A randomized clinical trial comparing basal insulin peglispro and insulin glargine, in combination with prandial insulin lispro, in patients with type 1 diabetes: IMAGINE 1.

AIMS: The primary objective was to demonstrate that basal insulin peglispro (BIL) was non-inferior compared with insulin glargine (GL) for haemoglobin A1c (HbA1c) at 26 weeks with a non-inferiority margin of 0.4%. MATERIALS AND METHODS: IMAGINE 1 was a Phase 3, open-label, parallel-arm study conducted in nine countries. Adults with type 1 diabetes (n = 455) were randomized (2:1) to bedtime BIL or GL in combination with prandial insulin lispro for 78 weeks, with a primary endpoint of 26 weeks. An electronic diary facilitated data capture and insulin dosing calculations for intensive insulin management. RESULTS: At 26 weeks, mean HbA1c was 7.06% ± 0.04% and 7.43% ± 0.06% for patients assigned to BIL (N = 295) and GL (N = 160), respectively (difference -0.37% [95% CI: -0.50 to -0.23], P < .001); more patients on BIL achieved HbA1c <7% (44.9% vs 27.5%, P < .001). Compared with GL, patients using BIL lost weight, with lower fasting serum glucose and between-day fasting blood glucose variability, and 36% less nocturnal hypoglycemia, 29% more total hypoglycemia and more severe hypoglycemia. Total and prandial insulin doses were lower with BIL; basal insulin doses were higher. Alanine aminotransferase increased with BIL, with more patients having elevations ≥3 × ULN. BIL treatment was associated with more frequent injection site reactions and an increase from baseline in serum triglycerides. CONCLUSIONS: In patients with type 1 diabetes, treatment with BIL compared to GL for 26 weeks was associated with lower HbA1c, less nocturnal hypoglycemia, lower glucose variability and weight loss. Increases in total and severe hypoglycemia, triglycerides, aminotransferases and injection site reactions were also noted.

Effects of luseogliflozin, a sodium-glucose co-transporter 2 inhibitor, on 24-h glucose variability assessed by continuous glucose monitoring in Japanese patients with type 2 diabetes mellitus: a randomized, double-blind, placebo-controlled, crossover study.

The aim of the present study was to determine the effects of luseogliflozin on 24-h glucose levels, assessed by continuous glucose monitoring, and on pharmacodynamic variables measured throughout the day. In this double-blind, placebo-controlled, crossover study, 37 patients with type 2 diabetes mellitus inadequately controlled with diet and exercise were randomized into two groups. Patients in each group first received luseogliflozin then placebo for 7 days each, or vice versa. After 7 days of treatment, the mean 24-h glucose level was significantly lower with luseogliflozin than with placebo [mean (95% confidence interval) 145.9 (134.4-157.5) mg/dl vs 168.5 (156.9-180.0) mg/dl; p < 0.001]. The proportion of time spent with glucose levels ≥70 to ≤180 mg/dl was significantly greater with luseogliflozin than with placebo [median (interquartile range) 83.2 (67.7-96.5)% vs 71.9 (46.9-83.3)%; p < 0.001] without inducing hypoglycaemia. The decrease in glucose levels was accompanied by reductions in serum insulin levels throughout the day.

Gender differences in the incidence and progression of diabetic retinopathy among Japanese patients with type 2 diabetes mellitus: a clinic-based retrospective longitudinal study.

A clinic-based retrospective longitudinal study conducted for 5.8 ± 2.5 years, including 383 (M/F 245/138) Japanese patients with type 2 diabetes mellitus showed that females exhibit a significantly higher prevalence of proliferative diabetic retinopathy (DR) at baseline and that female gender is an independent risk factor for the development of DR.

Analysis of vasopressin receptor type II (V2R) gene in three Japanese pedigrees with congenital nephrogenic diabetes insipidus: identification of a family with complete deletion of the V2R gene.

To investigate the association of mutations in the arginine vasopressin receptor type II (V2R) gene with congenital nephrogenic diabetes insipidus (CNDI) in the Japanese, we analyzed the V2R gene, located on the X chromosome, in three Japanese pedigrees with CNDI. In one pedigree, a large deletion spanning the entire coding region of the V2R gene was identified. In another pedigree, a G to A transition responsible for a substitution of Met88 (ATG) for Val88 (GTG) was detected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis revealed that this was a de novo mutation that had occurred in the proband's mother. Because CNDI was observed only in those with this mutation, the pathogenicity of this mutation seemed clear. In the last pedigree, only a silent mutation at Leu309 (CTA-->CTG) was found. All the individuals studied in this pedigree by allele-specific oligonucloetide-polymerase chain reaction (ASO-PCR) analysis showed a complete association of this mutation to the clinical symptoms. Because the silent mutation detected was unlikely to be a direct cause of CNDI, mutations in other regions of the V2R gene, such as a promoter region or other regulatory regions, may be responsible for the cause of CNDI in this pedigree. Thus, association of the V2R gene abnormality to clinical symptoms of CNDI was confirmed in three Japanese pedigrees, and a strong contribution of the V2R gene mutation to the development of CNDI was suggested.

Frequency of mutations of insulin receptor gene in Japanese patients with NIDDM.

To examine the prevalence of abnormalities in the insulin receptor structure gene in Japanese with non-insulin-dependent diabetes mellitus (NIDDM), a population of 51 patients with NIDDM was screened for mutations in this gene. Patient genomic DNAs of both alleles corresponding to 22 exons of the gene were amplified by polymerase chain reaction (PCR). The PCR products on pUC19 were sequenced. Three patients with heterozygous missense mutation Thr831-->Ala831 in exon 13 and one patient with heterozygous missense mutation Tyr1334-->Cys1334 in exon 22 of the beta-subunits were identified. Linkage analysis of one of the families plus statistical studies showed that the mutation Thr831-->Ala831 is possibly responsible for the onset of NIDDM. In COS cells transiently expressing both mutant receptor cDNAs and a cDNA of a M(r) 85,000 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), the mutation Tyr1334-->Cys1334 impaired binding of the receptor with the M(r) 85,000 subunit of PI 3-kinase, but linkage analysis of the family showed that the mutation did not cosegregate with NIDDM in the pedigree. Therefore, one missense mutation (Thr831-->Ala831) in the insulin receptor, as found in three patients, is possibly involved in the etiology of a subset of the 51 NIDDM patients.

Possible domains responsible for intracellular targeting and insulin-dependent translocation of glucose transporter type 4.

Translocation of the type 4 glucose transporter (GLUT4) to the cell surface from an intracellular pool is the major mechanism of insulin-stimulated glucose uptake in insulin-target cells. We developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We constructed c-myc epitope-tagged glucose transporter type 1 (GLUT1myc) and found that the GLUT1myc was also translocated to the cell surface of Chinese hamster ovary cells, 3T3-L1 fibroblasts and NIH 3T3 cells, in response to insulin, but the degree of translocation was less than that of GLUT4myc. Since GLUT1 and GLUT4 have different intracellular distributions and different degrees of insulin-stimulated translocation, we examined the domains of GLUT4, using c-myc epitope-tagged chimeric glucose transporters between these two isoforms. The results indicated that, (1) all the cytoplasmic N-terminal region, middle intracellular loop and cytoplasmic C-terminal region of GLUT4 have independent intracellular targeting signals, (2) these sequences for intracellular targeting of GLUT4 were not sufficient to determine GLUT4 translocation in response to insulin, and (3) the N-terminal half of GLUT4 devoid both of cytoplasmic N-terminus and of middle intracellular loop seems to be necessary for insulin-stimulated GLUT4 translocation.

Expression of insulin receptor on clonal pancreatic alpha cells and its possible role for insulin-stimulated negative regulation of glucagon secretion.

In pancreatic alpha cells, the existence and function of the insulin receptor has not yet been fully established. In this study, to confirm the expression of functional insulin receptors in pancreatic alpha cells, we performed: 1) insulin receptor binding assay, 2) Northern blot analysis and RT-PCR (reverse transcription-polymerase chain reaction) amplification of insulin receptor mRNA, 3) immunocytochemical staining, 4) biosynthetic labelling of insulin receptor protein using [35S]methionine, 5) analysis of insulin-stimulated autophosphorylation of the insulin receptor in glucagon secreting cell lines, In-R1-G9 and alpha TC clone 6 cells. Glucagon secretion decreased with the addition of insulin in both cells. The receptor binding studies using [125I-Tyr-A14] insulin revealed that both cells possessed a significant number of insulin receptors (In-R1-G9:K1 = 2.1 x 10(9) mol/l-1, K2 = 6.2 x 10(7) mol/l-1, R1 = 0.27 x 10(4), R2 = 1.86 x 10(4) sites/cell; alpha TC clone 6: K1 = 2.1 x 10(9) mol/l-1, K2 = 7.3 x 10(7) mol/l-1, R1 = 0.27 x 10(4), R2 = 1.95 x 10(4) sites/cell). Northern blot analysis as well as RT-PCR amplification showed the mRNA specific for insulin receptor in both cells. By immunocytochemical staining using anti-insulin receptor alpha-subunit antibody, positive immunostaining for insulin receptor was observed in both cells. [35S]Methionine labelling of both cells followed by immunoprecipitation using anti-insulin receptor antibody showed the correct size of the insulin receptor protein. The insulin receptor expressed in these cells underwent autophosphorylation by insulin stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Necrotizing lymphadenitis: a review of clinicopathological, immunohistochemical and ultrastructural studies.

Necrotizing lymphadenitis (NEL) has been reported to be a reactive process described under differing terminology by Fujimoto et al. (1972), Kikuchi (1972), Wakasa et al. (1973) and other Japanese pathologists. Recently, this type of lymphadenitis has also been reported in America and Europe. In Japan, NEL is observed more frequently in the northern area, however, no characteristic seasonal occurrence has been noted. The disease affects young females more than males, particularly from the third and fourth decades onwards. Common cold-like symptoms, lymphadenopathy of the cervical region and leukopenia are characteristic clinical findings in the early stages. Morphological features of the involved lymph nodes include the presence of numerous immunoblasts, histiocytes and macrophages, the latter with phagocytized nuclear debris derived from degenerated lymphocytes. However, granulocytes are generally absent. Tubular inclusions are observed ultrastructurally. Immunohistochemical studies of peripheral blood using monoclonal antibodies have revealed that the helper/suppressor (Leu 3a/2a) ratio increases gradually with the clinical course because of a decrease in Leu 2a + cells. The pathogenesis of NEL is uncertain, but it has been speculated that there is cytolytic infection of lymphocytes by a virus or other organism, accompanied by secondary blastic transformation of suppressor T-lymphocytes.

Differential effects of dietary eicosapentaenoic and docosahexaenoic fatty acids on lowering of triglyceride and cholesterol levels in the serum of rats on hypercholesterolemic diet.

This investigation was attempted in order to further clarify the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on lipid metabolism in rats. Ninety-three percent ethyleicosapentaenoate (EPAconc) and 91% ethyl-docosahexaenoate (DHAconc) were used and supplied at the 3% level to a hypercholesterolemic basal diet which contained 5% lard. Four experimental diets were prepared from (1) 5% olive oil, (2) 2% olive oil +3% ethyl-linoleate, (3) 2% olive oil +3% EPAconc, and (4) 2% olive oil +3% DHAconc plus hypercholesterolemic basal diet. Young male rats were fed on these diets for respective periods of 10 and 20 days. Concentrations of serum lipids in rats fed on the diets (2), (3) and (4) were compared to those of the control group (diet (1]. Serum total cholesterol level was significantly lower only in the animals fed on DHAconc, whereas serum triglyceride concentration was significantly lower only in the animals fed on EPAconc. Serum phospholipid level was significantly lowered both in the animals fed on either EPAconc or DHAconc. Serum lipid peroxide concentration was elevated in these two groups of animals, while serum alpha-tocopherol concentration was lower in both groups of animals fed on EPAconc and DHAconc, respectively, although these findings were less marked in the rats on the EPAconc diet. From these findings, it is postulated that EPA and DHA have different effects on lipid metabolism in rats with hypercholesterolemia experimentally induced.

Studies on the mechanism of synergistic action with synergisidin and imidazole antimycotics.

The investigation of mechanism of synergistic action with SYN and ECZ was performed using C. albicans SC5314 so that SYN was confirmed to show strong synergistic effects against Candida sp. in particular with addition of extremely small quantities under the MICs of imidazole antimycotics such as ECZ, MCZ and CTZ. The synergistic effect of antifungal activity against C. albicans SC5314 with a combination of SYN and ECZ (SYN + ECZ) showed fungistatic action. Effect of SYN + ECZ on osmotic resistance was not recognized and protoplast was not observed under a microscope. Accordingly, SYN + ECZ was considered not to take part in cell wall synthesis directly. For effect of SYN + ECZ on release of intracellular components, slow release of 260 nm-absorbing substances was occurred, so that SYN + ECZ was seemed not to affect cytoplasmic membrane damage directly. Also, it was suggested clearly that SYN + ECZ affected lipid metabolism and glycolysis including TCA cycle from the investigation on antagonism by growth recovery of C. albicans SC5314 by 106 kinds of substances such as fatty acids, isoprenoids, phospholipids, vitamins, amino acids, nucleic acid-related substances and TCA cycle-related substances. From the above results, it was suggested that the mechanism of synergistic action with SYN and ECZ against C. albicans SC5314 was due to affect the different reactions in lipid metabolism and the similar reactions in glycolysis including TCA cycle, respectively, in consideration of respective mechanism of actions of SYN alone and ECZ alone. A part of this work was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, 1981 (Kyoto).

Studies on synergisidin.

Penicillium sp. No. Y-11930 was isolated from soil sample collected at Shimouma, Setagaya, Tokyo in September 1978. Synergisidin produced by the strain was obtained with high yield in starch-corn steep liquor medium, extracted with ethyl acetate at pH 5.0 and crystallized from ethyl acetate after decolorization with active charcoal. The antibacterial activities of synergisidin against Gram-positive and Gram-negative bacteria and Mycobacterium were almost nothing but synergisidin showed weak activities against eumycetes with MICs of 6.25-100 microgram/ml. However, synergidisin was confirmed and discovered to show 30-125-fold strong synergistic effects against Candida sp. in particular with addition to extremely small quantities of imidazole antimycotics such as econazole, miconazole and clotrimazole. The acute toxicity was LD50 smaller than or equal to 250 mg/kg in mice (i.p.). the morphological degenerative effect on HeLA cells was observed in concentrations of more than 0.122 microgram/ml. The chemical structure of synergisidin was estimated as 7, 16-dihydroxy-2-methyl-4-oxo-3-oxabicyclo [10. 3. 1] hexadeca-5, 10-diene or 2, 15-dihydroxy-7-methyl-5-oxo-6-oxabicyclo (11. 3. 0] hexadeca-3, 11-diene (the same structure as decumbin, brefeldin A and ascotoxin) from various physiochemical properties but later, comparison with brefeldin A and ascotoxin revealed that synergisidin was identical with those.

Studies on terreic acid.

It was found that Aspergillus sp. No. Y-8980 which was isolated from a soil sample collected at Yoron Island in Kagoshima Prefecture belonged to Aspergillus terreus group by morphological observation. The active substance produced by the strain was obtained with a high yield in sucrose-yeast extract medium and extracted by chloroform, ethyl acetate and n-butanol at pH 2.4 approximately 2.6 from the culture broth. The substance was crystallized from chloroform and ethyl acetate after charcoal treatment of the crude crystal. From various physico-chemical properties, it was found that the substance was identical to terreic acid. Terreic acid showed MICs of 25 approximately 100 mcg/ml, 12.5 mcg/ml and 50 mcg/ml against Gram-positive and Gram-negative bacteria, Xanthomonas oryzae and Xanthomonas citri, respectively, but it did not control Pseudomonas, fungi and yeast. The LD50 was 75 mg/kg i.p. and i.v. in mice. With regards to the anti-tumor effect, the morphological degeneration on HeLa cells (human carcinoma cells) was observed in the concentrations of more than 6.25 mcg/ml of terreic acid. An increase of body weight of mice caused by Ehrlich ascites carcinoma cells was not definitely observed by the daily administration of 150 mcg of terreic acid per mouse for 8 consecutive days. Above showed the enough survival effect in dd mice implanted with Ehrlich ascites carcinoma cells, and the effect also was demonstrated by anatomies of mice.

The application of the sepharose bead immunofluorescence test for the detection of allergen-specific IgE and IgG antibodies in pollinosis.

A new application of the Sepharose bead immunofluorescence test for detection of allergen-specific IgE and IgG antibodies is described. Allergen extracts of four different grass pollens were coupled to CNBr-activated Sepharose 4B. Twenty normal and allergic sera were incubated with the allergen-coupled beads, washed and incubated with fluorescence-conjugated anti-gamma E and G globulins. After washing and staining with 0.5% trypan blue, the percentage of fluorescent beads was detected by fluorescence microscopy. In the IgG/anti IgG system, the smallest amount of IgG demonstrated was 20ng/ml; in the IgE/anti-IgE system it was 40ng/ml. Independent examination of tests gave a mean difference between observations of 4.1%. Reproducibility was also very good (var. coeff = 18%). The number of beads stained with IgE correlated well with intensity of skin reactions to the same extracts (r = 0.64; p = 5 x 10-7), the percentage of IgG stained beads being independent of skin reactivity. The Sepharose-IgE test allowed clear distinction between allergic and normal sera (p = 4 x 10-7), while the Sepharose IgG test did not distinguish between them at the time of diagnosis. However, the number of beads stained with IgG significantly increased in patients undergoing immunotherapy (p = 3.8 x 10-3). The Sepharose bead immunofluorescence test requires a very small amount of materials, is highly sensitive and easy to handle. It may be valuable in the "in vitro" diagnosis of grass pollen allergy and useful in evaluating immunotherapy.

Quantitative estimation of IgE and IgD by laser nephelometry.

The advantages and disadvantages of Laser Nephelometry (LN) in the determination of IgD and IgE are reported. Two laser nephelometer models (Behringwerke/Marburg), different batches of LN cuvettes, WHO reference standard sera, rabbit anti-human antisera and randomly selected allergic patients' sera were used for the standardization of the method. Cuvette blank values were significantly lower in the new model of laser nephelometer and the precision of these measurements was very high when two different cuvette charges were compared. In the determination of IgE by LN, it was possible to detect levels down to 125 IU/ml, the accuracy of the estimations varying between 4.8 and 8.2% and the repeatability between 3.2 and 24.4%, the highest variation coefficient being obtained in low level samples. The overall agreement between LN and RIST in 55 serum samples was 71%, and at concentrations below 200 IU/ml (normal) and above 400 IU/ml (increased) 80% and 85% respectively. In the determination of IgD by LN, the accuracy of the estimations was also very good (2.4 to 7.8%) and the variation coefficient varied between 2.8 and 13.3%. In the comparison of IgD estimations with LN and radial immunodiffusion in 27 samples a correlation coefficient of r = 0.82 was obtained. Although normal adult IgE values cannot be analysed, the clinically important increased IgE levels are correctly determined by LN. The method is more sensitive than the Mancini technique for IgE determination and in comparison with RIST, though low values are not obtained, LN is quicker, simpler and cheaper.

Effects of tetraacetyl-bis-dehydroascorbic acid, a derivative of ascorbic acid, on Ehrlich cells and HeLa cells (human carcinoma cells).

Derivatives of ascorbic acid were synthesized, and the studies were made on their effects in Ehrlich ascites carcinoma cells, in regard to the inhibition and the prolongation of survival time as well as on the morphological degeneration in HeLa cells. In a model infection study carried out by using tetraacetyl-bis-dehydroascorbic acid in dd mice infected with Ehrlich cells, it was proved that the prolongation of survival time was nearly double in comparison to the control group mice. Also, it was noted that hypertrophy due to abdominal dropsy and body weight were reduced much more than in the control group. From these results, the inhibiting effect of tetraacetyl-bis-dehydroascorbic acid was confirmed. While in the case of DHA and other derivatives, almost no inhibition and prolongation of survival time were observed. As for HeLa cells in a tissue culture, tetraacetyl-bis-DHA, in a dosage of 125-250 mug/ml, demonstrated definitely its morphological degeration. After 125 mug/ml of tetraacetyl-bis-DHA was added to a tissue culture solution of HeLa cells, the cells were washed and recultured. No growth of the cells was observed. Consequently, this substance was confirmed to be anti-HeLa substance with a low toxicity.