Early Enteral Feeding of Daikenchuto Stimulates Early Bowel Movement With Increased Portal Venous Blood Flow After Living Donor Liver Transplantation.

Daikenchuto (DKT), a Japanese Kampo medicine, had been reported to increase small intestinal blood flow after liver resection. The aim of this study was to evaluate the effects of early enteral feeding of DKT on portal venous flow and early bowel movement after living donor liver transplantation (LDLT) in an attempt to clarify whether these effects on bowel motility can prevent bacterial and/or fungal translocation. MATERIALS AND METHODS: Our prospective study included the consecutive 16 LDLT recipients at Mie University Hospital between June 2006 and September 2009. Sixteen patients were divided into the 2 groups according to enteral feeding starting postoperative day (POD) 1: 8 patients in DKT (15 g/d) administration (DKT group, for 1 week) and 8 patients in tepid water administration (non-DKT group, for 1 week). Portal venous flow, portal venous pressure, presence of fungal infection (serum level of ß-D-glucan and fungal polymerase chain reaction assay), time to first food intake, and time to first defecation were serially examined. RESULTS: Portal venous flow (mean [SD] velocity) was significantly increased in DKT group compared with non-DKT group: 47.5 (12.9) vs 31.8 (15.4) (P = .04) on POD 1, 46.8 (11.5) vs 28.8 (12.5) (P = .03) on POD 3, and 42.3 (17.2) vs 25.2 (9.0) (P = .05) on POD 5. However, mean (SD) portal venous pressures did not significantly change between the 2 groups. Between the 2 groups (DKT vs non-DKT), the day of first oral intake was not significantly different: 6.9 (2.5) vs 11.3 (8.7) (P = .061), but the mean (SD) day of first defecation was significantly shorter in the DKT group: 3.9 (1.1) vs 5.5 (2.6) (P = .02). Although fungal polymerase chain reaction assay was not significantly different between the 2 groups (4 vs 4 positive cases), the mean (SD) serum levels of ß-D-glucan were significantly lower in the DKT group than in the non-DKT group: 9.0 (7.4) vs 18.4 (15.9) pg/mL (P = .04). CONCLUSION: Early enteral feeding of DKT after LDLT increased portal vein blood flow without increasing portal vein pressure and stimulated early bowel movement, which in turn might prevent fungal translocation.

Supplementation of CD4+CD25+ regulatory T cells suppresses experimental autoimmune uveoretinitis.

AIMS: To investigate whether supplementation of natural CD4+CD25+ regulatory T cells ameliorates mouse experimental autoimmune uveoretinitis (EAU) induced by CD4+ T cell-dependent interphotoreceptor retinoid-binding protein (IRBP). METHODS: C57BL/6 mice were immunised with human interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP(1-20)), and IRBP(1-20)-sensitised T cells were obtained. CD4+CD25+ T cells derived from naive mice were cocultured with IRBP(1-20)-sensitised T cells, and their proliferation responses and cytokine production were measured. In addition, CD4+CD25+ T cells were transferred intravenously into mice 7 or 15 days after immunisation with IRBP(1-20), and the severity of EAU and T cell proliferation responses were evaluated. RESULTS: CD4+CD25+ regulatory T cells effectively inhibited both the proliferation of, and interleukin (IL)2, IL5 and interferon (IFN)gamma production by, IRBP(1-20)-sensitised T cells. Adoptive transfer of CD4+CD25+ regulatory T cells to IRBP(1-20)-immunised mice conferred considerable protection from EAU development and inhibition of T cell proliferation responses to IRBP(1-20). CONCLUSION: These findings show that natural CD4+CD25+ regulatory T cells possess the ability to inhibit activation of IRBP-reactive T cells that have been already sensitised in vivo, and adoptive transfer of these cells ameliorates EAU even in the effector phase. Supplementation of natural CD4+CD25+ regulatory T cells may have therapeutic potential for effective treatment of uveitis.

Differentiation of Th1 and Th2 cells in lymph nodes and spleens of mice during experimental autoimmune uveoretinitis.

PURPOSE: Experimental autoimmune uveoretinitis (EAU) is a T-cell-mediated autoimmune disease that can be elicited in susceptible rodent strains by immunization with a retinal autoantigen, such as interphotoreceptor retinoid-binding protein (IRBP). In this study, we investigated whether there is a correlation between inflammation in the eye and T-helper (Th)1- and Th2-type responses in the lymph nodes and the spleen after immunization of B10.A mice with IRBP. METHODS: B10.A mice were immunized with IRBP emulsified with complete Freund's adjuvant (CFA), and eyes were then enucleated for histological examination of EAU at 1, 2, 4, 6, or 8 weeks after immunization. In addition, lymph node cells and spleen cells were collected, and cultured with IRBP to measure T-cell proliferation responses and Th1-type (interleukin [IL]-2, interferon [IFN]-gamma), Th2-type (IL-4, IL-10) cytokine production. RESULTS: Pathologically, severe ocular inflammation occurred 2 weeks after IRBP immunization, persisted for 2 weeks, and then gradually resolved. Interleukin-2 and IFN-gamma production were observed in draining lymph node cells at 1 and 2 weeks after IRBP immunization. Those responses then diminished, whereas IFN-gamma production by spleen cells was observed from week 1, peaked at week 4, and gradually decreased. Alternatively, significant production of IL-4 or IL-10 by draining lymph node cells was not detected at any time point. Both IL-4 and IL-10 production by spleen cells was observed at week 6. CONCLUSIONS: Th1-type responses were observed early in draining lymph nodes, then in the spleen after IRBP immunization. The levels of IFN-gamma production by spleen cells reflected the severity of EAU, confirming their pathogenic role in this disease. Th2-type responses were generated in the spleen only as the disease receded, suggesting a role for Th2 cells in the spontaneous termination of EAU.

Radiofrequency ablation at the coronary sinus ostium interrupts the vagal efferent input to the atrioventricular node in the canine heart.

The fat pad at the junction of the inferior vena cava and inferior left atrium is the area of convergence of vagal projections into the atrioventricular node (AVN) region. The present study investigated whether radiofrequency (RF) ablation applied to the area around the coronary sinus (CS) ostium would impair vagal input to the AVN in the canine heart. Twenty-four dogs were anesthetized by sodium pentobarbital and RF energy was delivered at 20W for 5-10s. In the baseline state without vagal stimulation (10Hz, 2ms), the electrophysiological variables did not change significantly after RF ablation. Vagally induced changes in the sinus cycle length and effective refractory period of the right atrium and left ventricle did not differ after RF ablation. However, the effects of vagal stimulation on the AVN function were impaired after RF ablation to the CS area from the ostium to 10mm within the ostium. After ablation was applied to the fast pathway area, the vagally induced changes in the AVN function decreased, but these changes were not affected after RF ablation in the slow pathway area. RF ablation in the vicinity of the CS would attenuate vagal input to the AVN.

Identification of Th2-type suppressor T cells among in vivo expanded ocular T cells in mice with experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU), which is a T cell mediated organ specific autoimmune disease, is induced by immunization with interphotoreceptor retinoid binding protein (IRBP) in susceptible strains of mice. It has been found that IRBP-derived peptide 518-529 (p518-529) generates Th2-type responses and inhibits IRBP-induced EAU, indicating that the p518-529 might be an epitope for suppressor T cells in IRBP-induced EAU. First, we observed that there were T cells producing the Th2 type cytokines such as IL-4 and IL-10 in late phase of EAU. Furthermore, to examine whether p518-529-reactive T cells expand in the eye during EAU, T cell receptor (TCR) of ocular T cells was compared with that of p518-529 reactive T cells in spleen from mice with EAU by PCR-single strand conformation polymorphism (PCR-SSCP) and nucleotide sequence analysis. SSCP and sequence analyses indicated that p518-529 reactive TCR BV10+ T cells bearing amino acid motif(PWG) and TCR BV13+ T cells bearing amino acid motif(PGLGGY) in their complementary-determining region 3 (CDR3) region were clonally expanding in ocular tissues on day 28 after immunization, although these T cells were not detected on day 14. These findings demonstrate that p518-529 reactive Th2-type T cells expand oligoclonally in the uveitic eyes in the late stage of EAU and may function as Th2-type suppressor T cells for improvement of the disease.

Tetrahydrobiopterin restores endothelial function in long-term smokers.

OBJECTIVES: We sought to test whether tetrahydrobiopterin (BH4) supplementation improves nitric oxide (NO) bioactivity in smokers. BACKGROUND: In smokers, endothelium-derived NO bioactivity is impaired. BH4 is an essential cofactor of NO synthase, and its deficiency decreases NO bioactivity. METHODS: Sapropterin hydrochloride, an active analogue of BH4 (2 mg/kg body weight), was administered orally to healthy male smokers and age-matched nonsmokers. Before and 3 and 24 h after sapropterin, we measured plasma levels of BH4 and examined flow-mediated vasodilation (FMV) of the brachial artery by high resolution ultrasonography, a noninvasive test of endothelial function. RESULTS: Basal plasma levels of BH4 were not different between smokers and nonsmokers. Sapropterin administration increased plasma levels of BH4 by threefold at 3 h, which returned to the baseline at 24 h. Before sapropterin, FMV was significantly smaller in smokers (p = 0.0002). Sapropterin significantly augmented endothelium-dependent vasodilation in smokers, but did not affect it in nonsmokers (p = 0.001 by analysis of variance [ANOVA]). Coadministration of N(G)-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor (20 micromol), into the brachial artery completely abolished the vasodilatory effects of sapropterin (p = 0.002 by ANOVA). Endothelium-independent vasodilation by glyceryl trinitrate was not different between smokers and nonsmokers and was not altered by BH4. CONCLUSIONS: We demonstrated that BH4 supplementation improved bioactivity of endothelium-derived NO in smokers. These observations strongly suggest that decreased NO-dependent vasodilation in smokers could be related to reduced bioactivity of BH4.

Endothelial dysfunction by acute hyperhomocyst(e)inaemia: restoration by folic acid.

Recent evidence demonstrates that hyperhomocyst(e)inaemia is a novel risk factor for cardiovascular diseases. In patients with chronic hyperhomocyst(e)inaemia, endothelial function is impaired. However, whether hyperhomocyst(e)inaemia per se is a cause or an epiphenomenon of endothelial dysfunction remains unknown. In this study, we examined the effects of methionine-induced acute hyperhomocyst(e)inaemia on human endothelial function. In healthy volunteers we administered methionine (0.1 g/kg body weight, per os), a substrate of homocyst(e)ine, with or without folic acid (20 mg, per os) and examined flow-mediated vasodilatation of the brachial artery by high-resolution ultrasonography as a non-invasive measure of endothelial function. We also measured plasma levels of homocyst(e)ine before and 3, 8 and 24 h after methionine loading. Methionine administration increased plasma levels of homocyst(e)ine by four times the basal level at 8 h (P<0.0001, ANOVA). The plasma levels returned to baseline at 24 h. Flow-mediated vasodilatation was significantly decreased to half of the baseline value at 8 h and returned to baseline at 24 h (P<0.0001, ANOVA), whereas endothelium-independent vasodilatation by glyceryl trinitrate was not affected by the methionine loading. Co-administration of folic acid did not attenuate methionine-induced hyperhomocyst(e)inaemia but completely prevented endothelial dysfunction. Our results suggest that in humans a methionine-rich diet may acutely impair endothelial function, which can be prevented by folic acid supplementation.

Regulation of angiotensin II receptor expression by nitric oxide in rat adrenal gland.

We recently reported that administration of Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) production, activates the vascular and cardiac renin-angiotensin systems and causes vascular thickening and myocardial hypertrophy in rats with perivascular and myocardial fibrosis. It has been reported that aldosterone may contribute to the development of cardiac fibrosis, but it is not known whether inhibition of NO synthesis affects angiotensin II (Ang II) receptor gene expression and aldosterone secretion. The aim of this study was to investigate the effect of NO inhibition on the expression of Ang II receptors in the adrenal gland and on aldosterone secretion in rats. Wistar King A rats received normal water, L-NAME alone (1 mg/mL in the drinking water), or L-NAME and the alpha1-adrenergic receptor blocker bunazosin (0.1 mg/mL in the drinking water) for 1 week. After 1 week of treatment with L-NAME, systolic blood pressure, plasma aldosterone concentration (PAC), and mRNA level and number of Ang II type 1 receptor (AT1-R) were increased. Plasma renin activity, serum angiotensin-converting enzyme activity, and the number of AT2-R were unchanged. Although addition of bunazosin to L-NAME restored systolic blood pressure to the control level, PAC and AT1-R numbers remained significantly higher than those of control level. These results suggest that the increased AT1-R number and PAC induced by the inhibition of NO synthesis were independent of blood pressure and systemic renin-angiotensin system. Therefore, hypertension and myocardial fibrosis induced by NO blockade may be due in part to an elevation of PAC caused by increased AT1-R in the adrenal gland.

[Association of heat-shock protein and uveitis].

Heat-shock protein 60 derived from Yersinia enterocolitica (Yersinia HSP 60) and bovine retinal HSP (Retina HSP 60) were previously identified by immunological cross-reaction and a high degree of common antigenicity, and a specific antibody against both proteins was detected in the sera of uveitis patients. We report here an attempt to isolate and purify Retina HSP 60 and Yersinia HSP 60. Both Retina HSP 60 and Yersinia HSP 60 showed an enriched content of glycine of approximately 60 to 80%. Lewis rats were inculated with 50 or 100 micrograms of purified Retina or Yersinia HSP 60 emulsified in complete Freund's adjuvant. In 50 to 60% of those inoculated with Retina HSP 60, uveoretinitis was observed about 13 days after inoculation, with massive infiltration of lymphocytes and polymorphonuclear neutrophils in the iris, ciliary body, and retinal tissue. Rats inoculated with Yersinia HSP 60 did not develop ocular inflammation. Lymphocyte proliferation assay was performed to investigate cellular immunoresponses in the rats that developed ocular inflammation after immunization with Retina HSP 60. The results showed significantly higher response to the Retina and Yersinia HSP 60 than to either S-antigen or interphotoreceptor retinoid-binding protein (IRBP), which are known to induce ocular inflammation. Cross-reaction between Retina and Yersinia HSP 60 is suggested. This study suggests that the HSP 60 molecules may be involved in the pathogenesis of intraocular inflammation.

Intraocular cytokine quantification of experimental autoimmune uveoretinitis in rats.

Elevations of inflammatory cytokines have been implicated in the pathogenesis of experimental autoimmune uveoretinitis (EAU) in rats, although such analysis has relied on indirect methods of assessment such as measurement of mRNA content. In this study, we examined the feasibility of directly measuring cytokine concentrations in intraocular extracts prepared by ultrasonic disruption. Cytokines were measured by ELISA in eyes from EAU-induced Lewis rats immunized with interphotoreceptor retinoid-binding protein (IRBP), and compared to eyes from rats immunized with adjuvant only and from normal rats. Interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) were detectable in EAU eyes at near peak inflammation, with IFN-gamma achieving the highest mean concentration (331 pg/ml). In eyes from rats immunized with adjuvant only and in normal eyes, IFN-gamma, TNF-alpha, and IL-10 were nondetectable. IL-2 and IL-4 were detected at significantly lower mean concentrations (32.3 pg/ml and 69.4 pg/ml, respectively) compared to EAU eyes (217 pg/ml and 230 pg/ml, respectively); IL-4 was also detected in eyes from rats immunized with adjuvant alone (141 pg/ml). Thus, a direct method of measuring intraocular cytokine concentrations was successfully applied to reveal an elevation of IFN-gamma, TNF-alpha, IL-2, IL-4, and IL-10 in EAU eyes from rats immunized with IRBP, compared to rats immunized with adjuvant alone and to normal rats. These cytokine elevations reflect the local intraocular environment near peak inflammation, and suggest an important role for these cytokines in the mechanisms of onset and resolution of EAU in rats.

Regulation of cytokine production by sugi allergen-pullulan conjugate.

Sugi basic protein (SBP), a major allergen of Japanese cedar (Cryptomeria japonica) pollen, conjugated to pullulan (alpha-1,4'-, alpha-1,6'-glucan) reportedly suppresses IgE anti-SBP antibody production and enhances IgG anti-SBP antibody production in mice. We analyzed cytokine production by SBP-specific T cells after stimulation with an SBP-pullulan conjugate (SBP-P), native SBP, or a mixture of SBP and pullulan. When SBP-specific T cell lines were stimulated with the SBP-P conjugate in the presence of antigen-presenting cells (APC), the production of IFN-gamma, IL-4, IL-5, and IL-10 decreased compared with the cytokine levels produced by SBP-stimulated T cells. However, when these T cells were repeatedly stimulated with the SBP-P conjugate, the production of IFN-gamma increased progressively, while that of IL-4, IL-5, and IL-10 remained decreased compared with the T cells that were repeatedly stimulated with native SBP. Stimulation of the T cells with the mixture of SBP and pullulan showed little difference in the cytokine production profile from that observed after stimulation with native SBP alone. Interestingly, when the T cell lines stimulated repeatedly with SBP-P were subsequently stimulated with native SBP, a further increase in IFN-gamma production was observed, while IL-10 production decreased. Inhibition of IL-4 production was also observed when SBP-specific Th2 clones were stimulated with SBP-P. These results indicate that stimulation of T cells with SBP-P up-regulates Th1 cytokine production, while down-regulating that of Th2. It is, therefore, conceivable that immunotherapeutic treatment with the SBP-P conjugate rather than with conventional SBP solutions is preferable for improving Japanese cedar pollen allergy.

Cry j 2, a major allergen of Japanese cedar pollen, shows polymethylgalacturonase activity.

We examined Cry j 2, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, for polygalacturonase enzyme activity, since a nucleotide sequence of cDNA of Cry j 2 showed a significant homology with that of tomato polygalacturonase. Polygalacturonase is well known to depolymerize preferentially polygalacturonic acid (PGA) by hydrolysis. However, Cry j 2 did not act on PGA, but was found to depolymerize pectin and methylesterified PGA in a dose-dependent manner. The substrate specificity of Cry j 2 was different from that of polygalacturonase derived from Aspergillus niger. The depolymerizing activity of Cry j 2 reached a maximum at 50%-60% of methylesterification of PGA. In contrast, polygalacturonase showed its maximum activity of PGA, and the activity decreased as the degree of methylesterification increased. Interestingly, the pectin-depolymerizing activity of Cry j 2 was due to a hydrolysis, but not a lyase, activity which splits the glycosidic bonds by beta-elimination, since no unsaturated uronides were found by measurement of absorbance at 235 nm in the reaction mixture. The enzyme activity was markedly inhibited by anti-Cry j 2 antibodies. These results indicate that Cry j 2 probably has polymethylgalacturonase enzyme activity, as postulated by von Neukom in 1963, although existence of this activity has not yet been proven.

Carbohydrate structures of the glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen.

The glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen was treated with pepsin and glycopeptidase A to release asparagine-linked oligosaccharides. The reducing ends of the oligosaccharides were aminated with the fluorescent reagent 2-aminopyridine. The oligosaccharide derivatives were purified by gel permeation chromatography and reversed-phase HPLC. Their structures were determined by sequential exoglycosidase digestion and 500 MHz 1H-NMR spectroscopy. Four oligosaccharide structures, A, B, C, and D, were identified as the xylose-containing complex-type. They were present at a molar ratio of 8:1:6:1. By amino acid sequence analyses of the tryptic peptides, Asn-170 and Asn-333 of Cry j I were found to carry asparagine-linked oligosaccharides. [formula: see text]

Cry j I, a major allergen of Japanese cedar pollen, has pectate lyase enzyme activity.

In the course of analyzing the partial amino acid sequences of Cry j I, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, we found a peptide fragment which has a significant homology to some pectate lyase isozymes secreted by plant pathogenic bacteria. Therefore, we investigated whether Cry j I has pectate lyase activity. Cry j I reacted with polygalacturonic acid, resulting in the release of unsaturated uronide products. The optimum temperature and pH for the reaction were 60-70 degrees C and pH 10. The enzymatic reaction had an absolute Ca2+ ion requirement. These characteristics were very compatible with the character of the pectate lyase isozymes reported previously. These results clearly show that Cry j I has pectate lyase activity.

Molecular cloning of the second major allergen, Cry j II, from Japanese cedar pollen.

Cloning of a cDNA from Cry j II, the second major allergen from Japanese cedar (Cryptomeria japonica) pollen, is described. An isolated Cry j II cDNA contained an open reading frame coding for 514 amino acid residues. The mature Cry j II protein consisted of 388 amino acid residues (R46-S433). According to a homology analysis, no amino acid sequence homology was observed between Cry j II and Cry j I, another major allergen. But Cry j II showed homology with polygalacturonase (PG) derived from tomato (40% identity) at the amino acid level. The sequence information can potentially be used to devise an effective course of immunotherapy for Japanese cedar pollinosis.

[An investigation of factors in the pathogenesis of experimental autoimmune uveoretinitis (EAU) in congenic mice].

S-antigen or interphotoreceptor retinoid-binding protein (IRBP), when injected with Freund's complete adjuvant into mice, does not easily cause experimental autoimmune uveoretinitis (EAU). In this report, we describe the results of injecting IRBP with Freund's complete adjuvant, together with the intraperitoneal administration of Bordetella pertussis, into several types of congenic mice (B10, B10A, B10BR, B10D2). These congenic mice, of C57BL/10 (B10) origin, differ at the H-2 locus on chromosome 17. We were able to produce EAU in 38.5% of B10A mice, and 12.5% of B10BR mice, confirming that EAU can develop in these mice that carry the k genotype at the K, I-A, and I-E regions of the major histocompatibility complex (MHC) H-2 locus. We believe that the k genotype of the K, I-A, and I-E regions is important as a factor in the pathogenesis of EAU in congenic B10 mice.

[Enzyme-linked immunosorbent assay for the quantification of Cry j I and Cry j II].

Enzyme-linked immunosorbent assays (ELISA) were developed to specifically quantify the two major allergens from Japanese cedar pollen, Cry j I and Cry j II. Polystyrene microplates coated with antibodies specific for Cry j I or Cry j II were incubated with an allergen and then with biotinylated anti-Cry j I or Cry j II antibody. The bound allergen-biotin Ab complexes were detected with HRPO-conjugated streptavidin and an enzyme substrate. The working ranges of Cry j I ELISA and Cry j II ELISA were 0.3-20 ng/ml and 0.6-20 ng/ml, respectively. Intra- and inter-assay coefficients of variation for reproducibility were 1.5-10.3% and 0.9-12.9%. These ELISA systems showed no cross-reactivity between Cry j I and Cry j II and showed little cross-reactivity with pollen allergens of plants botanically related to the Japanese cedar. Using the Cry j I ELISA and the Cry j II ELISA, it was possible to quantify Cry j I and Cry j II easily and accurately. These ELISA systems will be useful in various fields, especially for the analysis and standardization of the allergens necessary for diagnosis and treatment of Japanese cedar pollinosis.

[Immunological and physicochemical properties of Cry j II, the second major allergen of Japanese cedar pollen (Cryptomeria japonica)].

Cry j II, the second major allergen of Japanese cedar (sugi, Cryptomeria japonica) pollen was examined for the allergenicity by intradermal test and RAST. Nineteen of the 25 allergic patients examined, showed positive reaction to the Cry j II. Contents of Cry j II in the extracts of the pollen collected in various regions from 1977 to 1991 showed yearly variation ranging from 2.9 to 14 mg/100 g pollen, whereas the amount of Cry j I in the extract was comparatively stable at about 35 mg/100 g pollen. Physicochemical treatments of Cry j I and Cry j II suggested that specific human IgE antibodies and some mAbs bind to conformational epitopes which are denatured and destroyed by certain treatments.

Epitopes on Cry j I and Cry j II for the human IgE antibodies cross-reactive between Cupressus sempervirens and Cryptomeria japonica pollen.

Forty sea from French patients allergic to Cupressus sempervirens pollen were tested for cross-reactivities against Cry j I, Cry j II (major allergens of Cryptomeria japonica pollen) and other pollen allergens from botanically related plants. Seventy-three per cent of the sera reacted with either Cry j I or Cry j II, or with both of them. These IgE cross-reactions were blocked effectively by mAb 046 (anti-Cry j I) or N26, T27 (anti-Cry j II), and weakly by mAbs 052, 027 and 026 (anti-Cry j I). Furthermore, the IgE antibodies in two sera, #40 and #11, bound to peptide fractions obtained from enzyme-digested Cry j I, and mAb 027 could also bind to the fractions. Analyses of the amino acid sequences of the peptides revealed that reactive peptides contained "NGNATPQLTKNAGVLTCSLSKR" sequence and the third residue N3 was glycosylated, however, when the N3 was not glycosylated, the IgE antibodies did not react, but mAb 027 could. The glycosylation of the N3 might be required for IgE-binding to the peptides. Sugar component on the N3 residue was found to be 0.4 mol galactose, 1.3 mol mannose, 0.8 mol fucose and 2.0 mol N-acetyl-glucosamine. Cross-reactivities against other pollen allergens from botanically related plants were found in most of the sera. However, many of these reactivities were detected by sandwich ELISA but not by an ELISA using allergen-coated plates, indicating that it is important to select an appropriate ELISA procedure in order to detect an allergen or an IgE antibody to an allergen.

[The onset mechanism of experimental autoimmune uveoretinitis induced by interphotoreceptor retinoid-binding protein].

In order to analyze the onset mechanism of experimental autoimmune uveoretinitis (EAU), two experimental models were used; one was EAU induced by one injection of purified bovine interphotoreceptor retinoid-binding protein (IRBP) with complete Freund's adjuvant in Lewis rat, and the other was an IRBP-induced autoimmune uveoretinitis that occurred spontaneously in nude (nu/nu) mice at 4 weeks of age reconstituted by the grafting of rat embryonic thymus (TG nude mouse). EAU develops when the IRBP-reactive lymphocytes in the regional lymph-nodes are activated. Activation begins when the T lymphocyte recognizes the peptide for the epitope bound to a major histocompatibility complex (MHC) molecule in the antigen-presenting cell by way of the T-cell receptor (TCR). In EAU, ten peptide residues p1182-1191 of the IRBP amino acid sequence, were revealed to be sufficiently capable of lymphocyte activation for EAU, and it was also shown that amino acid positions 1182W (tryptophane), 1185G (glycine), 1186V (valine) and 1188P (proline) of IRBP play important roles as the epitopes or agretopes in developing EAU. On the other hand, two amino acids of IRBP, amino acid positions 1182W (tryptophane) and 1194P (proline) were shown to be the agretopes inducing autoimmune uveoretinitis in the TG nude mouse. A study of the variable region of the TCR with a residual p1182-1194 specific T-cell line from the TG nude mouse revealed that as many as 96% utilized the T-cell receptor V beta 6 gene and that the peptide-MHC molecule complex was recognized by restricted receptors. Adhesion molecules such as ICAM-1 and LFA-1 were also found to play an important role as cofactors in activation of lymphocytes in the antigen-recognition process of EAU. Uveoretinitis seemed to result from an immune reaction in the eye occurring when the T lymphocyte arrives there, activating the immunological process. ICAM-1 and LFA-1 were also found to be involved in the infiltration process of inflammatory cells: our immunohistological examination revealed that ICAM-1 was present in the retinal pigment epithelium and epithelium of the ciliary body composing the blood-ocular barrier. In contrast, LFA-1 was expressed in the infiltrating cells. Finally, the tolerance of IRBP was discussed and it was experimentally demonstrated that the absence of IRBP-induced uveoretinitis in human beings and certain experimental animals resulted from endogenous IRBP serving as a tolerogen; we assumed that the breakdown of this self-tolerance would induce EAU due to thymic dysfunction or IRBP antigen injection.