Setsuro Komiya

Linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis

OBJECTIVEnTo examine the occurrence of apoptosis in human osteoarthritis (OA) cartilage, and to determine its relationship to cartilage degradation.nnnMETHODSnKnee cartilage was obtained from subjects at autopsy, from a tissue bank, and from OA patients undergoing total joint replacement surgery. Chondrocytes were isolated and the number of apoptotic cells was analyzed by flow cytometry. Apoptotic cells in cartilage sections were identified by the detection of DNA strand breaks. Electron microscopy was applied to demonstrate morphologic changes, and Safranin O staining was performed to analyze the relationship between apoptosis and proteoglycan depletion.nnnRESULTSnFlow cytometry on cell suspensions prepared from collagenase digests of cartilage showed that approximately 22.3% of OA chondrocytes and 4.8% of normal chondrocytes were undergoing apoptosis. Staining of cartilage sections demonstrated the presence of apoptotic cells in the superficial and middle zones. Cartilage areas that contained apoptotic cells showed proteoglycan depletion, and the number of apoptotic cells was significantly correlated with the OA grade.nnnCONCLUSIONnThese observations demonstrate increased chondrocyte apoptosis in OA cartilage. Chondrocyte apoptosis and proteoglycan depletion are anatomically linked and may be mechanistically related.

Detection of SYT-SSX Fusion Transcripts in Synovial Sarcoma by Reverse Transcription-Polymerase Chain Reaction Using Archival Paraffin-Embedded Tissues

The reciprocal translocation t(X;18)(p11;q11) is known to be highly characteristic of synovial sarcoma, and its consequence, an SYT-SSX fusion gene, is expected to be a diagnostic molecular marker. In this study, we conducted a reverse transcription-polymerase chain reaction-based assay to detect the SYT-SSX fusion gene transcripts using archival formalin-fixed, paraffin-embedded tumor specimens from a series of 32 synovial sarcoma cases including 6 tumors found in unusual anatomical sites. The SYT-SSX fusion transcripts could be detected in 30 of 32 paraffin-embedded specimens (94%). A subsequent sequence analysis using the polymerase chain reaction products confirmed that the detected messages were derived from either the SYT-SSX1 (22 cases) or SYT-SSX2 (8 cases) fusion gene. Of 23 SYT-SSX-positive monophasic tumors, 16 tumors had an SYT-SSX1 fusion transcript. Fusion transcripts were detectable in all the 7 biphasic tumors analysed, one of which had an SYT-SSX2 fusion transcript. All of the six tumors at unusual locations (lung; 3, metastasis to the abdominal cavity from a tumor of retroperitoneal origin; 1, sacral region; 1, iliopsoas muscle; 1) contained detectable messages. Our results indicate that this molecular assay can be applied to archival formalin-fixed, paraffin-embedded tumor tissues as a feasible and reliable molecular technique for the diagnosis of synovial sarcoma.

Asplenia and polysplenia syndrome.

This report described the morphological characteristics of seven cases of asplenia syndrome and three of polysplenia syndrome. Each syndrome has been characterized by a tendency for symmetric development of normally asymmetric organs, with varying degrees of cardiovascular anomalies. These latter anomalies are usually present in asplenia syndrome to a greater extent than in polysplenia syndrome. While, as observed in our material, the conotruncal anomalies were present more commonly in cases with asplenia, and absence of inferior vena cava with azygos continuation was seen specifically in all the cases with polysplenia. This evidence implied the presence of some pathogenetic distinction between the two syndromes. ACTA PATHOL. JPN. 32: 505∼511, 1982.

MORPHOLOGICAL AND BIOCHEMICAL EVIDENCE FOR APOPTOSIS IN THE TERMINAL HYPERTROPHIC CHONDROCYTES OF THE GROWTH PLATE

The purpose of this study was to investigate the mechanism of cell death in chondrocytes of the growth plate. In the degenerative chondrocyte zone of the growth plate, apoptotic chondrocytes were defeated by the in situ nick end labelling method, by DNA analysis in agarose gel, and by electron microscopy. The results of the in situ nick end labelling method and the occurrence of a ladder pattern of DNA in agarose gel analysis indicated the activation of endogenous endonucleases, resulting in DNA fragmentation. Electron micrographs showed the early morphological changes associated with apoptosis. This report presents both morphological and biochemical evidence for apoptosis in the terminal hypertrophic chondrocytes of the growth plate. These data suggest that apoptosis of degenerative chondrocytes may play an important role in the control of normal and pathological endochondral ossification.

CIS3 and JAB have different regulatory roles in interleukin-6 mediated differentiation and STAT3 activation in M1 leukemia cells

We have reported JAK-signaling modulators, CIS1 (cytokine-inducible SH2 protein-1), CIS3 and JAB (JAK2 binding protein), which are structurally related. In M1 myeloid leukemia cells, CIS3 was induced by neither interleukin 6 (IL6) nor interferon γ (IFNγ), while JAB was induced strongly by IFNγ and slightly by IL6 and leukemia inhibitory factor (ILF). Forced expression of CIS3 and JAB in M1 cells prevented IL6- or LIF-induced growth arrest and differentiation, even when their expression levels were comparable to endogenous ones in several cell lines such as HEL, UT-7, IFNγ-treated M1, and CTLL2 cells. Pretreatment of parental M1 cells with IFNγ but not IFNβ resulted in suppression of LIF-induced STAT3 activation and differentiation, further supporting that physiological level of JAB is sufficient to inhibit LIF-signaling. However, unlike JAB, CIS3 did not inhibit IFNγ-induced growth arrest, suggesting a difference in cytokine specificity between CIS3 and JAB. CIS3 inhibited STAT3 activation with slower kinetics than JAB and allowed rapid c-fos induction and partial FcγRI expression in response to IL6. In 293 cells, CIS3 as well as JAB bound to JAK2 tyrosine kinase domain (JH1), and inhibited its kinase activity, however, the effect of CIS3 on tyrosine kinase activity was weaker than that of JAB, indicating that CIS3 possesses lower affinity to JAK kinases than JAB. These findings suggest that CIS3 is a weaker inhibitor than JAB against JAK signaling, and JAB and CIS3 possess different regulatory roles in cytokine signaling.

APS, an adaptor protein containing PH and SH2 domains, is associated with the PDGF receptor and c-Cbl and inhibits PDGF-induced mitogenesis

Previously we cloned a novel adaptor protein, APS (adaptor molecules containing PH and SH2 domains) which was tyrosine phosphorylated in response to c-kit or B cell receptor stimulation. Here we report that APS was expressed in some human osteosarcoma cell lines, markedly so in SaOS-2 cells, and was tyrosine-phosphorylated in response to several growth factors, including platelet derived growth factor (PDGF), insulin-like growth factor (IGF), and granulocyte-macrophage colony stimulating factor (GM-CSF). Ectopic expression of the wild type APS, but not C-terminal truncated APS, in NIH3T3 fibroblasts suppressed PDGF-induced MAP kinase (Erk2) activation, c-fos and c-myc induction as well as cell proliferation. In vitro binding experiments suggest that APS bound to the β type PDGF receptor, mainly via phosphotyrosine 1021 (pY1021). Indeed, tyrosine phosphorylation of PLC-γ, which has been demonstrated to bind to pY1021, but not that of PI3 kinase and associated proteins, was reduced in APS transformants. PDGF induced phosphorylation of the tyrosine residue of APS close to the C-terminal end. In vitro and in vivo binding experiments indicate that the tyrosine phosphorylated C-terminal region of APS bound to c-Cbl, which has been shown to be a negative regulator of tyrosine kinases. Since coexpression of c-Cbl with wild type APS, but not C-terminal truncated APS, synergistically inhibited PDGF-induced c-fos promoter activation, c-Cbl could be a mechanism of inhibitory action of APS on PDGF receptor signaling.

Molecular detection of EWS-FLI1 chimeric transcripts in Ewing family tumors by nested reverse transcription-polymerase chain reaction : application to archival paraffin-embedded tumor tissues

Chromosomal translocations generating unique chimeric genes are highly characteristic of specific sarcomas, and their use as diagnostic markers has been suggested. From a diagnostic pathologic point of view, detection of such cytogenetic or molecular aberrations applicable to routinely processed archival tissue specimens is considered a powerful tool for tumor diagnosis. To assess the feasibility and reliability of the molecular detection of the transcript originating from the chimeric gene in paraffin‐embedded tumor specimens, we performed a nested reverse transcription‐polymerase chain reaction (RT‐PCR)‐based assay to detect the EWS‐FLI1 chimeric message in a series of Ewing family tumors. Of 24 paraffin‐embedded tumor specimens from 23 cases analyzed, the chimeric message was detectable in 20 (83%) specimens from 20 cases (87%) by this nested RT‐PCR assay, whereas none of 7 small round cell tumors not from this family (3 alveolar rhabdomyosarcomas, 2 neuroblastomas, 2 malignant lymphomas) showed detectable chimeric messages. In the sequence analysis of the PCR products, the amplified chimeric messages contained the junctions between exon 7 of the EWS gene and any one of exons 5, 6 and 8 of the FLI1 gene. The detection process was usually completed within 3 days, except for the subseqent sequence analysis. Our results endorse the use of this molecular assay as an ancillary technique in the diagnosis of Ewing family tumors using paraffin‐embedded material.

Correlation Between Vitamin D Receptor Genotypes and Bone Mineral Density in Japanese Patients with Osteoporosis

In order to better understand the pathogenesis of osteoporosis, we investigated the correlation between the vitamin D receptor (VDR) genotypes defined by BsmI restriction enzyme, as well as other related factors, and the bone mineral density (BMD) at the lumbar spine in 90 Japanese patients with osteoporosis. The same study was performed in 36 patients with osteoarthrosis of the hip joint and 92 healthy volunteers. The majority of the VDR genotypes were bb, and a few of the population showed either the BB or Bb genotype in all three groups. There was no statistical difference in the frequencies of these VDR genotypes in the three groups. The mean age-matched value of BMD (Z scores) at the lumbar spine in patients with osteoporosis was significantly lower than that in patients with osteoarthrosis or healthy volunteers. The mean Z scores of the healthy volunteers with bb genotype were significantly higher than those with BB genotype, whereas those of the osteoporosis patients with BB genotype were significantly higher than those with Bb genotype. There was no significant difference in the mean Z scores between bb and Bb genotypes in patients with osteoporosis and healthy volunteers. No significant difference was seen in the mean Z scores in patients with osteoarthrosis regardless of genotype. On the other hand, body weight significantly correlated with BMD in patients with osteoporosis by simple- and multiple-regression analysis. These results indicate that the BMD at the lumbar spine in Japanese patients with osteoporosis is affected by body weight, and might be affected partially by the VDR genotypes defined by BsmI.

Expression of a newly defined tumor-rejection antigen SART3 in musculoskeletal tumors and induction of HLA class I-restricted cytotoxic T lymphocytes by SART3-derived peptides.

We recently reported that a SART3 tumor‐rejection antigen possessing tumor epitopes is capable of inducing HLA class I‐restricted and tumor‐specific cytotoxic T lymphocytes in cancer patients. We studied the expression of the SART3 protein in musculoskeletal tumors to find a molecule for potential use in tumor‐specific immunotherapy. The SART3 was detected at protein levels in 100% of the osteosarcoma cell lines (n = 20), in 50% of the musculoskeletal tumor tissue specimens (n = 32), and at notable levels in 67% of osteosarcoma tissues (n = 9) and malignant fibrous histiocytosis tissues (n = 9), respectively. SART3‐derived peptides at positions 109‐118 and 315‐323 induced HLA‐A24‐restricted tumor‐specific cytoxic T lymphocytes from peripheral blood mononuclear cells of patients with osteosarcoma or malignant fibrous histiocytosis. These peptide‐induced cytotoxic T lymphocytes recognized HLA‐A24+ SART3+ osteosarcoma cells but not HLA‐A24− or SART3− cells. These results suggest that the SART3 protein and its derived peptides could be molecules appropriate for use in specific immunotherapies for approximately 60% of HLA‐A24+ patients with osteosarcoma or malignant fibrous histiocytosis.

Il-6 in a pleomorphic type of malignant fibrous histiocytoma presenting high fever

We describe a rare case of pleomorphic type of malignant fibrous histiocytoma (MFH) in the buttock that presented a systemic involvement. The case was of a 58-year-old woman presenting hepatic dysfunction and inflammatory reactions including fever, positive C-reactive protein (CRP), an elevated erythrocyte sedimentation rate, and high levels of platelets and ferritin. The fever of 3 months duration subsided on the first postoperative day. The MFH resection also brought rapid normalization in CRP, platelets, and leukocytes. The local and systemic productions of cytokines induced by this tumor were evaluated. In vivo and in vitro production of interleukin (IL)-6, IL-1beta, and tumor necrosis factor alpha by tumor cells were measured using enzyme-linked immunosorbent assay. Blood samples taken preoperatively, tumor tissues, and the primary culture medium showed extraordinarily high IL-6 levels. The plasma IL-6 level was normalized postoperatively. Immunohistochemistry showed the positivity of tumor cells for IL-6. The IL-6 produced by the tumor was concluded to have been responsible for the systemic illness.