Keiji Nakasho

The Involvement of IL-23 and the Th17 Pathway in Periodontitis

Interleukin (IL)-23 is an essential cytokine involved in expansion of the Th17 lineage, which is associated with many immune-related destructive tissue diseases. We hypothesized that the IL-23-induced Th17 pathway plays a role in periodontal pathology and examined the expression of cytokines, and related molecules, in periodontal lesions and control sites. IL-23 and IL-12 were expressed at significantly higher levels in periodontal lesions than in control sites. However, the relative expression of the IL-23 receptor compared with the IL-12 receptor β2 was significantly higher in periodontal lesions. Moreover, IL-17 expression was significantly higher in periodontal lesions, especially in the tissue adjacent to bone destruction, than in control sites. There was no significant difference in the expression levels of IFN-γ, an important cytokine inhibiting differentiation toward the Th17 pathway, between periodontal lesions and control sites. Together, these results suggest that the IL-23-induced Th17 pathway is stimulated in inflammatory periodontal lesions.

Polymorphisms in the 5′ flanking region of IL12RB2 are associated with susceptibility to periodontal diseases in the Japanese population

OBJECTIVESnThe expression of interleukin (IL)-12Rbeta2 molecule is a crucial regulatory factor in the T-helper type (Th) 1 differentiation of T cells. To elucidate the role of the cell-mediated immune (CMI) response in the pathogenesis of periodontitis, Japanese periodontal patients were subjected to single nucleotide polymorphism (SNP) analyses of the 5 flanking region of IL12RB2, whose variants are frequently detected in lepromatous leprosy patients, in which the very weak cellular immune response is caused by low expression of IL-12Rbeta2.nnnMATERIAL AND METHODSnThe gene polymorphisms of the 5 flanking region of IL12RB2 were examined in subjects with several types of periodontal disease and in healthy controls. Serum immunoglobulin (Ig) G antibody titres against periodontopathic bacteria were measured and compared in periodontal patients with and without variant alleles of IL12RB2.nnnRESULTSnThe frequencies of variant alleles of IL12RB2 were significantly higher in aggressive periodontitis patients as compared with healthy controls or chronic periodontitis patients. Serum IgG titres against all periodontal bacteria examined in subjects carrying variant alleles were higher than those in subjects without variant alleles.nnnCONCLUSIONnIL-12Rbeta2 SNPs could be useful as genetic markers to access the susceptibility of the general population to periodontal disease. Low CMI responses or high humoral responses are associated with the pathogenesis of inflammatory periodontal diseases.

Enhanced suppression of pulmonary metastasis of malignant melanoma cells by combined administration of α-galactosylceramide and interleukin-18

α‐Galactosylceramide (α‐GalCer) shows antitumor effects by activating natural killer (NK) cells indirectly through stimulation of the secretion of cytokines by NKT cells, whereas interleukin (IL)‐18 shows antitumor effects by activating NK cells directly. In the present study, we examined the antitumor effect of the combined administration of α‐GalCer and IL‐18. An injection of NK cell‐sensitive mouse B16 melanoma cells into a mouse tail vein produced pulmonary metastasis. The daily administration of α‐GalCer or IL‐18 alone for 4 days starting 1 day after the injection of B16 melanoma cells markedly suppressed the number of pulmonary metastatic foci, and their combined administration enhanced the antitumor effect compared with single administration. The antitumor effect of their combined administration was completely abolished by treatment of mice with anti‐asialo GM1 serum, which depletes NK cells but not NKT cells. Combined administration of α‐GalCer and IL‐18 enhanced the cytotoxicity of NK cells and increased the number of NK cells in the lung. Analysis of NKT cell‐dependent and NK cell‐independent secretion of cytokines, to which NK cells can respond, showed that the administration of α‐GalCer increased the secretion of IL‐2, IL‐4, interferon‐γ, IL‐12, granulocyte‐macrophage colony‐stimulating factor, tumor necrosis factor‐α, and IL‐10, and the combined administration of α‐GalCer and IL‐18 enhanced the secretion of IL‐2, IL‐4, interferon‐γ, and granulocyte‐macrophage colony‐stimulating factor further but only slightly. These results show that IL‐18 in combination with α‐GalCer exerts an antitumor effect on NK cell‐sensitive tumors primarily by the direct stimulation of NK cells by IL‐18 and the indirect stimulation of NK cells by α‐GalCer through its activation of NKT cells. (Cancer Sci 2008; 99: 113–120)

Transdifferentiation into biliary ductular cells of hepatocytes transplanted into the spleen

Aims: Transplantation of rat hepatocytes into the syngeneic rat spleen results in the appearance of cytokeratin (CK)7 and CK19 positive biliary cells that form ductules. We examined whether hepatocytes are the origin of these biliary ductular cells. Methods: We transplanted rat dipeptidyl peptidase IV (DPPIV) positive hepatocytes into the liver of retrorsine‐treated and partially hepatectomised DPPIV negative rats, which resulted in proliferation of DPPIV positive hepatocytes in the liver. Two months later, hepatocytes were prepared from chimaeric livers of these rats and transplanted into the spleen of DPPIV negative rats. Four weeks later, the expression of DPPIV in CK7 positive ductules in the spleen was examined by immunofluorescent double‐staining. Results: In the spleen of DPPIV negative rats transplanted with hepatocytes prepared from the chimaeric livers, DPPIV was found to be expressed in some CK7 positive biliary ductules where only a fraction of cells expressed DPPIV, whereas in the spleen of DPPIV negative rats transplanted with hepatocytes from livers of DPPIV positive rats, DPPIV was expressed in all CK7 positive biliary ductules. Conclusion: The present study indicates that hepatocytes transplanted into the spleen could transdifferentiate into biliary cells that aggregate to form ductular structures.

Sodium valproate, a histone deacetylase inhibitor, decreases the secretion of soluble Fas by human osteosarcoma cells and increases their sensitivity to Fas-mediated cell death

PurposeEffects of valproic acid (VPA), a histone deacetylase inhibitor, on the susceptibility to cell death induced by agonistic anti-Fas antibody were examined using four human osteosarcoma cell lines.MethodCell growth, secretion of soluble Fas, expression of cell-surface Fas, and sensitivity to Fas-mediated cell death were examined using cell proliferation assay, flow cytometry, enzyme-linked immunosorbent assay, and agonistic anti-Fas antibody, respectively.ResultsVPA suppressed the growth of all the four osteosarcoma cell lines and the secretion of soluble Fas from these cells. VPA showed no or slight suppressive effect on the expression of cell-surface Fas in the four osteosarcoma cell lines, but increased the sensitivity of three of four osteosarcoma cell lines to Fas-mediated cell death.ConclusionVPA enhances the susceptibility of human osteosarcoma cells to Fas-ligand-induced cell death by decreasing the secretion of soluble Fas and increasing the sensitivity to Fas-mediated cell death.

Differential effects of polymorphisms in the 5' flanking region of IL12RB2 on NK- and T-cell activity.

Single nucleotide polymorphisms (SNPs) in the 5 flanking region of IL12RB2 are frequently detected in lepromatous leprosy patients, and may be possible immunogenetic factors that reduce transcriptional activity of the IL-12Rbeta2 gene in Jurkat T cells. This study determined the functional effects of these SNPs on NK-cell activity, including IFN-gamma production and IL-12Rbeta2 gene expression. Reporter gene assays using the NK cell line NK3.3 revealed that transcriptional activities of the variant haplotypes were significantly higher in the NK cell line, in contrast to our previous results in Jurkat T cells. IFN-gamma production in activated T cells from donors was significantly lower than in cells from donors without the variant SNPs, while NK cells with these SNPs produced significantly higher amounts of IFN-gamma. These results suggest that these SNPs in IL12RB2 have differential effects on cellular activation of T cells and NK cells.

Pulmonary pleomorphic carcinoma producing hCG

An 80‐year‐old woman with a pleomorphic carcinoma (PC) producing hCG was admitted to Nippon Steel Hirohata Hospital because of an abnormal shadow on CT seen during a follow‐up examination after surgery for breast cancer. A right upper lobectomy was performed due to rapid growth of the shadow 3u2003months later. Macroscopically the tumor was a 4.8u2003×u20034.0u2003cm well‐circumscribed grayish‐white mass. On histology the tumor consisted mostly of intermingled spindle and polygonal cells, while evidence of poorly differentiated adenocarcinoma was seen in a few areas. A diagnosis of PC was made due to hCG expression in approximately 20% of the spindle and polygonal cells on immunohistology. Sixu2003months after the operation metastasis to the liver and adrenal gland was seen on CT. The patient died due to metastases 1u2003year after the operation, even though the patient had been at stage 1B at the time of the operation and appropriate chemotherapy had been given. PC patients with immunohistochemical hCG expression have elevated risk of local recurrence and metastasis.

Fibroblasts stimulated via HLA-II molecules produce prostaglandin E 2 and regulate cytokine production from helper T cells

Fibroblasts act as important immune regulatory cells via their ability to cross-talk with T cells accumulating in lesions. Our previous study showed that fibroblasts produce several cytokines and chemokines by crosslinking HLA class II (HLA-II) molecules with monoclonal antibodies or by making T-cell receptor–peptide–HLA complexes. It is thus conceivable that the interaction of T cells and fibroblasts via HLA-II affects fibroblast responses to stimuli. This study used human gingival fibroblasts (HGF) to investigate possible effects of these fibroblast-derived soluble factors on the differentiation of naïve T cells and on the subsequent fibroblast responses. After mixed lymphocyte reaction culture between naïve T cells and allogeneic dendritic cells in the presence of culture supernatant from HGF stimulated via HLA-DQ molecules (DQ-sup), but not via DR, T cells exhibited a Th2-shifted phenotype, thereby producing quantitatively more IL-13 and IL-5 compared with interferon-γ. Astonishingly, analyses to identify possible factors affecting the Th2 polarization secreted from HLA-II-stimulated HGF, prostaglandin E2, was detected only in DQ-sup. The Th2 polarization of naïve T cells was blocked in the presence of supernatants from indomethacin-treated HGF with HLA-DQ stimulation. In addition, we found that the culture supernatants of Th cells activated following mixed lymphocyte reaction culture in the presence of DQ-sup had the potential to induce gene expression of type I and III collagens in HGF. These results suggested that fibroblasts stimulated via HLA-DQ molecules promote Th2 polarization in Th-cell responses and showed the counter activation of collagen synthesis, implicating orchestrated responses among these cells in the fibrosis of chronic inflammatory lesions.

Immunotherapy with Interleukin-18 in Combination with Preoperative Chemotherapy with Ifosfamide Effectively Inhibits Postoperative Progression of Pulmonary Metastases in a Mouse Osteosarcoma Model

The effect of immunotherapy with interleukin-18 (IL-18) in combination with preoperative chemotherapy on the postoperative progression of pulmonary metastasis was examined using a spontaneous pulmonary metastasis model of mouse osteosarcoma. Mice were inoculated subcutaneously with highly metastatic murine osteosarcoma cells (LM8) and then underwent chemotherapy with ifosfamide (30 or 60 mg/kg body weight, days 14–16), immunotherapy with IL-18 (2 μg/mouse, days 18–24) or combined immunotherapy and chemotherapy. Tumors developed in mice were excised 21 days after cell inoculation when microscopic but not macroscopic pulmonary metastasis was observed in mice. Three weeks after the excision of the tumors, macroscopic pulmonary metastasis was observed on the surface of the lung. Administration of ifosfamide or IL-18 alone decreased the number of macroscopic pulmonary metastases, and combined administration of ifosfamide and IL-18 resulted in much greater inhibition of pulmonary metastasis. These results suggest that immunotherapy in combination with preoperative chemotherapy is highly effective in suppressing postoperative progression of pulmonary metastasis.

Poorly Differentiated Adenocarcinoma with Signet-Ring Cell Carcinoma in a Hyperplastic Polyp of the Stomach: Report of a Case

Hyperplastic polyps (HPs) of the stomach have been reported to be mostly benign. However, in rare cases, carcinomas have been found in HPs. We treated a 59-year-old Japanese male who underwent a total gastrectomy, and a gross examination of the resected stomach revealed a 4.8 × 3.8-cm polyp on the greater curvature of the antrum and multiple small polyps in the whole gastric mucosa. Histologically, the large polyp consisted mainly of hyperplastic foveolar epithelium, while the presence of variously colored lobules demonstrated a poorly differentiated adenocarcinoma mixed with signet-ring cell carcinoma. Hyperplastic polyps should therefore be carefully examined microscopically as a polypectomy specimen and in resected stomach specimens.