Corrigendum to "Long-Term Effects of Goshajinkigan in Prevention of Diabetic Complications: A Randomized Open-Labeled Clinical Trial".

[This corrects the article DOI: 10.1155/2014/128726.].

Long-term effects of goshajinkigan in prevention of diabetic complications: a randomized open-labeled clinical trial.

Objective. This clinical trial was designed to investigate whether goshajinkigan reduces the onset of diabetic complications or not. Materials and Methods. A total of 332 type 2 diabetic mellitus patients were registered from 9 clinical centers from March 2000 to August 2007. Patients were randomly assigned to take goshajinkigan extract powder, 2.5 grams for 3 times a day or no kampo therapy, additionally to the regular treatment. The primary endpoints were the onset of macrovascular diseases or progression of nephropathy or retinopathy. Statistical analysis was performed by the intention-to-treat method. Results. After 5 years of observation, 116 patients were submitted to analysis. Among them, no macrovascular events were observed in both groups. Although 43 participants had upstaging of retinopathy or nephropathy in total, there was no significant difference between goshajinkigan group and control group. Deterioration of ankle reflex was suppressed in goshajinkigan group. Also glycated hemoglobin, and fasting plasma glucose were decreased in the goshajinkigan group. Conclusion. Although the power of analysis was too low to demonstrate any effects of goshajinkigan on the progression of macrovascular diseases, retinopathy or nephropathy, goshajinkigan may be beneficial for diabetic neuropathy and glycemic control.

Association of increased reactive oxygen species production with abdominal obesity in type 2 diabetes.

OBJECTIVE: The close relationship between oxidative stress and abdominal obesity is well known, but the association is unclear in diabetic patients. The aim of this study was to confirm that increased reactive oxygen species (ROS) production is associated with abdominal obesity in diabetic patients. METHODS: ROS production was assayed in Epstein-Barr virus-transformed immortalized lymphoblasts by means of a cypridina luciferin analogue chemiluminescence method. We divided 96 Japanese male diabetic patients into 2 groups: patients with abdominal obesity according to the accepted Japanese criteria (waist circumference is more than 85 cm) (group AO, n = 36); and patients without abdominal obesity (group N, n = 60). Subjects with body mass index (BMI) in the normal range (21 ≤ BMI < 25 kg/m(2)) were then selected and assigned to 2 subgroups (group AOnormal-BMI [n = 13]; and group Nnormal-BMI [n = 35]); ROS production was compared between these 2 subgroups. RESULTS: Stimulation with arachidonic acid (AA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) increased ROS production in lymphoblasts, which was more greatly elevated in lymphoblasts derived from group AO than those from group N. Even in the subjects with normal BMI, AA- and TPA-stimulated ROS production in group AO was significantly higher than that in group N. CONCLUSIONS: These data suggest that increased ROS production is more closely associated with abdominal obesity than high BMI or insulin resistance in diabetic patients.

Induction of insulin production in rat pancreatic acinar carcinoma cells by conophylline.

We set up a screening system to detect low-molecular-weight compounds that induce insulin expression in pancreatic acinar carcinoma AR42J cells. They can differentiate into insulin-producing cells with neuron-like morphological change when treated with activin A. We employed this morphological change for the screening of beta-cell inducers among various signal transduction inhibitors. As a result, a vinca alkaloid, conophylline, induced neurite formation at 0.1 approximately 0.3 microg/ml in 72 h, like activin A. Conophylline-treated cells were found to express insulin as measured at both mRNA and protein levels. By RT-PCR analysis, conophylline-treated cells were shown to express neurogenin3 strongly. They also expressed Beta2/NeuroD and Nkx2.2, but not Pax4 and PP. Although activin A induces nuclear translocation of Smad2, conophylline did not. But the latter induced p38 activation, like activin A, as detected by phosphorylation. Pretreatment with a p38-specific inhibitor, SB203580, lowered the conophylline-induced insulin production. Therefore, p38 activation would be involved in the differentiation of AR42J cells into insulin-producing cells. Studies on structure-activity relationship with conophyllidine, conofoline, conophyllinine, and related monomer alkaloids showed that the dimeric aspidosperma structure with the dihydrofuran unit in its center was essential for the differentiation-inducing activity.

Antidiabetic effect of a nitrosamine-free dephostatin analogue, methoxime-3,4-dephostatin, in db/db mice.

Et-3,4-dephostatin, a protein-tyrosine phosphatase (PTPase) inhibitor, potentiates insulin-dependent signal transduction and shows an antidiabetic effect in mice. However, it contains a nitrosamine moiety that is often mutagenic and carcinogenic. Therefore, we previously designed and synthesized methoxime-3,4-dephostatin as a nitrosamine-free analogue of dephostatin. In the present paper, we studied in situ and in vivo antidiabetic effects of this PTPase inhibitor. Methoxime-3,4-dephostatin induced 2-deoxyglucose transport by mouse 3T3-L1 adipocytes and rat L6 myocytes without insulin. It also inhibited glucagon-induced glucose release from primary culture rat hepatocytes. When hepatocytes were prepared from starved rats, methoxime-3,4-dephostatin did not inhibit the release of glucose, indicating that the chemical may act on glycogenolysis. Oral administration of methoxime-3,4-dephostatin for 3-7 days inhibited the increase in the blood glucose level in type-2 diabetes model db/db mice. It also decreased food and water intakes of mice, but showed no liver or blood toxicity.

Quantitative analysis of mRNA expression of TIMPs in the periprosthetic interface tissue of loose hips by real-time PCR system.

Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), have been reported to play a critical role in extracellular degradation around artificial hip joints. Although messenger ribonucleic acid (mRNA) expression patterns of several MMPs and TIMPs were reported, there is no report of quantitative mRNA analysis of TIMPs in periprosthetic tissues. In this study, mRNA expression of four different types of TIMPs in periprosthetic interface tissue of loose hips was analyzed by a quantitative polymerase chain reaction system. The mRNA expression level of TIMP-1, -2, and -3 in periprosthetic interface tissue was significantly higher than that in control. In contrast, the mRNA expression level of TIMP-4 in the periprosthetic interface tissue was lower. This study suggested that increased levels of TIMP-1, -2, and -3, and decreased levels of TIMP-4 may contribute to pathologic extracellular matrix degradation in combination with MMPs, thus leading to prosthetic loosening and osteolysis around artificial total hip joints.

Balance of GAD65-specific IL-10 production and polyclonal Th1-type response in type 1 diabetes.

It has been proposed that cytokine responses of memory CD4+ cells change from a T-helper 2 (Th2)-to a T-helper 1 (Th1)-dominant response as the disease progresses in non-obese diabetic (NOD) mice. However, the regulation of Th1/Th2 balance in spontaneous diabetes development in this model is not well understood. In this study, higher glutamic acid decarboxylase 65 (GAD65)-specific IL-10 production was observed at 10-12 weeks in NOD mice, and a marked increase of Th1-type response (IFN-gamma production) upon polyclonal (anti-CD3 antibody) stimulation was observed just before diabetes development along with a decline of GAD65-specific IL-10 production. Moreover, there was a clear negative correlation between IL-10 level upon GAD65 stimulation and log(IFN-gamma) level upon anti-CD3 antibody stimulation (r=-0.999, p<0.001). These results suggest that the balance between GAD65-specific IL-10 production and polyclonal Th1-type response may regulate the onset of hyperglycemia in type 1 diabetes in NOD mice.

Secretion of GIP in responders to acarbose in obese Type 2(NIDDM) patients.

Acarbose has been shown to reduce postprandial hyperglycemia and to improve lipid parameters in diabetics via its inhibitory effects on intestinal alpha-glucosidases. Response to acarbose may therefore be dependent upon gastric or pancreatic hormone function. To test this hypothesis, we treated 27 mild type 2 (NIDDM) Japanese diabetics who were mildly obese with low-dose acarbose (150 mg/day) for 3 months. We then performed a responder analysis to determine specific hormonal responses that may be associated with a good response to acarbose. At the end of the treatment period, a total of 15 evaluable patients was grouped as responders (n=6) and nonresponders (n=9) based on an effective decrease in postprandial glucose levels (>30 mg/day) and glycosylated hemoglobin (HbA1c) levels (>0.5%). There were no differences between the two groups in demographic variables or mean postprandial glucose levels at baseline. There was a small but significant increase in postprandial cholecystokinin (CCK) in responders, and fasting gastric inhibitory peptide (GIP) levels were significantly increased in responders and all patients after treatment. Serum leptin levels were reduced by treatment in our mildly obese responders and this was associated with a significant decrease in body weight. These results suggest that treatment with low-dose acarbose may reduce hyperglycemia in mild type 2 Japanese patients and may improve metabolic control by regulating hormones involved in glycemic control and digestive absorption. Acarbose may provide a safe adjunct to help treat insulin resistance in type 2 patients.

Osteoprotegerin inhibits in vitro mouse osteoclast formation induced by joint fluid from failed total hip arthroplasty.

Osteoprotegerin (OPG) is a key regulator of osteoclastogenesis. We investigated the presence of OPG and bone-resorbing cytokines, the potential of osteoclastic differentiation in joint fluid from failed total hip arthroplasty (THA), and the inhibitory effect of OPG on osteoclast formation in vitro induced by the joint fluid. The study was aimed to clarify one important step in the cascade of periprosthetic osteolysis in the process of implant loosening. OPG levels in failed THA joint fluid of 20 cases were significantly lower than osteoarthritis (OA) joint fluid of 15 cases (p < 0.001). The levels of bone-resorbing cytokines, interleukin (IL)-1beta, and IL-6 were significantly higher in failed THA joint fluid than OA fluid (p < 0.001 and p = 0.001, respectively). Marked osteoclast formation was observed in the presence of failed THA joint fluid in the mouse coculture system, when compared to OA fluid (p < 0.001). The addition of 100 ng/mL OPG to the mouse coculture system completely inhibited osteoclast formation in the presence of failed THA joint fluid (p < 0.001). The data suggest that low levels of OPG combined with higher IL-1beta and IL-6 levels represent the potential of osteoclast differentiation and its activation in failed THA joint fluid. Inhibition of osteoclastogenesis in vitro by OPG suggests that a low level of OPG with elevated bone resorbing cytokines contributes to periprosthetic osteolysis via osteolytic joint fluid, thus leading to THA prosthesis loosening.

Potentiation of insulin-related signal transduction by a novel protein-tyrosine phosphatase inhibitor, Et-3,4-dephostatin, on cultured 3T3-L1 adipocytes.

We previously isolated dephostatin from Streptomyces as a novel inhibitor of CD45-associated protein-tyrosine phosphatase. We prepared Et-3,4-dephostatin as a stable analogue and found it to inhibit PTP-1B and SHPTP-1 protein-tyrosine phosphatases selectively but not to inhibit CD45 and leukocyte common antigen-related phosphatase ones effectively. Et-3,4-dephostatin increased the tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 with or without insulin in differentiated 3T3-L1 mouse adipocytes. The increase of tyrosine phosphorylation by Et-3,4-dephostatin was more prominent in 6-h than in 30-min incubation. It also increased phosphorylation and activation of Akt with or without insulin. Et-3,4-dephostatin also enhanced translocation of glucose transporter 4 from the cytoplasm to the membrane and 2-deoxy-glucose transport. Et-3,4-dephostatin-induced glucose uptake was inhibited by SB203580, a p38 inhibitor, but not by PD98059, a MEK inhibitor, or by cycloheximide as insulin-induced uptake. Interestingly, although LY294002, a phosphatidylinositol 3-kinase inhibitor, inhibited the insulin-induced glucose uptake completely, it only partially inhibited the Et-3,4-dephostatin-induced uptake. It also blocked insulin-induced glucose transporter 4 translocation but not the Et-3,4-dephostatin-induced one. The increase in c-Cbl tyrosine phosphorylation caused by Et-3,4-dephostatin was stronger than that in insulin receptor phosphorylation. These observations indicate that a phosphatidylinositol 3-kinase-independent pathway involving c-Cbl is more important in Et-3,4-dephostatin-induced glucose uptake than in insulin-induced uptake. Et-3,4-dephostatin showed an in vivo antidiabetic effect in terms of reducing the high blood glucose level in KK-A(y) mice after oral administration. Thus, Et-3,4-dephostatin potentiated insulin-related signal transductions in cultured mouse adipocytes and showed an antidiabetic effect in mice.

Cholesteryl ester transfer protein polymorphism associated with macroangiopathy in Japanese patients with type 2 diabetes.

A polymorphism in the gene for cholesteryl ester transfer protein (CETP) has been reported to be associated with serum cholesterol levels and risk for atherosclerotic vascular diseases, and to clarify the relationship between the gene polymorphism for CETP and macroangiopathy in diabetes mellitus, a cross-sectional study was performed. The subjects of the study were182 Japanese (age: 59.6+/-8.6 years) with type 2 diabetes and no signs of renal dysfunction, 24 of whom had macroangiopathy, and 158 of whom did not. The genotype of the subjects for the TaqIB polymorphism of CETP in intron one was analyzed by using polymerase chain reaction - restriction fragment length polymorphism. Serum CETP levels were significantly higher in the B1/B1 genotype than in the other genotypes (P<0.05). The serum CETP levels were correlated with the serum LDL cholesterol levels (P<0.01), but not with the HDL cholesterol levels. Macroangiopathy was more frequently observed in subjects with the B1/B1 genotype than in the other genotypes (odds ratio=2.953, 95% confidence interval=1.250-6.977, P=0.0136). Logistic regression analysis revealed that the CETP genotype was independently associated with macroangiopathy. The exact mechanism underlying the association remains unknown, but differences in serum CETP levels may be involved.

High-turnover periprosthetic bone remodeling and immature bone formation around loose cemented total hip joints.

Aseptic loosening and periprosthetic osteolysis are the major problems awaiting solution in total hip surgery. The clinical investigation focused on the analysis of periprosthetic bone remodeling to clarify one important key event in the cascade of periprosthetic connective tissue weakening and osteolysis around loose artificial hip joints. Twelve acetabular bone samples adjacent to granulomatous synovial-like membrane of loose hip prosthesis were retrieved at revision surgery and processed for Villanueva bone staining for morphological observation and bone histomorphometric analysis. Eight well-fixed bony samples were used as control. Although osteoclastic surface and eroded surface by osteoclasts were evident in the periprosthetic bone from loose hip joints (p = 0.003 and p = 0.027), increased osteoid/low-mineralized bone matrix (p < 0.001) and osteoid width (p < 0.001) also were significant findings in structural analysis. In addition, not only elevated mineral apposition rate (MAR; p = 0.044) but also increased mineralizing surface (p = 0.044) and bone formation rate (BFR; p = 0.002) in loose periprosthetic bones were shown in dynamic data analysis. These results were confirmed by precise morphological observation by confocal laser scanning microscopy. Active coupling of bone formation and resorption and increased osteocytes with abundant bone canalicular projections were found in combined with the presence of immature bone matrices (osteoid and low-mineralized bone areas) in periprosthetic bones from loose hip joints. These results indicated that active osteoclastic bone resorption and/or defective bone formation are coupled with monocyte/macrophage-mediated foreign body-type granuloma in the synovial-like interface membrane of loose hip joints. Thus, this unique high-turnover periprosthetic bone remodeling with bad bone quality probably is caused by the result of cellular host response combined with inappropriate cyclic mechanical loading. The fragile periprosthetic bone may contribute to hip prosthesis loosening.

Ruthenium-catalysed asymmetric hydrosilylation of ketoximes using chiral oxazolinylferrocenylphosphines.

Chiral ruthenium(II) complexes, RuCl2(PPh3)(oxazolinylferrocenylphosphine), have been found to be effective catalysts for asymmetric hydrosilylation of ketoximes to give the corresponding primary amines in good yields with high enantioselectivities (up to 89% ee) after acid hydrolysis.

Messenger ribonucleic acid expression of 16 matrix metalloproteinases in bone-implant interface tissues of loose artificial hip joints.

Matrix metalloproteinases (MMPs) have been reported to be the major factors responsible for aseptic loosening of artificial hip joints. So far, messenger ribonucleic acid (mRNA) expression patterns of seven MMPs have been reported, but that of many other MMPs which have been newly discovered or recently considered to be responsible for prosthetic loosening is still unknown. In this study, mRNA expression pattern of 16 different types of MMPs were analyzed to evaluate which MMPs were locally produced and contributed to prosthetic loosening. Synovium-like interface tissues between bone and prosthesis were collected from 18 cases of aseptic loose artificial hip joint at revision surgery. Six cases of normal synovium were used as controls. Total RNA was extracted by single-step acid guanidinium-thiocyanate-phenol-chloroform procedure. mRNA expression of MMPs was analyzed by semiquantitative reverse transcription-polymerase chain reaction. Based on local expression pattern of MMPs at the mRNA level, aseptic loose artificial hip joint was characterized by elevated expression of MMP-1, MMP-9, MMP-10, MMP-12, and MMP-13; moderate expression of MMP-2, MMP-7, MMP-8, MMP-11, membrane type (MT)1-MMP (MMP-14), MT2-MMP (MMP-15), MT3-MMP (MMP-16), MT4-MMP (MMP-17), and MMP-19; lower expression of MMP-3; and little significance of MMP-20. The MMPs detected in this study can potentially degrade almost all components of the periprosthetic extracellular matrix. Thus, many MMP type enzymes possibly contribute to prosthetic loosening and osteolysis through pathologic extracellular matrix degradation and connective tissue/bone remodeling around prostheses.

High macrophage-colony stimulating factor levels in synovial fluid of loose artificial hip joints.

OBJECTIVE: To clarify a macrophage-colony stimulating factor (M-CSF) related mechanism of aseptic loosening of artificial hip joints. METHODS: Synovium-like interface tissues between bone and prosthesis, regenerated pseudocapsular tissues, and synovial fluid (SF) were collected from 9 patients with loose artificial hip joint at revision surgery. Tissue distribution, production site, and SF level of M-CSF in loose hip joints were investigated by immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and ELISA, respectively. For a comparative assessment of the M-CSF level in loose hip joints, SF of active rheumatoid arthritis (RA) and mild osteoarthritis (OA) also were analyzed by ELISA. RESULTS: Immunohistochemical analysis showed the presence of M-CSF immunoreactive cells mainly in the interface tissues between bone and prosthesis and inner pseudocapsular tissues, both of which were in contact with joint fluid. RT-PCR analysis confirmed the local production of M-CSF in these periprosthetic tissues. Significantly higher M-CSF level in loose hip joint fluid than in active RA and mild OA fluid was revealed by ELISA. CONCLUSION: High M-CSF level in loose hip joint fluid suggests transportation of M-CSF from production sites to joint fluid. This indicates that not only polyethylene wear particles (reported to induce foreign body reaction at the bone-prosthesis interface), but also M-CSF, abundant in joint fluid, are transported to and affect the interface. Thus, M-CSF is locally produced in periprosthetic tissues of loose hip joints and possibly contributes to periprosthetic weakening and osteolysis via joint fluid, leading to prosthetic loosening.

Novel Catalytic Hydrogenolysis of Trimethylsilyl Enol Ethers by the Use of an Acidic Ruthenium Dihydrogen Complex.

The heterolytic cleavage of H(2) is the key to the novel catalytic hydrogenolysis of trimethylsilyl enol ethers catalyzed by [RuCl(eta(2)-H(2))(dppe)(2)]OTf (dppe = 1,2-bis(diphenylphosphanyl)ethane, OTf = trifluoromethanesulfonate), which results in the formation of a ketone and Me(3)SiH (see scheme). In addition, the stoichiometric, ruthenium-assisted protonation of a prochiral lithium enolate with H(2) gave a chiral ketone with high enantioselectivity (up to 75 % ee).

Effects of troglitazone on fat distribution in the treatment of male type 2 diabetes.

We investigated the efficacy of additional administration of 400 mg troglitazone (+T), which became available as a treatment for type 2 diabetes following the demonstration of its ability to reduce insulin resistance, in combination with diet (D + T) or sulfonylurea (S + T) therapy. Body fat area as determined by computed tomographic (CT) scanning at the umbilical level, as well as several clinical and biochemical parameters of glycemic control and lipid metabolism, were compared before and after 3 months of additional treatment with troglitazone. The body mass index (BMI) tended to increase in both groups (22.7 +/- 0.6 v 23.2 +/- 0.6 kg/m2 in D + T, nonsignificant [NS]; 22.2 +/- 0.5 v 22.3 +/- 0.5 kg/m2 in S + T, NS), while it tended to decrease in the control group (only diet therapy, 23.6 +/- 0.6 v 23.1 +/- 0.8 kg/m2, NS). Mean blood pressure ([BP] 96 +/- 3 v 89 +/- 4 mm Hg, P < .05) decreased significantly in the D + T group. Changes in the glycemic and lipid profile and leptin did not reach statistical significance. The D + T group showed a significant decline in immunoreactive insulin ([IRI] 12.4 +/- 1.2 v 8.0 +/- 1.0 microU/mL, P < .05), reflecting markedly reduced insulin resistance, as well as a significant increase in plasma insulin-like growth factor-1 ([IGF-1] 175.7 +/- 14.2 v 189.8 +/- 12.6 ng/mL, P < .05). A slight weight gain was associated with a tendency for subcutaneous fat to increase, while visceral fat decreased in both troglitazone-treated groups. The decrease in the visceral to subcutaneous fat ratio (V/S ratio) was statistically significant in the D + T group (1.09 +/- 0.11 v 0.94 +/- 0.09, P < .05), while the V/S ratio in the control group did not change. A notable finding of this study is the difference in the response to troglitazone between subcutaneous and visceral adipose tissue. It is suggested that troglitazone may exert beneficial effects by reducing visceral fat.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in joint fluid of the patients with loose artificial hip joints.

The pseudojoint cavity formed in patients undergoing total hip arthroplasty (THA) is later remodeled to synovial membrane-like tissue, which produces pseudosynovial fluid. This pseudosynovium also is an important source of matrix metalloproteinases (MMPs). As it is widely speculated that synovial fluid MMPs may contribute to local tissue degradation in rheumatoid arthritis (RA) and osteoarthritis (OA), we hypothesize that locally produced MMPs are found in the pseudosynovial fluid, via which they have access to the implant-host interface, and that if they retain their proteolytic potential, they might contribute to aseptic loosening. Enzyme-linked immunosorbent assay (ELISA), immunoblotting, and zymography were used to analyze MMPs and tissue inhibitors of metalloproteinases (TIMPs) in synovial fluid in aseptic loosening, which was compared to RA and OA. Pseudosynovial THA fluid was characterized using low levels of MMP-1 but moderate levels of MMP-13 and MT1-MMP (MMP-14). Due to the lack of an appropriate assay, MMP-13 and MT1-MMP were not similarly assessed, but the immunoblotting indicated that they were in the 56 kD intermediate proteolytically processed forms. The MMP-9 level was intermediate between RA and OA. MMP-2 was on a significant level, but there were no differences among study groups. The THA group also was characterized using relatively high levels of TIMP-1 and TIMP-2. Accordingly, MMP-9 and MMP-2 were found to occur in the 92 kD and 72 kD proenzyme form, respectively, with full activity retained in all study groups. The data suggest that proMMP-2-TIMP-2 and proMMP-9-TIMP-1 complexes are formed in the pseudosynovial fluid due to the excess of TIMPs over MMPs in aseptic loosening of THA. TIMP-complexed MMPs are resistant to MMP-mediated proteolytic activation, which may explain their latency and proenzyme zymogen form. Thus, formation of stabilizing proMMP-TIMP complexes enable transportation of proMMPs far from their original site of production. Due to motion-associated cyclic changes of the intra-articular pressure, fluid-phase MMPs stabilized by TIMPs might be absorbed to implant surfaces and interface tissues and help to dissect the implant/cement-to-bone interface in situ. Consequently, they may contribute to local proteolytic/tissue destructive events and aseptic loosening.

Systemic delivery of interleukin 10 by intramuscular injection of expression plasmid DNA prevents autoimmune diabetes in nonobese diabetic mice.

We previously demonstrated that intramuscular plasmid injection serves as a useful method of long-term systemic delivery of cytokines. In the present study, we assess intramuscular DNA injection as a means of systemically delivering interleukin 10 (IL-10), a cytokine with immunosuppressive properties, and preventing the progression of autoimmune diabetes in the nonobese diabetic (NOD) mouse, an excellent model for human insulin-dependent diabetes mellitus (IDDM). We injected IL-10 expression plasmid (pCAGGS-IL10) or a control pCAGGS plasmid into the muscles of NOD mice twice at 3 and 5 weeks of age. IL-10 was detectable by ELISA in the sera of mice injected with pCAGGS-IL10 for more than 2 weeks after the injection. Although the severity of insulitis at 13 weeks of age was not improved by the intramuscular injection of pCAGGS-IL10, the incidence of diabetes was markedly reduced in NOD mice injected with pCAGGS-IL10 as compared with those injected with pCAGGS or as compared with nontreated NOD mice. These results show that the progression of autoimmune diseases in mice can effectively be suppressed by intramuscular DNA injection, and suggest that this method is potentially applicable to the treatment of human autoimmune diseases.