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47th Annual AAEP convention
47TH ANNUAL AAEP CONVENTION Several unique in-depth sessions were presented at the Annual Convention of the American Association of Equine Practitioners, last November in San Diego. The session on lameness in the western performance horse was reviewed in the February issue of JEVS. Reviewed here are selected aspects of the session on current concepts in equine osteoarthritis. Most of the session was reports by researchers at Colorado State University, under the direction of Dr. Wayne McIlwraith. Research on measurement of synovial fluid and serum concentrations of chondroitin sulfate 846 epitope and carboxy propeptides of type II procollagen for diagnosis of osteochondral fragmentation in horses was discussed. This research has been ongoing for severa years at CSU. Using fluids taken from horses being operated for osteochondral chip fragmentation, a study showed that synovial fluid epitope 846 and total protein concentrations were significantly higher in joints with chip fragments than in unaffected joints but that C-propeptide of type II collagen (CPII) and keratan sulfate (KS) concentrations as well as white blood cell counts were not. Synovial fluid, total protein and 846 epitope concentrations were linearly related to the grade of chip fragment. Serum epitope 846 and CPII concentrations were significantly higher in horses with chip fragments than in control horses. Discriminate analysis allowed 27/34 (79%) horses to be correctly classified as having or not having osteochondral fragmentation on the basis of serum epitope 846 and CPII concentrations. These results suggest that serum and synovial fluid concentrations of epitope 846 and CPII are associated Volume 22, Number 3, 2002
with osteochondral fragmentation. It is felt that serum epitope 846 and CPII concentrations may be useful in the diagnosis of chip fragments in the horse. The results also suggest that increases in concentrations of epitope 846 and CPII suggest that increased synthesis of cartilage aggrecan and type 11 procollagen are associated with osteochondral chip fragmentation. Another project discussed was the use of collagen degradation marker COL2-3/4Cshort immunoassay to detect collagenase cleaved type H collagen fragments and the inhibitory effect of a novel nonpeptidic matrix metalloproteinase inhibitor. In an in vitro study, a new nonpeptidic matrix metalloproteinase inhibitor from Bayer was evaluated using interleukin-I produced cartilage degradation. Monitored were the effects of IL-1 on glycosaminoglycan release from articular cartilage explants as well as GAG concentrations in the cartilage using the dimethyl methylene blue dye assay. As well, collagenase-cleaved type II collagen release from the explants was measured with the COL2-3/4Cshort immunoassay. A significant inhibitory effect of the MMP inhibitor, BAY129566, on both glycosaminoglycan release from the articular cartilage as well as collagenase-cleaved type II collagen release from the articular cartilage explants was demonstrated. Also disscussed was the development of a new anti-neoepitope antibody
to identify type H collagen degradation in equine articular cartilage. Researchers have succeeded in raising antibodies specific for the C-terminus neoepitope of the 3/4 collagenase cleavage site of type II collagen in the horse. This work was necessary because existing antibodies either recognized both type I and type II collagen fragments or, if specific for type II, only weakly cross-reacted if at all with the cleaved type II collagen from the horse. Using sequence information, the appropriate peptide was chosen for immunization. The new antibody is described as 234CEQ. The generation and location of the neoepitope is illustrated in the figure as is a comparison with other species. This work is an important breakthrough in that researchers now have an equine-specific antibody to recognize early type II collagen degradation in articular cartilage. It was very important to have an antibody that was specific for type II collagen degradation as this is the collagen unique to articular cartilage. After discussing other new diagnostic techniques to better characterize osteoarthritis in the horse, Dr. McIlwraith commented on what may be available in the future: "Publications so far on the use of biomarkers for the diagnosis of monitoring equine joint disease have been previously discussed. Confounding effects that may influence the interpretation of marker levels have yet to be fully characterized. For example, because the liver and kidneys can play an important role in the metabolism and clearance of many biomarkers, the function of these organs must be assessed whenever fluid levels of biomarkers are being measured. Other factors that have been shown to influence biomarker levels are 111
ciceadian rhythms, intestinal peristalsis, physical activity, age, breed, diet, and sex as well as surgery and general anesthesia. Collection and storage of samples is also critical in assessing biomarker levels. “In October 2000, an industry sponsored panel of equine and human researchers convened in Northampton, England, to assess the state of molecular biomarker research of bone and cartilage metabolism in the horse and a report of this meeting and abstracts are forthcoming in Equine Veterinary Education and the Equine Veterinary Journal. This group met to try to unite the field in practical areas such as sample collection, the best set of biomarkers to assay and optimization of collaborative efforts. “The use of biomarkers for the detection of joint disease in the horse is relatively new and extremely exciting in its potential. However, one must be careful that as with any other emerging field, initial enthusiasm of the technologies not be translated into unrealistic goals. It is unlikely that one marker will be the “magic bullet” but with careful examination of a series of markers in light of specific disease processes, the use of markers for the diagnosis and monitoring of joint disease is not unrealistic.” Gene Therapy The term gene therapy is commonly understood to mean the use of molecular methods to replace defective or absent genes, or to counteract those that are
112
over-expressed. The key technologies needed for gene therapy are the methods by which genes are isolated (cloned), manipulated (engineered), and transferred (gene transfer) into host cells. Dr. David Fresbie said “The field of equine veterinary practice is in an ever-evolving state, requiring current technologies to be constantly evaluated for new applications. The field of molecular biology is already integrated into equine practice for use in blood typing, identification of Salmonella species from fecal samples, detecting bovine papillomavirus types 1 and 2 in sarcoids, 28 specific strains of equine herpesviruses, and virulence factors associated with Rhodococcus equi, to cite a few examples. The specific use of gene therapy in the horse is a novel application. The purpose of this review article is to help familiarize the equine practitioner with the concept of gene therapy, and introduce its utility as well as potential future benefits for the equine industry in the treatment of osteoarthritis.” He said that in 1944, DNA was first reported to carry genetic information. Since this time great attention has been devoted to unlocking the information encoded within DNA molecules, given the realization that DNA contained a genetic blueprint for each species and could hold key information for normal function as well as disease processes. The functional unit of DNA is the gene, which can be defined as the set of DNA sequences that are required to produce a single polypeptide. The gene sequence codes for a specific messenger RNA (mRNA) molecule that, in turn,
carries the genetic information from the nucleus to the cytoplasm for translation into an amino acid sequence (i.e., a protein). Many recognized disease states relate to a lack of, a defect in, or an imbalance of a particular protein(s). Since the gene is the basal unit ultimately responsible for protein production, it is also a logical therapeutic target. Currently, most gene therapy protocols are directed towards increasing levels of selected therapeutic proteins in an attempt to alter specific disease processes. Depending on the natural function of the particular protein, we may be able to enhance or repress certain direct effects on specific cellular processes. An example of an application of gene therapy being explored is the correction of abnormal proliferation of smooth muscle cells in coronary arteries causing narrowing and obliteration of coronary vessels following angioplasty. This holds great promise for patients with coronary heart disease. The technique uses the expression of the DNA encoding for thymidine kinase. This enzyme activates a prodrug (drug administered in an inactive form) capable of blocking cell proliferation when activated. Preliminary research involving pigs, whose arteries closely resemble humans showed that local arterial administration of the DNA coding for thymidine kinase concurrent with prodrug infusion followed by angioplasty procedure reduced proliferation of the vascular smooth muscle by 50-90% in the treatment group when compared to control animals.
JOURNAL OF EQUINE VETERINARY SCIENCE
with osteochondral fragmentation. It is felt that serum epitope 846 and CPII concentrations may be useful in the diagnosis of chip fragments in the horse. The results also suggest that increases in concentrations of epitope 846 and CPII suggest that increased synthesis of cartilage aggrecan and type 11 procollagen are associated with osteochondral chip fragmentation. Another project discussed was the use of collagen degradation marker COL2-3/4Cshort immunoassay to detect collagenase cleaved type H collagen fragments and the inhibitory effect of a novel nonpeptidic matrix metalloproteinase inhibitor. In an in vitro study, a new nonpeptidic matrix metalloproteinase inhibitor from Bayer was evaluated using interleukin-I produced cartilage degradation. Monitored were the effects of IL-1 on glycosaminoglycan release from articular cartilage explants as well as GAG concentrations in the cartilage using the dimethyl methylene blue dye assay. As well, collagenase-cleaved type II collagen release from the explants was measured with the COL2-3/4Cshort immunoassay. A significant inhibitory effect of the MMP inhibitor, BAY129566, on both glycosaminoglycan release from the articular cartilage as well as collagenase-cleaved type II collagen release from the articular cartilage explants was demonstrated. Also disscussed was the development of a new anti-neoepitope antibody
to identify type H collagen degradation in equine articular cartilage. Researchers have succeeded in raising antibodies specific for the C-terminus neoepitope of the 3/4 collagenase cleavage site of type II collagen in the horse. This work was necessary because existing antibodies either recognized both type I and type II collagen fragments or, if specific for type II, only weakly cross-reacted if at all with the cleaved type II collagen from the horse. Using sequence information, the appropriate peptide was chosen for immunization. The new antibody is described as 234CEQ. The generation and location of the neoepitope is illustrated in the figure as is a comparison with other species. This work is an important breakthrough in that researchers now have an equine-specific antibody to recognize early type II collagen degradation in articular cartilage. It was very important to have an antibody that was specific for type II collagen degradation as this is the collagen unique to articular cartilage. After discussing other new diagnostic techniques to better characterize osteoarthritis in the horse, Dr. McIlwraith commented on what may be available in the future: "Publications so far on the use of biomarkers for the diagnosis of monitoring equine joint disease have been previously discussed. Confounding effects that may influence the interpretation of marker levels have yet to be fully characterized. For example, because the liver and kidneys can play an important role in the metabolism and clearance of many biomarkers, the function of these organs must be assessed whenever fluid levels of biomarkers are being measured. Other factors that have been shown to influence biomarker levels are 111
ciceadian rhythms, intestinal peristalsis, physical activity, age, breed, diet, and sex as well as surgery and general anesthesia. Collection and storage of samples is also critical in assessing biomarker levels. “In October 2000, an industry sponsored panel of equine and human researchers convened in Northampton, England, to assess the state of molecular biomarker research of bone and cartilage metabolism in the horse and a report of this meeting and abstracts are forthcoming in Equine Veterinary Education and the Equine Veterinary Journal. This group met to try to unite the field in practical areas such as sample collection, the best set of biomarkers to assay and optimization of collaborative efforts. “The use of biomarkers for the detection of joint disease in the horse is relatively new and extremely exciting in its potential. However, one must be careful that as with any other emerging field, initial enthusiasm of the technologies not be translated into unrealistic goals. It is unlikely that one marker will be the “magic bullet” but with careful examination of a series of markers in light of specific disease processes, the use of markers for the diagnosis and monitoring of joint disease is not unrealistic.” Gene Therapy The term gene therapy is commonly understood to mean the use of molecular methods to replace defective or absent genes, or to counteract those that are
112
over-expressed. The key technologies needed for gene therapy are the methods by which genes are isolated (cloned), manipulated (engineered), and transferred (gene transfer) into host cells. Dr. David Fresbie said “The field of equine veterinary practice is in an ever-evolving state, requiring current technologies to be constantly evaluated for new applications. The field of molecular biology is already integrated into equine practice for use in blood typing, identification of Salmonella species from fecal samples, detecting bovine papillomavirus types 1 and 2 in sarcoids, 28 specific strains of equine herpesviruses, and virulence factors associated with Rhodococcus equi, to cite a few examples. The specific use of gene therapy in the horse is a novel application. The purpose of this review article is to help familiarize the equine practitioner with the concept of gene therapy, and introduce its utility as well as potential future benefits for the equine industry in the treatment of osteoarthritis.” He said that in 1944, DNA was first reported to carry genetic information. Since this time great attention has been devoted to unlocking the information encoded within DNA molecules, given the realization that DNA contained a genetic blueprint for each species and could hold key information for normal function as well as disease processes. The functional unit of DNA is the gene, which can be defined as the set of DNA sequences that are required to produce a single polypeptide. The gene sequence codes for a specific messenger RNA (mRNA) molecule that, in turn,
carries the genetic information from the nucleus to the cytoplasm for translation into an amino acid sequence (i.e., a protein). Many recognized disease states relate to a lack of, a defect in, or an imbalance of a particular protein(s). Since the gene is the basal unit ultimately responsible for protein production, it is also a logical therapeutic target. Currently, most gene therapy protocols are directed towards increasing levels of selected therapeutic proteins in an attempt to alter specific disease processes. Depending on the natural function of the particular protein, we may be able to enhance or repress certain direct effects on specific cellular processes. An example of an application of gene therapy being explored is the correction of abnormal proliferation of smooth muscle cells in coronary arteries causing narrowing and obliteration of coronary vessels following angioplasty. This holds great promise for patients with coronary heart disease. The technique uses the expression of the DNA encoding for thymidine kinase. This enzyme activates a prodrug (drug administered in an inactive form) capable of blocking cell proliferation when activated. Preliminary research involving pigs, whose arteries closely resemble humans showed that local arterial administration of the DNA coding for thymidine kinase concurrent with prodrug infusion followed by angioplasty procedure reduced proliferation of the vascular smooth muscle by 50-90% in the treatment group when compared to control animals.
JOURNAL OF EQUINE VETERINARY SCIENCE