Ryoko Yoshida

The Modulation of CD40 Ligand Signaling by Transmembrane CD28 Splice Variant in Human T Cells

The role of CD40 ligand (CD40L)/CD40 signaling in T cell–dependent B cell differentiation and maturation has been amply documented. The mechanism of CD40 signaling in B cells has been well established, whereas the signaling mechanism of CD40L in T cell costimulation remains unknown. In this study we show that CD28i, a transmembrane splice variant of CD28 costimulatory receptor, complexes with CD40L in human T cells. The cross-linking of CD40L resulted in the coendocytosis of CD28i with CD40L. The tyrosine phosphorylation of CD28i followed the cross-linking of CD40L, and the overexpression of CD28i augmented the c-Jun NH2-terminal kinase, p21-activated kinase 2, and nuclear factor κB activation. These data indicate that CD28i, by functioning as a signaling adaptor, transduces CD40L signaling as well as CD28 signaling in human T cells.

A novel mutation of CLCNKB in a Japanese patient of Gitelman-like phenotype with diuretic insensitivity to thiazide administration.

The clinical phenotypes of patients with Bartter syndrome type III sometimes closely resemble those of Gitelman syndrome. We report a patient with mild, adult-onset symptoms, such as muscular weakness and fatigue, who showed hypokalemic metabolic alkalosis, elevated renin–aldosterone levels with normal blood pressure, hypocalciuria and hypomagnesemia. She was also suffering from chondrocalcinosis. A diuretic test with furosemide and thiazide showed a good response to furosemide, but little response to thiazide. Although the clinical findings and diuretic tests predicted that the patient had Gitelman syndrome, genetic analysis found no mutation in SLC12A3. However, a novel missense mutation, p.L647F in CLCNKB, which is located in the CBS domain at the C-terminus of ClC-Kb, was discovered. Therefore, gene analyses of CLCNKB and SLC12A3 might be necessary to elucidate the precise etiology of the salt-losing tubulopathies regardless of the results of diuretic tests.

Clinical application of the different cross-reactivities of anti-insulin antibodies to insulin lispro to evaluate endogenous insulin secretion.

BACKGROUND Insulin analogs are often used to treat patients with diabetes. We evaluated the cross-reactivities of anti-insulin antibodies in two insulin immunoassay kits (Architect and ECLusys) against recombinant human insulin and insulin analogs, and measured insulin concentrations in the serum of the diabetic patients treated with only insulin lispro. METHODS Ten-fold dilutions of recombinant human insulins and insulin analogs were measured using Architect and ECLusys kits. The serum samples of 4 type 2 diabetic patients at fasting, and several time points after breakfast (25 kcal/kg) following subcutaneous injection of insulin lispro were measured by Architect, ECLusys and LISPro RIA kit. RESULTS The ECLusys kit could detect human insulin but not insulin analogs. The Architect kit detected human insulin and insulin analogs with similar recovery ratios. The difference in serum insulin concentrations measured by Architect and ECLusys assays reflected the concentration measured by LISPro insulin kit in the patients. The differences in the AUC between Architect and ECLusys assays were significantly correlated with the AUC for LISPro assay (p<0.01). CONCLUSIONS By exploiting the different cross-reactivities of anti-insulin antibodies to insulin analogs, it may be possible to measure the endogenous and exogenous insulin concentrations in diabetic patients treated with insulin analogs.

Adverse effects of obesity on β-cell function in Japanese subjects with normal glucose tolerance.

SUMMARY The purpose of the present study was to elucidate the role of obesity in both early- and late-phase insulin secretion during an oral glucose tolerance test (OGTT) performed with 75 g glucose in Japanese subjects. This was performed using indices of β-cell function adjusted for insulin sensitivity. Of 155 subjects assessed, 68 had normal glucose tolerance (NGT) and 87 had impaired glucose tolerance (IGT). We used the homeostasis model assessment-insulin resistance (HOMA-IR) index as an indicator of insulin sensitivity. As indicators of β-cell function, we used the HOMA-β index, an insulinogenic index (ΔI30/ΔG30), and ΔAUC I/G(0-120), which were obtained in the OGTT. We then reevaluated the results after adjusting the β-cell function for insulin sensitivity ([ΔI30/ΔG30]/HOMA-IR index and [ΔAUC I/G(0-120)]/HOMA-IR index). β-Cell function was observed to reduce as the glucose tolerance deteriorated from NGT to IGT. However, when the effects of obesity were considered, the obese subjects with NGT already showed a decline in the (ΔAUC I/G(0-120))/HOMA-IR index value when compared with the nonobese subjects with NGT, despite the fact these subjects did not differ with regard to (ΔI30/ΔG30)/HOMA-IR index. As the glucose tolerance deteriorated to IGT, both (ΔI30/ΔG30)/HOMA-IR index and (ΔAUC I/G(0-120))/HOMA-IR index decreased to an identical extent in both subgroups. These data indicate that obesity causes a decrease in insulin secretion, especially during the late phase following a glucose load, even if the glucose tolerance remains normal.:

A novel mitogen fusion protein against CD40+ cells with potent vaccine adjuvant properties.

A large number of infectious diseases caused by viral or bacterial infections are treatable and/or preventable by vaccination. In addition, ongoing research is aimed at the development of vaccines against other types of diseases, including almost all forms of cancer. The efficacy of a vaccine relies on the antigen-specific response by the entire repertoire of immune competent cells. Here, we have generated a powerful mitogen fusion protein, CD40L-FasL-IgFc, which stimulates CD40(+) cells robustly. We found that this specific cell activation is accompanied by increased expression of PRDI-BF1 (Blim-1) RNA, an indicator of terminal B-cell differentiation, in cultures stimulated with CD40L-FasL-IgFc. The addition of specific inhibitors of NF-kappaB and MEK1/2 partially suppressed the observed proliferative effects of CD40L-FasL-IgFc. When tested in vivo, the immune response to influenza HA vaccine was significantly increased by co-administration of CD40L-FasL-IgFc. Moreover, the co-administration of the cDNA expression plasmid encoding CD40L-FasL-IgFc significantly boosted the vaccine response. We now have a unique opportunity to evaluate our novel fusion protein adjuvant, and other similarly constructed fusion proteins, in both protein-based and genetic vaccines.