Fumiko Date

Orexin-A expression in human peripheral tissues

Orexin-A is a neuropeptide present in the brain and is known to regulate feeding and sleeping. In this study, we examined the systemic distribution of orexin-A in human tissues. Immunoreactivity for orexin-A was detected in ganglion cells of the thoracic sympathetic trunk, myenteric plexuses and endocrine cells of the gastrointestinal tract, islet cells of the pancreas and syncytiotrophoblasts and decidual cells of the placenta. In the gastrointestinal tract, orexin-A immunoreactivity was detected in the myenteric plexuses from 26 gestational weeks to birth. In double immunostaining in the pancreas, a great majority of insulin-positive cells was simultaneously positive for orexin-A. mRNA expression for prepro-orexin was also detected in the kidney, adrenal gland, pancreas, placenta, stomach, ileum, colon and colorectal epithelial cells. These results suggest the production of orexin-A in various human peripheral tissues and orexin-A may also play important roles in some peripheral organs.

Double immunostaining for c-erbB-2 and p53 in human stomach cancer cells

We examined 32 cases of surgically resected stomach cancer for the immunohistochemical expression of p53 and c-erB-2 protein and correlated the findings with clinical outcome and established prognostic factors. p53 Was observed in the nuclei of tumor cells in 11 of 32 cases (33%) and c-erbB-2 immunoreactivity was observed in nine of 32 cases (27%). In eight cases both p53 and c-erbB-2 were positive; however, double immunostaining revealed that less than a third of the same tumor cells were positive for both p53 and c-erbB-2 in these cases. The immunohistochemical localization patterns of p53 and c-erbB-2 were not related to the histopathologic differentiation of the carcinoma cells. No correlation was observed between expression of p53 and/or c-erbB-2 and the clinical outcome and established prognostic factors, including clinical stage and histologic types of the tumor examined. These results indicate that abnormalities of p53 and c-erbB-2 may not play major roles in the biologic behavior of human stomach cancer and that abnormalities in p53 and c-erbB-2 do not necessarily occur simultaneously in the development of stomach cancer.

Intraoral reconstruction with innervated forearm flap: A comparison of sensibility and reinnervation in innervated versus noninnervated forearm flap

OBJECTIVE To evaluate the cutaneous sensibility and sensory reinnervation in patients who underwent intraoral reconstruction with an innervated or noninnervated forearm flap. STUDY DESIGN Results of the use of innervated forearm flaps in oral reconstruction was compared with the use of noninnervated flaps. The evaluation of sensibility and reinnervation comprised clinical sensibility tests and immunohistochemical investigation of postoperative biopsy specimens against S-100 and neurofilament. RESULTS The innervated flaps (4 patients) provided earlier and qualitatively better recovery of sensation than the noninnervated flaps (9 patients). Immunohistochemical investigation revealed the existence of a larger number of regularly arranged sensory nerve fibers in the cutaneous tissue of the innervated flaps than in the noninnervated flaps. Examination with an electron microscope found the structure of these nerve fibers to be well preserved in the innervated flaps, whereas nerve fibers in the noninnervated flaps were degenerative. CONCLUSION These findings suggest (1) that the innervated flaps are superior to the noninnervated flaps not only for the repair of defects but also for the restoration of function and (2) that the innervated flaps contribute to the improvement of the quality of life for patients.

Characterization of lymphangiogenesis in various stages of idiopathic diffuse alveolar damage

In pulmonary fibrosis, an abnormal healing process, is believed to be involved in the damage to lung tissue. This process has not been correlated with lymphangiogenesis, which has garnered current interest in relation to wound healing. The aim of the present study was to clarify the characteristics of lymphangiogenesis in pulmonary fibrosis associated with idiopathic diffuse alveolar damage. Formalin-fixed and paraffin-embedded lung tissues from 13 autopsy cases with idiopathic diffuse alveolar damage were used. Antibodies specific to CD34 and D2-40 were used to detect blood vessels and lymphatics, respectively, and immunohistochemical examinations and morphometry analyses were performed. The standardized density of capillaries was increased significantly in the exudative stage of diffuse alveolar damage, whereas that of the lymphatics remained unchanged. In the proliferative stage, new lymphatics emerged, primarily in the intra-alveolar fibrotic lesions where capillaries were absent. In the fibrotic stage, in which the lung was shrunken, as revealed by the elevated density of pulmonary arteries, the standardized density of capillaries was reduced significantly. The standardized area density of the interstitium was elevated in the proliferative stage and subsequently reduced in the fibrotic stage. Three-dimensional reconstruction of images revealed that some new lymphatics lacked connection to existing lymphatics. During the progression of diffuse alveolar damage, lymphangiogenesis occurs independent of capillary angiogenesis.

Macrophages participate in lymphangiogenesis in idiopathic diffuse alveolar damage through CCL19-CCR7 signal

An abnormal healing process is believed to be involved in lung tissue damage in pulmonary fibrosis. Lymphangiogenesis has been determined to play a role in structural remodeling in diffuse alveolar damage. However, the mechanism of lymphangiogenesis remains unclear. The aim of this study is to investigate the cellular mechanisms of lymphangiogenesis in diffuse alveolar damage, focusing on the roles of macrophages. Formalin-fixed and paraffin-embedded lung tissues from 13 autopsy cases with idiopathic diffuse alveolar damage were used. Antibodies specific for D2-40 and CD68 were mainly used as the markers for lymphatics and macrophages, respectively, and immunohistochemical examinations and morphometric analyses were performed. Immunohistochemistry showed the aggregation of numerous CD68+ macrophages around newly formed lymphatics in the intraalveolar fibrotic lesions in the proliferative stage, and some of the CD68+ macrophages were colocalized with the lymphatic endothelium. These macrophages were characterized by the expression of vascular endothelial growth factor-C. Among the 3 stages of diffuse alveolar damage, the number of Ki-67+ cells on the lymphatic endothelium tended to increase in newly formed lymphatics of the proliferative stage. In addition, the number of CD68+ macrophages on the lymphatic endothelium was significantly increased in the newly formed lymphatics of the proliferative stage more than those of the fibrotic stage. The CD68+ macrophages around the newly formed lymphatics coexpressed CCR7. Dual immunostaining showed the coexpression of CCL19-a ligand for CCR7-on the lymphatic endothelium. Thus, macrophages may participate in lymphangiogenesis in diffuse alveolar damage, which is facilitated by CCL19 and CCR7.

Angiomyofibroblastoma of the vulva: Case report with immunohistochemical, ultrastructural and DNA ploidy studies and a review of the literature

A case of anglomyofibroblastoma of the vulva in a 49‐year‐old woman was examined. The tumor measured 3×2.5×2 cm and appeared light gray to tan In color on the cut surface. Light microscopic examinations revealed that spindle or oval shaped tumor cells were arranged in loose edematous stroma with numerous thin‐walled vessels. Ultrastructurally, cell organellae were not well developed but intracytoplasmic filaments of intermediate size were abundant in the tumor cells. Desmin, CD34 and vimentin immunoreactivity were detected in almost all of the tumor cells. Both estrogen and progesterone receptors were diffusely expressed In the tumor, suggestive of the sex sterold‐dependency of this tumor. The Ki‐67 labeling index was less than 1% and the DNA content of the tumor cells, which was examined by image cytometry, demonstrated diploidy (DNA index=0.97). These findings may reflect the quiescent or slow growing features of angiomyofibroblastoma.

The definition of fibrogenic processes in fibroblastic foci of idiopathic pulmonary fibrosis based on morphometric quantification of extracellular matrices

There is limited information regarding the process of tissue remodeling in fibroblastic foci associated with idiopathic pulmonary fibrosis. The aim of this study was to identify the different pathologic stages of tissue remodeling in fibroblastic foci based on the histopathologic differences in the glycosaminoglycan distribution and collagen deposition. In addition, we also aimed at clarifying the stage-specific characteristics by taking into consideration the expression pattern of matrix metalloproteinase and angiogenesis. Lung biopsies of 16 patients with idiopathic pulmonary fibrosis were used. The presence of glycosaminoglycans was detected by Alcian blue staining, and type I collagen was detected by immunohistochemical analysis with a primary antibody specific to the cross-linked carboxyterminal telopeptide of type I collagen. The fibroblastic foci characterized by the expression intensity of Alcian blue and telopeptide of type I collagen were divided into 3 groups, namely, Alcian blue(+)telopeptide of type I collagen(weak), Alcian blue(+)telopeptide of type I collagen(+), and Alcian blue(weak)telopeptide of type I collagen(+); consequently, 3 new stages were defined--stages I, II, and III, respectively. A significant inverse correlation was observed between the area densities of Alcian blue(+) and telopeptide of type I collagen(+) in fibroblastic foci. Stage I was characterized by the expression of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloprotease-2 in fibroblasts and the overlying epithelium of fibroblastic foci, and also the absence of capillary angiogenesis. In contrast, the expression of these proteins was attenuated in stage III, except for that of matrix metalloproteinase-2 in fibroblasts. In stages II and III, capillary angiogenesis was observed. Lymphangiogenesis was undetected in all the 3 stages. Thus, pathologic staging helps understand the roles of the factors involved in tissue remodeling in idiopathic pulmonary fibrosis.

Interleukin-17 is a critical target for the treatment of ankylosing enthesitis and psoriasis-like dermatitis in mice

Abstract Ankylosis is a major pathological manifestation of spondyloarthropathy. The aim of this study was to evaluate the effects of anti-IL-17 therapy on spontaneous ankylosing enthesitis in mice. In this study, we used male DBA/1 mice as a spontaneous ankylosis model. Serum IL-17 concentrations were determined using enzyme-linked immunosorbent assay. Male DBA/1 mice from different litters were mixed and caged together preceding the treatment at 10 weeks (wk) of age (prophylaxis) or 21 wk of age (intervention). Treatment with anti-IL-17 antibodies or saline was initiated after caging in groups of mice and administered weekly. The onset of tarsal ankylosis was assessed by ankle swelling and histopathological examination. Pathological changes and mRNA expression levels were assessed in joints and ears obtained at the experimental end-point. We found that circulating IL-17 increased with the onset of ankylosis in male DBA/1 mice, coinciding with the onset of dermatitis. The symptoms of dermatitis corresponded to the pathological characteristics of psoriasis: acanthosis with mild hyperkeratosis, scaling, epidermal microabscess formation and augmented expression of K16, S100A8 and S100A9. Prophylactic administration of anti-IL-17 antibodies significantly prevented the development of both ankylosis and dermatitis in male DBA/1 mice caged together. On the other hand, administration of anti-IL-17 antibodies after disease onset had a lesser but significant effect on ankylosis progression but did not affect dermatitis progression. In conclusion, IL-17 is a key mediator in the pathogenic process of tarsal ankylosis and psoriasis-like dermatitis in male DBA/1 mice caged together. Thus, IL-17 is a potential therapeutic target in ankylosing enthesitis and psoriasis in humans.

Induction of lipocalin-type prostaglandin D synthase in mouse heart under hypoxemia

Hypoxemia is a common manifestation of various disorders and generates pressure overload to the heart. Here we analyzed the expression of lipocalin-type prostaglandin D synthase (L-PGDS) in the heart of C57BL/6 mice kept under normobaric hypoxia (10% O2) that generates hemodynamic stress. Northern and Western blot analyses revealed that the expression levels of L-PGDS mRNA and protein were significantly increased (> twofold) after 14 days of hypoxia, compared to the mice kept under normoxia. Immunohistochemical analysis indicated that L-PGDS was increased in the myocardium of auricles and ventricles and the pulmonary venous myocardium at 28 days of hypoxia. Moreover, using C57BL/6 mice lacking heme oxygenase-2 (HO-2(-/-)), a model of chronic hypoxemia, we showed that the expression level of L-PGDS protein was twofold higher in the heart than that of wild-type mouse. L-PGDS expression is induced in the myocardium under hypoxemia, which may reflect the adaptation to the hemodynamic stress.

Indispensable roles of OX40L-derived signal and epistatic genetic effect in immune-mediated pathogenesis of spontaneous pulmonary hypertension

BackgroundPulmonary hypertension (PH) refers to a spectrum of diseases with elevated pulmonary artery pressure. Pulmonary arterial hypertension (PAH) is a disease category that clinically presents with severe PH and that is histopathologically characterized by the occlusion of pulmonary arterioles, medial muscular hypertrophy, and/or intimal fibrosis. PAH occurs with a secondary as well as a primary onset. Secondary PAH is known to be complicated with immunological disorders. The aim of the present study is to histopathologically and genetically characterize a new animal model of PAH and clarify the role of OX40 ligand in the pathogenesis of PAH.ResultsSpontaneous onset of PAH was stably identified in mice with immune abnormality because of overexpression of the tumor necrosis factor (TNF) family molecule OX40 ligand (OX40L). Histopathological and physical examinations revealed the onset of PAH-like disorders in the C57BL/6 (B6) strain of OX40L transgenic mice (B6.TgL). Comparative analysis performed using different strains of transgenic mice showed that this onset depends on the presence of OX40L in the B6 genetic background. Genetic analyses demonstrated a susceptibility locus of a B6 allele to this onset on chromosome 5. Immunological analyses revealed that the excessive OX40 signals in TgL mice attenuates expansion of regulatory T cells the B6 genetic background, suggesting an impact of the B6 genetic background on the differentiation of regulatory T cells.ConclusionPresent findings suggest a role for the OX40L-derived immune response and epistatic genetic effect in immune-mediated pathogenesis of PAH.