Examination of in vivo gelatinolytic activity in rheumatoid arthritis synovial tissue using newly developed in situ zymography and image analyzer.

OBJECTIVE: The aim of this study was to examine in vivo gelatinolytic activity of rheumatoid arthritis (RA) synovium using a newly developed in situ zymography (ISZ) method and pathological image analyzer, and to evaluate the relationship between this activity and several features on RA. METHODS: A total of 8 samples of synovium were obtained from RA patients during surgery, and 8 samples from osteoarthritis (OA) patients were examined as controls. Furthermore, total 14 samples of syovium were obtained for comparison among radiographical classifications as Larsen grade (4 cases of grade III, 5 cases of grade IV and 5 cases of grade V). These specimens were frozen with OCT compound immediately after surgery. Frozen sections were applied to a newly developed gelatin-coated FIZ film (Fuji Film Co.Tokyo.Japan) designed for use ISZ, and incubated at 37 degrees C for 6 hours. Using an image analyzer (image processor for analytical pathology; IPAP), two variables were measured as indicators of in vivo gelatynolytic activity: optical density of gelatinolyzed area (ODG), and ratio of gelatinolyzed area (RGA). Also, we investigated the relationship between these indicators and the following variables: radiographic changes (Larsen grades), clinical data (C-reactive protein concentration), histological score of synovial tissue (modified Rooney's score), and expression of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 (assessed by immunohistochemistry). RESULTS: RA synovium had significantly higher RGA and lower ODG than OA, indicating higher gelatinolytic activity in RA. Synovium from cases with Larsen grade IV or V had significantly lower ODG than cases with grade III, but there was no significant difference in RGA between grades. There was no significant correlation between gelatinolytic activity (ODG or RGA) and either CRP or modified Rooney's Histological Score. The results of ISZ indicate that the gelatinolyzed areas were mainly localized in the lining area, with a small amount scattered throughout the stroma. The results of immunohistochemistry indicate that MMP-2, MMP-9, TIMP-1 and TIMP-2 were expressed in areas of gelatinolysis. CONCLUSIONS: The present results indicate that in vivo gelatinolytic activity of synovium is stronger in RA than in OA. They also indicate that gelatinolytic activity of RA synovial cells is stronger in cases with Larsen grade IV or V than in cases with grade III, although the gelatinolyzed area is similar. Gelatinolytic activity, as indicated by optical density and the gelatinolyzed area, differed between regions, even within the same specimen, suggesting an imbalance between production of proteinases and their inhibitors. We believe that the present zymography method can contribute to the elucidation of biological enzymatic activity of RA synovium.

Downregulation of inhibitor of apoptosis proteins in apoptotic human chondrocytes treated with tumor necrosis factor-alpha and actinomycin D.

OBJECTIVE: Apoptosis of chondrocytes plays a pivotal role in cartilage degeneration. Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine and has been assumed to cause the degradation of human cartilage. To investigate the mechanisms of TNF-alpha-mediated apoptosis of human chondrocytes from a point of view of the balance between the caspase-cascade and the expression of inhibitor of apoptosis proteins (IAPs), although both of them are induced with TNF-signals. METHODS: The expression of TNF-receptors (TNF-Rs) in normal human articular chondrocyte (NHAC-kn) was examined with immunocytochemistry. Subconfluent cultures of NHAC-kn were tested with TNF-alpha and/or actinomycin D (actD), and the induction of apoptosis was evaluated by the frequency of apoptotic cells visualized with nuclear staining using Hoechst 33342. The activation of caspases and the expression of IAPs were examined with Western blot analyses. RESULTS: NHAC-kn expressed TNF-R1 and -R2. When NHAC-kn was treated with TNF-alpha (10 ng/ml) and actD (0.2 microg/ml) for 24 h, the frequency of apoptotic cells increased to more than 25%. TNF-alpha alone, however, induced the apoptosis insufficiently (up to 8.3%), even when used at the concentration of 100 ng/ml for 48 h. In apoptotic human chondrocytes induced with TNF-alpha (10 ng/ml) and actD (0.2 microg/ml), the caspase-3, -8, and -9 were activated and the protein expression of XIAP and c-IAP1 decreased. CONCLUSIONS: In apoptotic human chondrocytes induced with TNF-alpha and actD, the balance between caspase activation and IAPs' expression lay with the executioner caspase (caspase-3) and led to decreased expression of XIAP and c-IAP1.

Induction of mononuclear cell infiltration into liver by Japanese herbal medicine.

Juzen-Taiho-To (JTT) is a Japanese herbal medicine that has been administered mainly to patients weakened by long illness. Currently, it has also been used for cancer patients and showed antitumor effects that have been reported as phagocytosis enhancement, cytokine induction and antibody production. In this study, we examined the effect of oral administration of JTT in mice on the immunological restoration of the liver, especially focused on natural killer (NK) T-cell induction. Mice were grouped to receive JTT or placebo orally for a period of 1, 3 and 7 days. After sacrifice, the liver tissue was fixed, embedded and stained with hematoxylineosin and some antibodies by common staining methods. Transmission electron microscope (TEM) observation was also carried out. Although the JTT-treated mice had the same appearance as the non-JTT-treated mice, their livers were infiltrated by massive mononuclear cells, some of which were aggregated in clusters. Immunohistochemical staining revealed that there was abundant cytokine expression of interleukin (IL)-12 and massive infiltration of mononuclear cells with large granules in the liver of JTT-treated mice. Oral administration of JTT may induce the expression of IL-12 and be followed by immunological restoration such as NK T-cell induction in liver

The role of TNF-alpha in the pathogenesis of inflammation and joint destruction in rheumatoid arthritis (RA): a study using a human RA/SCID mouse chimera.

OBJECTIVE: In order to elucidate which cytokine preferentially stimulates the synovium in patients with rheumatoid arthritis (RA), we investigated the roles of tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) using SCID mice engrafted with human RA tissue (SCID-HuRAg). METHODS: The SCID-HuRAg mice were prepared according to our previously described method. First, SCID-HuRAg mice were treated with chimeric anti-TNF-alpha monoclonal antibody (mAb, 100 microg/mouse) and histological changes were examined 4 weeks after the initial treatment. Secondly, a total of 100 microg of recombinant TNF-alpha or IL-6 (0.6 microg/h) was administered daily to mice using an osmium pump. The histological changes and serum cytokine levels were examined 4 weeks after the initial administration. Human immunoglobulin G (IgG) was administered to mice as a control. RESULTS: Synovial inflammatory cells were significantly decreased after the anti-TNF-alpha mAb treatment; conversely, the degree of synovial inflammation was significantly exacerbated by TNF-alpha administration. The levels of both IL-6 and TNF-alpha in sera were significantly increased by recombinant TNF-alpha administration, while TNF-alpha levels were unchanged by IL-6 administration. This suggests that TNF-alpha controls IL-6 production. Despite the profound changes in inflammation, we found no effects on bone and no articular cartilage damage was produced by TNF-alpha. CONCLUSION: This study provides strong evidence that TNF-alpha is a key molecule in the control of the inflammatory changes that occur in the RA synovium. In addition, TNF-alpha regulates IL-6 production. However, other inflammatory pathways independent of TNF-alpha may contribute to the bone and cartilage damage seen in RA.

Development of large pseudocysts adjacent to the knee joint in rheumatoid arthritis. Assessment of radiological and histopathological approaches.

Abstract This study examined the pathogenesis of large pseudocysts adjacent to knee joints in rheumatoid arthritis (RA). The radiological and histopathological features of 17 large subarticular pseudocysts in 12 knee joints of 10 patients were analyzed. Nine of the 10 patients were classified as class 2 according to Steinbrocker's functional class. Eight large pseudocysts were located at the lateral femoral condyle, seven were at the proximal part of the tibia, one was at the medial femoral condyle, and one was at the patella. The large pseudocysts were divided into two groups according to whether they did or did not connect with the joint cavity. Serial radiographs revealed that all large pseudocysts in communication with the joint cavity had enlarged gradually over the past several months. They extended from the subarticular area toward the bone marrow. Histopathological findings confirmed that holes allowing communication were located at a transitional zone between the ligament and the hyaline cartilage, and that rheumatoid granulation tissue invaded the large pseudocyst through these holes. The results of this study indicate that large pseudocysts are formed by the extension of articular inflammation. Moreover, repeated extrinsic mechanical stress due to walking and the aggressive inflammatory nature of rheumatoid arthritis play important roles in the formation of large pseudocysts.

[Expression of urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) plays a role in destruction of joint tissue in rapidly destructive coxarthropathy (RDC)].

OBJECTIVE: Proliferation of small vessels including capillaries in joint tissues is one of the significant histopathological features of rapidly destructive coxarthropathy (RDC). To examine the relationship between the angiogenesis and bone destruction, expression of urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR), which are necessary for angiogenesis was histochemically investigated. MATERIALS AND METHODS: Synovia and cartilage-bone tissues of femoral head were obtained from RDC patients at total hip replacement (THR). These joint tissues were fixed with 4% paraformaldehyde, and cartilage-bone tissues were decalcified, additionally. Immunohistochemical staining was performed on paraffin sections, using monoclonal antibodies against human uPA and uPAR. RESULT: There was a difference between expression of uPA and that of uPAR. Expression of uPA was observed in many types of cells such as osteoclasts, giant cells, fibroblast-like cells and macrophages, of which osteoclasts and giant cells were most prominent. On the other hand, expression of uPAR was detected mainly in fibroblast-like cells and macrophages and rarely seen in osteoclasts and giant cells. CONCLUSION: These results indicate that uPA-plasmin system contributes to bone destruction in RDC, and furthermore, that fibroblast-like cells and macrophages play an important role in activation of uPA-plasmin system.

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.

[Dynamics of interleukin (IL)-18 in serum, synovial fluid and synovial membrane in the patients with rheumatoid arthritis].

OBJECTIVES: IL-18 is a novel cytokine that plays an important role in the Th1 response. The aim of this study is to investigate the dynamics of IL-18 in serum, synovial fluid and synovial membrane in the patients with rheumatoid arthritis. MATERIALS AND METHODS: The serum, synovial fluid and synovial membrane were obtained from RA patients at operation. The levels of IL-18 in the serum and synovial fluid were measured by ELISA. We then examined the expression of IL-18 in synovial tissues using anti-human IL-18 monoclonal antibody in immunohistochemical study. RESULTS: The levels of IL-18 in serum and synovial fluid in RA patients were 193.7 +/- 109.7 pg/ml and 258.8 +/- 238.0 pg/ml, respectively. Compared with OA patients and normal volunteers, the level of IL-18 in RA patients was higher in both serum and synovial fluid. (P < 0.05) In synovial membrane, the cells positive for anti IL-18 antibody were confirmed not only in RA (n = 26) but also in OA (n = 7) patients. The positive cells were the synovial lining cells, macrophages, fibroblasts and endothelial cells. However, a large number of positive cells were demonstrated in synovial tissues in RA compared with OA patients.

Type II alveolar epithelial cells and interstitial fibroblasts express connective tissue growth factor in IPF.

Connective tissue growth factor (CTGF) is a growth and chemotactic factor for fibroblasts encoded by an immediate early gene that is transcriptionally activated by transforming growth factor-beta. Previous studies have shown that both CTGF messenger ribonuclear acid (mRNA) and protein are expressed in renal fibrosis and bleomycin-induced pulmonary fibrosis in mice. The aim of the present study was to investigate the localization of CTGF protein and its mRNA expression in the fibrotic lung tissue of patients with idiopathic pulmonary fibrosis (IPF). Using human fibrotic lung tissue obtained from eight autopsy cases and four biopsy cases with IPF, immunohistochemical staining, in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) were performed. The cellular immunoreactivity for CTGF was markedly increased in the lung tissue of patients with IPF, compared to normal lungs. The immunolocalization of CTGF was confined predominantly to proliferating type II alveolar epithelial cells and activated fibroblasts. In the normal lung, type II alveolar epithelial cells stained for CTGF were sparsely distributed. CTGF mRNA was localized in proliferating type II alveolar epithelial cells and activated fibroblasts in the interstitium of fibrotic lung tissues. RT-PCR analysis showed that CTGF mRNA was expressed at a higher level in fibrotic lungs than in normal lungs. In both an autocrine and a paracrine manner, type II alveolar epithelial cells and activated fibroblasts may play a critical role in pulmonary fibrosis by producing connective tissue growth factor which modulates fibroblast proliferation and extracellular matrix production.

Methotrexate inhibits rheumatoid synovitis by inducing apoptosis.

OBJECTIVE: To clarify the pharmacological action of methotrexate (MTX) on the synovium of patients with rheumatoid arthritis (RA) using severe combined immunodeficient (SCID) mice in which human RA synovial tissue had been grafted (SCID-HuRAg). METHODS: One month after engraftment of human RA tissue into SCID mice, MTX (0.3 mg/kg) was administered orally, then the appearance of apoptosis in the grafted tissue was examined by TdT mediated dUTP nick end labeling (TUNEL) staining and electron microscopy at various time points after MTX administration. In cultured synovial cells, synovial apoptotic changes after MTX treatment were studied by agarose gel electrophoresis and flow cytometric analysis. To compare the histological changes induced by MTX with those induced by other disease modifying antirheumatic drugs (DMARD) and a nonsteroidal antiinflammatory drug, histological examination of the grafted synovial tissues from SCID-HuRAg mice was conducted after 4 weeks of oral administration of MTX (0.3 mg/kg/week), salazosulfapyridine (30 mg/kg/day), auranofin (0.2 mg/kg/day), bucillamine (10 mg/kg/day), or indomethacin (2 mg/kg/day). RESULTS: A significant decrease in the number of inflammatory cells was observed in the grafted synovial tissue of MTX treated SCID-HuRAg. A similar antiinflammatory effect was not observed with the other DMARD. Induction of apoptosis was noted with MTX treatment but not with the others. The pro-apoptotic effect of MTX was also observed in synovial cell cultures. CONCLUSION: MTX induces apoptosis in RA synovium that, in turn, may contribute to its antiinflammatory effect on RA synovitis.

Urocortin in the synovial tissue of patients with rheumatoid arthritis.

Urocortin is a newly identified member of the corticotropin-releasing factor (CRF) neuropeptide family, and is known to be involved in the modulation of the inflammatory process. We examined the expression of urocortin, CRF and their receptors (CRF receptor; CRF-R) in the synovial tissue of patients with rheumatoid arthritis (RA) in order to study the possible biological roles of urocortin. Synovial tissues/fluids were obtained from 38 patients with RA, nine patients with osteoarthritis and four with trauma. We studied the concentration of urocortin in the synovial fluid using RIA, and the expression of urocortin in synovial tissue using immunohistochemistry, mRNA in situ hybridization and reverse transcriptase-PCR (RT-PCR). In addition, we examined the immunolocalization of CRF and the expression of CRF-R1, -R2-alpha and -R2-beta mRNAs utilizing RT-PCR in these synovial tissues. Urocortin concentrations in synovial fluid were higher in RA patients (79.8+/-154 pg/ml) than in control patients (12.3+/-4.8 pg/ml; P< or =0.05). Urocortin immunoreactivity and mRNA signals were both detected in synovial cells, lymphocytes, fibroblasts and macrophages. The number of urocortin-positive cells in the synovium was significantly higher in RA (73.1+/-32.1 cells per high-power field) than in control (18.4+/-10.4 cells per high-power field) patients. In addition, both urocortin immunoreactivity and mRNA signals in the synovium reached maximum levels in the active stage of RA inflammation. Moreover, the number of immunoreactive urocortin-positive cells was significantly correlated with the urocortin concentration in synovial fluid (r=0.705; P<0.001) and with histologically defined local inflammatory activity (r=0.641; P<0.001). The distribution and number of immunoreactive CRF-positive cells in synovial tissue were similar to those of urocortin-positive cells (r=0.701; P<0.001). Urocortin, CRF-R1 and CRF-R2-alpha mRNAs detected by RT-PCR were expressed in in the synovium of 10/10, 10/10 and 2/10 RA patients respectively, but CRF-R2-beta was not expressed. Urocortin was actively synthesized in the synovium of RA patients. The present study suggests that urocortin may play an important role as an autocrine and/or paracrine regulator of synovial inflammation in RA.

Synchronous multifocal osteosarcoma with lymphatic spread in the lung: an autopsy case report.

Synchronous multifocal/multicentric osteosarcoma (MOS) is a rare variant of osteosarcoma. We report here an autopsy case of a 15-year-old boy with MOS. Radiological examinations showed multiple sclerotic lesions in the left distal femur and in the ipsilateral proximal tibia without pulmonary metastasis at the first examination. Histological examination showed osteoblastic-type osteosarcoma. Despite high-dose chemotherapy the patient died of multiple bone and lung involvements 6 months after the initial diagnosis. Autopsy examination revealed prominent invasion of the tumor cells into lymphatic vessels and pleural dissemination without the formation of bulky, nodular metastasis in the lungs. Metastases in pulmonary hilar lymph nodes were noted without metastasis in other organs. Immunohistochemistry revealed that p53 protein was positive in most of the tumor cells. In summary, the present case was characterized by multiple bone involvement and prominent lymphatic spread of sarcoma cells in the lungs.

The SCID-HuRAg mouse as a model for rheumatoid arthritis.

Abstract Many animal models have been developed for a study of rheumatoid arthritis (RA). However, RA animal models are not always similar to RA patients in their response to antirheumatic drugs. Recently, humanized monoclonal antibody (mAb) has been developed for the treatment of RA, but at present there is no animal model on which to screen this mAb therapy because of problems with cross-reactivity. We therefore considered the development of a novel animal model for the screening of antirheumatic drugs using the severe combined immunodeficiency (SCID) mouse in order to prevent the rejection of human transplant cells. Following subcutaneous implantation of synovial tissue in the SCID mouse, all target cells within the SCID-HuRAg mouse were of human origin, having migrated from the implanted tissue. Moreover, human interlukin-6 and rheumatoid factor were detected in this mouse serum. We therefore propose that this SCID-HuRAg mouse is a novel, useful animal model for the study and development of new drugs for RA patients. This novel RA animal model is reviewed in this chapter.

[Telepathology at presence and in the future].

Telepathology is the performance of pathology at distance using available telecommunication links such as optic fiber, communication satellite, and integrated services digitized network (ISDN). The main applications of telepathology are to provide frozen section service, consultation between pathologists at a distance, and conducting conferences using displays. These activities are required due to the shortage and disproportional distribution of pathologists. Telepathology for frozen section service is fairly effective in providing indications for surgical procedures by discriminating malignant tumor from benign tumor, confirming metastasis to distant organs or lymph nodes, and decisions regarding the surgical margin. However, there are several public insurance problems in the spread and practice of telepathology. To solve these problems, not only from a medical approach but also from the development of mechanical engineering, economic and legal issues must be considered.

Preoperative anxiety and volume and acidity of gastric fluid in children.

Forty-three patients aged 3-6 years, undergoing minor surgery were studied. Parents staying with their children were asked to evaluate the anxiety of their children and themselves by a visual-analogue scale the night before surgery (VAS-N) and just before premedication in the morning (VAS-M). After induction, gastric fluid was collected and the volume and pH were measured. Patients with a VAS-M lower than 5 were considered the low-anxiety group (L-group; n=24) and the remainder comprised the high-anxiety group (H-group; n=19). The gastric volume of the H-group was significantly lower than that of the L-group. No difference was found in pH. A significant overall correlation of VAS-N was found between patients and their parents. These results suggest that the low level anxiety of children and their parents could not reduce the volume and acidity of gastric fluid and consequently the risk of aspiration pneumonia.

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.

Relationship between Ca-dependent change of serum PTH and extracellular Ca2+-sensing receptor expression in parathyroid adenoma.

Abnormal PTH secretion and cell growth in hyperparathyroid tissues are accompanied with reduced expression of Ca2+-sensing receptor (CaR) which plays a key role in Ca-regulated PTH release. In this study, we examined the receptor expression in parathyroid adenomas using specific anti-CaR antibody and investigated relationship between CaR expression in adenomatous tissues and parameters of Ca-dependent change of serum PTH. The results show a considerable variation in the number of CaR positive cells among the adenomatous tissues. Expression of the receptor protein was not related to set-point error but was more reduced in the patients with more elevated minimum or baseline levels of serum PTH. CaR expression was severely reduced in the patients with highly elevated maximum serum PTH, while the receptor expression was also decreased in some patients with normal maximum serum PTH. Baseline level / maximum level ratio of serum PTH was increased in these patients. In conclusion, reduced CaR expression is related to abnormality in three parameters of PTH secretion (minimum serum PTH, maximum serum PTH, and baseline level / maximum level ratio of serum PTH) and may contribute to hypersecretion from parathyroid adenomas.