Tetsuo Hamada

Pathologic evidence of degeneration as a primary cause of rotator cuff tear.

Histopathologic, histochemical, and morphometric studies were done on 80 medial stumps of torn rotator cuff tendons to clarify the cause of tears. A high prevalence and diffuse distribution of degenerative changes were observed in the rotator cuff tendons including thinning and disorientation of collagen fibers, myxoid degeneration, hyaline degeneration, chondroid metaplasia, calcification, vascularproliferation, and fatty infiltration. No distinct inflammatory reaction was observed. Thinning and disorientation of collagen fibers, myxoid degeneration, and hyaline degeneration were seen in all cases. All changes except vascular proliferation and fatty infiltration were more pronounced in the middle to deep layers of the tendons than in the superficial layer. The collagen fibers were disoriented in the deep layer of the tendons, shown by microscopic image analysis. The frequency and distribution of thinning and disorientation of collagen fibers, myxoid degeneration, and hyaline degeneration suggest that these are early degenerative processes. Chondroid metaplasia and calcification may be chronic pathologic changes that occur after tearing regardless of the type of tear. Preexisting degenerative change in the middle and deep layers of the tendon in association with microtrauma seems to be the main cause of rotator cuff tears.

Shortened microsatellite d(CA)21 sequence down-regulates promoter activity of matrix metalloproteinase 9 gene.

One characteristic elements in the promoter of the matrix metalloproteinase 9 (MMP‐9) gene is the d(CA) repeat. To investigate whether this element regulates the transcription of the MMP‐9 gene and its enzymatic activities, we sequenced the promoter region isolated from esophageal carcinoma cell lines. TE9 cells with low MMP‐9 enzymatic activity had the number of d(CA) repeats shortened from 21 to 14 or 18. TE8, TE10 and TE11 cells with high MMP‐9 activities had 21 or 23 d(CA) repeats. Luciferase assays using MMP‐9 promoter containing 18, 14 or 0 d(CA) repeats showed transcriptional activities which were 50, 50 or 5%, respectively, of the level achieved with promoter containing 21 d(CA) repeats. Sequence analysis of the promoter of 223 Japanese subjects revealed that most had two alleles with 20, 21 or 22 d(CA) repeats, whereas six had one or two alleles with 14, 18 or 19 d(CA) repeats. We postulate that length alteration of the d(CA) repeat causes phenotypic differences among carcinoma cells and that microsatellite instability may contribute to the polymorphism of d(CA) repeat length.

Cell death and regeneration of renal proximal tubular cells in rats with subchronic cadmium intoxication

Male Sprague-Dawley rats were injected subcutaneously with 0.6 mg cadmium (Cd)/kg/day for 8 wk. The subsequent changes in renal proximal tubules were studied histologically, histochemically, and ultrastructurally. The urinary and tissue Cd concentrations were determined by atomic absorption spectrophotometry. After 4 wk of exposure, apoptosis was observed predominantly in segment S3 along with epithelial regeneration in the affected tubules, and these changes gradually became more pronounced as the experimental period was prolonged. The apoptotic cells were shed into the lumen and were found to contain a large quantity of Cd. Apoptotic cells were counted in paraffin sections after various periods of exposure to Cd. Nuclear bromodeoxyuridine uptake, mitotic count, and nuclear density were used as indicators of tubular regeneration. A correlation was found between the numerical increase of apoptotic cells and the rate of urinary Cd excretion, and the rate of increase in the tissue Cd concentration had a tendency to reduce after 4 wk as the rate of urinary Cd increased. These observations suggest that apoptosis might be helpful for the efficient excretion of Cd into urine. Progressive increases in the preceding indicators of regeneration were observed. From our results, it appears that Cd-induced tubular damage, i.e., cell deletion due to apoptosis, is reversible as a result of marked epithelial regeneration. On the basis of these histological changes, the critical concentration of Cd required to produce renal tubular damage was estimated to be 600 μg/g dry tissue.

Switch of histamine receptor expression from H2 to H1 during differentiation of monocytes into macrophages

It is known that histamine suppresses gene expression and synthesis of tumor necrosis factor alpha (TNF‐α) induced by lipopolysaccharide (LPS) in human peripheral blood mononuclear monocytes (HPM) or alveolar macrophages via histamine H2 receptors. We investigated the effect of histamine and differentiation in macrophages on the expression and secretion of TNF‐α, TNF‐α‐converting enzyme (TACE), and histamine H1 and H2 receptors by use of a leukemia cell line, U937, and HPM. Differentiation of U937 and HPM cells with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) enhanced the H1 receptor expression and rather suppressed the H2 receptor, resulting in up‐regulation of the histamine‐induced expression and secretion of TNF‐α, modulated via TACE. Therefore, histamine failed to inhibit up‐regulated expression of TNF‐α induced by LPS in macrophages. The switch from H2 to H1 receptors during differentiation in the monocyte/macrophage lineage could participate in the pathogenic processes of atherosclerosis and inflammatory reactions in the arterial wall.

Association of microRNA-21 expression with its targets, PDCD4 and TIMP3, in pancreatic ductal adenocarcinoma.

Since the discovery of small non-coding RNAs, the analyses of microRNA (miRNA) expression patterns in human cancer have provided new insights into cancer biology. miRNA-21 has been suggested to be one of the miRNAs that have an important role in the development or biological behavior of a variety of malignancies, including pancreatic cancer. This study was conducted to evaluate the relationship between the expression of miRNA-21 and that of its molecular targets, programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinase (TIMP3), in pancreatic ductal adenocarcinoma. The study included 65 pancreatic ductal adenocarcinomas and 5 normal pancreatic tissue specimens for comparison. The miRNA expression profiling of five selected pancreatic ductal adenocarcinomas and five normal pancreatic specimens was performed using a microarray platform, and was evaluated by a hierarchical clustering analysis. The miRNA most highly expressed in pancreatic ductal adenocarcinomas (ie, miRNA-21) was further assessed by quantitative real-time reverse transcription PCR (RT-PCR) assays in the 65 pancreatic ductal adenocarcinoma cases. The expression pattern of its molecular targets (eg, PDCD4 and TIMP3) in pancreatic ductal adenocarcinoma was examined immunohistochemically. In the microarray analyses, 28 miRNAs were upregulated in pancreatic ductal adenocarcinoma compared with normal pancreatic tissue, whereas 48 miRNAs were downregulated. miRNA-21 was the most significantly overexpressed miRNA in the pancreatic ductal adenocarcinomas analyzed, and was also highly expressed in 75% of the 65 pancreatic ductal adenocarcinomas examined by real-time RT-PCR. High miRNA-21 expression was correlated with a worse prognosis in the pancreatic ductal adenocarcinoma patients (P=0.045). The immunohistochemical expression patterns of PDCD4 (reduced nuclear staining pattern) and TIMP3 (downregulated expression) were significantly associated with both the upregulated miR-21 expression (P<0.05) and the poor survival of the patients (P<0.001 and P=0.001, respectively). Our data suggest that an overexpression of miRNA-21 is, therefore, associated with the biological behavior of pancreatic ductal adenocarcinoma via the downregulation of the expression of tumor suppressors, PDCD4 and TIMP3, thus resulting in tumor progression and the adverse clinical course of pancreatic ductal adenocarcinoma.

Effects of histamine and interleukin-4 synthesized in arterial intima on phagocytosis by monocytes/macrophages in relation to atherosclerosis

We investigated the localization of histidine decarboxylase (HDC), which is the rate‐limiting enzyme that generates histamine from histidine, in human aorta/coronary artery. RT‐PCR and immunohistochemical staining revealed that the HDC gene was expressed in monocytes/macrophages and T cells in the arterial intima but not in smooth muscle cells in either the arterial intima or the media. A luciferase promoter assay with U937 and Jurkat cells demonstrated that interleukin‐4 (IL‐4) inhibited the expression of the HDC gene. In contrast, among a scavenger receptor family, IL‐4 as well as histamine up‐regulated U937 cells to express the LOX‐1 gene but not the SR‐A gene, which genes encode receptors that scavenge oxidized lipids. These findings suggest that histamine synthesized in the arterial wall participates in the initiation and progression of atherosclerosis and that IL‐4 can act as an important inhibitory and/or stimulatory factor in the function of monocytes/macrophages modulated by histamine in relation to the process of atherosclerosis.

A role for interleukin 4 in production of matrix metalloproteinase 1 by human aortic smooth muscle cells

Effect of interleukin 4 (IL-4) on the production of matrix metalloproteinase 1 (MMP-1) by normal and immortalized human intimal smooth muscle cells (SMC) was investigated. The production of the precursors of MMP-1 by intimal SMC was enhanced in a dose-dependent manner by addition of IL-4 to the culture medium, whereas the cytokine also showed an inhibitory effect on DNA synthesis in the cells. In addition, mRNA of IL-4 was found in the atherosclerotic and nonatherosclerotic areas of the intima. Although the production of MMP-1 and the proliferation of SMC are thought to play an important role in reconstruction of the intima during atherogenesis, our results suggest a possible role of IL-4 induced MMP-1 in inhibiting tissue remodeling caused by a variety of arterial disorders including atherosclerosis.

Pathological Study on Beagles after Long-term Oral Administration of Cadmium*

Histopathological, histochemical, and electron microscopic examinations were performed on beagles after a long-term oral cadmium (Cd) administration of > 8 years. Although renal atrophy was remarkable in groups receiving doses of 50 and 100 mg/kg body weight/day, bone lesions could not be demonstrated by roent-genological and histopathologic examination. It was noticed that concomitant regeneration or recovery and cell death of the epithelium occurred in the proximal convoluted tubules. The cell death was consistent with apoptosis, a special feature of cell death, which was shown to play a major part in the tubular damage of cadmium by electron microscopic examination. Fatty degeneration of the pars recta tubules was seen to show dose-dependence. The intrarenal cadmium was localized predominantly in the cytoplasm of the proximal tubular epithelium by histochemical and ultraccntrifugal cell fractionation examinations. Although no remarkable changes were found in the other organs, aggregates of siderophages in the liver and focal hemorrhage in the spleen, known as spontaneous lesions, might be related to Cd intoxication. In conclusion, the present study revealed that no bone lesions occur with Cd administration in adult beagles in spite of long-term administration. An excessive cell death to regeneration or recovery in the proximal tubules might result in the renal cortical atrophy. No remarkable changes were seen in the glomeruli and distal nephrons, which were in good agreement with Cd distribution.

Endometrial stromal sarcoma of the uterus with rhabdoid features

A case of endometrial stromal sarcoma of the uterus with rhabdoid features, occurring in a 57 year old woman is reported. Electron microscopy and immunohistochemistry revealed that the rhabdoid cells contained Intermediate filaments which were positive for vimentln, cytokeratin, α‐smooth muscle actin, and muscle specific actin, but not for myoglo‐bin and desmin. This indicated that the tumor in this case differed somewhat from the three rhabdoid tumors and an endometrial stromal sarcoma with rhabdoid differentiation previously reported and that, therefore, these tumors were heterogeneous.

Cytopathological Changes Induced by Cadmium-Exposure in Canine Proximal Tubular Cells: A Cytochemical and Ultrastructural Study

A highly sensitive cytochemical method for demonstrating intracellular Cd using 8-hydroxyquinoline was developed and applied in the cytopathological study of primary-cultured renal tubular cells from beagle kidneys. The Cd-8-hydroxyquinoline emitted a yellowish-green fluorescence which first appeared in the cytoplasm within 30 min and in the nucleus about 60-90 min after exposure to 100 mM CdCl2. It was noteworthy that intranuclear Cd was stained in the nucleolar regions. The sensitivity of the cytochemical method for Cd was estimated to be about 1.0 pg Cd/cell. Ultrastructural features of the dead cells were consistent with those of apoptosis. We conclude that Cd absorbed by proximal tubular cells rapidly reaches to the nuclei and affects nuclear as well as cytoplasmic metabolism.