Use of monoclonal antibodies to locate the chondroitin sulfate chain(s) in type IX collagen

Recent results show that type IX collagen isolated from chicken cartilage is associated with one or perhaps two chondroitin sulfate chains. To locate the chondroitin sulfate chain(s) along the type IX collagen molecule, rotary shadowing was performed in the presence of monoclonal antibodies which recognize stubs of chondroitin sulfate generated after chondroitinase ABC digestion. Monoclonal antibodies 9-A-2 and 2-B-6 which recognize stubs of chondroitin 4-sulfate were found to bind specifically to the NC3 domain of type IX collagen, and this binding was dependent on prior digestion of the preparation with chondroitinase ABC. Monoclonal antibody 1-B-5, which recognizes unsulfated stubs of chondroitin sulfate, did not show any specific binding to type IX collagen either with or without chondroitinase ABC digestion. As a control, monoclonal antibody 2C2 was used, which in previous work was shown to bind specifically to an epitope located close to or at the NC2 domain. Binding of this antibody to NC2 was unaffected by chondroitinase ABC digestion, and no specific binding of the antibody to the NC3 domain was detected either before or after chondroitinase ABC digestion.     

Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule. The three native fragments were designated (F1)2F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8 , IIB12 , and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules. Monoclonal antibody IIB12 , in contrast, recognized an epitope located only 73 nm from the 7S domain. This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed to binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [alpha 1(IV)]2 alpha 2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2 , were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by alpha-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.     

Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues

Monoclonal antibodies were produced against the recently described short chain cartilage collagen (type X collagen), and one (AC9) was extensively characterized and used for immunohistochemical localization studies on chick tissues. By competition enzyme-linked immunosorbent assay, antibody AC9 was observed to bind to an epitope within the helical domain of type X collagen and did not react with the other collagen types tested, including the minor cartilage collagens 1 alpha, 2 alpha, 3 alpha, and HMW-LMW. Indirect immunofluorescence analyses with this antibody were performed on unfixed cryostat sections from various skeletal and nonskeletal tissues. Only those of skeletal origin showed detectable reactivity. Within the cartilage portion of the 13-d-old embryonic tibiotarsus (a developing long bone) fluorescence was observed only in that region of the diaphysis containing hypertrophic chondrocytes. None was detectable in adjacent regions or in the epiphysis. Slight fluorescence was also present within the surrounding sleeve of periosteal bone. Consistent with these results, the antibody did not react with the cartilages of the trachea and sclera, which do not undergo hypertrophy during the stages examined. It did, however, lightly react with the parietal bones of the head, which form by intramembranous ossification. These results are consistent with our earlier biochemical analyses, which showed type X collagen to be a product of that subpopulation of chondrocytes that have undergone hypertrophy. In addition, either it or an immunologically cross-reactive molecule is also present in bone, and exhibits a diminished fluorescent intensity as compared with hypertrophic cartilage.     

Developmental acquisition of type X collagen in the embryonic chick tibiotarsus

The temporal and spatial distribution of short chain skeletal (Type X) collagen was immunohistochemically examined in the chick tibiotarsus from 6 days of embryonic development to 1 day posthatching. The monoclonal antibody employed (AC9) was recently produced and characterized as being specific for an epitope located within the helical domain of the type X collagen molecule (T. M. Schmid and T. F. Linsenmayer, J. Cell Biol., in press). The earliest detectable appearance of type X collagen was at 7.5 days, at which time it was restricted to a middiaphyseal location (i.e., in the primary center of ossification). This was in marked contrast to type II collagen, which appears earlier and is distributed throughout the cartilaginous anlagen. With increasing embryonic age, the reactivity with the type X antibody progressively extended toward the epiphyses, lagging somewhat behind the progression of chondrocyte hypertrophy. The anti-type X collagen antibody also reacted with the bony matrix itself, but the immunofluorescent signal produced by this source was considerably less than that produced by cartilage. At 19 days of development, a new small site of type X deposition was initiated in an epiphyseal location, which subsequently enlarged in circumference. These results are consistent with our previous biochemical studies suggesting that, in cartilage, type X collagen is specifically a product of that population of chondrocytes which have undergone hypertrophy.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.     

Intrinsic and extrinsic controls of the hypertrophic program of chondrocytes in the avian columella

Immunohistochemical studies of the chick columella have shown that the extracellular matrix of this ossicular cartilage template is composed largely of type II collagen. As development proceeds, synthesis of type X collagen, a hypertrophic cartilage-specific molecule, is initiated by endochondral chondrocytes within the zone of cartilage cell hypertrophy. Subsequently, these cells and their surrounding extracellular matrix are removed, resulting in marrow cavity formation. We have examined which of these processes are programmed within the columella chondrocytes themselves, and which require involvement of exogenous factors. Prehypertrophic columella from 12-day chick embryos were grown either in organ culture on Nuclepore filters or as explants on the chorioallantoic membrane of host embryos. Chondrocytes from the same source were grown in monolayer cell cultures. In both organ culture and cell culture, chondrocytes developed to the stage at which some of them entered the hypertrophic program and initiated the production of type X collagen as determined by immunofluorescence histochemistry with a monoclonal antibody specific for that collagen type. The organ cultures, however, did not progress to the next stage, in which detectable removal of the type X collagen-containing matrix occurs. When identical columella were grown on the chorioallantoic membrane of host chicks, the type X collagen-containing matrix which formed was rapidly removed, resulting in the formation of a marrow cavity. Thus, progression of endochondral chondrocytes to the deposition of type X collagen-containing matrix seems to be programmed within the cells themselves. Subsequent removal of this matrix requires the involvement of exogenous factors.     

Syngeneic anti-idiotypic monoclonal antibodies against an anti-human chorionic gonadotrophin antibody

Monoclonal antibody designated 1B10 (Mab 1B10) has been shown to be highly specific for the beta-chain of human chorionic gonadotrophin (HCG). We used this antibody to investigate its paratope using anti-idiotypic antibodies. Purified Mab 1B10 has been used to immunize syngeneic BALB/c mice to produce anti-idiotypic monoclonal antibodies. An enzyme immunoassay (ELISA) on Mab 1B10 coated plate was employed to screen the supernatants of growing hybridomas. The specificity of each antibody selected was assessed using an inhibition ELISA and immunoblotting. Monoclonal antibodies belonging to two categories were selected. (a) Those (designated Mab 4F8 and Mab 7G9) recognizing epitopes of the Ig molecule located in/or near the antigen-binding site of Mab 1B10. In ELISA these antibodies were shown to inhibit in a dose-dependent manner, the reaction of Mab 1B10 with its specific antigen; (b) those (Mab 2B8, Mab 3B8) reacting with epitopes located outside of the antigen binding site of the antiHCG antibody molecule and did not influence the reactions of Mab 1B10 and its antigen. Following immunization of syngeneic BALB/c mice monoclonal antibodies (Mab 4F8, Mab 7G9) were produced which recognized epitopes located on the variable region of Mab 1B10 since they did not react with other marine monoclonal antibodies of the same isotype. These antibodies inhibited the binding of Mab 1B10 to its corresponding epitope on the molecule of HCG and they can be defined as syngeneic anti-idiotypic antibodies.     

Immunoelectron microscopy of type X collagen: supramolecular forms within embryonic chick cartilage

To determine the supramolecular forms in which avian type X collagen molecules assemble within the matrix of hypertrophic cartilage, we performed immunoelectron microscopy with colloidal gold-labeled monoclonal antibodies. In addition double-labeled analyses were performed for the molecule and type II collagen, employing two monoclonal antibodies attached to different size gold particles. Both in situ limb cartilages and the extracellular matrix of chondrocyte cultures were examined. We observed in both systems that the type X collagen is present in two forms. One is as fine filaments (less than 5 nm in diameter) within mats which are found predominantly in the pericellular matrix of the hypertrophic chondrocytes. The second form is in association with the fibrils (10-20 nm in diameter) which also react with the antibody for type II collagen. It seems that the filamentous mats represent a form in which the type X collagen is initially secreted from the cell. The type X associated with the striated fibrils most likely represents a secondary association of the molecule with preexisting type II/IX/XI fibrils. The data are consistent with our previously proposed hypothesis that type X collagen is involved in, and perhaps even "targets," certain matrix components for degradation and removal.     

The structure of human collagen type IX and its organization in fetal and infant cartilage fibrils

Human collagen type IX was isolated from the media of organ cultures of fetal or infant hyaline cartilage. It consisted of three distinct, disulfide-bonded polypeptides of 115, 84, and 72 kDa, respectively. Digestion with chondroitinase ABC reduced the apparent molecular mass of the 115-kDa chain to about 65 kDa demonstrating that also human collagen type IX is a proteoglycan. In the electron microscope, the molecule had a rigid rod-like structure with characteristic kinks and with a globular domain at one end. Digestion of human collagen type IX with pepsin leads to somewhat heterogeneous fragments. Affinity-purified antibodies to the mixture of fragments specifically reacted with the fragment HMW without cross-reaction with chicken HMW. LMW of both species were recognized to the same low extent. Mechanically generated fibril fragments from human fetal cartilage were heterogeneous in diameter. Significantly, they could be immunostained for collagen type IX in a D-periodic pattern and regardless of the fibril diameter. Some fibrils were poorly labeled, again independently of the diameter. Therefore, the role of collagen type IX in cartilage probably is not to control directly the lateral growth during fibrillogenesis but rather to stabilize the fibril network.     

Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan

Monoclonal antibodies were raised against proteoglycan core protein isolated after chondroitinase ABC digestion of human articular cartilage proteoglycan monomer. Characterization of one of the monoclonal antibodies (1/20/5-D-4) indicated that it specifically recognized an antigenic determinant in the polysaccharide structure of both corneal and skeletal keratan sulfate. Enzyme immunoassay analyses indicated that the mouse monoclonal IgG1 recognized keratan sulfate in native proteoglycan aggregate and proteoglycan monomer preparations isolated from hyaline cartilages of a wide variety of animal species (human, monkey, cow, sheep, chicken, and shark cartilage). The 1/20/5-D-4 monoclonal antibody did not recognize antigenic determinants on proteoglycan isolated from Swarm rat chondrosarcoma. This finding is consistent with several biochemical analyses showing the absence of keratan sulfate in proteoglycan synthesised by this tissue. A variety of substructures isolated after selective cleavage of bovine nasal cartilage proteoglycan (Heineg?rd, D., and Axelsson, J. (1977) J. Biol. Chem. 252, 1971-1979) were used as competing antigens in radioimmunoassays to characterize the specificity of the 1/20/5-D-4 immunoglobulin. Substructures derived from the keratan sulfate attachment region of the proteoglycan (keratan sulfate peptides) showed the strongest inhibition. Both corneal and skeletal keratan sulfate peptides as competing antigens in radioimmunoassays showed similar inhibition when compared on the basis of their glucosamine content. Therefore, the 1/20/5-D-4 monoclonal antibody appears to recognize a common determinant in their polysaccharide moieties. Chemical desulfation of the keratan sulfate reduced the antigenicity of the glycosaminoglycan. The antibody did not recognize determinants present in dermatan sulfate, heparin, heparin sulfate, or hyaluronic acid.     

Characterization of the structural proteins of the murine coronavirus strain A59 using monoclonal antibodies

Monoclonal antibodies reacting with the A59 strain of mouse hepatitis virus (MHV-A59) were characterized and those specific to the E2 major envelope glycoprotein were studied in detail. Antibodies were tested for their ability to neutralize viral infectivity (N+ characteristic) and prevent viral-induced cell-to-cell fusion (F+ characteristic). All four possible combinations of activities reflecting E2 functions were found, i.e., N+F+, N-F-, N+F-, and N-F+. In addition, competitive binding studies with these monoclonal antibodies revealed two nonoverlapping antigenic regions. The first region, designated A, was recognized by antibodies which included each of the four functional types. Region B was identified by a single monoclonal antibody with N-F- activities. The existence of antibodies which only neutralize virus or only block viral-induced fusion implies that the structures on E2 which serve as targets for neutralization and which induce fusion are not identical. The critical determinants for neutralization and fusion must be closely related topographically on E2 since both N+F- and N-F+ antibodies recognize the same antigenic region.     

Modulated Expression of Type X Collagen in the Meckel's Cartilage with Different Developmental Fates

Mammalian Meckel's cartilage undergoes regionally diverse histodifferentiation: the caudal end of Meckel's cartilage extends to the developing ear and gives rise to malleus and incus through endochondral ossification while its major distal region differentiates into sphenomandibular ligament and the anterior ligament of the malleus tympanic plate through fibrous transformation. Since the entire Meckel's cartilage develops up to chondrocyte hypertrophy, the regional extracellular matrix components in the hypertrophic Meckel's cartilage may differ in association with the diverse developmental fates. In this project, the expressions of cartilage collagens were investigated in developing rat Meckel's cartilage and particular interest was given to type X collagen. A cDNA, HP114, encoding the NC1 domain of rat α1(X) collagen was cloned, and a synthetic peptide based on the sequence deduced from HP114 was used to generate a monospecific antibody. In situ hybridization of newborn rat condylar and angular cartilages undergoing endochondral ossification showed restricted labeling with the α1(X) collagen probe in the hypertrophic chondrocyte layer. In contrast, the α1(X) collagen probe totally failed to label the major distal portion of Meckel's cartilage even in the hypertrophic cartilage zone. Immunohistochemistry using the anti-type X collagen monospecific antibody consistently failed to recognize the epitope in the corresponding portion of Meckel's cartilage throughout the experimental periods of gestational Day 17, newborn, and Postnatal Day 7, while the strictly localized positive staining was found in the posterior part of Meckel's cartilage which gave rise to malleus and incus. Since major cartilage collagens type II and type IX were found to be present throughout Meckel's cartilage, we postulate that the regulatory molecular mechanism of type X collagen expression may be closely associated with the developmental fates of fibrous transformation and endochondral ossification in mammalian Meckel's cartilage.     

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.     

Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen

A monoclonal antibody, IV-IA8, generated against chicken type IV collagen has been characterized and shown to bind specifically to a conformational-dependent site within a major, triple helical domain of the type IV molecule. Immunohistochemical localization of the antigenic determinant with IV-IA8 revealed that the basement membranes of a variety of chick tissues were stained but that the basement membrane of the corneal epithelium showed little, if any, staining. Thus, basement membranes may differ in their content of type IV collagen, or in the way in which it is assembled. The specificity of the antibody was determined by inhibition ELISA using purified collagen types I-V and three purified molecular domains of chick type IV collagen ([F1]2F2, F3, and 7S) as inhibitors. Only unfractionated type IV collagen and the (F1)2F2 domain bound the antibody. Antibody binding was destroyed by thermal denaturation of the collagen, the loss occurring at a temperature similar to that at which previous optical rotatory dispersion studies had shown melting of the triple helical structure of (F1)2F2. Such domain-specific monoclonal antibodies should prove to be useful probes in studies involving immunological dissection of the type IV collagen molecule, its assembly within basement membranes, and changes in its distribution during normal development and in disease.     

Detection of phenylalanine hydroxylase protein in human platelets. Immunochemical detection

A protein reactive with anti-phenylalanine hydroxylase monoclonal antibody PH8 has been recovered from human platelet extracts. Two bands corresponding to molecular masses of about 60 kDa and 55 kDa were revealed by immunoblotting after electrophoresis according to Laemmli. Using the same antibody, a single band with a molecular mass of 60 kDa was demonstrated in extracts from human pineal gland; two similar antigens were found in human liver extracts and no antigen was found in adrenal gland extracts. Monoclonal antibodies, PH1 and PH3, did not react with these antigens during immunoblotting. Monoclonal antibodies, PH7 and PH9, reacted with the 55 kDa antigen in platelet extracts. The antigen content in platelet extracts was measured by ELISA with monoclonal antibodies relative to its content in the liver. The antigen content in platelet extracts was about 100 times as low as that in liver extracts and amounted to 100 ng/mg of protein. These findings suggest that the phenylalanine hydroxylase antigen is present in human platelets.     

Specific identification of tissue kallikrein in exocrine tissues and in cell-free translation products with monoclonal antibodies.

A panel of six mouse monoclonal antibodies (IgG1) has been prepared against purified rat urinary kallikrein (EC 3.4.21.35) and characterized. In radioimmunoassay, the antibody titres of ascitic fluid giving 50% binding to 125I-kallikrein range from 1:2 X 10(3) to 1:1 X 10(6). Antibodies from four of the clones show no cross-reactivity with human urinary kallikrein, rat urinary esterase A or tonin. However, antibodies from a fifth clone cross-react with tonin and, from a sixth, with both urinary esterase A and tonin. Three of the kallikrein affinity-purified monoclonal antibodies inhibited, whereas one of the antibodies stimulated, kallikrein activity. Tissue kallikrein from rat submandibular-gland and pancreatic extracts and urine were labelled with [14C]di-isopropyl phosphofluoridate, immunoprecipitated with each of the six monoclonal antibodies and identified to be 38 kDa proteins, similar in size to purified rat urinary kallikrein. Western-blot analysis shows that 125I-labelled kallikrein monoclonal antibodies (V4D11) bind directly to a 38 kDa protein in submandibular-gland and pancreatic extracts and urine. Cell-free translation products of submandibular-gland polyadenylylated[poly(A)+]mRNA were immunoprecipitated with affinity-purified sheep anti-kallikrein antibodies and three monoclonal antibodies (V4D11, V4G6 and V1C3). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these immunoprecipitates revealed that two kallikrein precursors with Mr values of 37 000 and 35 000 are encoded by submandibular-gland mRNA. The third monoclonal antibody, V1C3, which binds to active kallikrein, did not recognize either precursor form. Collectively, the data show that these monoclonal antibodies comprise a set of powerful and specific reagents for studies of tissue kallikreins.     

Monoclonal antibody that recognizes an epitope of the sperm equatorial region and specifically inhibits sperm-oolemma fusion but not binding.

Balb/c mice were immunized with purified hamster sperm heads for induction of antisera and the production of monoclonal antibodies that recognize preferentially the equatorial segment. Twenty-six hybridoma clones secreted monoclonal antibodies with strong affinity for spermatozoa. The supernatants of 16 clones contained antibodies against the equatorial segment, of which 11 were specific to this region. Five supernatants (M1-M5) containing antibodies that bind to various regions of the sperm head were selected and assessed for the ability to inhibit hamster fertilization in vitro using intact and zona-free oocytes. All the supernatants inhibited fertilization compared with the control. However, M1 supernatant specifically inhibited sperm-egg fusion in a concentration-dependent manner, while sperm-oolemma binding and sperm motility remained unaffected. M1 supernatant recognized an epitope that is exclusive to the equatorial segment and expression of this epitope increased after capacitation and the acrosome reaction. Preliminary immunoblot analysis indicated that M1 monoclonal antibody recognized two protein bands of 37.5 and 34.0 kDa.     

Immunochemistry of human Lp[a]: characterization of monoclonal antibodies that cross-react strongly with plasminogen.

Forty different monoclonal antibodies were produced from hybridomas that were raised against human Lp[a]. Of these, 14 strongly cross-reacted with plasminogen on ELISA screening assays while 16 clearly did not and 10 were only marginally cross-reactive. We took advantage of the homology between plasminogen and apo[a] to define the epitopes of 8 strongly cross-reacting monoclonal antibodies. We were able to subdivide these into four general categories based upon site competition assays (using both plasminogen and Lp[a]), and their reactivity with elastolytically derived plasminogen fragments. Group A monoclonal antibodies (F1 1E3, F2 3A3) recognized epitopes within the kringle 5 and protease domains (miniplasminogen) of plasminogen. The group B monoclonal antibody (F6 1A3) reacted solely with plasminogen kringle 4-like domains and appeared to recognize a limited number of sites on Lp[a]. Group C monoclonal antibodies (F6 1B5, F6 1G9) recognized a second, more frequently distributed site within these kringle 4-like domains. The final group, D, monoclonal antibodies (F6 2C3, F6 2G2, F6 3F4) reacted with a cluster of sites found associated with kringle 4-like domains but also reacted with the miniplasminogen domain. Interestingly, only the members of this group were able to interfere with the proteolytic activity of plasmin. Neither periodate treatment of Lp[a] nor incubation of Lp[a] with epsilon-aminocaproic acid affected the binding of any of our monoclonal antibodies.     

Detection of collagenase-induced damage of collagen by 9A4, a monoclonal C-terminal neoepitope antibody.

To determine whether the collagen network is compromised by collagenase during acute inflammation, a monoclonal antibody (9A4) was developed with specificity for the C-terminal neoepitope sequence generated by collagenase-cleavage of type II collagen (Gly-Pro-Pro-Gly-Pro-Gln-Gly-COOH). 9A4 was shown to detect the collagen collagenase-cleavage neoepitope with a K = 1.7 x 10(-7) M (type II) and K = 2 x 10(-6) M (type I). It does not recognize uncleaved native or denatured collagen. Articular cartilage from control animals is unstained by 9A4. During acute inflammation elicited in hamsters by intra-articular LPS, positive staining for the 9A4 neoepitope indicated the collagen was damaged. Wheel running exercise was used to apply stress to control cartilage and cartilage from animals with damaged collagen. After 6 months of running, the cartilage from normal animals was unaffected. By contrast, in the group with damaged collagen, the cartilage was fibrillated in all animals and in half of those, the cartilage failed and bony eburnation resulted.     

Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage

The levels of type X collagen in mineralizing normal chicken epiphyses and nonmineralizing rachitic chicken tibial epiphyses were measured and compared. Qualitative immunoperoxidase studies with anti-chick type X collagen monoclonal antibodies on sections from normal and rachitic cartilage demonstrated that the type X collagen levels in rachitic growth plates are reduced. Northern hybridization of mRNA and biosynthetic studies have confirmed that type X collagen synthesis in rickets is also decreased. In hypocalcemic rickets, the level of type X collagen mRNA is reduced by 80% whereas the level of type X collagen mRNA is only reduced by 50% in normocalcemic rickets. These observations provide additional evidence that type X collagen is involved in the process of cartilage mineralization and also suggest that the partial recovery of type X collagen synthesis in normocalcemic rickets may be related to the elevated plasma concentration of calcium. Calcium concentration may therefore play an important role in the control of type X collagen synthesis.