In vivo mechanical condition plays an important role for appearance of cartilage tissue in ES cell transplanted joint.

The objective of this study was to evaluate the effects of the mechanical environment on the formation of cartilage tissue in transplanted embryonic stem (ES) cells. Full-thickness osteochondral defects were created on the patella groove of SD rats, and ES cells (CCE ES cells obtained from 129/Sv/Ev mice and Green ES FM260 ES cells obtained from 129SV [D3] - Tg [NCAG-EGFP] CZ-001-FM260Osb mice) were transplanted into the defects embedded in collagen gel. The animals were randomly divided into either the joint-free group (JF group) or the joint-immobilized group (JI group) for 3 weeks after a week postoperatively. The results showed that cartilage-like tissue formed in the defects of the JF group whereas large teratomatous masses developed in the defects of the JI group. Some parts of the cartilage-like tissue and the teratomatous masses were positively stained with immunostain for GFP when the Green ES FM260 ES cells were transplanted. It is suggested that the environment plays an important role for ES cells in the process of repairing cartilage tissue in vivo.

Detection of gene expression in synovium of patients with osteoarthritis using a random sequencing method.

BACKGROUND: The etiology of osteoarthritis (OA) is multifactorial and current research attributes it to a complex network of biochemical factors. We attempted to identify important molecules in OA joint destruction. PATIENTS AND METHODS: Synovium was collected from 2 women with hip OA. Total RNA was extracted from the combined synovium. Messenger RNAs (mRNAs) were randomly sequenced for identification with the oligo-capping method. mRNA expression of 9 genes that were found to be frequently expressed was compared in synovium from 7 OA patients and 2 control patients with no signs of arthritis. RESULTS: We sequenced 7,339 mRNAs in total and identified 4,247 different kinds, which were ranked in order of frequency. Fibronectin was the protein most frequently expressed (230/7,339), followed by matrix metalloproteinases (MMPs) 1 and 3. The 9 genes selected were those encoding fibronectin 1, MMP1, MMP3, tissue inhibitor of metalloproteinase 3, apolipoprotein L-I (APOL1), syndecan binding protein, insulin-like growth factor binding protein 5, heat shock protein 90, and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5). We investigated expression of these 9 genes in synovium from the 7 individual patients with OA. All 9 genes were expressed in OA and control synovium. Expression of MMP1 mRNA was weak in OA samples, however, while expression of ADAMTS5 and APOL1 mRNAs was weak in the controls and some of the OA samples. INTERPRETATION: ADAMTS5 and APOL1 may have important roles in the mechanism of OA.

Matrix morphogenesis in cornea is mediated by the modification of keratan sulfate by GlcNAc 6-O-sulfotransferase.

Matrix assembly and homeostasis in collagen-rich tissues are mediated by interactions with proteoglycans (PGs) substituted with sulfated glycosaminoglycans (GAGs). The major GAG in cornea is keratan sulfate (KS), which is N-linked to one of three PG core proteins. To ascertain the importance of the carbohydrate chain sulfation step in KS functionality, we generated a strain of mice with a targeted gene deletion in Chst5, which encodes an N-acetylglucosamine-6-O-sulfotransferase that is integral to the sulfation of KS chains. Corneas of homozygous mutants were significantly thinner than those of WT or heterozygous mice. They lacked high-sulfated KS, but contained the core protein of the major corneal KSPG, lumican. Histochemically stained KSPGs coassociated with fibrillar collagen in WT corneas, but were not identified in the Chst5-null tissue. Conversely, abnormally large chondroitin sulfate/dermatan sulfate PG complexes were abundant throughout the Chst5-deficient cornea, indicating an alteration of controlled PG production in the mutant cornea. The corneal stroma of the Chst5-null mouse exhibited widespread structural alterations in collagen fibrillar architecture, including decreased interfibrillar spacing and a more spatially disorganized collagen array. The enzymatic sulfation of KS GAG chains is thus identified as a key requirement for PG biosynthesis and collagen matrix organization.

Overexpression of LAMP3/TSC403/DC-LAMP promotes metastasis in uterine cervical cancer.

LAMP3 (DC-LAMP, TSC403, CD208) was originally isolated as a gene specifically expressed in lung tissues. LAMP3 is located on a chromosome 3q segment that is frequently amplified in some human cancers, including uterine cervical cancer. Because two other members of the LAMP family of lysosomal membrane glycoproteins, LAMP1 and LAMP2, were previously implicated in potentially modulating the interaction of vascular endothelial and cancer cells, we hypothesized that LAMP3 might also play an important part in metastasis. To clarify the metastatic potential of LAMP3 in cervical cancers, we transfected a LAMP3 expression vector into a human uterine cervical cancer cell line, TCS. In an in vitro invasion assay, the migration of LAMP3-overexpressing TCS cells was significantly higher than in control TCS cells. In an in vivo metastasis assay, distant metastasis was detected in 9 of 11 LAMP3-overexpressing TCS cell-injected mice and in only 1 of 11 control mice. Histologic study showed that LAMP3-overexpressing cells readily invaded into the lymph-vascular space. In clinical samples, quantitative real-time reverse transcription-PCR (RT-PCR) analyses showed that LAMP3 mRNA was significantly up-regulated in 47 of 47 (100%) cervical cancers and in 2 of 15 (13%) cervical intraepithelial neoplasias, compared with a low level of LAMP3 mRNA expressed in normal uterine cervixes. Interestingly, high LAMP3 expression was significantly correlated with the overall survival of patients with stage I/II cervical cancers. These findings indicate that LAMP3 overexpression is associated with an enhanced metastatic potential and may be a prognostic factor for cervical cancer.

Mutated G-protein-coupled receptor GPR10 is responsible for the hyperphagia/dyslipidaemia/obesity locus of Dmo1 in the OLETF rat.

1. We have confirmed the Diabetes Mellitus OLETF type I (Dmo1) effect on hyperphagia, dyslipidaemia and obesity in the Otsuka Long-Evans Tokushima Fatty (OLETF) strain. The critical interval was narrowed down to 570 kb between D1Got258 to p162CA1 by segregation analyses using congenic lines. 2. Within the critical 570 kb region of the Dmo1 locus, we identified the G-protein-coupled receptor gene GPR10 as the causative gene mutated in the OLETF strain. The ATG translation initiation codon of GPR10 is changed into ATA in this strain and, so, is unavailable for the initiation of translation. 3. The GPR10 protein has a cognate ligand, namely prolactin-releasing peptide (PrRP). Centrally administered PrRP suppressed the food intake of congenic rats that have a Brown Norway derived Dmo1 region (i.e. with wild-type GPR10), but did not suppress that of the OLETF strain, indicating that GPR10 is without function and could explain hyperphagia in the OLETF strain. 4. Moreover, when restricted in food volume to the same level consumed by the congenic strain, OLETF rats showed few differences in the parameters of dyslipidaemia and obesity compared with congenic strains. 5. Taken together, these results demonstrate that the mutated GPR10 receptor is responsible for the hyperphagia leading to obesity and dyslipidaemia in the obese diabetic strain rat.

Use of bone morphogenetic protein 2 and diffusion chambers to engineer cartilage tissue for the repair of defects in articular cartilage.

OBJECTIVE: To examine the ability of cartilage-like tissue, generated ectopically in a diffusion chamber using recombinant human bone morphogenetic protein 2 (rHuBMP-2), to repair cartilage defects in rats. METHODS: Muscle-derived mesenchymal cells were prepared by dissecting thigh muscles of 19-day postcoital rat embryos. Cells were propagated in vitro in monolayer culture for 10 days and packed within diffusion chambers (10(6)/chamber) together with type I collagen (CI) and 0, 1, or 10 microg rHuBMP-2, and implanted into abdominal subfascial pockets of adult rats. Tissue pellets were harvested from the diffusion chambers at 2 days to 6 weeks after implantation, and examined by histology, by reverse transcription-polymerase chain reaction (PCR) for aggrecan, CII, CIX, CX, and CXI, MyoD1, and core binding factor a1/runt-related gene 2, and by real-time PCR for CII. Tissue pellets generated in the chamber 5 weeks after implantation were transplanted into a full-thickness cartilage defect made in the patellar groove of the same strain of adult rat. RESULTS: In the presence of 10 microg rHuBMP-2, muscle-derived mesenchymal cells expressed CII messenger RNA at 4 days after transplantation, and a mature cartilage mass was formed 5 weeks after transplantation in the diffusion chamber. Cartilage was not formed in the presence of 1 microg rHuBMP-2 or in the absence of rHuBMP-2. Defects receiving cartilage engineered with 10 microg rHuBMP-2 were repaired and restored to normal morphologic condition within 6 months after transplantation. CONCLUSION: This method of tissue engineering for repair of articular defects may preclude the need to harvest cartilage tissue prior to mosaic arthroplasty or autologous chondrocyte implantation. Further studies in large animals will be necessary to validate this technique for application in clinical practice.

Embryonic stem cells form articular cartilage, not teratomas, in osteochondral defects of rat joints.

Embryonic stem (ES) cells are considered to be a potential tool for repairing articular cartilage defects, but so far it has been impossible to cause these cells to differentiate into chondrocytes exclusively, either in vivo or in vitro. To explore a potential new cell source of cell transplantation for articular cartilage defects, we transplanted ES cells into articular cartilage defects in immunosuppressed rats. ES cells (AB2.2 or CCE cells) were transplanted into articular cartilage defects in the patellar groove of immunosuppressed rats treated with cyclosporine. The cells were histologically observed until 8 weeks after transplantation. To determine whether the repair tissue in the defect in the AB2.2-transplanted group was derived from the transplanted cells, the neomycin-resistant gene, which had been transfected into AB2.2 cells but does not exist in rat cells, was used for detection. The cells produced cartilage, resulting in repair of the defects from 4 weeks until 8 weeks after the transplantation without forming any teratomas. The neomycin-resistant gene was detected in every sample, demonstrating that the repair tissue in the AB2.2-transplanted group was derived from the transplanted AB2.2 cells. The environment of osteochondral defects is chondrogenic for ES cells. ES cells may thus be a potential tool for repairing articular cartilage defects.

A flow cytometric method for determination of the interferon receptor IFNAR2 subunit in peripheral blood leukocyte subsets.

INTRODUCTION: It is expected that expression levels in the peripheral blood mononuclear cells (PBMC) of IFNAR2, a subunit of the interferon (IFN) receptor, may be a marker for predicting IFN response. In the present study, we have established a rapid and convenient method for assaying IFNAR2, using flow cytometry. METHODS: Fifty microliters of whole blood from healthy volunteers was treated with an anti-IFNAR2 antibody and stained with a Fluorescein isothiocyanate (FITC)-conjugated secondary antibody. In addition, the cells were stained with subset-specific antibodies conjugated with phycoerythrin (PE) and PE covalently linked to cyanin 5 at the same time. The mean FITC-fluorescence intensities were analyzed separately by gating on subset-specific regions. RESULTS: IFNAR2 was detected in most lymphocytes, monocytes, and granulocytes, although IFNAR2 expression was higher in the monocytes and granulocytes than in the lymphocytes. The intra- and interdaily variations of IFNAR2 in lymphocytes, monocytes, and granulocytes were small. Among the lymphocyte subsets, IFNAR2 showed high expression in natural killer (NK) cells and low expression in T lymphocytes. The effect of IFN-alpha on IFNAR2 expression was examined in vitro. A down-regulation of IFNAR2 was observed by IFN-alpha above 100 IU/ml. DISCUSSION: This assay may be useful for examining IFNAR2 in various leukocyte subsets, separately, as well as providing a rapid and easy method for monitoring expression of type I IFN receptors.

A < 1.7 cM interval is responsible for Dmo1 obesity phenotypes in OLETF rats.

1. Dmo1 (Diabetes Mellitus OLETF type I) is a major quantitative trait locus for dyslipidaemia, obesity and diabetes phenotypes of male Otsuka Long Evans Tokushima Fatty (OLETF) rats. 2. Our congenic lines, produced by transferring Dmo1 chromosomal segments from the non-diabetic Brown Norway (BN) rat into the OLETF strain, have confirmed the strong, wide-range therapeutic effects of Dmo1 on dyslipidaemia, obesity and diabetes in the fourth (BC4) and fifth (BC5) generations of congenic animals. Analysis of a relatively small number of BC5 rats (n = 71) suggested that the critical Dmo1 interval lies within a < 4.9 cM region between D1Rat461 and D1Rat459. 3. To confirm the assignment of the Dmo1 critical interval, we intercrossed BC5 animals to produce a larger study population (BC5:F1 males; n = 406). For the present study, we used bodyweight at 18 weeks of age as an index of obesity; this phenotype is representative of the closely associated dyslipidaemia and hyperglycaemia phenotypes. 4. Interval mapping assigned logarithm of odds (LOD) peaks at the D1Rat90 marker (LOD = 9.11). One LOD support interval lies within the < 1.7 cM region between D1Rat461 and D1Rat459. 5. This large intercross study confirms that Dmo1 is likely localized within the interval.

Immunohistochemical and genetic analysis of mandibular cysts in heterozygous ptc knockout mice.

BACKGROUND: Alterations of human patched (ptc) homolog have been proven to be responsible for basal cell nevus syndrome (BCNS). Mandibular cysts in heterozygous ptc knockout mouse (ptc+/- mouse) were microradiologically, histologically, immunohistochemically, and genetically examined to investigate the possible role of the ptc gene and its associates in the jaw cysts. METHODS: The mandibular bones were prepared from 63 ptc+/- mice and 6 ptc+/+ mice. Soft X-ray radiographs and histological sections were examined for detection of the presence of mandibular cysts. The mandibular cysts were immunohistochemically investigated using anti-ptc, shh, and smo antibodies. PCR analysis of loss of heterozygosity (LOH) of ptc was performed in genomic DNA from the mandibular cysts. RESULTS: Six ptc+/+ mice showed no pathologic change in any examinations. Microradiologically, ptc+/- mice did not show any apparent lesion. Mandibular cysts were often multiple, and were histologically detected in the alveolar bones or periodontal ligaments of the molars in 16 (25.4%) ptc+/- mice. The mandibular cysts were lined by thin parakeratotic stratified squamous epithelium and contained keratinized materials. Immunohistochemical examination showed sonic hedgehog (shh) protein mainly in cyst lining epithelium, and ptc and smoothened (smo) proteins in cyst lining epithelium, and surrounding fibrous connective tissue. Expression of ptc protein in the cyst lining epithelium tended to be weak as compared with incisor enamel organs and gingival stratified squamous epithelium. LOH of the ptc gene couldn't be found in lining epithelium of mandibular cysts in any ptc+/- mice. CONCLUSIONS: Ptc+/- mouse is a useful model of BCNS from the standpoint of occurrence of jaw cysts, and downregulation of ptc protein in cyst lining epithelium caused by gene targeting would be associated with formation of jaw cysts in ptc+/- mice.

Molecular cloning, mRNA expression and chromosomal localization of mouse angiotensin-converting enzyme-related carboxypeptidase (mACE2).

We isolated two mouse cDNA clones which show significant similarities with human angiotensin-converting enzyme-related carboxypeptidase (ACE2). The cDNAs were 2746 and 1995 bp in length and seemed to arise from the same gene by alternative splicing. The longer cDNA encoded a 798-amino acid protein containing the sequence motif conserved among zinc metallopeptidases. Mouse ACE2 showed 83% identity with human ACE2. Northern blot analysis revealed that 2.8- and 2.0-kb transcripts were expressed mainly in the kidney and the lungs. The mouse ACE2 gene was mapped to chromosome X 70.5 cM.

OASIS is a transcriptional activator of CREB/ATF family with a transmembrane domain.

Murine OASIS is a putative CREB/ATF family transcription factor that is induced in gliosis, but its molecular role has not been determined. We have isolated the human OASIS gene and investigated the potential of OASIS protein as a transcriptional activator. We found that OASIS can activate transcription through box-B elements but not through the somatostatin CRE. OASIS contains a putative C-terminal hydrophobic transmembrane domain, a typical structural feature for the transcription factors activated by regulated intramembrane proteolysis. Truncation of the OASIS transmembrane domain resulted in a significant increase in transcriptional activity and altered its subcellular localization from the endoplasmic reticulum to the nucleus. Western blot analysis of transfected cells identified OASIS polypeptides of 82 and 66 kDa. These results suggest that the transmembrane domain plays an important role in the regulation of transcriptional activation by OASIS.

Molecular and immunohistochemical analysis of signaling adaptor protein Crk in human cancers.

Crk is a signaling adaptor protein which is mostly composed of SH2 and SH3 domains, and has been shown to play a pivotal role in cell proliferation, differentiation, and migration. Because Crk was originally isolated as an avian sarcoma virus CT10 encoding oncoprotein v-Crk, we examined a potential role for c-Crk in the carcinogenesis of human cancers. First, to analyze gene mutations of c-Crk, we isolated a human bacterial artificial chromosome clone containing Crk genome and exon/intron structures. However, polymerase chain reaction-single strand conformation polymorphism methods failed to show any genomic mutations in the Crk exon which could be related to carcinogenesis. Second, immunohistochemical analysis of c-Crk-II demonstrated that the levels of c-Crk-II were significantly elevated in most of the tumors, particularly in the colon and lung cancers. Furthermore, immunoblot analysis using human lung cancer cell lines revealed that the expression levels of c-Crk-II were correlated to growth rates of cells. The elevated expression levels of c-Crk-II might be related to the development of human cancers.