Miwa Uzuki

Aromatase in Human Bone Tissue

Peripheral aromatization of androgens exert estrogenic actions in many tissues. Recently in situ production of estrogens by aromatase was detected in human bone and cultured osteoblasts and has been proposed to participate in the maintenance of bone mass. We examined aromatase expression by immunohistochemistry and mRNA in situ hybridization in 16 cases of tibia (female 2 male, 14 female, 62 ± 5.2 years old) and quantified the level of aromatase mRNA in 28 cases of rib, femur, and lumbar vertebrae (16 male, 12 female, 58.0 ± 11.3 years old) by reverse transcriptase‐polymerase chain reaction (RT‐PCR) in order to study whether or not and in which cell types aromatase was expressed in human bone tissues. We also studied alternative use of multiple exons 1 of its gene and immunolocalization of type I 17β‐hydroxysteroid dehydrogenase (HSD), which converts estrone produced by aromatase to estradiol. Strong aromatase immunoreactivity and mRNA hybridization as well as type I 17β‐HSD immunoreactivity were detected in lining cells, osteoblasts, chondrocytes of articular cartilage, and adipocytes adjacent to bone trabeculae in all the cases examined. Amounts of aromatase mRNA varied greatly among the subjects (11.25 ± 9.77, 0.61–42.84 attomol/ng of total RNA). The amount of aromatase expression was not correlated with age or gender of the subjects but positively correlated with the degree of osteroporotic changes evaluated by radiological findings of lumbar vertebrae. Analysis of multiple exons 1 revealed that 1b or fibroblast type was predominantly (23/26) utilized as a promoter of aromatase gene expression. These results demonstrated that aromatase is expressed widely in human bone tissue and may play important roles in maintenance of human bone tissue.

Pancreatectomy combined with superior mesenteric-portal vein resection for adenocarcinoma in pancreas

The aims of this study were to investigate morbidity, mortality, and survival of patients with ductal adenocarcinoma of the pancreas who underwent pancreatectomy without (group 1) or with (group 2) en bloc portal vein resection and to study the degree of carcinoma invasion of the portal vein in group 2. The medical records of 46 and 28 patients in groups 1 and 2, respectively, were reviewed. In addition, the degree of invasion of the wall of the portal vein was categorized histologically into three types: type I, transmural invasion involving the intima; type II, invasion of the wall of the vein without intimal involvement; and type III, compression of the wall of the vein by surrounding carcinoma without true invasion. The morbidity and mortality in group 1 (26% and 4%) were not different from those in group 2 (32% and 4%). Similarly, there was no difference in survival between the two groups. Survival tended to vary directly with the depth of invasion of the wall of the portal vein: type I 6.8 ± 1.9 months; type II 15.3 ± 6.4 months; type III 20.6 ± 13.0 months. These findings suggest that en bloc resection of the pancreas and the portal vein does not increase mortality and morbidity after pancreatectomy; survival after en bloc resection was similar to that of patients not requiring portal vein resection. Combined resection of the pancreas with the portal vein could be an option in the treatment of pancreatic cancer with direct invasion of the portal vein.

Mixed Osteoclastic/pleomorphic-type Giant Cell Tumor of the Pancreas with Ductal Adenocarcinoma: Histochemical and Immunohistochemical Study with Review of the Literature

We described a rare form of giant cell tumor of the pancreas composed of mixed osteoclastic/pleomorphic-type giant cell tumor with ductal adenocarcinoma. Immunohistochemical study showed positive staining of cytokeratin and epithelial membrane antigen for pleomorphic-type giant cells and ductal adenocarcinoma but negative for osteoclastic-type giant cells. Vimentin stained positive in both types of giant cells but negative in adenocarcinoma. Osteoclastic-type giant cells were strongly positive for CD68 and tartarate-resistant acid phosphatase was present in these cells, suggesting the osteoclast-like character. CD68 was negative for both pleomorphic-type giant cells and ductal adenocarcinoma. From these findings, we consider that this tumor might be a carcinosarcoma-like neoplasm consisting of both an epithelial and a histiocytic-mesenchymal component.

Sex steroid receptors in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a disease characterized primarily by chronic inflammatory synovitis and is well-known to be associated with significant sex differences in its prevalence and clinical features. Sex steroids have been proposed to be involved in the pathogenesis of RA, but details pertaining to the expression of sex steroid receptors in RA synovial tissue have yet to be fully characterized. In the present study, we examined oestrogen receptor (ER) alpha, ERbeta, progesterone receptor (PR) and androgen receptor (AR) mRNA expression using real-time reverse transcriptase-PCR (RT-PCR) in eight female RA synovial tissues and six female synovial tissues without inflammation, and determined immunolocalization of ERalpha, ERbeta, PR-A, PR-B and AR using immunohistochemistry in synovial tissues obtained from 22 RA patients. Real-time RT-PCR analysis demonstrated the expression of ER, PR and AR mRNAs in both RA and non-inflamed synovial tissues. Relative abundance of ER mRNAs was significantly higher in RA synovial tissue than non-inflamed synovial tissue (P<0.05). In addition, the relative ERalpha/ERbeta mRNA expression ratio was significantly lower in RA than non-inflamed synovial tissue (RA, 2.34 +/- 1.60; and non-inflamed, 20.7 +/- 19.1; P<0.05). There were no significant differences in relative abundance of PR mRNA. Relative abundance of AR mRNA was significantly lower in RA (P<0.05). Immunoreactivity for ERalpha, ERbeta, PR-B and AR was detected in the lining cells, inflammatory cells and fibroblasts in all the patients examined. The labelling indices for ERbeta and PR-B were more abundant in both lining cells (ERbeta, 54.2 +/- 12.2%; PR-B, 73.6 +/- 18.9%) and inflammatory cells (ERbeta, 74.6 +/- 16.2%; PR-B, 75.9 +/- 16.1%) than in fibroblasts (ERbeta, 36.5 +/- 15.6%; PR-B, 49.4 +/- 18.0%). Labelling indices for ERalpha and AR were significantly higher in lining cells (ERalpha, 14.4 +/- 8.6%; AR, 31.2 +/- 11.3%) and fibroblasts (ERalpha, 12.1 +/- 7.5%; AR, 20.1 +/- 9.6%) than those in inflammatory cells (ERalpha, 5.7 +/- 3.3%; AR, 9.2 +/- 4.4%). There were significant differences (P<0.05) in the labelling indices for ERalpha, ERbeta and PR-B between men and women under 50 years of age in fibroblasts of RA synovial tissues. These results indicate that sex steroid receptors are present in RA and non-inflamed synovial tissues, including inflammatory cells in RA, and suggest that sex steroids may play important roles in the regulation of inflammation of RA synovial tissue.

Angiogenesis and inflammatory cell infiltration in lumbar disc herniation.

Study Design. Immunohistochemical study of the angiogenesis and inflammatory cell invasion in lumbar disc herniation. Objectives. To observe the blood vessel formation within the extracellular matrix in lumbar disc herniation, and to elucidate the role of angiogenesis in the natural shrinking of hernias. Summary of Background Data. There have been few reports of detailed observation of blood vessel formation within the extracellular matrix, and the role that angiogenesis plays in the natural shrinking of hernias has not been elucidated. Methods. Twenty tissue samples surgically removed from 17 patients with herniated discs were studied (9 men, 8 women, 23–58 years old, 11 extrusion type, 9 sequestration type). In the immunohistochemical study, an anti-CD34 antibody for vascular endothelial cells, an anti-CD68 for macrophages, and an anti-vascular endothelial growth factor antibody was used for vascular endothelial growth factor. Results. Many spindle-shaped cells expressing vascular endothelial growth factor were seen inside granulation tissue infiltrating the cartilage matrix, and the number of vascular endothelial growth factor–positive cells and the number of CD34+ cells were positively correlated (R = 0.73, P < 0.001). In the area surrounding CD34+ cells that had formed a lumen, many CD68+ cells were observed, and the number of CD34+ cells and the number of CD68+ cells were positively correlated (R = 0.66, P < 0.001). Conclusions. The results suggest that the vascular endothelial growth factor produced by the spindle-shaped cells acts to promote angiogenesis inside granulation tissue infiltrating the cartilage matrix, and that new blood vessels play an important role as a passage for macrophages into the degenerated matrix.

Histopathological predictors of regional lymph node metastasis at the invasive front in early colorectal cancer

Akishima‐Fukasawa Y, Ishikawa Y, Akasaka Y, Uzuki M, Inomata N, Yokoo T, Ishii R, Shimokawa R, Mukai K, Kiguchi H, Suzuki K, Fujiwara M, Ogata K, Niino H, Sugiura H, Ichinose A, Kuroda Y, Kuroda D & Ishii T
(2011) Histopathology59, 470–481

Treatment of rheumatoid synovitis with anti‐reshaping human interleukin‐6 receptor monoclonal antibody: Use of rheumatoid arthritis tissue implants in the SCID mouse model

OBJECTIVE To examine the effect of anti-reshaping human interleukin-6 receptor monoclonal antibody (anti-rsHuIL-6R mAb) on patients with rheumatoid arthritis (RA), using SCID mice in which human RA synovial tissue has been grafted (SCID-HuRAg). METHODS Tissue from human RA pannus was implanted subcutaneously in the backs of 69 SCID mice. Differences from human RA were examined pathologically. Anti-rsHuIL-6R mAb (100 microg) was administered intraperitoneally to mice once a week for 4 weeks. The implanted tissue was removed from the SCID-HuRAg mice on the fifth week after the initial treatment and examined pathologically. A group of SCID-HuRAg mice treated with control mAb, an auranofin-treated group, and an untreated group were used as controls. A total of 32 mice (8 in each group) were studied. RESULTS Histologic characteristics of the implanted tissues in SCID-HuRAg mice were very similar to those of human RA even 2 months after implantation. In addition, the presence of CD4-, CD8-, CD20-, IL-6-, tumor necrosis factor alpha-, tartrate-resistant acid phosphatase (TRAP)-, matrix metalloproteinase 1 (MMP-1)-, and MMP-9-positive cells was confirmed by immunohistochemical staining. A significant decrease in the number of inflammatory cells, MMP-positive cells, and TRAP-positive cells was observed in the anti-rsHuIL-6R mAb treatment group as compared with the control groups. CONCLUSION The SCID-HuRAg mouse is a useful model for evaluating the effectiveness of antirheumatic drugs. Anti-rsHuIL-6R mAb may have an antiinflammatory effect on RA synovitis and an inhibitory effect on osteoclasts.

MICRODETERMINATION OF PROSTAGLANDIN E2 IN JOINT FLUID IN RHEUMATOID ARTHRITIS PATIENTS USING GAS CHROMATOGRAPHY/SELECTED ION MONITORING

We devised an effective purification for the microdetermination of prostaglandin E2 (PGE2) in human joint fluid using gas chromatography/selected ion monitoring and determined PGE2 in the joint fluid in rheumatoid arthritis (RA) patients using this method. The methyl estermethoxime-tert-butyldimethylsilyl ether derivative was prepared, then gas chromatography/selected ion monitoring was carried out by monitoring the ion at m/z 566.4 for PGE2 and at m/z 570.4 for the internal standard (PGE2-d4). A good linear response over the range of 10 pg to 50 ng was demonstrated. We detected PGE2 to a level of about 46 pg/ml in the joint fluid of RA patients. The level of PGE2 in RA patients was significantly higher than that in osteoarthritis patients used as controls. Moreover, we measured inflammatory cytokine (IL-1beta, TNFalpha, IL-6 receptor) levels in joint fluid by using enzyme-linked immunosorbent assay. A relationships between the PGE2 level in joint fluid and these cytokines or biochemical data as the indicator of RA disease was not observed. We found that the PGE2 level in each patient was influenced by therapeutic drugs. The PGE2 level in RA patients with non-steroidal anti-inflammatory drugs was lower than with steroids.

Interleukin-6 upregulates expression of ADAMTS-4 in fibroblast-like synoviocytes from patients with rheumatoid arthritis

Aim:  A disintegrin‐like and metalloproteinase with thrombospondin type 1 motif (ADAMTS)‐4 and ADAMTS‐5 play crucial roles in the cleavage of aggrecan. Several recent studies have demonstrated the effect of cytokines such as interleukin (IL)‐1β, tumor necrosis factor‐α and transforming growth factor‐β on the expression of ADAMTS‐4 and ADAMTS‐5 in fibroblast‐like synoviocytes (FLS). However, the effect of IL‐6 remains unclear. The aim of this study is to investigate the expression of ADAMTS‐4 and ADAMTS‐5 in FLS of rheumatoid arthritis (RA) patients after IL‐6 stimulation.

The mechanisms underlying fibroblast apoptosis regulated by growth factors during wound healing

While investigating the mechanisms underlying cell death during wound healing processes, we uncovered the pro‐apoptotic effects of basic fibroblast growth factor (bFGF) on granulation tissue fibroblasts following pretreatment with transforming growth factor (TGF)‐β1 in vitro. bFGF induced caspse‐3 activation and apoptosis in TGF‐β1‐pretreated granulation tissue‐derived fibroblasts (GF‐1) following bFGF treatment for 48 and 96 h. In contrast, fibroblasts that had been treated in the same manner and that originated from the uninjured dermis did not display apoptosis, indicating that the mechanisms underlying apoptosis events in fibroblasts that originate from normal dermal and wound tissues differ. In this process, we also found that bFGF inhibited Akt phosphorylation at serine 473 and induced a rapid loss of phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 in pretreated GF‐1 cells, an event that coincided with the dissociation of phosphorylated FAK from the focal adhesions. Therefore, inhibition of survival signals relayed via the disrupted focal adhesion structures and inactivated Akt following bFGF treatment may lead to apoptosis in GF‐1 cells pretreated with TGF‐β1. Pretreatment of GF‐1 with TGF‐β1 followed by the addition of bFGF resulted in significantly greater inhibition of phosphorylation of Akt and FAK compared to treatment with TGF‐β1 or bFGF alone. The combinatorial treatment also led to proteolysis of FAK and inhibition of FAK and Akt protein expression in GF‐1 cells. These findings demonstrated a significant role for the two cytokines in apoptosis of granulation tissue fibroblasts during wound healing. In vivo studies also confirmed a marked decline in phosphorylation and protein expression of Akt and FAK in bFGF‐injected skin wounds. These results led to the hypothesis that temporal activation of TGF‐β1 and bFGF at the injury site promotes apoptosis in granulation tissue fibroblasts, an event that is critical for the termination of proliferative granulation tissue formation. Copyright