The role of the ICOS/B7RP-1 T cell costimulatory pathway in murine experimental autoimmune uveoretinitis

ICOS/B7RP-1 is a new member of the CD28/B7 family of costimulatory molecules and plays differential roles in autoimmune diseases. In this study, we examined the role of ICOS/B7RP-1 pathway in the pathogenesis of mouse experimental autoimmune uveoretinitis (EAU), an animal model of human autoimmune uveitis. ICOS expression was found on infiltrating CD4+ T cells in the region of the retina in EAU-induced mice. The anti-B7RP-1 monoclonal antibody (mAb)-treated or ICOS-deficient mice showed a substantial reduction of disease scores. Blockade of ICOS/B7RP-1 interaction during the effector phase ameliorated the disease, whereas its blockade during the induction phase exhibited no significant effect. Moreover, administration of anti-B7RP-1 mAb effectively ameliorated the disease induced by adoptive transfer of pathogenic T cells. The anti-B7RP-1 mAb treatment inhibited the expansion and/or effector function of pathogenic T cells, given that proliferative response and IFN-gamma production by lymph node cells were reduced upon restimulation with the antigen peptide in vitro. These results suggest that the ICOS/B7RP-1 interaction plays a critical role in the pathogenesis of uveitis. We also indicated that ICOS-mediated costimulation plays differential roles in EAU and experimental autoimmune encephalomyelitis, which is also a Th1 disease induced in the same manner as EAU.     

Morphology of the lingual papillae in the raccoon dog and fox

The dorsal lingual surfaces of the raccoon dogs (Nyctereutes procyonoides) and fox (Vulpes vulpes japonica) were examined by scanning electron microscopical (SEM) observations. The distribution and type of the lingual papillae found in the raccoon dog were similar to those in the fox. Filiform, fungiform, foliate and vallate papillae were observed. The filiform papillae were distributed over the entire dosal surface of the tongue. Each filiform papilla on the apical surface of the tongue had several pointed processes. The filiform papillae of the lingual body consisted of a main papilla and some secondary papillae. The fungiform papillae were present rounded bodies, and more densely distributed on the lingual apex. The foliate papillae were seen on the dorsolateral aspect of the tongue. The vallate papillae were located on both sides of the posterior end of the lingual body. Each papilla was surrounded by groove and crescent pad. On the periphery of the papillae, large conical papillae were observed.     

Determination of Phthalates in Diet and Bedding for Experimental Animals Using Gas Chromatography-Mass Spectrometry

We have developed a gas chromatography–mass spectrometry method to measure five phthalates (dibutyl phthalate, butylbenzyl phthalate, di-2-ethylhexyl phthalate, diisooctyl phthalate, and diisononyl phthalate) in diets and beddings for experimental animals. The recoveries from diets and beddings spiked with five phthalates were 98.8%–148% with coefficients of variation of 0.4%–7.8% for diets and 94.7%–146% with coefficients of variation of 1.0%–5.0% for beddings. We analyzed commercial animal diets and beddings, and found that the levels of phthalates varied from sample to sample; the concentrations of five phthalates were 141–1,410 ng/g for diets and 20.5–7,560 ng/g for beddings.     

The spectrum of micronodular thymic epithelial tumours with lymphoid B-cell hyperplasia

AIMS: A rare type of thymoma, micronodular thymoma with lymphoid B-cell hyperplasia, was recently reported by Suster and Moran. Thymic epithelial tumours with a similar pattern but with varied cytological features of the tumour cells are analysed. METHODS AND RESULTS: A total of 11 cases of thymic epithelial tumours characterized by micronodular proliferation of tumour cells separated by abundant lymphoid stroma with prominent germinal centres were reviewed clinicopathologically and examined immunohistochemically. The presence of Epstein-Barr virus (EBV) genome was also examined by in-situ hybridization. Based on the morphology of tumour epithelial cells, cases were subdivided into four groups: group 1 (two cases) having spindle epithelial cells; group 2 (two cases) showing an admixture of spindle and polygonal epithelial cells; group 3 (five cases) having polygonal epithelial cells, with mild to moderate cytological atypia in four cases, and group 4 (two cases) representing lymphoepithelioma-like carcinoma. The degree of cytological atypia and the number of tumour cells positive for MIB-1 and p53 gradually increased towards group 4. The abundant lymphoid stroma in all cases contained many CD20-positive B-cells and CD3 and CD45RO-positive T-cells. CD99-positive immature T-cells were present in all cases of groups 1 and 2 and in most cases of group 3, but not in both cases of group 4 tumours. IgG, IgM and IgD-positive plasma cells and lymphocytes were also present in all cases, more prominent in those of groups 3 and 4. The EBV genome was detected in only a few lymphocytes in five cases. CONCLUSIONS: The tumours in this series belong to a distinct category of thymic epithelial tumours and each of the above groups may constitute a spectrum in the continuum of cytological atypia. The aetiological relationship of EBV with these tumours could not be proved. The lymphoid B-cell hyperplasia may result from a host immune response and may suggest a favourable clinical course of this type of tumour.     

Effects of site-directed mutagenesis in the cysteine residues and the N-glycosylation motif in recombinant Der f 1 on secretion and protease activity

BACKGROUND: The group 1 allergens from mite feces, which belong to the papain-like cysteine protease family, are the most significant in-door allergens. In this study, we analyzed the contribution of the cysteine residues and N-glycosylation in Der f 1, the group 1 allergen from Dermatophagoides farinae, to secretion and maturation by using systems for expression of recombinant Der f 1 (rDer f 1). METHODS: The rDer f 1 and its mutants were expressed in yeast Pichia pastoris and insect SF9 cells. Secretion of their proforms was checked by SDS-PAGE or immunoblotting. Protease activities of the secreted proform of a mutant and the mature form were compared with that of native Der f 1. RESULTS: The proform of a mutant Der f 1, pro-N53Q, whose consensus motif for N-glycosylation was disrupted, was not secreted in insect SF9 cells although secreted in P. pastoris. Indirect evidence was obtained to support the disulfide bond formation between Cys4 and Cys118, which were not conserved in papain. A mutant for Cys35 in the catalytic site of the cysteine protease, pro-C35S/N53Q, was secreted, but the other mutants for cysteines concerning intramolecular disulfide bonds were not secreted in P. pastoris. The prosequence of pro-C35S/N53Q was removed by an in vitro activation process. The mature C35S/N53Q showed low protease activity. CONCLUSION: N-glycosylation is essential for secretion in insect SF9 cells but not in P. pastoris. Disulfide bonds are essential for secretion in P. pastoris. A mutation in the catalytic site, C35S, is not completely critical to removal of the prosequence and protease activity. The findings are useful for future design of recombinant products for application in immunotherapy.     

Evaluation of the Role of Keratan Sulphate as a Molecular Marker to Monitor Cartilage Metabolism in Horses

The role of keratan sulphate (KS) as a metabolic marker of cartilage was evaluated using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n = 3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor-Iα (IGF-Iα) or interleukin-1α (IL-1α) for 2 weeks. The concentrations of sulphated glycosaminoglycans (GAG) and KS in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition enzyme-linked immunosorbent assay using a 1/20/5D4 antibody, respectively. The concentration of GAG was significantly increased both in the media of pellets stimulated by IGF-Iα and in those stimulated by IL-1α. KS concentration was significantly increased in those stimulated by IL-1α, while no significant change was found in those stimulated by IGF-Iα. A high correlation between GAG and KS concentrations was found in the media of pellets stimulated by IL-1α (r= 0.84), but not in those stimulated by IGF-Iα (r= 0.59). The results suggest that the concentration of KS reacting to 1/20/5D4 mirrors the GAG concentration during the stage of cartilage catabolism, but not during the cartilage anabolic stage. The KS concentration in biological fluids could therefore be a useful marker to understand further the cartilage catabolic process. It may also represent some aspects of the cartilage anabolic process.     

Postoperative radiation therapy after complete resection of thymoma has little impact on survival

BACKGROUND:

Postoperative radiation therapy for thymoma is widely used, although the clinical benefits are controversial. Furthermore, to the authors' knowledge, the relation between postoperative radiation therapy and cell type classified by the World Health Organization (WHO) is not known.

METHODS:

The records of 324 patients (ages 17‐83 years; mean, 51 years; 160 males and 164 females) who underwent complete resection of a thymoma between 1970 and 2005 were reviewed. Mediastinum postoperative radiation therapy was performed for 134 patients. Survival rates and patterns of recurrence were determined according to Masaoka stage and WHO cell type.

RESULTS:

The 10‐year disease‐specific survival rates for patients with and without postoperative radiation therapy were 92.8% and 94.4%, respectively (P = .22). Subset analyses after stratifying by Masaoka stage and WHO cell type demonstrated that the 10‐year disease‐specific survival rate for patients without postoperative radiation therapy with Masaoka stage I and II, as well as those with WHO cell types A, AB, or B1, was 100%, which was satisfactory. Furthermore, the rates for patients with Masaoka stage III/IV and those with WHO cell types B2/B3 with or without postoperative radiation therapy were not found to be significantly different. In 24 patients with disease recurrence, pleural dissemination was observed most often, followed by distant metastases; local disease recurrence without other recurrence occurred in 2.

CONCLUSIONS:

The authors concluded that surgical resection alone is sufficient for thymoma patients with Masaoka stage I and II, and those with WHO cell types A, AB, and B1. Furthermore, an optimal treatment strategy should be established for patients with Masaoka stage III/IV and WHO cell type B2/B3 thymomas. Cancer 2009. © 2009 American Cancer Society.     

Quantitative analysis of NK1 receptor in the human brain using PET with 18F-FE-SPA-RQ

18F-fluoroethyl-SPA-RQ (18F-FE-SPA-RQ) was recently developed as a radioligand for the measurement of neurokinin 1 (NK1) receptor with PET. In this study, we used 18F-FE-SPA-RQ with PET to visualize and quantify NK1 receptor in the human brain. METHODS: PET scans were performed on 7 healthy men after intravenous injection of 18F-FE-SPA-RQ. Binding potential (BPND) was calculated by the indirect kinetic, simplified reference tissue model (SRTM), and ratio methods. The indirect kinetic method was used as the gold standard method and was compared with the SRTM method, with scan times of 180, 270, and 330 min, and with the ratio method, with time integration intervals of 120-180, 210-270, and 300-330 min. The cerebellum was used as the reference brain region. RESULTS: Regional radioactivity was highest in the caudate head and putamen; mid level in the parahippocampus, cerebral cortex, and thalamus; and lowest in the cerebellum. BPND values by the indirect kinetic method were 3.15 +/- 0.36, 3.11 +/- 0.66, 1.17 +/- 0.25, and 0.46 +/- 0.14 in the caudate, putamen, parahippocampal region, and thalamus, respectively. For cerebral cortical regions, BPND values by the indirect kinetic method were 0.94 +/- 0.23, 0.82 +/- 0.15, 0.76 +/- 0.15, and 0.69 +/- 0.16 in the occipital, temporal, frontal, and anterior cingulate cortices, respectively. BPND values by the SRTM and ratio methods were in good agreement with those by the indirect kinetic method (r = 0.94-0.98). CONCLUSION: The regional distribution of 18F-FE-SPA-RQ was in agreement with previous PET studies and postmortem studies of NK1 receptor in the human brain. The ratio method will be useful for clinical research of psychiatric disorders, for the estimation of NK1 receptor without arterial blood sampling and long dynamic PET.     

Angiotensin II further enhances type IV collagen production stimulated by platelet-derived growth factor and fibroblast growth factor-2 in cultured human mesangial cells

SUMMARY: Angiotensin II (Ang II) is considered to play a role in the development of glomerulosclerosis, which is characterized by excessive accumulation of mesangial matrix after mesangial cell proliferation. We have reported that platelet-derived growth factor (PDGF) and fibroblast growth factor-2 (FGF-2) stimulate type IV collagen production by cultured human mesangial cells (HMC). Although Ang II is well known to have a mitogenic effect in various kinds of cells, its role in the production of extracellular matrix is still undetermined. This study was designed to examine the effect of Ang II and its interaction with PDGF and FGF-2 in type IV collagen production by HMC. Cultured HMC were incubated with Ang II with or without PDGF or FGF-2 for 72 h and type IV collagen, fibronectin and laminin in the cell supernatants were measured. Ang II (10⁻⁶–10⁻⁸) itself did not change the production of type IV collagen, fibronectin, laminin and transforming growth factor-β. PDGF and FGF-2 enhanced type IV production, although they did not stimulate the production of fibronectin and laminin. Ang II further increased the stimulating effect of PDGF and FGF-2 in type IV collagen production in a dose-dependent manner. This effect of Ang II was completely blocked by Ang II type I receptor antagonist (Losartan). These data imply that Ang II is a potent stimulator of type IV collagen production by HMC in the presence of growth factors.