Identification and characterization of a WT1 (Wilms Tumor Gene) protein-derived HLA-DRB1*0405-restricted 16-mer helper peptide that promotes the induction and activation of WT1-specific cytotoxic T lymphocytes

Effective tumor vaccine may be required to induce both cytotoxic T lymphocyte (CTL) and CD4+ helper T-cell responses against tumor-associated antigens. CD4+ helper T cells that recognize HLA class II-restricted epitopes play a central role in the initiation and maintenance of antitumor immune responses. The Wilms tumor gene WT1 is overexpressed in both leukemias and solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for cancer immunotherapy. In this study, we identified a WT1 protein-derived 16-mer peptide, WT1(332)(KRYFKLSHLQMHSRKH), which was restricted with HLA-DRB1*0405, one of the most common HLA class II types in Japanese, as a helper epitope that could elicit WT1-specific CD4+ T-cell responses. We established a WT1(332)-specific CD4+ helper T-cell clone (E04.1), which could respond to both HLA-DRB1*0405-positive, WT1-expressing transformed hematopoietic cells and autologous dendritic cells pulsed with apoptosis-induced WT1-expressing cells, indicating that the WT1(332) was a naturally processed helper epitope. Stimulation of peripheral blood mononuclear cells with both the CTL epitope (WT1(235)) and the helper epitope (WT1(332)) in the presence of WT1(332)-specific TH1-type CD4+ T cell clone strikingly enhanced the induction and the functional activity of WT1(235)-specific CTLs compared with that of peripheral blood mononuclear cells with the WT1(235) alone. These results indicated that a helper epitope, WT1(332) should be useful for improvement of the efficacy of CTL epitope-based cancer vaccine targeting WT1 in the clinical setting.     

Combined genotypes of ACE and NADPH oxidase p22phox associated with somatic mutation of mtDNA and carotid intima-media thickness in Japanese patients with type 2 diabetes mellitus

Background: We previously reported that the carotid artery intima-media thickness (IMT) increased with age and that patients with type 2 diabetes mellitus (DM) had a significantly larger IMT than did age-matched nondiabetic subjects with normal glucose tolerance. Although the exact mechanism behind the increase in IMT in diabetic patients has not been determined, data obtained from in vivo and in vitro studies suggest that hyperglycemia-induced oxidative stress may lead to atherogenesis.Objective: The aim of this single-center study was to determine whether long-term oxidative stress and the carotid IMT are influenced by differences of the angiotensin-converting enzyme insertion/deletion (ACE I/D) and NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase p22phox C242T genotypes.Methods: Eligible subjects were Japanese patients with type 2 DM. Polymorphism of the ACE I/D and p22phox gene was investigated using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism, respectively. The rate of an acquired mutation of mitochondrial DNA—that is, A-to-G substitution at position 3243 (mtDNA A3243G)—was determined by real-time PCR. As a marker of early atherosclerosis, the carotid artery IMT was measured using high-resolution B-mode ultrasonography.Results: A total of 262 Japanese patients (173 men, 89 women; mean [SEM] age, 58 [0.6] years [range, 18-80 years]) were recruited and enrolled for study. An ACED-positive (DD or DI) and p22phox 242T-negative genotype (CC) was associated with a significantly higher mtDNA A3243G mutation rate than the other 3 possible genotypes (0.0219% [0.0028%] vs 0.0097% [0.0012%]; P < 0.05). This genotype also had higher maximum and mean IMT values than the other genotypes (1.13 [0.048] mm vs 0.99 [0.03] mm and 1.01 [0.036] mm vs 0.92 [0.023] mm; P < 0.05). These parameters were similar among the other 3 genotypes.Conclusion: In this study, the ACED-positive and p22phox 242T-negative genotype showed higher rates of somatic mtDNA mutation (mtDNA A3243G) and higher carotid mean and maximum IMT levels.     

Identification of specific gene expression profiles in human mast cells mediated by Toll-like receptor 4 and FcepsilonRI

Rodent mast cells (MCs) are reported to play a pivotal role in both innate and adaptive immunity. However, there is so far no evidence that human MCs are involved in innate immunity. We found that a functional Toll-like receptor 4 (TLR4) was expressed on human MCs when it was up-regulated by interferon gamma (IFN-gamma). To systematically explore how human MCs modulate the immune system following TLR4-mediated activation and FcepsilonRI aggregation, we used high-density oligonucleotide probe arrays (GeneChip) to compare the lipopolysaccharide (LPS)-induced gene expression profile with the IgE/anti-IgE-mediated profile in MCs. Both a shared core response, and LPS- or anti-IgE-specific programs of gene expression were observed in MCs. Furthermore, MCs exhibited an antiviral response gene program in response to IFN-gamma, and LPS sustained that expression. Compared with the LPS-stimulated gene expression profile of human peripheral blood mononuclear cells, LPS-stimulated MCs specifically induced a subset of genes that included a Th2 cytokine and chemokines that recruit Th2 cells and eosinophils. These results reveal that human MCs express tailored pathogen- and antigen-specific immune responses and that human MCs may play important roles in innate and adaptive immunity.     

Clinical characteristics of Japanese reflux esophagitis patients as determined by Los Angeles classification

BACKGROUND: Recent studies have shown that the number of patients with reflux esophagitis is increasing in Japan, but the prevalence and risk factors associated with reflux esophagitis in Japanese patients are not well defined. METHODS: By using all endoscopic records in the Katta General Hospital from April through to September 1999, we identified 392 patients. We examined the Los Angeles classification, peptic ulcer, gastric mucosal atrophy, hiatal hernia and other medical variable factors for their contribution to esophagitis in the patients. RESULTS: Patients (13.8%) were diagnosed as having reflux esophagitis with a mucosal break. In a multivariate analysis, reflux esophagitis was associated with hiatal hernia (odds ratio (OR) 2.276, 95% confidence interval (CI) 1.164-4.450), with patients over 65 years of age (OR 2.521, 95% CI 1.238-5.134) and the open type of gastric mucosal atrophy (OR 0.420, 95% CI 0.225-0.785). There was no significant difference between esophagitis and Helicobacter pylori infection and peptic ulcer. CONCLUSIONS: We observed that age, hiatal hernia and a lower rate of gastric mucosal atrophy were associated with the proportion of mucosal breaks accompanying esophagitis.     

Quantitative CT measurements of small pulmonary vessels in chronic obstructive pulmonary disease: do they change on follow‐up scans?

The aims of this study were to perform a longitudinal evaluation of the cross‐sectional area (CSA) of small pulmonary vessels and the extent of emphysema measured on computed tomography (CT) scans of patients with chronic obstructive pulmonary disease (COPD), and to correlate the pulmonary vascular measurements with extent of emphysema. The institutional review board approved this retrospective study and waived the need for patients' informed consent. Seventy‐four patients with COPD who underwent both initial and follow‐up CT scans at an interval of ≥12 months were analysed. The CSA of small pulmonary vessels <5 mm² was measured, and the percentage of total CSA of the area of the lung (%CSA_<5) was calculated. The extent of emphysema was assessed as the percentage of low attenuation area (%LAA, <5 and %LAA during the follow‐up period was assessed using the Spearman rank correlation. The %LAA increased significantly on follow‐up CT scans (P<0·0001). The %CSA_<5 was slightly decreased on follow‐up scans, but the difference was not significant. Although longitudinal change in %LAA was positively correlated with duration of follow‐up period (ρ = 0·505, P<0·0001), longitudinal change in %CSA_<5 was not. In conclusion, there was a progressive increase in the extent of emphysema over time, but no significant decrease in the CSA of small pulmonary vessels over the same time period.     

Prostaglandin D2-induced eosinophilic airway inflammation is mediated by CRTH2 receptor

Mast cell-derived prostaglandin D(2) (PGD(2)) is one of the essential modulators of eosinophilic airway inflammation in asthma and allergic rhinitis. Two G protein-coupled receptors for PGD(2), prostaglandin D(2) receptor (DP) and chemoattractant receptor-homologous molecule expressed on Th(2) cells (CRTH2), are both expressed on the surface of eosinophils, and CRTH2 has been demonstrated to mediate PGD(2)-induced eosinophil mobilization in vitro. However, it has not yet been determined whether PGD(2) and its receptors mediate in vivo eosinophil trafficking into the airways or other organs. We demonstrated that intratracheal administration of PGD(2) in rats pretreated with systemic interleukin-5 (IL-5) injection induced marked airway eosinophilia, determined by the differential counts of cells in bronchoalveolar lavage (BAL) fluid and lung histology, within 2 h. Systemic IL-5 alone significantly increased the number of eosinophils in the peripheral blood but showed no effect on airway eosinophilia. Three CRTH2-specific agonists (13,14-dihydro-15-keto-PGD(2), 11-deoxy-11-methylene-15-keto-PGD(2), and indomethacin) demonstrated equivalent induction of BAL eosinophilia to that of PGD(2), but a DP agonist (BW 245C [5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)-hydantoin]) or a thromboxane A(2) receptor (TP) agonist ([1S-1alpha,2beta(5Z), 3alpha(1E,3R*),4alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid) showed no effect. PGD(2) or CRTH2 agonist-induced BAL eosinophilia was almost completely inhibited by pretreatment with a CRTH2/TP antagonist, ramatroban [BAY-u3405; (+)-(3R)-3-(4-fluorobenzenesulfonamido)-1,2,3,4-tetra-hydrocarbazole-9-propionic acid], whereas a TP-specific antagonist, SQ29,548 (5-heptenoic, 7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]-hept-2-yl]-[1S-[1alpha,2alpha(Z),3alpha,4alpha]]), or a DP-specific antagonist, BW A868C [3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexy-2-hydroxyethylamino)-hydantoin], did not inhibit the effects of PGD(2). These results suggest that CRTH2 plays a significant role in the eosinophil trafficking from the bloodstream into the airways in PGD(2)-related airway inflammation.