Anti-A and anti-B titers, age, gender, biochemical parameters, and body mass index in Japanese blood donors.

It has been reported that anti-A and anti-B (ABO antibody) titers decrease with age, but little is known about the association between ABO antibody titers and physiologic/biochemical parameters such as body mass index (BMI), gamma-glutamyl transpeptidase (GGT), and total cholesterol (T-Cho). We investigated the present situation of ABO antibody titers among healthy blood donors in Japan and the physiologic/biochemical factors that may be associated with changes in ABO antibody titers. Plasma from 7450 Japanese blood donors was tested for ABO antibody titers using ABO reverse typing reagents by an automated microplate system; donor samples were classified into low, middle, and high titers according to the agglutination results obtained with diluted plasma samples. Multivariate regression analysis was performed to analyze the association between ABO antibody titers and age, gender, biochemical parameters (alanine transaminase [ALT], GGT, globulin, T-Cho, and glycosylated albumin [GA]), and BMI according to the ABO blood groups. A significant correlation between ABO antibody titers and age/gender, except for gender in anti-A of blood group B donors, was observed. BMI showed significant but negative correlations with anti-A and anti-B (ß = -0.085 and -0.062, respectively; p < 0.01) in blood group O donors. In addition, significant but negative correlations between GGT and T-Cho with anti-B of blood group A donors (ß = -0.055 and -0.047, respectively; p < 0.05) were observed. Although differences existed among the ABO blood groups, ABO antibody titers seem to be associated with physiologic and biochemical parameters of healthy individuals.

Surgical resection of primary tumor in the extremities improves survival for metastatic soft-tissue sarcoma patients: a population-based study of the SEER database

Purpose The objectives of this study were to clarify whether resection of primary tumor in the extremities for patients with metastatic soft-tissue sarcoma (STS) improves survival, and to clarify patient groups for whom primary tumor resection should be considered. Methods/patients Using the surveillance, epidemiology, and end results database, we identified 1453 patients with metastatic STS of the extremities at initial presentation between 1983 and 2016. Of these 1453 patients, 898 patients underwent primary tumor resection (Surgery group), and 555 patients did not (No-surgery group). Results After adjusting for patient background by propensity score matching, a total of 804 patients were included for analysis. Patients in the Surgery group showed improved survival (cancer-specific survival (CSS) hazard ratio (HR) = 0.59, 95% confidence interval (CI) 0.50–0.71 overall survival rate (OS) HR = 0.60, 95% CI 0.51–0.70). In subclass analysis, patients with high-grade STS, undifferentiated pleomorphic sarcoma, leiomyosarcoma, or synovial sarcoma showed improved survival in the Surgery group (high grade—CSS HR = 0.57, 95% CI 0.45–0.72, OS HR = 0.58, 95% CI 0.48–0.71; undifferentiated pleomorphic sarcoma—CSS HR = 0.60, 95% CI 0.42–0.84, OS HR = 0.61, 95% CI 0.46–0.82; leiomyosarcoma—CSS HR = 0.50, 95% CI 0.33–0.75, OS HR = 0.50, 95% CI 0.35–0.72; synovial sarcoma—CSS HR = 0.46, 95% CI 0.31–0.68, OS HR = 0.43, 95% CI 0.30–0.62). Conclusions Our results indicated that primary tumor resection in metastatic STS exerts positive impacts on survival. Further clinical research is needed to confirm these results (AU)

Surgical resection of primary tumor in the extremities improves survival for metastatic soft-tissue sarcoma patients: a population-based study of the SEER database.

PURPOSE: The objectives of this study were to clarify whether resection of primary tumor in the extremities for patients with metastatic soft-tissue sarcoma (STS) improves survival, and to clarify patient groups for whom primary tumor resection should be considered. METHODS/PATIENTS: Using the surveillance, epidemiology, and end results database, we identified 1453 patients with metastatic STS of the extremities at initial presentation between 1983 and 2016. Of these 1453 patients, 898 patients underwent primary tumor resection (Surgery group), and 555 patients did not (No-surgery group). RESULTS: After adjusting for patient background by propensity score matching, a total of 804 patients were included for analysis. Patients in the Surgery group showed improved survival (cancer-specific survival (CSS) hazard ratio (HR) = 0.59, 95% confidence interval (CI) 0.50-0.71 overall survival rate (OS) HR = 0.60, 95% CI 0.51-0.70). In subclass analysis, patients with high-grade STS, undifferentiated pleomorphic sarcoma, leiomyosarcoma, or synovial sarcoma showed improved survival in the Surgery group (high grade-CSS HR = 0.57, 95% CI 0.45-0.72, OS HR = 0.58, 95% CI 0.48-0.71; undifferentiated pleomorphic sarcoma-CSS HR = 0.60, 95% CI 0.42-0.84, OS HR = 0.61, 95% CI 0.46-0.82; leiomyosarcoma-CSS HR = 0.50, 95% CI 0.33-0.75, OS HR = 0.50, 95% CI 0.35-0.72; synovial sarcoma-CSS HR = 0.46, 95% CI 0.31-0.68, OS HR = 0.43, 95% CI 0.30-0.62). CONCLUSIONS: Our results indicated that primary tumor resection in metastatic STS exerts positive impacts on survival. Further clinical research is needed to confirm these results.

Coordinated existence of multiple gangliosides is required for cartilage metabolism.

OBJECTIVE: Gangliosides, ubiquitously existing membrane components that modulate transmembrane signaling and mediate cell-to-cell and cell-to-matrix interactions, are key molecules of inflammatory and neurological disorders. However, the functions of gangliosides in the cartilage degradation process remain unclear. We investigated the functional role of gangliosides in cartilage metabolism related to osteoarthritis (OA) pathogenesis. DESIGN: We generated knockout (KO) mice by targeting the ß1, 4-N-acetylgalactosaminyltransferase (GalNAcT) gene, which encodes an enzyme of major gangliosides synthesis, and the GD3 synthase (GD3S) gene, which encodes an enzyme of partial gangliosides synthesis. In vivo OA and in vitro cartilage degradation models were used to evaluate the effect of gangliosides on the cartilage degradation process. RESULTS: The GalNAcT and GD3S KO mice developed and grew normally; nevertheless, OA changes in these mice were enhanced with aging. The GalNAcT KO mice showed significantly enhanced OA progression compared to GD3S mice in vivo. Both GalNAcT and GD3S KO mice showed severe IL-1α-induced cartilage degradation ex vivo. Phosphorylation of MAPKs was enhanced in both GalNAcT and GD3S KOs after IL-1α stimulation. Gangliosides modulated by GalNAcT or GD3S rescued an increase of MMP-13 induced by IL-1α in mice lacking GalNAcT or GD3S after exogenous replenishment in vitro. CONCLUSION: These data show that the deletion of gangliosides in mice enhanced OA development. Moreover, the gangliosides modulated by GalNAcT are important for cartilage metabolism, suggesting that GalNAcT is a potential target molecule for the development of novel OA treatments.

Silent KEL alleles identified from Japanese individuals with the Ko phenotype.

BACKGROUND AND OBJECTIVE: The rare Ko phenotype lacks all 36 antigens in the Kell blood system. The molecular basis of the Ko phenotype has been investigated, and more than 40 silent KEL alleles are reported by many investigators. The majority of silent alleles are the KEL*02 background. Here, we report molecular genetic analysis of the KEL gene in Japanese individuals with the Ko phenotype. MATERIALS AND METHODS: The Ko phenotype was screened from Japanese blood donors for several years using monoclonal anti-Ku or anti-K14 by an automated blood grouping system PK7300. Kell-related antigens were typed by standard tube tests. Genomic DNA was extracted from the blood samples, and KEL gene was analysed by polymerase chain reaction (PCR) and Sanger sequencing. RESULTS: We collected 35 Ko blood samples with K-k-, Kp(a-b-), Js(a-b-) and K14-. PCR and sequence analysis revealed that 11 individuals were homozygous for a mutant KEL allele with a c.299G>C (p.Cys100Ser) mutation (rs. 200268316). Three individuals were homozygous for the KEL*02N.24 allele that is c.715G>T (p.Glu239*), and one individual was homozygous for the KEL*02N.40 allele that is c.1474C>T (p.Arg492*). Five individuals were homozygous for novel KEL alleles with single-nucleotide mutations, four individuals had a c.2175delC (p.Pro725 fs*43), and one individual had a c.328delA (p.Arg110 fs*79). The remaining 15 individuals were compound heterozygous, and eight new alleles were identified from them. CONCLUSIONS: We identified three known and ten new silent KEL alleles from Japanese individuals with the Ko phenotype. The KEL allele with the c.299G>C (p.Cys100Ser) mutation was the most frequent.

Proliferative Enteropathy caused by Lawsonia intracellularis in Chickens.

Proliferative enteropathy (PE) is an infectious disease caused by Lawsonia intracellularis (Li), an obligate intracellular bacterium. PE is endemic in swine herds and has been reported in a variety of mammals including horses, hamsters, rabbits, rats, guinea pigs, ferrets, foxes, dogs, sheep, deer and non-human primates. Avian cases are reported only in ratite birds, including emus and ostriches. Some studies show an absence of Lawsonia spp. infection in chickens. In this study, we performed morphological and bacteriological examinations on the intestines of two broiler chickens that had been condemned at a poultry slaughter plant in Japan due to intestinal haemorrhage, which was a result of focal coccidial enteritis. Histopathology revealed proliferation of the villous epithelium in the small and/or large intestines, especially the caeca, regardless of coccidial lesions. Warthin-Starry silver staining and immunohistochemistry using anti-Li monoclonal antibody revealed numerous bacteria and/or antigens in the villous epithelium. Scanning electron microscopy and transmission electron microscopy revealed the presence of curved rods, morphologically compatible with Li, in the apical cytoplasm of the epithelium. Polymerase chain reaction products specific for Li were amplified from DNA samples extracted from formalin-fixed and paraffin wax-embedded tissue. These results suggest that Li can cause PE, characterized by proliferation of the villous epithelium, in chickens.

A novel DO null allele with a c.268C>T (p.Gln90Stop) mutation in Japanese.

The Dombrock blood group system consists of two antithetical antigens, Do(a) (DO1) and Do(b) (DO2), and seven high-prevalence antigens, Gy(a) (DO3), Hy (DO4), Jo(a) (DO5), DOYA (DO6), DOMR (DO7), DOLG (DO8) and DOLC (DO9). Do(a) /Do(b) polymorphism is associated with c.793A>G (p.Asn265Asp) in exon 2 of the DO (ART4) gene, and the corresponding alleles are named DO*01 and DO*02. The rare Donull or Gy(a-) phenotype lacks all Dombrock antigens, and the DO null alleles vary with both DO*01 and DO*02 backgrounds. We report a novel DO null allele, which has a c.268C>T (p.Gln90Stop) nonsense mutation with a DO*02 background identified from four unrelated Gy(a-) Japanese individuals.

Production of human monoclonal anti-Jk3, recognising an epitope including the Jk(a) /Jk(b) polymorphic site of the Kidd glycoprotein.

BACKGROUND AND OBJECTIVES: The Kidd blood group system consists of polymorphic antigens, Jk(a) (JK1) and Jk(b) (JK2), and a high-incidence antigen, Jk3. Anti-Jk3 is often observed in immunised Jk(a-b-) individuals. In this study, we aimed to establish a human hybridoma cell line secreting monoclonal anti-Jk3 (HIRO-294). MATERIALS AND METHODS: Peripheral blood lymphocytes of a Filipino woman with the Jk(a-b-) phenotype having anti-Jk3 were transformed with Epstein-Barr virus and then hybridised with the myeloma cell line JMS-3 using the polyethylene glycol (PEG) method. The reactivity and specificity of the anti-Jk3 were examined by serology and flow cytometry. RESULTS: Four hybridoma clones secreting anti-Jk3 were established and the antibody from one of these clones, HIRO-294, was examined. The reactivity of HIRO-294 was positive with 227 Jk(a+b-) red blood cells (RBCs), 298 Jk(a-b+) RBCs, and 1043 Jk(a+b+) RBCs, but was negative with 21 Jk(a-b-) RBCs. Eluates from Jk(a+b-) RBCs and Jk(a-b+) RBCs sensitised with the anti-Jk3 were cross-reacted with Jk(a-b+) RBCs and Jk(a+b-) RBCs, respectively. The reactivity of HIRO-294 was enhanced by the treatment of RBCs with ficin, trypsin, pronase and α-chymotrypsin, but was not changed by their treatment with neuraminidase, dithiothreitol and ethylenediaminetetraacetic acid (EDTA) glycine acid (GA). The RBCs sensitised by the anti-Jk3 were not agglutinated with the commercial reagents of anti-Jk(a) and anti-Jk(b) by saline test, whereas the nonsensitised RBCs or those sensitised by monoclonal anti-D [HIRO-3, immunoglobulin G (IgG) class] were agglutinated with those reagents. CONCLUSIONS: We established a human hybridoma cell line secreting monoclonal anti-Jk3 (HIRO-294). This antibody had unique specificity, recognising the Kidd glycoprotein including the Jk(a) /Jk(b) polymorphic site.

JK null alleles identified from Japanese individuals with Jk(a−b−) phenotype.

The Kidd blood group system consists of three common phenotypes: Jk(a+b−), Jk(a−b+) and Jk(a+b+), and one rare phenotype, Jk(a−b−). Jka/Jkb polymorphism is associated with c.838G>A (p.Asp280Asn) in exon 9 of the JK (SLC14A1) gene, and the corresponding alleles are named JK*01 and JK*02. The rare phenotype Jk(a−b−) was first found in a Filipina of Spanish and Chinese ancestry, and to date, several JK null alleles responsible for the Jk(a−b−) phenotype have been reported. We report seven novel JK null alleles, 4 with a JK*01 background and 3 with a JK*02 background, identified from Jk(a−b−) Japanese.

Sugarcane molasses-based bio-ethanol wastewater treatment by two-phase multi-staged up-flow anaerobic sludge blanket (UASB) combination with up-flow UASB and down-flow hanging sponge.

This study was designed to evaluate a treatment system for high strength wastewater (vinasse) from a sugarcane molasses-based bio-ethanol plant in Thailand. A laboratory-scale two-phase treatment system composed of a sulfate reducing (SR) tank and multi-staged up-flow anaerobic sludge blanket (MS-UASB) reactor was used as the pre-treatment unit. Conventional UASB and down-flow hanging sponge (DHS) reactors were used as the post-treatment unit. The treatment system was operated for 300 days under ambient temperature conditions (24.6-29.6 °C). The hydraulic retention time (HRT) in each unit was kept at 25 h for the two-phase system and 23 h for the UASB&DHS. The influent concentration was allowed to reach up to 15,000 mg chemical oxygen demand (COD)/L. COD removal efficiency (based on influent COD) of the two-phase MS-UASB and the UASB&DHS was 54.9 and 18.7%, respectively. Due to the effective removal of sulfide in the SR tank, the MS-UASB achieved a high methane conversion ratio of up to 97%. In DHS, nitrification occurred at the outside portion of the sponge media while denitrification occurred at the inside. Consequently, 27% of the total nitrogen (TN) was removed. An amount of 32% of residual nitrogen (28 mgN/L) was in the form of nitrate, a better nitrogen state for fertilizer.

Depletion of gangliosides enhances cartilage degradation in mice.

OBJECTIVE: Glycosphingolipids (GSLs) are ubiquitous membrane components that play a functional role in maintaining chondrocyte homeostasis. We investigated the potential role of gangliosides, one of the major components of GSLs, in osteoarthritis (OA) pathogenesis. DESIGN: Both age-associated and instability-induced OA models were generated using GM3 synthase knockout (GM3S(-/-)) mice. A cartilage degradation model and transiently GM3S-transfected chondrocytes were analyzed to evaluate the function of gangliosides in OA development. The amount of each series of GSLs in chondrocytes after IL-1α stimulation was profiled using mass spectrometry (MS). RESULTS: OA changes in GM3S(-/-) mice were dramatically enhanced with aging compared to those in wild-type (WT) mice. GM3S(-/-) mice showed more severe instability-induced pathologic OA in vivo. Ganglioside deficiency also led to the induction of matrix metalloproteinase (MMP)-13 and ADAMTS-5 secretion and chondrocyte apoptosis in vitro. In contrast, transient GM3S transfection of chondrocytes suppressed MMP-13 and ADAMTS-5 expression after interleukin (IL)-1α stimulation. GSL profiling revealed the presence of abundant gangliosides in chondrocytes after IL-1α stimulation. CONCLUSION: Gangliosides play a critical role in OA pathogenesis by regulating the expression of MMP-13 and ADAMTS-5 and chondrocyte apoptosis. Based on the obtained results, we propose that gangliosides are potential target molecules for the development of novel OA treatments.

Viral gene transfer to developing mouse salivary glands.

Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs, and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have been powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs. Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we have identified new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs. We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day 12 or 13 SMGs. We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Transduction of SMG epithelia with self-complementary (sc) AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis. Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial vs. mesenchymal cells in an organ undergoing branching morphogenesis.

Evaluation of sludge properties in a pilot-scale UASB reactor for sewage treatment in a temperate region.

In this study, continuous operation of a pilot-scale upflow anaerobic sludge blanket (UASB) reactor for sewage treatment was conducted for 630 days to investigate the physical and microbial characteristics of the retained sludge. The UASB reactor with a working volume of 20.2 m(3) was operated at ambient temperature (16-29 °C) and seeded with digested sludge. After 180 days of operation, when the sewage temperature had dropped to 20 °C or lower, the removal efficiency of both total suspended solids (TSS) and total biochemical oxygen demand (BOD) deteriorated due to washout of retained sludge. At low temperature, the cellulose concentration of the UASB sludge increased owing to the rate limitation of the hydrolytic reaction of suspended solids in the sewage. However, after an improvement in sludge retention (settleability and concentration) in the UASB reactor, the process performance stabilized and gave sufficient results (68% of TSS removal, 75% of total BOD removal) at an hydraulic retention time (HRT) of 9.7 h. The methanogenic activity of the retained sludge significantly increased after day 246 due to the accumulation of Methanosaeta and Methanobacterium following the improvement in sludge retention in the UASB reactor. Acid-forming bacteria from phylum Bacteroidetes were detected at high frequency; thus, these bacteria may have an important role in suspended solids degradation.

Simulating the bovine spongiform encephalopathy situation in Japan.

Despite various measures taken by the Japanese government to protect the cattle population from exposure to the bovine spongiform encephalopathy (BSE) agent, the first case of BSE was detected in September 2001. Subsequently, BSE surveillance was enhanced, involving mandatory reporting and investigation of all clinical BSE suspects, and testing of fallen stock and all cattle slaughtered for human consumption. Tests on over nine million cattle led to the detection of 35 additional cases by the end of May 2009. Using the surveillance data and other information as input variables, models were developed to explore the possible source of introduction of BSE into Japan, evaluate the effectiveness of control measures, estimate the prevalence of BSE in different birth cohorts, predict a future BSE epidemic, and simulate the impact of changes in surveillance strategies. Despite difficulties associated with the availability and uncertainty of some of the input variables, these models provided an objective insight into the BSE situation in Japan.

Epidemiological features of the bovine spongiform encephalopathy epidemic in Japan.

On 10 September 2001, the first case of bovine spongiform encephalopathy (BSE) in Japan was confirmed in a five-year-old dairy cow born in Hokkaido and raised in Chiba prefecture. Subsequently, BSE surveillance was enhanced. As a result, 35 additional cases were detected by the end of March 2009, with two to ten cases being detected each year up to the end of 2007. The epidemic appeared to peak in 2006. Cases are detected mostly in dairy cattle; in cattle born in 1996 and 2000; and in cattle born in Hokkaido. Two were atypical cases of BSE and the remainder classical cases. This paper describes and discusses the demographic structure of dairy and beef cattle, surveillance programmes in place and the epidemiological features of the BSE epidemic in Japan.

Recent outbreaks of avian influenza in Japan.

Recently Japan had three outbreaks of avian influenza (Al) in 2004, 2005 and 2007. An outbreak of highly pathogenic avian influenza (HPAI) was recorded in early 2004, the first for 79 years, with four farms being infected with HPAI virus subtype H5N1. In 2005, 41 farms were found to be infected with AI virus subtype H5N2. In early 2007, four farms were infected with HPAI virus subtype H5N1 again. In all of these outbreaks, the disease was eradicated without resorting to vaccination, through a campaign of culling, movement control of chickens in areas around infected premises, and intensive clinical and serological surveillance. This paper describes the nature of the outbreaks, the eradication measures implemented, clinical and serological surveillance techniques used, and the possible sources of infection.

Frequency of rate of body temperature chart at mid cycle in pregnant women and the subsequent effect on pregnancy.

PURPOSE OF INVESTIGATION: To determine if changes in basal body temperature (BBT) during the ovuratory phase are related to subsequent effects on pregnancy. METHODS: BBT records from 216 pregnant women in a spontaneous cycle or a clomiphene citrate cycle during a recent 6-year period were studied. The last day of low phase (LDLP) and the number of days until high phase (NDHP) were determined for all subjects. RESULTS: In the spontaneous cycle group, medium-cycle cases were most frequent and long-cycle cases were most frequent in the clomiphene cycle group. The NDHP ranged between one and three days in 82.8% of the subjects in the spontaneous cycle group and in 86.1% of the subjects in the clomiphene cycle group. CONCLUSIONS: Our findings demonstrate the importance of properly evaluating an NDPH of two or even three days in a BBT-based assessment of ovarian function in the ovulatory phase.

Cellular prion protein prevents brain damage after encephalomyocarditis virus infection in mice.

Cellular prion protein (PrP(C)), a cell-surface glycoprotein normally associated with neurons, is also expressed in other cell types such as glia and lymphocytes. To further elucidate these roles of PrP(C), wild-type prion protein gene (Prnp(+/+)) mice and Prnp-deficient (Prnp(-/-)) mice were infected with encephalomyocarditis virus B variant (EMCV-B) via an intracranial route. EMCV-B causes encephalitis and apoptotic cell death in vivo. Histopathological studies revealed that Prnp(+/+) mice infected with 600 pfu of EMCV-B showed more severe infiltration of inflammatory cells, accompanied by higher activation of microglia cells around the hippocampus, than Prnp(-/-) mice; viz., no differences in the brain virus titer between these two lines of mice. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP, nick end-labeling (TUNEL) staining of the brain specimens revealed that the CA1 hippocampal pyramidal cells showed a larger number of apoptotic neurons in Prnp(-/-) than Prnp(+/+) mice. Based on all these findings, PrP(C) may play certain roles in the induction of inflammation and inhibition of apoptosis in vivo.

Extracellular matrix modulates expression of cell-surface proteoglycan genes in fibroblasts.

Cell-surface proteoglycans are involved in many functions, including interactions with components of the extracellular microenvironment. They also act as coreceptors that bind and modify the actions of various growth factors, cytokines, and the extracellular matrix (ECM). This study investigated the regulation by the ECM of the expression of cell-surface proteoglycans (CD44, syndecan-1-4, betaglycan, glypican-1). We examined the changes in the expression levels of cell-surface proteoglycan genes in intact tendon, monolayer culture, and under various culture conditions. There was a significant increase in the expression of CD44 and syndecan-4 mRNAs during cell isolation from the tendon. With the switch to a 3D culture environment, there was a significant increase in the expression of CD44 at each passage point relative to its expression in 2D at those passage points. Syndecan-4 mRNA also increased steadily at each passage point in 3D culture environment. This influence on cell surface proteoglycans gene expression may indicate that collagen gel culture mimics in vivo tendon environment. This study provides further insight into the regulation of cell-surface proteoglycans in ligament and tendon fibroblasts by the ECM and 3D culture conditions.

Elimination of CD4+ CD25+ regulatory T cells breaks down reovirus type 2-triggered and CpG ODN-induced prolonged mild autoimmune insulitis in DBA/1 mice.

We have reported previously that subclinical prolonged mild T helper (Th) 1-dependent autoimmune insulitis with impaired glucose tolerance in wealing DBA/1J mice, which is induced by the combined effects of reovirus type 2 (Reo-2) and synthetic 20-base oligodeoxynucleotides with CpG motifs (CpG ODN) (control mice). Compared with the control mice, newborn mice treated with monoclonal antibody (MoAb) against mouse CD25(+) CD4(+) T cells together with Reo-2 and CpG ODN greatly reduced the absolute number of splenic CD25(+) T cells and resulted in the development of severe insulitis, leading to an overt early diabetes. Moreover, the treatment of the MoAb increased production of interferon-gamma (IFN-gamma) and decreased that of interleukin-4 (IL-4) and transforming growth factor-beta1 (TGF-beta1) and developed high titre of autoantibodies against pancreatic islet cells. These evidences suggest that CD4(+) CD25(+) T cell may, at least in part, maintain tolerance to Reo-2-triggered and CpG ODN-induced prolonged mild Th1-dependent autoimmune insulitis, leading to the overt disease. This system may give a novel model to elucidate the mechanisms of the development of overt diabetes from borderline subclinical diabetes in virus-triggered autoimmune type I diabetes in human.