A promiscuous T cell hybridoma restricted to various I-A molecules

In a previous study, we identified T cell receptor and major histocompatibility complex (MHC) contact sites on the pigeon cytochrome c p43-58 peptide. Positions 46 and 54 of p43-58 were shown to be the MHC-binding sites. Specific amino acids were identified on the MHC-binding sites which bound to the relevant I-A molecule. In the present study, using NOD (I-A^g7) mice, we established a T cell hybridoma, NOE33-1-2, specific for a p43-58 analog 46R50E54A with arginine (R) and alanine (A) at positions 46 and 54, respectively. Interestingly, NOE 33-1-2 recognized 46R50E54A in the presence of not only I-A^g7, but also I-A^{d, s, u and v}. In contrast to previous reports that promiscuous T cells were able to recognize peptide antigens with various HLA-DR or I-E molecules consist of monomorphic α and polymorphic β chains, the promiscuous T cell clone NOE33-1-2 recognized peptides with various I-A molecules lacking the monomorphic chain.     

Induction of hRAD9 is required for G2/M checkpoint signal transduction in gastric cancer cells.

DNA damage triggers the activation of checkpoints that delay cell cycle progression to allow for DNA repair. Loss of G2 checkpoints provides a growth advantage for tumor cells undergoing aberrant mitosis. However, the precise mechanisms of G2 checkpoints acting in gastric cancer are unknown. Here, we analyzed the G2 checkpoint function in two gastric cancer cells, MKN-28 cells containing a mutant p53 gene and MKN-45 cells which have wild-type p53. Two agents damaging DNA, camptothecin (CPT) or ultraviolet light (UV), were utilized to trigger a G2 phase cell cycle checkpoint response in these tumor cells. Both CPT and UV inhibited the growth of MKN-45 cells, whereas they did not affect the growth of MKN-28 cells. CPT induced cell cycle arrest at the G2/M phase and enhanced the expression of human RAD9 (hRAD9) in MKN-45 cells. In addition, hRAD9 showed perinuclear staining and similar localization with Bcl-2 in MKN-45 cells but not in MKN-28 cells after having applied CPT or UV light. These results suggest that besides p53 activity, the induction of hRAD9 is required for G2/M checkpoint signal transduction in gastric cancer cells.     

Norepinephrine reduces heat responses of cutaneous C-fiber nociceptors in Sprague-Dawley rats in vitro

Nociceptors are excited or sensitized by many inflammatory mediators as well as by elevation of tissue temperature. We have shown that there is a facilitatory synergistic interaction between norepinephrine (NE) and bradykinin (BK) on cutaneous C-fiber nociceptors in normal Lewis rats. These interactions may play an important role in the mechanism of sympathetically maintained pain. In the present experiment, using skin-saphenous nerve in vitro preparations, we tested the effect of NE on the activity of nociceptive fibers and their response to heat in normal Sprague-Dawley rats. For comparison with the previous data on Lewis rats, we also examined the effect of NE on BK response. NE (10(-5) or 10(-6) M) did not excite nociceptive fibers before repeated heat stimuli or BK superfusion (10(-5) or 10(-6) M) to the receptive field. In contrast, after a few applications of heat or BK, NE excited 20-43% of nociceptive fibers to similar magnitudes. We also found that NE sensitized subsequent BK responses, but somewhat unexpectedly that it suppressed subsequent heat responses. This occurred regardless of the presence or absence of NE-induced excitation. These results suggest different mechanisms of NE modification to the BK and heat responses of cutaneous C-fiber nociceptors.     

Fc receptor-mediated accumulation of macrophages in crescentic glomerulonephritis induced by anti-glomerular basement membrane antibody administration in WKY rats

Anti-glomerular basement membrane (GBM) glomerulonephritis induced in WKY rats is characterized by glomerular accumulation of CD8(+) T cells and monocytes/macrophages, followed by crescent formation. The mechanism of leukocyte accumulation after antibody binding to GBM is still unclear. To unveil an involvement of Fcgamma receptors (FcgammaR) in leukocytes recruitment we examined the expression of FcgammaR in glomeruli and the effects of the administration of F(ab')(2) fragment of anti-GBM antibody or FcgammaR blocking on the initiation and progression of this model. A gradual increase of FcgammaR mRNA expression in glomeruli during the time course of disease suggested their significance in the development of glomerulonephritis. Glomerular lesions and proteinuria were induced only in rats injected with intact IgG of anti-GBM antibody, but not with the F(ab')(2) fragment. In vivo blocking of FcgammaR by administering heat-aggregated IgG led to the decrease of mRNA expression for all types of FcgammaR (types 1, 2 and 3) and a significant amelioration of glomerulonephritis manifestations. By flow cytometry and immunohistochemistry FcgammaR2-expressing cells in glomeruli were identified as macrophages, but not CD8(+) T cells. The expression of FcgammaR1 and 3 was significantly decreased, and that of FcgammaR2 became undetectable in CD8(+) T cell-depleted rats. Thus, CD8(+) T cells may stimulate FcgammaR expression on macrophages, contributing to their glomerular accumulation and injury. These studies provide direct evidence for a crucial involvement of IgG Fc-FcgammaR interaction in glomerular recruitment of macrophages and following induction of anti-GBM glomerulonephritis in WKY rats.     

Spinocerebellar ataxia 27 with a novel nonsense variant (Lys177X) in FGF14

Spinocerebellar ataxia 27 (SCA27) is an autosomal dominant SCA caused by variants in the fibroblast growth factor 14 (FGF14) gene. We examined a Japanese SCA patient whose deceased father also suffered from SCA. The patient was a 63-year-old male. He graduated from junior high school but received no further education. The predominant complaint was slowly progressive dysarthria and gait disturbance, which appeared at age 47. He showed pathological saccadic dysmetria, saccadic intrusions into smooth pursuit eye movements, dysarthria, and limb and truncal ataxia. His gait was wide-based but he did not require a walking stick. Limb muscle strength was intact. Deep tendon reflexes were normal or slightly reduced. Pathological reflexes were absent. He demonstrated mildly impaired vibration sense in the lower limbs. There was no urinary dysfunction. Brain MRI showed cerebellar atrophy without brainstem involvement. We first confirmed the absence of repeat expansion in genes known to be responsible for SCAs 1–3, 6–8, 10, 12, 17, 36 and dentatorubral-pallidoluysian atrophy. By exome analysis, we identified a novel heterozygous variant (NM_004115, c.529A>T; Lys177X) in exon 4 of the FGF14 gene. This variant is expected to generate a truncated FGF14 protein lacking the heparin binding sites, those are likely to modify the activity of FGF14. We confirmed the absence of the variant in 502 healthy Japanese individuals by Sanger sequencing. There is no record of the variant in public databases. We conclude that the novel variation in FGF14 is causative for SCA27 in this patient.     

Novel mutations in TNFRSF1A in patients with typical tumor necrosis factor receptor-associated periodic syndrome and with systemic lupus erythematosus in Japanese

Molecular defects of TNFRSF1A was investigated in members of a family presenting with typical phenotypes of tumor necrosis factor receptor-associated periodic syndrome (TRAPS) and in patients with the autoimmune disorders, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Genomic DNA from the members of a family with typical TRAPS, as well as from 100 patients with SLE, 100 patients with RA and 100 healthy individuals, was studied for mutations in exons 2, 3 and 4 of the TNFRSF1A gene. All individuals were Japanese. Three novel missense mutations were identified in the TNFRSF1A. The C70G mutation was identified in family members with typical TRAPS, which was the second case in eastern Asian population. In addition, the T61I and R104Q mutations were each identified in 2 of the 100 SLE patients. The T61I mutation was identified in one of the 100 healthy individuals. No mutations were identified in the 100 RA patients. Functional analysis revealed that PMA-induced shedding of TNFRSF1A from PBMCs was impaired in a patient carrying T61I. A larger scale of study will clarify whether these two mutations, T61I and R104Q, are associated with chronic inflammatory disorders, such as SLE, or not.     

Effects of drug adherence on patient outcomes to early treatment for Japanese cedar pollinosis]

Drug adherence is one of the important aspects in caring for patients with allergic rhinitis. To improve clinical efficacy of early treatment for Japanese cedar pollinosis (JCP), we evaluated the effect of drug adherence on patients' outcomes. Patients were randomly selected from 16 ENT clinical sites in Osaka and Wakayama between February 24 and March 8, 2003 (peak pollen season). Efficacy was assessed using patients' ratings of nasal and ocular symptoms and overall assessment in their condition compared with previous season ones. Costs include direct costs of the drugs used for treatment to JCP from January to February. Five hundred one patients taking early treatment were enrolled. Compared to low adherence patients, those who reported higher level of adherence significantly improved overall health condition, and achieved better symptom relief of rhinorrhea and nasal congestion. In multiple logistic regression analysis, the following factors were independent risk factors for low adherence: student (p=0.002), using OTC medications (p=0.006), and short-duration of medication (p=0.001). Low costs were also risk factor for low adherence. We conclude that taking medications for JCP for 22-28 days is the best way to enhance patients' outcomes.     

Conformation and gas permeability of poly(α-amino acid) membranes

The permeability of some poly(?-amino acid) (PAA) membranes like such of poly(N,δ-carbobenzoxy-L -lysine) (PCBL), poly(N,δ-carbobenzoxy-L -ornithine) (PCBO) as well as of copolymers of L -lysine and carbobenzoxy-L -lysine to oxygen and carbon dioxide were determined at different water content especially with regard to their use for artificial lungs. The conformations of these polymer membranes were studied by means of IR-spectra and X-ray diffraction. The results on the diffusion coefficient obtained for PCBL and PCBO were analysed in terms of Eyring's theory. They suggest that the microvoids in the interstices between the α-helices, assumed on the basis of X-ray analyses, play an important role, especially below the glass transition temperature T_g of the side-chains. This seems also to contribute partly to the diffusion of gas through the membrane even above T_g and results in an abnormally high “apparent activation energy” of diffusion in the wet state below T_g. It was found that the permeability of the membrane of partially decarbobenzoxylated PCBL for oxygen in a certain temperature range is higher than that for carbon dioxide. This has never been observed for any other synthetic polymer, because usually carbon dioxide has a higher solubility in these materials than oxygen. In the case of the partially decarbobenzoxylated PCBL membranes, however, the diffusion coefficient of carbon dioxide, and therefore the permeation of this gas, decreases much more than that of oxygen with increasing decarbobenzoxylation accompanied by the formation of β-structure and concomitantly the formation of intermolecular hydrogen bonds. Obviously, this phenomenon may be responsible for the permselectivity of these membranes for oxygen compared with carbon dioxide.