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Cyclic nigerosyl nigerose (CNN) is a cyclic tetrasaccharide that exhibits properties distinct from other conventional cyclodextrins. Herein, we demonstrate that treatment of B16 melanoma with CNN results in a dose-dependent decrease in melanin synthesis, even under conditions that stimulate melanin synthesis, without significant cytotoxity. The effects of CNN were prolonged for more than 27 days, and were gradually reversed following removal of CNN. Undigested CNN was found to accumulate within B16 cells at relatively high levels. Further, CNN showed a weak but significant direct inhibitory effect on the enzymatic activity of tyrosinase, suggesting one possible mechanism of hypopigmentation. While a slight reduction in tyrosinase expression was observed, tyrosinase expression was maintained at significant levels, processed into a mature form, and transported to late-stage melanosomes. Immunocytochemical analysis demonstrated that CNN treatment induced drastic morphological changes of Pmel17-positive and LAMP-1-positive organelles within B16 cells, suggesting that CNN is a potent organelle modulator. Colocalization of both tyrosinase-positive and LAMP-1-positive regions in CNN-treated cells indicated possible degradation of tyrosinase in LAMP-1-positive organelles; however, that possibility was ruled out by subsequent inhibition experiments. Taken together, this study opens a new paradigm of functional oligosaccharides, and offers CNN as a novel hypopigmenting molecule and organelle modulator.

A 61-year-old man with high myopia who had received a systemic α1A-adrenoceptor antagonist had phacoemulsification and in-the-bag intraocular lens implantation in the right eye. One day postoperatively, marked pigment dispersion in the anterior chamber, posterior bowing of the iris, and iridodonesis were noted associated with a subsequent elevation in intraocular pressure (IOP). Pharmacological pupil dilation was effective in reducing pigment dispersion and IOP, and laser peripheral iridotomy was performed to alleviate posterior bowing of the iris. We hypothesize that dynamic changes in the aqueous humor flow by cataract surgery and latent flaccidity of the iris due to the systemic α1A-adrenoceptor antagonist caused reverse pupillary block. High myopia may be another risk factor for this complication.

We previously reported that oral administration of NK-4, a criptocyanine dye, enhances interleukin (IL)-12-depend- ent interferon (IFN)-γ production by lipopolysaccharide (LPS)-stimulated mouse splenocytes. These findings raised a possibility that NK-4 potentiated IFN-γ production by T cells, natural killer (NK) cells or natural killer T (NKT) cells in response to IL-12 produced by macrophage and dendritic cells. To explore this possibility, we first analyzed percentages of T, NK or NKT cells in splenocytes of mice that were administered NK-4 orally for three days. The percentage of NKT cells in splenocytes from NK-4-treated mice was significantly (p

Aim: To evaluate the relationship between vision-related quality of life (VR-QOL) and visual function in patients undergoing vitrectomy, gas tamponade and cataract surgery for macular hole (MH). Methods: The 25-item National Eye Institute Visual Function Questionnaire (VFQ-25) was self-administered by 32 patients with MH (age 66.2 (SD 5.4) years) preoperatively and at 3 months postoperatively. Clinical data were collected, including logarithm of minimum angle of resolution (logMAR) best corrected visual acuity (BCVA), severity of metamorphopsia and letter contrast sensitivity. The severity of metamorphopsia was evaluated by the M-Charts. MH index was measured using optical coherence tomography. The presence and severity of cataract were graded using the Lens Opacities Classification System III reference standards. Multiple regression analysis was performed to investigate the relationship between various explanatory variables and VFQ-25 questionnaire scores. Explanatory variables tested were the severity of metamorphopsia, visual acuity, letter contrast sensitivity, MH index and grade of cataract. Results: Vitrectomy for MH significantly improved VFQ-25 composite score as well as subscale scores, including general vision, near activities, distance activities, social functioning, mental health and dependency (p

We have recently shown that royal jelly has potent antiallergic properties in a mouse model of immediate hypersensitivity. However, it is still unclear which components of royal jelly exhibit antiallergic activity. In this study, we have screened for antiallergic factors in royal jelly based on inhibition of IL-4 production by anti-CD3 stimulated spleen cells derived from OVA/alum-immunized mice. Using a series of column chromatographies, we purified a 70 kDa glycoprotein, major royal jelly protein 3 (MRJP3), that suppresses IL-4 production. In in vitro experiments, MRJP3 suppressed the production of not only IL-4 but also that of IL-2 and IFN-gamma by T cells concomitant with inhibition of proliferation. The MRJP3-mediated suppression of IL-4 production was also evident when lymph node cells from OVA/alum-immunized mice were stimulated with OVA plus antigen presenting cells. We next examined the purified suppressive factor on OVA/alum-induced allergic responses in mice. Interestingly, in spite of the antigenicity of MRJP3 itself as an extraneous foreign protein, intraperitoneal administration of MRJP3 inhibited serum anti-OVA IgE and IgG1 levels in immunized mice. In addition, heat-treated soluble MRJP3 treatment reduced its antigenicity while maintaining its inhibitory effects on antibody responses to OVA. These results indicate that MRJP3 can exhibit potent immunoregulatory effects in vitro and in vivo. Furthermore, considering the intriguing immunomodulatory effects of MRJP3, it may be of clinical significance to design MRJP3-derived antiallergic peptides by identifying the associated polypeptide regions.

The therapeutic effects of tryptanthrin (TRYP), a natural product from the medicinal plant Polygonum tinctorium, were examined in a murine model of inflammatory bowel disease (IBD). Colitis was induced by 5% dextran sodium sulfate (DSS) in drinking water for 7 days from day 0. TRYP (100 mg/kg) was administered orally suspended in 5% arabia gum everyday from day 3 for 5 days. Histopathological analysis showed reduced colon damage in TRYP-treated mice on day 6; however, colon injury resumed after treatment was stopped. The production of prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) by untreated and treated mouse colon tissues cultured in vitro were mostly unchanged by TRYP treatment. However, mitogen-stimulated spleen cells from TRYP-treated colitic mice produced less interleukin 2 (IL-2) and less interferon-gamma (IFN-gamma) than untreated colitic mouse spleen cells, early after induction of colitis. When colitis was induced with 5% DSS for 7 days and TRYP was given to the mice for 8 days from day 3, TRYP enhanced the survival of the mice but results were not significant. A significant reduction of weight loss was observed in TRYP-treated mice with colitis induced by 5% DSS for 4 days as compared to control mice. Remarkably, whereas 90% of the vehicle-treated mice died from wasting disease, all the TRYP-treated mice survived, suggesting that TRYP may have a therapeutic effect on colitis.

Lumin was orally administered to mice daily for 3 d, and on the day following the final administration, mice were sacrificed and splenocytes were stimulated with lipopolysaccharide (LPS). Splenocytes obtained from lumin-treated mice showed enhanced production of interferon gamma (IFN-gamma) and increased percentages of CD3+ cells. Although T cells are considered to be the source of IFN-gamma, it is unlikely that LPS directly stimulates T cells. Next we performed neutralization experiments using a monoclonal antibody (mAb) against interleukin (IL-)12 because this cytokine, which is produced by macrophages, has the direct ability to induce IFN-gamma production and the proliferation of activated T cells. This antibody inhibited IFN-gamma production by splenocytes. We thus show that orally administered lumin enhances IFN-gamma production by splenocytes when the latter are stimulated with LPS, a phenomenon that was observed in correlation with activation of T cells by IL-12, that is produced by macrophages.

We evaluated the effect of tryptanthrin and kaempferol, both isolated from Polygonum tinctorium Lour., against Helicobacter pylori colony formation in vitro and in H. pylori-infected Mongolian gerbils. H. pylori suspension was mixed with solution of tryptanthrin and/or kaempferol and placed onto agar plates. These plates were incubated at 37 degrees C, under 10% CO2 for 5 days, and the H. pylori colonies were counted. For the in vivo experiment, Mongolian gerbils were inoculated with H. pylori ATCC 43504 orally. After 4 weeks, the infected gerbils were given tryptanthrin and/or kaempferol, administered orally, twice a day for 10 days. The animals were killed and the number of live H. pylori in their stomachs was determined. In vitro both tryptanthrin and kaempferol significantly decreased the numbers of H. pylori colonies a dose-dependent manner. An additive effect on colony formation was observed with the combined use. In the in vivo experiment, oral administration of tryptanthrin and/or kaempferol significantly decreased the numbers of colonies in the gerbils' stomachs. We concluded that tryptanthrin and kaempferol were effective against H. pylori in vivo.

Interleukin-18 (IL-18) was identified as a molecule that induces IFN-gamma production and enhances NK cell cytotoxicity. Characterization of the receptor for human IL-18 (hIL-18R) is important for investigating the physiological role of IL-18 in nature. In the present study, we describe a monoclonal antibody (mAb) against hIL-18R (mAb No. 117-10C). This mAb inhibited the binding of 125I-labeled hIL-18 to IL-18R-expressing L428 cells. This mAb also neutralized hIL-18-induced T helper 1 type cytokine (IFN-gamma and GM-CSF) production by Con A-stimulated PBMC. PBMC were examined for the expression of IL-18R by two-color flow cytometry. Most CD19(+) B cells and a percentage of CD8(+) T cells were found to constitutively express IL-18R. Treatment of PBMC with IL-12 preferentially induced IL-18R expression on CD56(+) NK cells regardless of costimulation with mitogen. IL-18R expression on CD4(+) T cells was induced weakly by IL-12 treatment and moderately by PHA stimulation. However, neither could IL-12 treatment nor PHA stimulation induce IL-18R expression on CD8(+) T cells. Costimulation with both IL-12 and PHA was necessary for optimal IL-18R expression on CD8(+) T cells as well as on CD56(+) NK cells, CD4(+) T cells, and CD19(+) B cells. These results support the growing number of reports showing that IL-18 has modulatory effects on T, B, and NK cells.

Interleukin (IL)-18 was identified as a molecule that induces IFN-γ production and enhances NK cell cytotoxicity. In this paper, we report upon the purification and characterization of human IL-18 receptor (hIL-18R). We selected the Hodgkin’s disease cell line, L428, as the most strongly hIL-18R-expressing cell line based on the results of binding assays. This binding was inhibited by IL-18 but not by IL-1β. The dissociation constant (K d) of125I-IL-18 binding to L428 cells was about 18.5 nm, with 18,000 binding sites/cell. After immunizing mice with L428 cells and cloning, a single monoclonal antibody (mAb) against hIL-18R was obtained (mAb 117-10C). Sequentially, hIL-18R was purified from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-extracted L428 cells by wheat germ lectin-Sepharose 4B chromatography and mAb 117-10C-Sepharose chromatography. The internal amino acid sequences of hIL-18R all matched those of human IL-1 receptor-related protein (IL-1Rrp), the ligand of which was unknown to date. When expressed in COS-1 cells, the cDNA of IL-1Rrp conferred IL-18 binding properties on the cells and the capacity for signal transduction. From these results, we conclude that a functional IL-18 receptor component is IL-1Rrp.

Deficiencies of Bruton's tyrosine kinase (Btk) have been implicated in the pathogenesis of human X-linked agammaglobulinemia (XLA). The distinctive phenotype observed in B-cell deficiency indicates the crucial role of Btk in B-cell development. This report describes a nationwide study of Btk deficiency in Japan, covering 51 XLA patients (35 independent families). Along with the identification of mutations, the resulting protein products were characterized by an in vitro kinase assay and a Western blot analysis. Thirty-one of the families were found to have mutations in the coding region of Btk. Although mutations were not found in the cDNA of 4 families, the Btk transcripts of these patients were greatly reduced. The identification of several novel missense mutations, in combination with the result of other studies, clarified the presence of two (missense) mutation hot spots, one in the SH1 and the other in the PH domain. The absence of kinase activity seen in 32 of the families underscored the importance of Btk protein analysis as a diagnostic indicator of XLA. The protein analysis also clarified the different effects of missense mutations on kinase activity and protein stability.

症例は68歳, 女性. 右手の腫脹・疼痛が出現, CK上昇は認められなかったがGottron徴候陽性よりamyopathic dermatomyositisを疑われた. 間質性肺炎の所見がないにも関わらずKL-6が高値で, かつ症状も急速に進行したため, 悪性腫瘍の検索を行ったところ卵巣癌が発見された. 腫瘍摘出術と化学療法を行った結果, 症状の改善と平行してKL-6は減少した. 間質性肺炎のないKL-6上昇を認めた場合には悪性腫瘍の検索が必要である.

The immuno-potentiating effects of the antler-shaped fruiting body of Ganoderma lucidum (Rokkaku-Reishi, RR), which has been used as a traditional supplement for human health, were investigated in mice. BALB/c mice were administered orally with RR for 3 days at a dose of 50 mg/kg or 500 mg/kg, and interferon-gamma (IFN-gamma) production by splenocytes in response to lipopolysaccharide (LPS) was examined on day 4. The oral administration of 500 mg/kg of RR resulted in a significant increase (p0.05) in IFN-gamma production. Stimulation of splenic adherent cells from these mice with LPS also resulted in a significant increase (p0.05) in interleukin-12 (IL-12) production compared with that from the control mice, suggesting that splenic macrophages were activated by RR administration. Furthermore, 500 mg/kg of RR administered for 14 days resulted in a significant increase (p0.05) in IFN-gamma production by splenocytes in response to both LPS and concanavalin A (Con A). These results suggest that not only splenic macrophages but also T cells were activated by the long-term treatment with RR in vivo. On the other hand, the production of interleukin-4 (IL-4), which is known as an allergic disease-related cytokine, was not affected by the long-term treatment with RR. Our results suggest that the oral administration of RR resulted in Th1-associated immuno-potentiating activities in vivo.

In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.

Tryptanthrin, a biologically active compound found in the medicinal plant Polygonum tinctorium, reportedly has several biological activities. We investigated the effects of tryptanthrin on cytokine production by lymphocytes in response to staphylococcal enterotoxin B (SEB), which causes a variety of disorders in humans based on its induction of large amounts of immunostimulatory cytokines. Tryptanthrin dose-dependently inhibited interferon-gamma (IFN-gamma) and interleukin-2 production by mouse spleen cells and Peyer's patch (PP) lymphocytes in vitro. The efficacy of tryptanthrin was further studied in a mouse model in vivo. Tryptanthrin was administered orally 2 h after an oral challenge with SEB. Nineteen hours after SEB administration, PP lymphocytes were prepared, and IFN-gamma production by PP lymphocytes was examined. The production of IFN-gamma increased after SEB administration, and the elevated IFN-gamma production was significantly inhibited by tryptanthrin treatment. These results suggest that tryptanthrin may be effective in the treatment of disorders of the intestines, such as food poisoning, that are associated with activated lymphocytes.

Polygonum tinctorium Lour. (P. tinctorium) is known to have the ability to suppress inflammation. We attempted to isolate the active compounds from P. tinctorium based on their inhibitory effects on the production of interferon-gamma, which is a well-known inflammatory cytokine. We thus isolated indirubin, an isomer of indigo. Indirubin exerted its inhibitory effects not only on interferon-gamma production by human myelomonocytic HBL-38 cells but also on interferon-gamma and interleukin-6 production by murine splenocytes with no influence on the proliferation of either cells. Because of its inhibitory activity on interferon-gamma production, we further investigated the effects of indirubin on 2,4, 6-trinitro-l-chlorobenzene (TNCB)-induced delayed-type hypersensitivity as a representative inflammatory reaction. When injected intraperitoneally, indirubin significantly inhibited the ear swelling of TNCB-elicited mice. The amount of interferon-gamma in the culture supernatants of elicited mouse lymphocytes was inhibited by indirubin treatment. These results suggest that indirubin is a compound with anti-inflammatory effects.

Insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetes (NOD) mouse model is thought to be an autoimmune CD4 Th1-like cell-mediated disease. We tested the efficacy of oral use of interferon-alpha (IFN-alpha) therapy on IDDM in NOD mice. Using urine and blood sugar levels as indicators of IDDM, oral administration of murine IFN-alpha (100 IU/body) to NOD mice significantly delayed the onset of symptomatic diabetes. However, oral use of IFN-alpha did not prevent diabetic NOD mice from losing weight once NOD mice were symptomatic, suggesting that orally administered IFN-alpha is a prophylactic rather than therapeutic approach to the management of IDDM.

Bruton’s tyrosine kinase (Btk) has been shown to play a role in normal B-lymphocyte development. Defective expression of Btk leads to human and murine immunodeficiencies. However, the exact role of Btk in the cytoplasmic signal transduction in B cells is still unclear. This study represents a search for the substrate for Btk in vivo. We identified one of the major phosphoproteins associated with Btk in the preB cell line NALM6 as the Wiskott-Aldrich syndrome protein (WASP), the gene product responsible for Wiskott-Aldrich syndrome, which is another hereditary immunodeficiency with distinct abnormalities in hematopoietic cells. We demonstrated that WASP was transiently tyrosine-phosphorylated after B-cell antigen receptor cross-linking on B cells, suggesting that WASP is located downstream of cytoplasmic tyrosine kinases. An in vivo reconstitution system demonstrated that WASP is physically associated with Btk and can serve as the substrate for Btk. A protein binding assay suggested that the tyrosine-phosphorylation of WASP alters the association between WASP and a cellular protein. Furthermore, identification of the phosphorylation site of WASP in reconstituted cells allowed us to evaluate the catalytic specificity of Btk, the exact nature of which is still unknown.

Protein interaction cloning method was used to identify a novel molecule, Sab, which binds to the SH3 domain of Bruton's tyrosine kinase (Btk), the deficient cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia and murine X-linked immunodeficiency. Immunoprecipitation using the anti-Sab antibody identified the protein product of the gene as a 70 kDa molecule. While Sab does not have a proline-rich sequence, it was shown to bind to Btk through the commonly conserved structure among SH3 domains. Remarkably, Sab exhibited a high preference for binding to Btk rather than to other cytoplasmic tyrosine kinases, which suggests a unique role of Sab in the Btk signal transduction pathway.

Interleukin-18 (IL-18)/interferon- γ -inducing factor (IGIF) is a novel cytokine, which is a potent inducer of IFN- γ production and plays an important role in Th1 responses. In order to develop a specific ELISA for the measurement of human IL-18, we established 13 anti-human IL-18 monoclonal antibodies and characterized them. 7 murine anti-human IL-18 mAbs and 6 rat anti-human IL-18 mAbs were obtained by fusion of splenocytes from mice or rats immunized with human IL-18, with SP2/0 myeloma cells. These antibodies were classified into 4 groups according to competitive binding ELISAs to the human IL-18 molecule. 1 murine mAb and all 6 rat mAbs neutralized IFN- γ production induced by IL-18. A specific human IL-18 ELISA was developed using two neutralizing mAbs (#125-2H and #159-12B). This ELISA detects human IL-18 with a minimum detection limit of 10 pg/ml, but does not react with heat-denatured human IL-18. The ELISA does not show any cross-reactivity with other cytokines. Using this assay, human IL-18 was measurable in the plasma of leukemia patients. This ELISA would become a powerful tool for investigating the relationship between IL-18 and various diseases or analyzing the control mechanisms of IL-18 production from IL-18 producing cells.