HER2 oncogene plays an important role in the oncogenesis and clinical behavior of 25-30% of human breast cancers. Recently, a recombinant humanized monoclonal antibody against HER2 protein, trastuzumab, has been administrated to patients with HER2 positive breast carcinoma. This antibody-based therapy is only effective in HER2-positive carcinoma cases, and therefore, it is very important to evaluate HER2 status with accuracy in order to determine the appropriateness of trastuzumab treatment for the breast cancer patients. Evaluation of HER2 status is mainly performed in the primary breast tumor tissues and several pathological methods, including immunohistochemistry, fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) which has been newly developed, are currently used in diagnostic laboratories. In this review, we describe the recent studies about these methods and discuss their reliability and possible further application to various pathological specimens obtained from breast carcinoma patients.

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The collection of homogeneous or pure cell populations for molecular analyses has been a difficult challenge for many years. Many different approaches have been tried unsuccessfully to obtain pure populations of pituitary cells. Recent developments in laser capture microdissection have facilitated the process of acquiring homogeneous cell populations. Some investigators have combined LCM with immunophenotyping before performance of the LCM procedure. With this combined approach, immunophenotypically homogeneous cell population can be readily collected for molecular analyses. We will review the general approaches to immuno-LCM and show how these methods have been applied recently for the analysis of folliculostellate cells in the pituitary gland.

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Sarcoidosis, of unknown etiology, may result from exposure of a genetically susceptible subject to a specific environmental agent(s), possibly an infectious one, although none has been identified. Propionibacterium acnes is so far the only bacterium to be isolated from sarcoid lesions. Genomes of P. acnes have been detected in large numbers in sarcoid lymph nodes by the quantitative polymerase chain reaction. By in situ hybridization, P. acnes genomes were found in sarcoid lymph nodes in and around sarcoid granulomas. These results point to an etiological link between P. acnes and some cases of sarcoidosis. Host factors may be more critical than agent factors in the etiology of sarcoidosis, as already suggested from the phenomenon of the Kveim test, in which a suspension of sarcoid tissues injected intracutaneously causes sarcoid granulomas in patients with sarcoidosis but not in healthy people or patients with other diseases. A recombinant trigger-factor protein, RP35, from P. acnes causes a cellular immune response in some patients with sarcoidosis, but not in subjects without sarcoidosis. RP35 caused pulmonary granulomas in mice sensitized with the protein and adjuvant. Sarcoid granulomas may form during hypersensitivity to antigens of P. acnes indigenous to or proliferating in the affected organ.

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There is considerable interest in tumor angiogenesis and its inhibition, because tumor angiogenesis plays an important role in tumor growth and distant metastasis, and is highly associated with overall survival and relapse free-survival in cancer patients.
This study was performed to determine the mechanisms underlying tumor angiogenesis and its inhibition using the angiogenesis inhibitor TNP 470 and antimitotic drug, docetaxel (Taxotere). Variable 3D image analysis of tumor vasculature after chemotherapy was performed by CLSM using the DMBA-induced rat breast cancer model.
3D images of the vasculature in DMBA-induced rat breast cancer were characterized by increased microvessel density and anastomosing, irregularly branched microvessel networks. After administration of TNP-470 or docetaxel, tumor microvessel density was markedly decreased and tumor microvessel networks were markedly destroyed. Furthermore, tumor microvessels less than 30 μm in diameter were not found. The remaining tumor microvessels (30-60 μm in diameter) were mainly localized in the periphery of the tumor. These observations suggest that docetaxel might have the ability to inhibit tumor angiogenesis as well as TNP-470. CLSM with the FI method is a valuable technique for estimation of the efficacy of anti-angiogenic chemotherapy.

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We examined the nerve fibers among the cochlear supporting cells (NFSCs) in paraffin- and celloidin-embedded sections stained by the use of sodium retrieval immunoperoxidase technique (SRT) and/or tyramine amplification immunoperoxidase technique (TAT). SRT or TAT showed clear immunostaining of the radial nerve fibers and spiral ganglions, but a combination of the both techniques resulted in a higher density of the immunostained components at an amplification of 300-1,000 fold compared with the conventional immunoperoxidase technique (CIT). The presence of NFSCs was also clear with the combined use. This innervation was seen in the apical turns but not in the basal turns, similar to the location of lipid droplets among the Hensen cells, which are proposed to provide a high-energy source. Although the physiological significance and role of these nerve fibers has not yet been clearly defined, the coexistence of NFSCs and lipid droplets suggests that NFSCs may relate to energy metabolism. Our study also showed that SRT and/or TAT markedly enhanced the signal strength. These methods should allow the detection of very weak immunohistochemical signals under the use of stronger fixation or harder embedding.

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The purpose of the present study was to investigate the relationship between the neuropeptide calcitonin gene-related peptide-immunoreactive (CGRP-IR) nerves and the differentiation of undifferentiated mesenchymal cells into fibroblast-like cells and odontoblasts during the healing process after a pulpotomy. The first maxillary molars from 56-day-old Wistar rats (n=60) were used. The rats were sacrificed to undergo an immunoelectron microscopic examination at 1, 3, 7, 14, and 28 days, postoperatively. In 1 to 3 postoperative days, numerous unmyelinated degenerated CGRP-IR nerve terminals were observed. In 7 postoperative days, regenerated terminals were found to contain numerous large granular vesicles, small clear vesicles, a few mitochondria and a labeled organelle. Certain terminals were found to be attached with the cell bodies of fibroblast-like cells and their processes at the fibrous matrix layer of the dentin bridge during healing process following a pulpotomy. In 14 to 28 postoperative days, CGRP-IR nerve terminals had come into contact with the differentiating odontoblasts at the odontoblast layer of the dentin bridge. These findings demonstrate strong evidence that CGRP may be related to both the proliferation and cytodifferentiation process as well as to the active function of the renewed odontoblasts in dentin bridge formation.

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Phospholipase D (PLD), one of the signal transducing enzymes, is recognized to play a role in the pancreatic exocrine and endocrine secretion, and yet the distribution of PLD in the pancreas has remained unclarified. We investigated the expression and localization of PLD isoforms, PLD1 and PLD2, in the rat pancreas. Western blot analysis showed that PLD1-immunoreactive band was observed in the islets, but not in the acinar cells. In contrast, PLD2-immunoreactive band was observed in both the islets and the acinar cells. In the immunohistochemistry, PLD1 was evenly distributed throughout the islets while PLD2 immunoreactivity was more intense in the periphery than in the central portion of the islets. To further elucidate the cell-specific localization of PLD2 in the islets, double immunofluorescent staining was performed. PLD2 was mainly localized in A and PP cells, which secrete glucagon and pancreatic polypeptide, respectively. PLD2 was also detected in acinar cells, ductal epithelial cells and intrapancreatic nerve fibers. Meanwhile, PLD1 was detected in ductal epithelial cells and intrapancreatic ganglia, but not in acinar cells. On the basis of this heterogeneity in the distribution of PLD isoforms, it is suggested that PLD might play a specific role in the pancreatic exocrine and endocrine function.

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Vascular endothelial growth factor (VEGF) plays a very important role in vessel invasion during the process of endochondral ossification. Although grafted periosteum shows endochondral ossification, little is known about the role of VEGF in this process. Additionally, some reports have suggested that bone morphogenetic protein-4 (BMP-4) and VEGF work cooperatively in some way during endochondral ossification. In the present study we investigated, using immunohistochemical, histochemical, ultrastructural, and radiographic techniques, the role of VEGF and BMP-4 in bone formation from periosteal grafts taken from Japanese white rabbit tibia. By 14 days after grafting, fibroblasts in the grafted periosteum differentiated into chondrocytes to form cartilage. Some chondrocytes showed VEGF expression. Subsequent vessel invasion into cartilage from the VEGF immunopositive area coincided with commencement of endochondral ossification. Cartilage was replaced by newly formed bone by 35 days. Soft X-ray findings indicated calcification at 35 days after grafting. Chondrocytes and cartilage matrix were both BMP-4 positive. In newly formed bone, BMP-4 and VEGF expression was demonstrated in osteoblasts. These findings suggest that VEGF are related to vascular invasion into the grafted periosteum during endochondral ossification. Further, VEGF production in chondrocytes and osteoblasts may be related to BMP-4 production.

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We studied the role of infiltrating cells and cytokines synthesis in oxazolone (OXA)-induced contact hypersensitivity (CHS) of C57BL/6 mouse ear model. The cell mediate lesion was induced by the OXA challenge after abdominal skin sensitization, and those of infiltrating cells were examined at various time points (0-168 hr). The CHS lesions were indicated as an ear swelling at 24 to 48 hr after the challenge that was caused mainly by infiltration of mononuclear cells expressing CD90⁺, CD4⁺ and CD8⁺. However, NK cells by means of asialo GM1⁺ cells, were detected in the lesions as early as 2 hr after challenge. The infiltration of NK cells peaked at 8 hr and replaced in the predominant accumulation of polymorphonuclear (PMN) cells and some of CD90⁺ cells. Cytokine expression of the infiltrating cells by RT-PCR, revealed a high level of IL-2 expression at 8, 12, and 24 hr and then reduced expression in the following times, while, IFN-γ was constantly detected from 2 hr to 168 hr.
Deletion of the NK cell activity by administration of the anti-asialo GM1 antibody resulted in significantly suppressed cell infiltration and ear swelling. IL-2 mRNA expression was undetected in the infiltrating cells of deletion mouse, whereas IFN-γ was detected at 12, 24, and 48 hr. The presence of NK cells as the first infiltrating cells in the lesion and IL-2 and INF-γ synthesis from the infiltrating cells may play an important role in the elicitation phase of CHS.

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cDNAs encoding facilitated-diffusion glucose transporters GULT1, GULT3, GULT4, and GULT5 were introduced into the rat pancreas by in-vivo electroporation method, and their expression and localization in pancreatic acinar cells were examined immunohistochemically. GLUT1 was localized at the basolateral membrane, whereas GLUT3 and GLUT5 were at the apical membrane. Restriction of GLUT3 and GLUT5 to the apical membrane makes a marked contrast to their localization to the entire plasma membrane when expressed in hepatocytes in situ. Such differential localization may be due to differential apical targeting mechanism: direct targeting in acinar cells and indirect transcytotic delivery in hepatocytes in situ. GLUT4 was present in the membrane of zymogen secretory granules in the cytoplasm in acinar cells. This observation suggests that GLUT4 is segregated to the regulated secretory pathway. Expression of glucose transporters in situ is a useful method in analyzing the targeting mechanism of membrane proteins in cells of tissues in situ.

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We investigated histopathological and immunohistochemical properties of the ligamentum flavum of the lumbar spine with calcium crystal deposition. Ligamentum flavum of the lumbar spine containing calcium deposits were harvested from 41 surgical cases. Sections of the ligaments were immunostained for estrogen receptor (ER), progesterone receptor (PR), and transforming growth factor (TGF)-β. The results were compared with those of ligaments without calcium deposits. The elastic fibers of the ligament with calcium deposits showed marked degeneration (irregular arrangement and fragmentation of fiber bundles) and nodular lesions. Deposits of calcium crystals were present in these areas where elastic fibers were degenerated. Immunostaining for ER was positive in these areas, and PR as well as TGF-β-containing chondrocytes were detected around and within the calcified areas. TGF-β, ER and PR-containing chondrocytes appeared to precipitate deposition of calcium crystals in the nodular lesions of the degenerated lumbar ligamentum flavum. Our results suggest that TGF-β and sex hormones play a role in the calcification of the lumbar ligamentum flavum.

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