Bovine bone implant with bovine bone morphogenetic protein in healing a canine ulnar defect.

Xenograft is considered an alternative material for bone transplantation, but its bone healing capacity is inferior compared to that of autografts and allografts. Here, we tested whether bone morphogenetic protein (BMP) addition enhances the suitability of demineralized xenogeneic bovine bone for bone grafting in dogs, and whether xenogeneic bone is a suitable carrier material for BMPs. The capacity of demineralized bovine bone implants, with and without native partially purified bovine BMP, to heal a 2-cm ulnar defect was determined in six dogs over a follow-up time of 20 weeks. No instances of bone union were seen, but there was slightly more bone formation in the xenografts with BMP, though the difference was not statistically significant. The ulnas treated with an implant with BMP were also mechanically stronger, but the difference was not significant. Computed tomography scans showed no differences in the implant area in bone density, bone mineral content, or bone cross-sectional area. It is concluded that native, partially purified BMP does not sufficiently improve the suitability of bovine demineralized xenografts as a bone substitute material for dog. Demineralized xenogeneic bone does not seem to be a feasible carrier material for BMP.

Soluble immunological parameters and early prognosis of renal cell cancer patients.

In local or metastatic cancer, a prognostic tumour marker could be a valuable tool in the selection of different treatments. In renal cell cancer (RCC) no such markers have been available. We therefore evaluated the association between several pretreatment serum markers, tumour classification and short term survival in RCC patients. Serum samples were collected before surgery and three months thereafter from 24 RCC patients. Interleukin-6 (IL-6), IL- 12, soluble IL-2 receptor (sIL-2R) and intercellular adhesion molecule-1 (sICAM-1) were measured in serum samples using specific commercial enzyme immunoassay kits. Serum IL-6, sIL-2R and sICAM-1 levels before nephrectomy were significantly higher in non-local tumours than in local ones (mean IL-6 53 pg/ml versus 6.3 pg/ml, and sICAM-1 443 ng/ml versus 290 ng/ml, sIL-2R 3779 pg/ml versus 1796 pg/ml). In contrast, IL-12 levels were higher in local tumours (148 versus 102 pg/ml) and the levels increased significantly (P < 0.005) after removal of the primary tumour in patients with local disease. All patients with local tumours had normal IL-6 values, while only one with a non-local tumour had IL-6 levels below 10 pg/ml. In addition, IL-6 and sICAM-1 levels before operation were significantly higher in patients with short (less than one year) survival (p=0.007 to IL-6 and p=0.006 to sICAM-1). In contrast, patients with shorter survival had significantly lower IL-12 (p=0.03) levels. Our findings suggest that RCC induces changes in several immunological parameters. These soluble immunological factors, IL-6, IL-12, sIL-2R and sICAM-1, might have a role as prognostic factors in RCC.

Probiotics reinforce mucosal degradation of antigens in rats: implications for therapeutic use of probiotics.

The effects of probiotics, administered with different diets, i.e., unhydrolyzed or hydrolyzed dietary antigens, on macromolecular degradation in the gut mucosa were studied. Rat pups were divided into five feeding groups at the age of 14 d. In addition to maternal milk, the milk group was gavaged daily with cows' milk and the hydrolysate group with extensively hydrolyzed whey formula, while controls received sterile saline. In addition to these diets, the milk-GG group and the hydrolysate-GG group were given probiotic bacteria, Lactobacillus GG ATCC 53103 (10(10) colony-forming units per day). At 21 d, the absorption of macromolecules, horseradish peroxidase and beta-lactoglobulin across patch-free jejunal segments was studied in Ussing chambers. The degree of macromolecular degradation was studied by means of HPLC gel filtration. The absorption rate of intact horseradish peroxidase differed among the feeding groups (P = 0.038). This was due to the high median (interquartile range) absorption of intact horseradish peroxidase (ng x h-1 x cm-2) in the milk group [255 (14-1332)] and supplementation with L. GG in the milk-GG group [35 (8-233)] restoring the status to the control level [22 (0-116)]. A parallel effect was seen in the hydrolysate group [100 (9-236)] vs. the hydrolysate-GG group [1 (0- 13)]. A gel filtration study confirmed that larger molecules were absorbed across the mucosa in the milk group compared to the other groups. The absorption of degraded horseradish peroxidase differed between the feeding groups (P = 0. 005). L. GG had a distinct effect when administered with unhydrolyzed, native protein vs. hydrolyzed protein: it increased absorption of degraded horseradish peroxidase in the milk-GG group [7310 (4763-8228)] vs. the milk group [3726 (2423-5915)], while reducing it in the hydrolysate-GG group [2051 (1463-2815)] vs. the hydrolysate group [4573 (3759-9620)]. Our results showed that probiotics not only restore aberrant macromolecular transport, but they also have a specific effect on mucosal degradation depending on dietary antigen: adjuvant-like properties (unhydrolyzed antigen) and immunosuppressive-like properties (hydrolyzed antigen). The antigenicity of the diet therefore should be taken into consideration, when introducing novel probiotic functional foods for the management of gastrointestinal disorders.

Extraction and characterization of native canine bone morphogenetic protein (cBMP) qualified with osteoinductivity.

Bone morphogenetic protein (BMP) was extracted from canine bone matrix, partially purified and tested for osteoinductivity. A radiographically and histologically detectable ectopic bone formation was induced by 6.0 mg canine (cBMP) in muscle pouch of BALB mouse at 21 days post implantation. Characterization of the cBMP preparation by a gel filtration chromatography defined that the material consisted of proteins or protein complexes with molecular weights from 4 to 120 kD. Isoelectric focusing showed that the molecules were acidic with isoelectric points of 4.6-5.6.

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 Wharton's 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 Wharton's 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: Wharton's 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.

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.

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.

Human fibroblast-derived molecules as antigens in enzyme-linked immunosorbent assay for coeliac disease-specific IgA.

We have recently shown that cultured human fibroblasts synthesize and secrete protein molecules that bind to IgA-class anti-reticulin and anti-endomysium antibodies but not to anti-gliadin antibodies in coeliac disease patient sera. In the present report, we describe a reproducible method for purification of these antigen molecules from fibroblast culture medium. Using reversed-phase chromatography as the final purification step, four different protein molecules reacting with coeliac disease patient sera IgA were obtained. In enzyme-linked immunosorbent assay (ELISA) for coeliac disease-specific IgA, a mixture of 0.5 microgram of the four reversed-phase-separated molecules was used as antigen. The optical density values in ELISA of the sera from newly diagnosed coeliac disease patients (n = 34) were 0.740-3.400 (mean 1.830) and in control patients (n = 66) 0.090-0.850 (mean 0.320). Using an arbitrary cut-off level of 0.700, the sensitivity of the present autoantibody test was 100%, specificity 91% and positive predictive value 85%. Our identified autoantigens may generate the production of the classical tissue antibodies, known as anti-reticulin and anti-endomysium antibodies, and may be used as antigen in an immunoassay for the antibodies.

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

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.

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.

Fibroblasts and transforming growth factor beta induce organization and differentiation of T84 human epithelial cells.

BACKGROUND & AIMS: The gut epithelium in the crypt-villus axis represents a continuous developmental system in which the role of fibroblast-epithelial interactions is obvious. The aim of this study was to establish an in vitro method whereby fibroblast-guided differentiation of crypt-like gut epithelial cells can be studied. METHODS: Intestinal epithelial cells (T84 and HT-29) were cultured within type I collagen gel together were fibroblasts without cell-to-cell contact. T84 cells were also grown in the presence of transforming growth factor beta and hepatocyte growth factor. The gels were studied using light and electron microscopy and histochemical and immunohistochemical methods. RESULTS: The epithelial cells formed unorganized cell clusters within the gels, but when given fibroblast support, 76% of the T84 cell colonies (not HT-29) organized into luminal formations, and basement membranes including laminin were well deposited. The cells in the columnar single cell-layer luminal formations (49% of all colonies) were differentiated, showing microvilli, up-regulated alkaline phosphatase brush border activity, and mucin profiles typical for small intestine. This fibroblast-induced organization and differentiation was induced by transforming growth factor beta. CONCLUSIONS: Crypt-like T84 epithelial cells are able to differentiate when grown three-dimensionally together with fibroblasts or transforming growth factor beta. This method may be used for mesenchymal-epithelial cell cross-talk studies.

The in vitro response to human fibroblast-derived extracellular matrix proteins is restricted by specific HLA class II genes. Relevance for coeliac disease.

Coeliac disease is an immunologic disease of the small intestine which is caused by ingestion of wheat gliadin, the disease-promoting agent. The disease associates strongly with the particular HLA type, HLA-DQA1*0501, DQB1*0201 alleles. Further specific autoantibodies against reticulin and endomysium are found in patients; these autoantibodies appear to be disease specific. An extracellular matrix noncollagenous protein reacts specifically with CD patients' serum immunoglobulin A and is the target of antireticulin antibodies. In this study the immune response to this matrix protein was analyzed in vitro in normal, healthy individuals. Our study shows that the immune response to Fb-CDAP is strictly regulated by the HLA-DR3, DQA1*0501, DQB1*0201 alleles, and that only those cells which were positive for these alleles produced an immune response. On the other hand, half of the cells positive for these HLA alleles were responders. Monoclonal antibodies to DR and DQ inhibited the response in an additive way, showing that both DR and DQ can act as an antigen-presenting structure. The immune response to gliadin has been shown to associate with the same HLA type as CD, but the association is not as strong. Our results show that the immune responses to Fb-CDAP can be generated in vitro in genetically predisposed persons in the absence of CD.

Circulating autoantibodies to a 240 kD fetal brain protein.

Antibodies (IgG and IgM) recognizing a 240 kD antigen of the cat fetal brain were found in sera of healthy people and in sera (IgG) obtained at uncomplicated delivery from the umbilical cord of the newborn infant. The method applied was immunoblotting. Using the same method, the 240 kD antigen could not be detected in the adult brain or other fetal tissues. It seems that the antigen is specific for the fetal brain. The role of the antigen and the origin of generation and significance of function of the antibodies in the circulation are the objects of our further studies.

Partial purification and characterization of bone morphogenetic protein from bone matrix of the premature moose (Alces alces): degradation of bone-inducing activity during storage.

In spite of the advances in recombinant techniques in the production of bone morphogenetic proteins (BMPs), the best clinical results so far have been obtained with human and animal source-extracted BMPs. Also, the poor availability of recombinant products gives rise to continued research with different extracted and purified proteins. In a search for a new source of bone-matrix-derived BMP with high osteoinductive activity, BMP was extracted from fresh bone matrix of the premature moose (Alces alces). Bone-inducing activity was investigated by implanting 0.5-20 mg of BMP into thigh muscle pouches of BALB mice. Radiologically detectable formation of new bone required 2.0 mg of partially purified BMP. Immediately after the extraction, an analytic chromatogram with known molecular weight (MW) markers showed three fractions with different MWs. After 15 months of storage at +1 degree C lyophilized and desiccated, BMP was fractionated by HPLC gel filtration and bioassayed. New bone formation was evaluated qualitatively by histology and quantitatively by radiomorphometry, the quantity of calcified tissue per milligram of implanted agent being determined. Fractions I and III, with high (100-700 kD) and low MW (15-25 kD), respectively, were apparently more effective inducers of new bone than the second-time-tested partially purified BMP complex, the activity of which had significantly (p < 0.05) decreased during 15 months of storage compared to initial results after extraction. However, the bone-inducing activity of fractions I and III corresponded closely to the initial activity of the BMP complex. Fraction II, with medium MW (25-55 kD), caused an apparent inflammatory reaction and no bone formation, and was though to be immunogeneic. Fraction III was considered to include the dominant BMP component with MW 18.5 and fraction I an association of BMP with other non-collagenous bone matrix proteins after one-step gel filtration. The results suggest that BMP from the premature moose has high bone-forming activity. With identification and removal of apparently immunogenic protein fractions, the inflammatory reaction and inhibitory effect on bone induction could be eliminated, and still higher bone-forming activity was attained. Acid protease enzymes were assumed to be responsible for the observed decline in the inductive activity of semi-purified BMP after 15 months of storage, as both osteoinductive fractions proved to be acidic in isoelectric focusing.

Composites of bone morphogenetic protein (BMP) and type IV collagen, coral-derived coral hydroxyapatite, and tricalcium phosphate ceramics.

Few studies have been performed to investigate the characteristics of native Bone Morphogenetic Protein (BMP). We derived 5.85 g of native moose BMP from 102 kg of fresh moose bone and performed chromatographic studies to characterise its structure. We found the BMP molecule to have a molecular weight of 11-700 kDa. To investigate the ideal local delivery system for BMP we used porous discs as a carrier. These were implanted into 132 mice. The volume of new bone formed was evaluated by means of histological specimens, radiographs and isotope studies. The best results were obtained by implanting porous discs (coral and ceramic) with type IV collagen and BMP. We suggest that this is the best delivery system for future experiments regarding the acton of BMP.

Bovine bone morphogenetic protein (bBMP/NCP)-induced repair of skull trephine defects in pigs.

Bovine bone morphogenetic protein and associated noncollagenous proteins (bBMP/NCP) were implanted in skull trephine defects, 14 and 22 mm in diameter, in adult minipigs. The defects were larger than the critical size for repair of cranial bones in pigs observed for a period of six to 16 weeks. The trephines were placed either in the nasal bone at eye level, or in the frontal bone. In minipigs the repair was more advanced in trephines in the frontal bone than in the nasal region, but was incomplete in either bone bed. As in dogs, implants of bBMP/NCP in heterotopic sites were resorbed before a competent cell population could respond. Serum anti-BMP antibody was elevated. The IgG levels of the antibody titer showed a clearcut dose-response; the level was significantly higher in one pig with implants of 85 mg of bBMP/NCP proteins in a frontal bone trephine.

In vivo glucose uptake and glucose transporter proteins GLUT1 and GLUT4 in heart and various types of skeletal muscle from streptozotocin-diabetic rats.

The in vivo glucose uptake and the levels of two glucose transporter proteins (GLUT1 and GLUT4) were measured in heart and in various types of skeletal muscle from streptozotocin-diabetic rats. Diabetes (12-16 weeks) reduced the in vivo glucose uptake (glucose metabolic index, GMI), and the levels of GLUT1 and GLUT4 in heart by 75%, 60% and 70%, respectively. In diaphragm consisting of approximately equal amounts of type I (slow-contracting oxidative), IIa (fast-contracting oxidative) and IIb (fast-contracting glycolytic) fibers, GMI and GLUT4 levels were reduced by 60% and 40%, respectively, with no change in GLUT1 levels. In muscle consisting mainly of type I fibers (e.g., m. soleus), GMI and GLUT4 levels were reduced by 60% and 30%, respectively, whereas GLUT1 levels were unaltered. In mixed-type muscle consisting of type IIa and IIb fibers (e.g., m. plantaris and red part of m. gastrocnemius), GMI and GLUT1 levels were unchanged, whereas GLUT4 levels were decreased by 45%. In contrast, GMI was increased by 100% in type IIb fibers (e.g., the white part of m. gastrocnemius), probably reflecting the 4-fold increase in blood glucose levels, whereas GLUT4 levels were lowered by 55% with no change in GLUT1 levels. These data demonstrate a marked difference in the response of in vivo glucose uptake to long-term hypoinsulinemia between oxidative (type I) and glycolytic (type IIb) fibers. Furthermore, in contrast to the GLUT4, GLUT1 levels are regulated differentially in heart and skeletal muscle in response to streptozotocin-induced diabetes.

Partially purified reindeer (Rangifer tarandus) bone morphogenetic protein has a high bone-forming activity compared with some other artiodactyls.

Noncollagenous proteins, including bone morphogenetic protein (BMP), were extracted in 4 mol/l guanidinium hydrochloride (GuHCl) from the pulverized and HCl-demineralized matrix of reindeer, bovine, sheep and porcine bone. To remove water-soluble material, the GuHCl solution was dialyzed against water and water-insoluble material and redissolved in 4 mol/l GuHCl. Gelatin peptides were removed by extraction in 0.25 mol/l citrate buffer (pH 3.1). The yield consisted mostly of large complexes and protein molecules of molecular weight less than 35,000 daltons. Isoelectric focusing of the material showed three to four different protein molecules: three acidic and one neutral. Bone-forming activity was investigated by implanting 0.6-15.0 mg of partially purified protein preparation into the thigh muscles of BALB mice. Radiologically detectable formation of new bone required 0.6 mg of reindeer BMP, 2.5 mg of bovine BMP, 5.1 mg of sheep BMP, and 8.0 mg of porcine BMP. A rough estimate of the area of the deposits showed that reindeer BMP had the highest bone formation activity, and porcine had the lowest. The formation of new bone was confirmed histologically. It is suggested that the differences in osteogenic activity are due to quantitative differences in BMP constituents or in the degree of complex formation in the protein preparations. Also immune and other defense mechanisms may generate differences in osteogenic response.

Purification of fibroblast-derived celiac disease autoantigen molecules.

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.

Purification of monocomponent bovine bone morphogenetic protein in a water-soluble form.

Noncollagenous protein material was extracted from HCl-demineralized bovine bone particles in 4 M guanidinium hydrochloride. Water- and citrate buffer-insoluble material was collected, solubilized in 6 M urea and fractionated by preparative isoelectric focusing using a running voltage of 5000 V. The material removed from the area between pH 4.7 and 5.7 of the isoelectric focusing gel was osteoinductive (identified by its capacity to induce bone development). This was solubilized in 6 M urea and dialyzed against 0.2 M Tris buffer. The Tris buffer-soluble material was fractionated by HPLC gel filtration. The water- and citrate buffer-insoluble material contained mainly high-molecular-weight protein complexes which were osteoinductive, and < 5% of the material was osteoinductive monocomponent bone morphogenetic protein. The Tris buffer-soluble material contained only two polypeptides: an osteoinductive peptide of molecular weight 18,500 and a non-osteoinductive peptide of molecular weight 8,000. The very high voltage used during the isoelectric focusing caused a slow break-down of the urea-soluted protein complexes, which significantly increased the yield of monocomponent bone morphogenetic protein. By the present method it is possible to prepare Tris buffer solution containing up to 2 mg/ml of pure monocomponent bone morphogenetic protein.