Hairy/enhancer-of-split related with YRPW motif protein 1 promotes osteosarcoma metastasis via matrix metallopeptidase 9 expression.

BACKGROUND: Activation of the Notch pathway has been reported in various types of cancers. However, the role of the hairy/enhancer-of-split related with YRPW motif protein 1 (HEY1) in osteosarcoma is unknown. We examined the function of HEY1 in osteosarcoma. METHODS: Expression of HEY1 was studied in human osteosarcoma. The effects of HEY1 in osteosarcoma were evaluated in vitro and in a xenograft model. Moreover, we examined the function of matrix metallopeptidase 9 (MMP9) as a downstream effector of HEY1. RESULTS: HEY1 was upregulated in human osteosarcoma. Knockdown of HEY1 inhibited the invasion of osteosarcoma cell lines. In contrast, the forced expression of HEY1 increased the invasion of mesenchymal stem cell. In addition, lung metastases were significantly inhibited by the knockdown of HEY1. We found that MMP9 was a downstream effector of HEY1 that promotes the invasion of osteosarcoma cells. Knockdown of HEY1 decreased the expression of MMP9. Addition of MMP9 rescued the invasion of osteosarcoma cells that had been rendered less invasive by knockdown of HEY1 expression. CONCLUSIONS: Our findings suggested that HEY1 augmented the metastasis of osteosarcoma via upregulation of MMP9 expression. Therefore, inhibition of HEY1 may be a novel therapeutic strategy for preventing osteosarcoma metastasis.

Direct search for a ferromagnetic phase in a heavily overdoped nonsuperconducting copper oxide.

The doping of charge carriers into the CuO(2) planes of copper oxide Mott insulators causes a gradual destruction of antiferromagnetism and the emergence of high-temperature superconductivity. Optimal superconductivity is achieved at a doping concentration p beyond which further increases in doping cause a weakening and eventual disappearance of superconductivity. A potential explanation for this demise is that ferromagnetic fluctuations compete with superconductivity in the overdoped regime. In this case, a ferromagnetic phase at very low temperatures is predicted to exist beyond the doping concentration at which superconductivity disappears. Here we report on a direct examination of this scenario in overdoped La(2-x)Sr(x)CuO(4) using the technique of muon spin relaxation. We detect the onset of static magnetic moments of electronic origin at low temperature in the heavily overdoped nonsuperconducting region. However, the magnetism does not exist in a commensurate long-range ordered state. Instead it appears as a dilute concentration of static magnetic moments. This finding places severe restrictions on the form of ferromagnetism that may exist in the overdoped regime. Although an extrinsic impurity cannot be absolutely ruled out as the source of the magnetism that does occur, the results presented here lend support to electronic band calculations that predict the occurrence of weak localized ferromagnetism at high doping.

Functional folate receptor beta-expressing macrophages in osteoarthritis synovium and their M1/M2 expression profiles.

OBJECTIVE: The distribution of folate receptor (FR)-ß+ macrophages and their M1/M2 expression profiles were examined in osteoarthritis (OA) synovial tissues, and compared to those in rheumatoid arthritis (RA) synovial tissues and CD163+ macrophages in both OA and RA synovial tissues. METHOD: The phenotypes and fluorescein isothiocyanate (FITC)-folate uptake of FR-ß+ synovial macrophages were analysed by flow cytometry. The distribution of FR-ß+ macrophages in OA and RA synovial tissues was examined by immunofluorescent microscopy. Tumour necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), interleukin (IL)-10, and transforming growth factor (TGF)-ß expression in FR-ß+ macrophages was detected by double-immunostaining in both OA and RA synovial tissues. RESULTS: FR-ß+ macrophages were predominantly present in the synovial lining layer in OA patients. The proportion of CD163-FR-ß+ cells in synovial mononuclear cells (MNCs) was increased in OA compared to RA synovial tissues. FR-ß(high) macrophages from OA synovial tissues represented the majority of folic acid-binding cells. Although FR-ß+ or CD163+ macrophages in the synovial tissues of OA and RA patients expressed a mixed pattern of M1 and M2 macrophage markers, there were more M2 markers expressing synovial macrophages in OA than in RA patients. CONCLUSIONS: The distribution and M1/M2 expression profiles of FR-ß+ synovial macrophages were different between OA and RA synovial tissues. Thus, the findings underscore that the M1/M2 paradigm using surface markers FR-ß and CD163 is an oversimplification of macrophage subsets. Functional FR-ß present on OA synovial macrophages provides a potential tool for the diagnosis and treatment of OA.

Conservative treatment with hyperbaric oxygen therapy for cervical spondylotic amyotrophy.

STUDY DESIGN: Small case series of patients with cervical spondylotic amyotrophy (CSA) managed by conservative treatment with hyperbaric oxygen (HBO) therapy. OBJECTIVE: To study the effects of conservative treatment with HBO therapy of CSA patients. SETTING: Department of Orthopaedic Surgery, Imakiire General Hospital, Kagoshima, Japan. METHODS: This study included 10 patients with CSA who underwent rehabilitation, including cervical traction and muscle exercise, for some period of time but did not respond well to it, and were then managed by additional HBO therapy for rehabilitation. Information was obtained on the duration of symptoms and strength of the most atrophic muscle, intramedullary high-signal-intensity changes on T2-weighted magnetic resonance imaging, presence of 'snake-eyes' appearance and the number of stenotic canal levels. RESULTS: The mean duration of symptoms before HBO treatment was 3.1 months. The axial T2-weighted magnetic resonance images of all 10 patients showed a 'snake-eyes' appearance. The mean number of stenotic canal levels was 0.3. There was marked improvement on manual muscle testing from a mean of 1.9 pretreatment to a mean of 4.4 at the last follow-up after HBO therapy. The outcomes of all 10 patients, whose results were classified as excellent or good, were considered clinically satisfactory. CONCLUSION: To our knowledge, conservative treatment with HBO therapy for CSA patients has not previously been described. It appears that HBO therapy might improve ischemic injury of the anterior horns in CSA patients with short duration of symptoms.

Inhibition of Notch pathway prevents osteosarcoma growth by cell cycle regulation.

The study shows constitutive activation of the Notch pathway in various types of malignancies. However, it remains unclear how the Notch pathway is involved in the pathogenesis of osteosarcoma. We investigated the expression of the Notch pathway molecules in osteosarcoma biopsy specimens and examined the effect of Notch pathway inhibition. Real-time PCR revealed overexpression of Notch2, Jagged1, HEY1, and HEY2. On the other hand, Notch1 and DLL1 were downregulated in biopsy specimens. Notch pathway inhibition using gamma-secretase inhibitor and CBF1 siRNA slowed the growth of osteosarcomas in vitro. In addition, gamma-secretase inhibitor-treated xenograft models exhibited significantly slower osteosarcoma growth. Cell cycle analysis revealed that gamma-secretase inhibitor promoted G1 arrest. Real-time PCR and western blot revealed that gamma-secretase inhibitor reduced the expression of accelerators of the cell cycle, including cyclin D1, cyclin E1, E2, and SKP2. On the other hand, p21(cip1) protein, a cell cycle suppressor, was upregulated by gamma-secretase inhibitor treatment. These findings suggest that inhibition of Notch pathway suppresses osteosarcoma growth by regulation of cell cycle regulator expression and that the inactivation of the Notch pathway may be a useful approach to the treatment of patients with osteosarcoma.

Tumour formation by single fibroblast growth factor receptor 3-positive rhabdomyosarcoma-initiating cells.

BACKGROUND: The hypothesis that malignant tumours are generated by rare populations of cancer stem cells that are more tumourigenic than other cancer cells has gained increasing credence. The objective of this study was to identify and characterise a subpopulation of human sarcoma-initiating cells. METHODS: We examined established rhabdomyosarcoma cell lines by flow cytometry. Tumourigenesis was examined by xenograft models. Real-time PCR and immunohistochemistry were performed to examine the gene expression using cell lines and biopsy specimens. RESULTS: Rhabdomyosarcoma cell lines included small populations of fibroblast growth factor receptor 3 (FGFR3)-positive cells. FGFR3-positive KYM-1 and RD cells were more strongly tumourigenic than FGFR3-negative cells. In addition, xenoengraftment of 33% of single FGFR3-positive KYM-1 cells yielded tumour formation. Stem cell properties of FGFR3-positive cells were further established by real-time PCR, which demonstrated upregulation of undifferentiated cell markers and downregulation of differentiation markers. We showed that in the absence of serum, addition of basic fibroblast growth factor maintained and enriched FGFR3-positive cells. On the other hand, ciliary neurotrophic factor reduced the proportion of FGFR3-positive cells. Real-time PCR and immunohistochemical examination revealed that embryonal rhabdomyosarcoma patient biopsy specimens were found to over-express FGFR3. CONCLUSIONS: Our findings suggest that rhabdomyosarcoma cell lines include a minor subpopulation of FGFR3-positive sarcoma-initiating cells, which can be maintained indefinitely in culture and which is crucial for their malignancy.

Traumatic spinal-cord herniation associated with pseudomeningocele after lower-thoracic nerve-root avulsion.

BACKGROUND: Traumatic spinal-cord herniation after nerve root avulsion is rare. We report on the first patient with spinal-cord herniation associated with pseudomeningocele in the lower conus medullaris region after nerve avulsion. CASE: This 72-year-old man presented with progressive pain in the left leg and motor weakness after two traumatic accidents. Constructive interference in steady-state (CISS) imaging showed the attachment of the spinal cord to the wall of a herniated pseudomeningocele and associated syringomyelia at the level of T12. At the time of surgery, a herniated pseudomeningocele was observed. The lateral portion of the spinal cord that had herniated into the pseudomeningocele was detached from its wall; this was followed by repair of the dural defect. A redundant nerve root was observed inside the pseudomeningocele, suggesting nerve root avulsion as the primary lesion. To facilitate cerebrospinal fluid drainage from the syringomyelia, we next performed dorsal root entry zone (DREZ)tomy to the pseudomeningocele. Postoperatively, he manifested significant clinical improvement. CONCLUSIONS: This is the first report of spinal cord herniation after nerve root avulsion in the conus medullaris region. CISS imaging is highly useful for the demonstration of spinal cord herniation, syringomyelia and pseudomeningocele. To restore neurological function in patients with progressive symptoms, we recommend surgical treatment.

Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis.

Immune and inflammatory systems are controlled by multiple cytokines, including ILs and INFs. These cytokines exert their biological functions through Janus tyrosine kinases and STAT transcription factors. One such cytokine, IL-6, has been proposed to contribute to the development of rheumatoid arthritis (RA). We found that STAT3 was strongly tyrosine phosphorylated in synovial tissue of RA patients, but not those with osteoarthritis. Blockade of the IL-6-gp130-JAK-STAT3-signaling pathway might therefore be beneficial in the treatment of RA. We show here that the mRNA for the endogenous cytokine signaling repressor CIS3/SOCS3 is abundantly expressed in RA patients. To determine whether CIS3 is effective in treating experimental arthritis, a recombinant adenovirus carrying the CIS3 cDNA was injected periarticularly into the ankle joints of mice with antigen-induced arthritis or collagen-induced arthritis (CIA). Periarticular injection of CIS3 adenovirus drastically reduced the severity of arthritis and joint swelling compared with control groups. CIS3 was more effective than a dominant-negative form of STAT3 in the CIA model. Thus, induction of CIS3 could represent a new approach for effective treatment of RA.

A Kirschner wire as a transverse-axis guide to improve acetabular cup positioning.

PURPOSE: To compare cup-positioning accuracy in total hip arthroplasty (THA) with or without use of a Kirschner wire as a transverse-axis guide for pelvic alignment. METHODS: Records of 18 men and 73 women (mean age, 60 years) who underwent primary THA with (n=49) or without (n=42) use of a Kirschner wire as a transverse-axis guide for pelvic alignment were reviewed. A 2.4-mm Kirschner wire as a transversea-xis guide was inserted to the anterior superior iliac spine and was parallel to a line linking the left and right anterior superior iliac spine. The safe zone for cup positioning was defined as 30º to 50° abduction and 10º to 30º anteversion. Of the 5 operative surgeons, 2 were classified as experienced (total surgical volume >300) and 3 as inexperienced (total surgical volume of <50). The proportion of patients with the cup in the safe zone was compared in patients with or without use of the transverse-axis guide and in experienced and inexperienced surgeons. RESULTS: For inexperienced surgeons, the use of the transverse-axis guide significantly improved the proportion of patients with the cup in the safe zone from 90% to 100% for abduction, from 50% to 82.4% for anteversion, and from 40% to 82.4% for both. Patients with the cup inside or outside the safe zone were comparable in terms of body height, weight, BMI, subcutaneous fat thickness, incision length, and acetabular cup size. CONCLUSION: The use of the transverse-axis guide improved the accuracy of cup positioning by inexperienced surgeons.

Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation.

Stimulation of B lymphocytes through their antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation of a number of proteins, which leads to a cascade of biochemical changes that initiates B cell proliferation and differentiation or growth inhibition. A novel cDNA, designed APS, encoding an adaptor protein with a Pleckstrin homology (PH) domain, Src homology 2 (SH2) domain, and a tyrosine phosphorylation site was cloned from a B cell cDNA library using a yeast two hybrid system. APS is structurally similar to SH2-B, an SH2 protein that potentially binds to the immunoreceptor tyrosine-based activation motif (ITAM) as well as Lnk which is postulated to be a signal transducer that links T-cell receptor to phospholipase Cgamma, Grb2 and phosphatidylinositol 3-kinase. APS expressed only in human Burkitt's lymphoma cells among cell lines we examined and tyrosine phosphorylated in response to BCR stimulation. APS bound to Shc irrespective of stimulation and bound to Grb2 after stimulation, suggesting that it plays a role in linkage from BCR to Shc/Grb2 pathway. These results indicate that APS, SH2-B and Lnk form a new adaptor family that links immune receptors to signaling pathways involved in tyrosine-phosphorylation.

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 gamma (IFNgamma), while JAB was induced strongly by IFNgamma 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, IFNgamma-treated M1, and CTLL2 cells. Pretreatment of parental M1 cells with IFNgamma but not IFNbeta 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 IFNgamma-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 FcgammaRI 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 beta type PDGF receptor, mainly via phosphotyrosine 1021 (pY1021). Indeed, tyrosine phosphorylation of PLC-gamma, 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.

APS, an adaptor protein containing Pleckstrin homology (PH) and Src homology-2 (SH2) domains inhibits the JAK-STAT pathway in collaboration with c-Cbl.

We cloned a novel adaptor protein, APS (adaptor molecule containing Pleckstrin homology (PH) and Src Homology-2 (SH2) domains), which was tyrosine phosphorylated in response to c-kit or B cell receptor stimulation. Here, we report that APS was tyrosine phosphorylated by Janus kinase-2 (JAK2) at its C-terminal tyrosine residue and interacted with c-Cbl. Forced expression of APS in an erythropoietin (EPO)-dependent hematopoietic cell line resulted in reduced activation of STAT5 but not cell proliferation in response to EPO. APS bound to the phosphorylated tyrosine residue, Y343 of the erythropoietin receptor cytoplasmic domain. Co-expression of APS and c-Cbl, but not expression of either alone inhibited EPO-dependent STAT5 activation in 293 cells. This required the C-terminal phosphorylation site, as well as PH and SH2 domains of APS. Therefore, one of the major functions of APS is in recruitment of c-Cbl into the receptor/JAK complex, thereby inhibiting JAK signaling activity.

Functional impact of human collagen alpha2(XI) gene polymorphism in pathogenesis of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is the leading cause of myelopathy in Japan. In earlier studies, we provided genetic linkage and allelic association evidence of distinct differences in the human collagen alpha2(XI) gene (COL11A2) that might constitute inherited predisposition to OPLL. In the present study, a strong allelic association with non-OPLL (p = 0.0003) was observed with an intron 6 polymorphism [intron 6 (-4A)], in which the intron 6 (-4A) allele is more frequently observed in non-OPLL subjects than in OPLL patients. In addition, a newly identified polymorphism in exon 6 [exon 6 (+28A)] was in linkage disequilibrium with the intron 6 (-4A). The functional impact of the polymorphisms was analyzed by comparing the differences in messenger RNA (mRNA) splicing by reverse-transcription polymerase chain reaction (RT-PCR) analysis in cultured cells from the interspinous ligament and an in vitro exon trapping study. The intron 6 (-4A) allele resulted in skipping exon 6 and retaining exon 7, while the exon 6 (+28A) allele was not associated with alteration in mRNA splicing. Similar mRNA species were observed in undifferentiated osteoblast (Ob) cells and in cells from posterior longitudinal ligament of non-OPLL subjects. The region containing exons 6-8 is an acidic subdomain presumably exposed to the surface that could interact with molecules of the extracellular matrix. Accordingly, retaining exon 7 together with removal of exon 6 observed in intron 6 (-4A) could play a protective role in the ectopic ossification process because the same pattern was observed in undifferentiated Ob cells and nonossified posterior longitudinal ligament cells.

Expression of the SART1 tumor-rejection antigen in human osteosarcomas.

We recently reported a tumor-rejection antigen, SART1259, possessing tumor epitopes capable of inducing cytotoxic T lymphocytes (CTLs). This study investigated the expression of SART1259 antigen in osteosarcoma and other skeletal malignant tumors to explore for a potential molecule for use in specific immunotherapy. The SART1259 antigen was detected in the cytosol fraction of 13 of 21 (62%) osteosarcoma cell lines and 3 of 8 (38%) osteosarcoma tissues, and 3 of 10 (30%) malignant fibrous histiocytoma (MFH) tissues. The HLA-A24+ and SART1259+ osteosarcoma cells were recognized by the HLA-A24 restricted and SART1 specific CTLs. These results raise a possibility that the SART1259 would be an appropriate molecule for use in specific immunotherapy of approximately one-third of HLA-A24+ patients with osteosarcoma and MFH.

Modulation of fas-mediated apoptosis in osteosarcoma cell lines.

Fas/APO-1 (or CD95) is a 45-kDa cell surface glycoprotein that transduces cellular death signals for apoptosis upon cross-linking with the Fas ligand, which is experimentally replaced by anti-Fas antibodies. We examined the Fas expression at the protein and mRNA levels, and susceptibility to anti-Fas-mediated apoptosis, in 14 osteosarcoma cell lines. Ten of 14 ostesarcoma cell lines constitutively expressed detectable levels of Fas on the cell surface with positive cell rates ranging from 10.2 to 72.4%. Four other cell lines expressed Fas almost exclusively in the cytoplasm. An antibody against Fas was able to induce Fas-mediated cell death only in two cell lines with high levels of surface Fas. In the presence of the protein synthesis inhibitor cyclohexamide, antibody to Fas led to an induction of apoptosis in ten of the osteosarcoma cell lines antibody-concentration dependently. These data suggest that Fas is a potential protein related to apoptosis in osteosarcomas when the sensitizing activity of cyclohexamide on anti-Fas-mediated cytotoxicity was exerted.

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.

Transcriptional activation of p21 by vitamin D(3) or vitamin K(2) leads to differentiation of p53-deficient MG-63 osteosarcoma cells.

p21 (WAF1/CIP1) is a downstream effector of p53 and mediates growth arrest by inhibiting the action of G(1) cyclin-dependent kinases. However, it has been reported that the p21 expression was triggered by multiple differentiation-inducing agents by a p53-independent pathway. These agents induced expression of p21 by binding to specific DNA elements and modulating transcriptional initiation. We demonstrated that the gene encoding p21 was not only a vitamin D(3) target gene but also a vitamin K(2) target gene in the cells and that their differentiation was well related to the transcriptional activation of the p21 gene. Transient overexpression of p21, using adenovirus-driven p21 expression plasmid, in MG-63 cells in the absence of vitamins D(3) and K(2) resulted in their differentiation. The transcriptional activation of p21 by vitamin D(3) or vitamin K(2) in p53-deficient osteosarcoma cells demonstrated the p53-independent role of p21 in human osseous differentiation. HUM PATHOL 32:410-416.

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.

Histamine-stimulated production of matrix metalloproteinase 1 by human rheumatoid synovial fibroblasts is mediated by histamine H1-receptors.

The purpose of this study was to investigate the role of histamine in human rheumatoid synovial fibroblasts in the production of factors responsible for tissue remodelling and cartilage breakdown in rheumatoid arthritis. We examined the effects of histamine of tritiated thymidine incorporation, production of matrix metalloproteinase-1 (MMP-1), histamine H1-receptor expression, phosphoinositide metabolism and intracellular calcium ion concentration ([Ca2+]i) in human rheumatoid synovial fibroblasts. Tritiated thymidine incorporation studies demonstrated that histamine markedly stimulated the proliferation of rheumatoid synovial fibroblasts. Immunofluorescence and Northern blot analyses revealed that proMMP-1 production was also stimulated by histamine. The levels of inositol phosphates and [Ca2+]i in the cells were elevated in response to histamine, indicating that the cells expressed histamine H1-receptors; and Northern blot analysis indicated that these H1-receptors were up-regulated by histamine. In in situ hybridization, large amounts of histamine H1-receptor mRNA were also detected in rheumatoid synovial tissue. These results suggest that the interaction between H1-receptor expression in rheumatoid synovial fibroblasts and histamine secretion by mast cells and macrophages in the affected sites is an important event responsible for tissue remodelling and joint destruction in rheumatoid arthritis.