Aulis Marttinen

Measurement of total and local bone morphogenetic protein concentration in bone tumours.

Summary. Bone morphogenetic protein (BMP) has been shown to be one of the significant factors in the prognosis of bone tumours. In normal development BMP induces new bone formation and later takes part in fracture healing, but its function in malignant tumours is not known. In this study the concentration of bone morphogenetic protein was measured in primary bone tumours by two methods. Local staining intensity was detected immunohistologically by the avidin-biotin-peroxidase method determining the highest dilution of anti-serum against bovine bone morphogenetic protein. The total amount of BMP in a tumour sample was measured by an enzyme-linked immunosorbent assay technique after digesting the tissue with collagenase to remove proteins from the connective tissue. Immunohistochemical staining showed that bone morphogenetic protein was present in the cytoplasm and in reactive bone formed by malignant cells. The local concentration was highest in the tissue of giant cell tumours compared to chondrosarcoma, osteosarcoma and benign bone tumours. The total amount in malignant bone tumours was 2.4 times higher compared to benign bone tumours.Résumé. Il a été démontré que la protéine morphogénétique osseuse (BMP) est un facteur important dans le pronostic des affections osseuses. Dans un développement osseux normal, cette protéine induit la formation de nouveau tissu osseux et contribue, par exemple, dans les fractures, à la consolidation. Dans les affections malignes, la fonction précise de la BMP reste inconnue. Dans la présente étude, la concentration de protéine morphogénétique osseuse a été mesurée dans des tumeurs osseuses primaires à l’aide de deux méthodes. L’intensité de coloration locale de la BMP a été détectée immunohistochimiquement par la méthode avidine-biotine-péroxydase, déterminant le titre le plus élevé de dilution d’antisérum par rapport à la BMP bovine (bBMP); le résultat de la coloration a été positif. Le total de protéine morphogénétique osseuse présent dans un échantillon de tumeur a été mesuréà l’aide d’un dosage enzymatique immuno-absorbant (ELISA) aprés digestion du tissu avec de la collagénase pour retirer les protéines du tissu conjonctif. La coloration immunohistochimique a montré que la BMP se localisait dans les cytoplasmes et dans le tissu osseux réactif formé par les cellules malignes. La plus forte concentration locale de BMP a été trouvée dans le tissu des cellules géantes de la tumeur dans chondrosarcome, l’ostéosarcome et les tumeurs osseuses bénignes. Le total de BMP présent dans les tumeurs osseuses malignes était 2.4 fois supérier à celui décelé dans les tumeurs osseuses bénignes.

Early dietary antigens delay the development of gut mucosal barrier in preweaning rats.

ABSTRACT: To determine the effects of early antigen exposure on the maturation of the gastrointestinal mucosal barrier, rat pups were divided into three groups at the age of 14 d. In addition to normal maternal milk, group CM (n = 24) received daily a gavage feed of cows milk and group PH (n = 20) a whey protein hydrolysate during the experimental feeding period (14–20 d). Controls (n = 15) remained on maternal milk only. At 21 d, when “gut closure” normally occurs, intestinal absorption of horseradish peroxidase (HRP), was examined in vitro in Ussing chambers. The absorption of intact HRP [geometric mean (95% confidence interval)] was significantly higher in group CM [35.3 (16.7, 74.7) ng.h−1.cm−2] than in group PH [5.2 (1.4, 19.5) ng.h−1cm−2] and in controls [3.4 (0.8, 15.1) ng.h−1. cm−2; F = 5.54, p = 0.006]. The absorption of degraded HRP was comparable in all groups. There were no modifications in electrical parameters in association with increased mucosal permeability to HRP. Furthermore, in group CM electron-microscopic studies disclosed accumulation of HRP in the cytoplasm of the epithelial cells and in the intercellular spaces where cell junctions remained unaltered. These results indicate that early administration of antigens delays the process of gut closure. They further suggest that continuously enhanced endocytosis of macro-molecules is induced by an insult to the mucosa as part of the host response to these antigens, irrespective of the protection afforded by maternal milk.

Purification of fibroblast-derived celiac disease autoantigen molecules

ABSTRACT: We have recently purified autoantigen polypeptides reacting with celiac disease patient sera IgA from the extracellular noncollagenous matrix compartment of fetal lung tissue. These molecules trigger the production of different tissue antibodies, the so-called antireticulin and antiendomysium antibodies in celiac disease. In the present report, we show that fibroblasts synthesize and secrete celiac disease autoantigen molecules. The secretion product, reactive with IgA from celiac disease patients, is a large-molecular-weight protein aggregate. When the protein complex was treated with 4 M guanidinium hydrochloride and 0.1% SDS, 11 monocomponent polypeptides could be detected by PAGE. Of these, four single polypeptides with molecular weights of 17 000–39 500 and isoelectric points of 5.0–7.0 were observed to react with IgA separated from sera of children with celiac disease. The polypeptide molecules produced by fibroblasts in vitro bound to antireticulin and antiendomysium antibodies but not to antigliadin antibodies. The present observations show that tissue antibodies found to be specifically associated with celiac disease are generated against a synthesis product of fibroblasts, a cell-type known to synthesize a number of biologically active polypeptides. The fibroblast-derived extracellular matrix proteins and the formed autoantibodies may be important in the pathogenesis of gluten-sensitive enteropathy.

Internalization and Intracellular Processing of Bone Morphogenetic Protein (BMP) in Rat Skeletal Muscle Myoblasts (L6)

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily capable of inducing bone and cartilage formation in ectopic extraskeletal sites and transducing their effects through binding to serine-threonine kinase receptors. In this study, the fate of 125I-labelled native BMP after binding to cell surface receptors on L6-myoblasts was examined with both continuous and intermittent exposure of the ligand. BMP was readily internalized in L6 cells at +37 degrees C, and the internalization reached a plateau in 2 h. Intracellular degradation of 125I-labelled BMP was established, and degradation products were also detected in binding buffer, indicating exocytosis of the processed ligands. BMP receptors were shown to be subject to acute down-regulation by the ligand, and receptors were completely recycled in 3 h. Hence, we conclude that BMP receptors, like receptors for various other polypeptide ligands, have the ability to mediate intracellular delivery and degradation of the ligand.

USE OF MYOBLASTS IN ASSAYING THE OSTEOINDUCTIVITY OF BONE MORPHOGENETIC PROTEINS

A novel, time- and BMP-saving in vitro method for the detection and quantitation of bone morphogenetic protein (BMP) activity was developed based on the measurable effects of BMP on rat skeletal muscle myoblasts (L6). Calcium incorporation, stimulation of alkaline phosphatase activity and production of osteocalcin were used as markers of bone cell metabolism and on-going morphogenesis. The morphological change was confirmed by Chlorantine fast red and von Kossa staining. The response of various BMPs was purity-dependent and consistent with intramuscular implantations of the same materials. Neither TGF-beta1 nor insulin could induce the same actions. The data from this study indicate that at least in part in vivo implantations of BMP extracts can be replaced by in vitro measurement of osteoinductivity. Considerable saving of time, BMP and experimental animals can be achieved using cell culture conditions for the determination of bone-forming activity.

Autoantibodies in celiac disease: importance of fibroblasts.

BACKGROUND Serum reticulin and endomysium autoantibodies are highly celiac disease-specific, and the autoantigens have been shown to be derived from human fibroblasts. Among human tissues, the umbilical cord also expresses these antigens. This study was conducted to compare different autoantibody tests and especially to elucidate whether human umbilical cord is a suitable substrate in tests and whether the cord jelly-derived fibroblasts express the antigens. METHODS The indirect immunofluorescence method was used to detect the tissue and Whartons jelly-derived fibroblast antibodies in 334 celiac disease and control sera samples. Affinity chromatography studies were used to show the correlation between human fibroblast-derived autoantigens and tissue and gliadin antibodies. The jelly-derived fibroblasts were used as antigen in a whole-cell enzyme-linked immunosorbent assay. RESULTS Celiac disease patient sera showed IgA-class human umbilical cord antibody with high sensitivity (100%) and specificity (99%). All celiac disease patient sera tested showed in indirect immunofluorescence the molecules expressed by Whartons jelly-derived fibroblasts. The whole-cell fibroblast autoantibody enzyme-linked immunosorbent assay had a sensitivity of 100% and a specificity of 81%. Human fibroblast-derived celiac disease autoantigens absorbed most of the IgA responsible for human umbilical cord antibodies but not the IgA responsible for gliadin antibodies in the same sera. CONCLUSIONS Whartons jelly-derived fibroblast autoantibodies tested in a novel whole-cell enzyme-linked immunosorbent assay correlated well with the human umbilical cord but not with gliadin antibodies.

Soluble Factors from Human Saos-2 Osteosarcoma Cells Induce Ectopic Bone Formation and Osteoblastic Differentiation of Cultured Mesenchymal Cells

Freeze-dried cells of the human osteosarcoma cell line Saos-2 have the capacity to induce bone formation. We studied the bone-inductive capacity of factors expressed by Saos-2 cells and secreted to a growth medium. When lyophilized Saos-2-conditioned medium was implanted in a mouse hamstring muscle pouch, new bone formation was observed. Unlike the radially symmetric cartilage usually seen in induced ossicles, an asymmetric morphology of the newly formed bone was observed, with hyaline cartilage on one side and fibrocartilage on the other side. The new bone area resembled histologically osteochondroma, a benign bone tumor, with flat chondrocyte-like cells arranged in rows, endochondral ossification and red bone marrow. Osteoinductivity was further confirmed in in vitro tests with mesenchymal cell lines. A serum-free ten-fold concentrated Saos-2 growth medium increased mineralization, measured as 45Ca incorporation, and also stimulated alkaline phosphatase activity in cultured rat myoblasts and mouse embry...

Bone Morphogenetic Protein in Bone Neoplasms: Comparison of Different Detection Methods

The presence of bone morphogenetic proteins (BMPs) in 13 primary bone neoplasms was investigated with conventional bioassay method and immunohistochemically using antiserum against highly purified mixture of bovine BMPs as antibody. In conventional bioassay after implantation of lyophilized bone tumor tissues into mouse muscle pouches 9/13 samples turned out positive by radiography and 10/13 histologically. By immunohistochemical staining, using the avidin-biotin-peroxidase method, signs of BMPs could be verified in all cases investigated. Microscopical scoring showed the local concentration of BMPs to be especially high in sections from giant cell tumors when compared to other bone neoplasms.

Naturally occurring human autoantibodies recognize a fetal brain antigen identified as microtubulus associated Protein 1B

We have recently reported naturally occurring autoantibodies against a large fetal brain antigen (FBA). Now we describe the process of purification and identification of this particular FBA. The brains of newborn rabbits were solubilized and purified with preparative gel electrophoresis. The protein fractions were concentrated and desalted and the fractions were tested by a known positive serum. On membrane digestion of the FBA-band gave a twelve amino acid sequence that resulted in best identity score for mouse, rat and human microtubule-associated protein (MAP) 1B: a member of the microtubule-associated protein family. Monoclonal anti-MAP1B recognized a band in immunoblots of the brain homogenate and of the partially purified fractions with the same electrophoretic mobility as that recognized by a known anti-FBA positive serum. When adult rabbit brain was used as an antigen, the anti-MAP1B failed to recognize any bands on immunoblots. MAP lB has not been previously known as an autoantigen, even though many structural proteins of the neuronal cytoskeleton are known to be targets of naturally occurring autoantibodies. MAP 1B is a functionally important regulatory protein in the developing brain; thus autoantibodies against MAP1B may affect the normal development.

INFLUENCE OF ETHYLENE OXIDE STERILIZATION ON NEW BONE FORMATION INDUCED BY BOVINE BONE MORPHOGENETIC PROTEIN

Bone morphogenetic proteins (BMPs) have the capacity to induce and accelerate bone regeneration. Before experimental and clinical settings, BMP must be sterilized. Ethylene oxide (EO) gas sterilizations at different temperatures are commonly used but the effects of that on the osteoinductive capacity of BMP have been the subject of controversy. Here, we investigated the effects of three different EO sterilization methods on the osteoinductivity of partially purified native bovine BMP (bBMP). Gelatin capsules containing 3 mg of bBMP were sterilized as follows: (i) manually inside a dessicator with 12% EO spray (20°C, exposure time 2 h); (ii) with an EO gas sterilizer (Steri-Vac 4XL, temperature 29°C, exposure time 4 h 10 min, ethylene oxide concentration 860 mg/l); (iii) with an EO gas sterilizer (Steri-Vac 5XL, temperature 42°C, exposure time 3 h, ethylene oxide concentration 700 mg/l). The sterilization processes were monitored with samples of Bacillus subtilis (3M, Attest 1264). Osteoinductivity of bBMP...