rössler apparatus. Tranilast (200 mg/kg, p.o. 1 h before ET) inhibited ET-induced increased inflation pressure. ET-induced bronchoconstriction detected as an increase in inflation pressure was clearly inhibited by the administration of indomethacin (used as a reference drug) at doses of 1 and 5 mg/kg 30 min prior to onset of the reaction. In addition to the above in vivo experiments, tranilast at concentrations between 10(-5) and 10(-4) g/ml inhibited ET-induced contraction of isolated guinea pig tracheal muscle, whereas indomethacin did not affect this in vitro response. In order to elucidate the inhibitory mechanism of tranilast on ET-induced bronchoconstriction, the effects in Ca(2+)-free medium and on ET-induced histamine and prostaglandin F2 alpha release from tracheal muscle were investigated. Consequently, tranilast did not affect ET-induced contraction of guinea pig tracheal muscle in Ca(2+)-free Tyrode's solution and ET induced neither histamine nor PGF2 alpha release. These results suggest that tranilast inhibits ET-induced bronchoconstriction by inhibiting the ET-induced calcium influx into tracheal muscle.

The anti-allergic and anti-asthmatic activities of three potassium (K+) channel openers, cromakalim, pinacidil, and nicorandil, were investigated. 1) Forty-eight-hour homologous passive cutaneous anaphylaxis (PCA) in mice was not affected by cromakalim, pinacidil, or nicorandil. Ketotifen significantly inhibited the reaction. 2) Antigen-induced histamine release from sensitized guinea pig lung tissue was not affected by cromakalim, pinacidil or nicorandil (except for 10(-4) M nicorandil). Salbutamol inhibited the release of histamine. 3) Histamine, serotonin and LTC4-induced vasculitis in rat back skin was not affected by any of these three K+ channel openers. 4) Antigen-induced constriction of isolated sensitized guinea pig tracheal muscle was relaxed by each of the K+ channel openers. 5) Constrictions of isolated guinea pig tracheal muscle caused by high potassium, histamine, LTC4, or U-46619 were clearly relaxed by each of the three K+ channel openers. 6) Increases of airway resistance caused by histamine, LTD4, or U-46619 in guinea pigs in vivo were inhibited by administration of each of the three K+ channel openers. 7) Experimental asthma caused by the IgE antibody and antigen system in guinea pigs was inhibited by each of the three K+ channel openers.

The effect of (9, 11), (11, 12)-didedoxa-9 alpha, 11-alpha-dimethylmethano-11,12-methano-13,14-dihydro-13-aza-14-oxo -15-cyclo-pentyl-16, 17, 18, 19, 20-pentanor-15-epi-TxA2 (ONO-3708) on 9,11-methanoepoxy-prostaglandin H2 (U-46619)-induced contraction of airway smooth muscle in the guinea pigs and human in vitro and bronchoconstriction in guinea pigs in vivo was investigated. In in vitro experiments, ONO-3708 inhibited the U-46619-induced contraction of isolated guinea pig and human tracheal smooth muscle in a dose related fashion (guinea pig; pA2=7.78, human; pA2 = 7.43). The contractions of guinea pig tracheal muscle caused by histamine and leukotriene D4 (LTD4) were not inhibited by ONO-3708. In in vivo experiments, intravenous injection of ONO-3708 at doses between 1 and 20 mg/kg inhibited the U-46619-induced increase of airway insufflation pressure as measured by Konzett-Rössler method. In addition, ONO-3708 inhibited the U-46619-induced increase in airway reactivity to acetylcholine. These data suggest that ONO-3708 has possible therapeutic utility for asthma in which TxA2 participates.

The aim of the present study is to investigate the effect of ketanserin, a potent and selective S2-serotonergic antagonist, on some anaphylactic reactions in mice, rats and guinea-pigs. Ketanserin (1 and 5 mg/kg) inhibited both IgE antibody-mediated 48 hr homologous passive cutaneous anaphylaxis and IgG antibody-mediated 1.5 hr homologous passive cutaneous anaphylaxis in mice. In histamine-, serotonin- and LTC4-induced skin reactions in mice, ketanserin inhibited an increase of capillary permeability caused by each mediator. Cyproheptadine, at doses of 0.1 and 0.5 mg/kg, inhibits 48 hr and 1.5 hr homologous passive cutaneous anaphylaxis and histamine-induced capillary permeability increase in mice ear. The inhibitory activity of cyproheptadine on these reactions is more potent than that of ketanserin. However, ketanserin showed a more potent inhibition than cyproheptadine for serotonin-induced capillary permeability. In rat passive peritoneal anaphylaxis, ketanserin had little effect on the release of histamine from peritoneal mast cells. Ketanserin inhibited an antigen-induced contraction of the sensitized guinea-pig trachea at an early period (within 5 min after treatment with antigen) but not at a late period (between 5 to 15 min). Cyproheptadine had little effect on this antigen-induced contraction of sensitized guinea-pig trachea. Moreover, ketanserin inhibited both histamine- and serotonin-induced contractions of the guinea-pig trachea, but not the contraction caused by carbachol and LTC4. When Forssman antibody was injected into the guinea-pigs, a biphasic increase of airway resistance was observed. Ketanserin inhibited an increase of airway resistance at late phases. These results suggest that ketanserin inhibited some anaphylactic reactions in mice and guinea-pigs probably due to the antagonistic action of histamine and serotonin.

The immunotoxicity of triphenyltin chloride (TPTC) in mice was investigated. 1) When TPTC was injected intraperitoneally (i.p.) into mice at doses between 1 and 10 mg/kg for 14 d, a reduction in the weights of thymus and spleen was noticed, however, the body weight was not changed significantly. 2) The production of hemolytic plaque forming cells in the spleen of mice immunized with sheep red blood cells (SRBC) was inhibited by the administration of 10 mg/kg of TPTC. 3) The i.p. injection of TPTC at a dose of 10 mg/kg for 5 d after the primary immunization of mice with dinitrophenylated ascaris antigen resulted in suppression of immunoglobulin E antibody formation in primary immune response, but did not affect the secondary immune response. 4) The production of antibody against polyvinylpyrrolidone (T cell independent antigen) was not affected by the administration of TPTC. 5) The production of effector T cells which are able to cause SRBC- or tuberculin-induced footpad reaction in mice and the induction of cytotoxic T cell in local graft versus host reaction in mice were also inhibited by TPTC. These results indicate that TPTC inhibits both the T cell dependent humoral and cellular immune responses in mice.

A solid-phase enzyme immunoassay for the measurement of anti-insulin antibodies in the sera of patients with diabetes mellitus was developed. Porcine insulin conjugated with bovine serum albumin was used for coating microtiter plates. This assay was as sensitive as the conventional radioimmunoassay. Anti-insulin antibody titers measured by the enzyme immunoassay and conventional radioimmunoassay correlated well and the correlation coefficient was 0.920, which was statistically significant (P less than 0.001). The enzyme immunoassay detected anti-insulin antibody in 23 out of 35 sera of patients with insulin-dependent diabetes mellitus. The present enzyme immunoassay does not require radioactive materials, is less expensive and is concluded to be practically useful.