CD137-guided isolation and expansion of antigen-specific CD8 cells for potential use in adoptive immunotherapy

The efficient isolation and ex vivo expansion of antigen-specific T cells are crucial for successful adoptive immunotherapy against uncontrollable infections and cancers. Several methods have been reported for this purpose, for example, employing MHC-multimeric complexes, interferon-gamma secretion, and antibodies specific for molecules expressed on T-cell surfaces, including CD25, CD69, CD107a, CD137, and CD154. Of the latter, CD137 has been shown to be one of the most promising targets since it is only expressed on CD8⁺ T cells early after encountering antigen, while being almost undetectable on resting cells. However, detailed comparisons between CD137-based and other methods have not yet been conducted. In this study, we therefore compared three approaches (with CD137, CD107a, and tetramers) using HLA-A24-restricted CMV pp65 and EBV BRLF1 epitopes as model antigens. We found that the CD137-based isolation of antigen-stimulated CD8⁺ T cells was comparable to tetramer-based sorting in terms of purity and superior to the other two methods in terms of subsequent cell expansion. The method was less applicable to CD4⁺ T cells since their CD137 upregulation is not sufficiently high. Collectively, this approach is most likely to be optimal among the methods tested for the isolation and expansion of antigen-specific CD8⁺ cells. K.W. and S.T. are employees of Medical Biological Laboratories Co., Ltd. S.S. is a representative executive of T Cell Technologies, Inc. Y.A. has received financial support through collaboration with Medical Biological Laboratories Co., Ltd.     

Prophylactic Effect of Irsogladine Maleate Against Indomethacin-Induced Small Intestinal Lesions in Rats

The effect of irsogladine maleate, a widely used antiulcer drug in Japan, on indomethacin-induced small intestinal lesions was examined in rats. Animals without fasting were given indomethacin (10 mg/kg, s.c.) and sacrificed 24 h later. Irsogladine (1-10 mg/kg) or 16,16-dimethyl prostaglandin E(2) (dmPGE(2) 0.03 mg/kg) was given p.o. twice, 0.5 before and 6 h after indomethacin, while ampicillin (800 mg/kg) was given twice, 18 and 0.5 h before. Indomethacin caused severe lesions in the small intestine, mainly the jejunum and ileum, accompanied by intestinal hypermotility, the up-regulation of inducible nitric oxide synthase (iNOS) expression, and an increase of myeloperoxidase (MPO) activity as well as enterobacterial invasion in the mucosa. These events were all prevented by both dmPGE(2) and ampicillin, except the intestinal hypermotility which was only prevented by dmPGE(2). Likewise, irsogladine also significantly and dose-dependently prevented these lesions at >1 mg/kg. This agent alone increased mucus secretion and significantly suppressed the decreased mucus response to indomethacin, resulting in a suppression of the bacterial invasion as well as the increase in MPO activity and iNOS expression. The protective effect of irsogladine was mimicked by isobutylmethylxanthine, a nonselective inhibitor of phosphodiesterase (PDE), as well as rolipram, a selective PDE4 inhibitor. These results suggest that irsogladine protects the small intestine against indomethacin-induced lesions, and this effect may be associated with the increased mucus secretion, probably due to the inhibitory actions of PDE, resulting in suppression of enterobacterial invasion and iNOS expression.     

Early-stage primary malignant melanoma of the esophagus detected simultaneously with esophageal squamous cell carcinoma

Primary malignant melanoma of the esophagus is a rare disease. The majority of patients are diagnosed at an advanced stage, and only a few are detected at an early stage. We herein describe a case of early-stage primary malignant melanoma of the esophagus that was detected simultaneously with early-stage primary esophageal squamous cell carcinoma. Both tumors were detected during esophagogastroduodenoscopy for heartburn. The malignant melanoma tumor was a nevus-like flat-type lesion in the upper thoracic esophagus, and the squamous cell carcinoma was a slightly depressed lesion in the abdominal esophagus. The tumor was resected by thoracoscopic esophagectomy. Histologically, the invasion of both tumors was limited to the mucosal layer, and no lymph node metastasis was detected. Immunohistochemically, the malignant melanoma cells were strongly positive for HMB-45, melan-A, and S-100 protein. The patient has survived without recurrence for 17?months after the operation.     

Treatment of cartilage defects by subchondral drilling combined with covering with atelocollagen membrane induces osteogenesis in a rat model

Background

The coverage of the atelocollagen membrane at the chondral defect after subchondral drilling might improve the beneficial effects for cartilage repair because of the prevention of scattering and accumulation of cells and growth factors from bone marrow within the chondral defect. On the other hand, it might block cells and factors derived from the synovium or cause high pressure in the chondral defect, resulting in prevention of cells and growth factors gushing out from the bone marrow, which leads to disadvantages for cartilage repair.

Method

We tested this hypothesis in a 2-mm-diameter chondral defect created in the articular cartilage of the patellar groove in a rat models. Defects were left untreated, or were drilled or drilled and covered with an atelocollagen membrane; healing was evaluated by histology and gene expression analysis using real-time polymerase chain reaction and immunohistochemistry.

Results

Membrane coverage induced bone tissue ingrowth into the punched chondral defect. At 1 week, expression of TGFβ, Sox9, Runx2, osteocalcin, Col1a1, and Col2a1 in the drilling group was significantly higher than in the covering group. At 4 weeks, expressions of TGFβ, Runx2, and Col1a1 were all significantly higher in the drilling group, while Sox9, osteocalcin, and Col2a1 were significantly higher in the covering group. Immunohistochemistry demonstrated Sox9, osteocalcin, and type II collagen on the bony reparative tissue in the covering group.

Conclusions

These results suggest that the atelocollagen membrane coverage resulted in inhibition of cartilage repair.     

Involvement of Rev1 in alkylating agent‐induced loss of heterozygosity in Oryzias latipes

Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C‐terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C‐terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild‐type fish. There was no significant difference in the frequency of DENA‐induced mutations between mutant with reduced dCMP transferase activity and wild‐type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.