ICRS 2019 - Conference Calendar
5.0.1 - Monoclonal Antibodies Against Glycosaminoglycan & Proteoglycan Epitopes & Neoepitopes: Science, Luck & Magic
Abstract
Introduction
I started to make monoclonal antibodies (mAbs) at the University of Alabama in Birmingham (UAB) in the early 1980’s when an Australian Dentist/Immunologist colleague and friend of mine (John F. Kearney) convinced me that I should quit making polyclonal antibodies in rabbits and use a new and exciting method of mAb production where he had personally been involved in its discovery and further development (1). Thus, the key and most important message/information from me in this presentation is that if anyone wants to produce mAbs they should use the draining Lymph Nodes (i.e. not lymphocytes from the Spleen) as their source of activated lymphocytes for cell fusion and subsequent mAb hybridoma production. The reason for using the draining lymph nodes (rather than the Spleen), as the source of antigen-activated lymphocytes, is that many of these cells are capable of producing mAbs that recognise epitopes (both protein and carbohydrate) in biological molecules that have been highly conserved in Nature and therefore their epitopes are common to proteoglycan structural domains from many different animal species. However, if one uses the Spleen as the source of lymphocytes for mAb production, these lymphocytes that they are harvesting are predominantly making antibodies that recognise epitopes that are different from (i.e. not identical to) those in the mouse amino-acid sequences and carbohydrate antigenic domains of the immunogenic molecule, so as to not produce an autoimmune response to these similar molecules that are in the cells and tissues from the immunised mouse; i.e. the process of “self/non-self recognition” in antibody production. Furthermore, this ‘draining lymph node method’ has the additional advantage that the immunisation time is only 5 injections in a 2 week immunisation period and in just 2 months after the first injection one can have prepared purified mAbs that are ready for the many different mAb experimental analyses we basic scientists want to perform (see reference (2) below for more details of this immunisation procedure).Content
Experimental Methods: The details of how to perform this ‘draining Lymph node lymphocyte method’ for mAb production are described in more detail in one of my earlier papers (2). Consequently, over several years of research, our laboratory has made numerous mAbs that detect epitopes and neo-epitopes (i.e. enzymatic digestion generated ‘new’ epitopes) on numerous glycosaminoglycan and proteoglycan molecules that, in general, give positive results with similar molecules in tissues and organs derived from a wide variety of different animal species.Results & Discussion: In my presentation I will provide examples of how these mAbs have been used in a variety of different immuno-chemical analyses; i.e. quantification & location of intracellular, cell surface and extracellular matrix proteoglycans derived from several different tissues and animal species including humans. Also, I will present examples of their use in studying connective tissue proteoglycan metabolism in arthritis. More complete details of these studies can be found in the full paper and review publications (2)-(11) cited below.
References
References:(1)Hammerling, G.J., Hammerling V. & Kearney J.F. Eds (1981). Monoclonal Antibodies & T-cell Hybridomas. New York: Elsevier/North Holland.
(2) Caterson, B., Christner, J.E. & Baker, J.R. (1983). Identification of a monoclonal antibody that specifically recognises Corneal and Skeletal Keratan Sulphate. Journal of Biological Chemistry 258, 8848-8854
(3) Caterson, B., Christner, J.E., Baker, J.R. & Couchman, J.R. (1985). Production and characterisation of monoclonal antibodies directed against connective tissue proteoglycans. Fed. Proc. 44, 386-393
(4) Caterson. B., Griffin, J., Mahmoodian, F. & Sorrell, J.M. (1990). Monoclonal antibodies against chondroitin sulphate isomers: their use as probes for investigating proteoglycan metabolism. Biochem. Soc. Trans. 18, 820-823
(5) Hughes, C.E., Caterson, B., White, R.J., Roughley, P.J. & Mort, J.S. (1992). Monoclonal antibodies recognising protease-generated neo-epitopes from cartilage proteoglycan degradation. Journal of Biological Chemistry 267, 16011-16014
(6) Visco, D.M., Johnstone, B., Hill, M.A., Jolly, G.A. & Caterson, B. (1993). Immunohistochemical analysis of 3-B-3(-) and 7-D-4 epitope expression in canine osteoarthritis. Arthritis & Rheumatism 36, 1718-1725
(7) Johnstone, B., Markopoulos M., Neame, P. & Caterson, B. (1993). Identification and characterisation of glycanated and non-glycanated forms of Biglycan and Decorin in human intervertebral disc. Biochem. J. 292, 661-666
(8) Caterson, B., Hughes, C.E., Roughley, P. & Mort, J.S. (1995). Anabolic and catabolic markers of proteoglycan metabolism in osteoarthritis. Acta Orthop Scand 66, 121-124
(9) Hughes, C.E., Caterson, B., Fosang, A.J., Roughley, P.J. & Mort, J.S. (1995). Monoclonal antibodies that specifically recognise neoepitope sequences generated by ‘aggrecanase’ and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro. Biochem. J. 305, 799-804
(10) Hayes, A.J., Hughes, C.E. & Caterson, B. (2008). Antibodies and immuno-histochemistry in extracellular matrix research. Science Direct: Methods 45, 10-21
(11) Bondeson, J., Wainwright, S., Hughes, C. & Caterson, B. (2008). Clin. Exp. Rheumatology 26, 139-145
5.0.2 - Horses and Humans: Building a Translational Research Program in Osteoarthritis and Cartilage Repair
Abstract
Introduction
Introduction: The author’s interest in orthopaedic research developed alongside specialty training in equine surgery, and a research project during his large animal surgery residency at Purdue in experimentally induced synovitis leading to early osteoarthritic change. Despite human osteoarthritis (OA) literature suggesting that synovitis was secondary to articular cartilage debris from articular cartilage degradation we clearly showed that equine synovitis without any trauma or instability to the joint could produce typical changes of early OA. Interestingly, this research project on synovitis led to an inquiry regarding the potential of diagnostic arthroscopy and its potential in the horse. This led to a “parallel” career path, starting with attending a human course in diagnostic arthroscopy of the knee and progressed into the development of diagnostic and surgical arthroscopic techniques in the horse. As in human orthopaedics, arthroscopic surgery revolutionized our ability to treat equine athletes as it did in people, limitations including acute articular cartilage loss and progressive osteoarthritis were soon recognized and led to the development of our research program at the Orthopaedic Research Center at Colorado State University.
Content
Post Traumatic Joint Disease is a common clinical problem in the horse with surveys estimating that up to sixty percent of lameness is related to OA. The most common syndrome is traumatically induced disease manifesting as synovitis primarily and progressing to morphologic damage progressing to OA. While we get osteochondral fragmentation as well as intraarticular ligament and meniscal injuries as primary conditions that need arthroscopic surgery, the majority of clinical OA in the equine athlete is treated by non-surgical intervention with synovitis as the main target.
The CSU Osteochondral Fragment Model was developed to test putative treatments for equine OA.The majority of our controlled based testings with different treatments have been done using the equine osteochondral fragment model. The model is based on an equine clinical problem (distal radial carpal bone fragmentation in the carpus) and is a consistent mild model of OA that can characterize both symptom modifying and disease-modifying effects. We have twenty-one published papers and the potential as a model of OA in people has been reviewed. (1)
Treatments for OA in the horse validated (or otherwise) with this model include underwater treadmilling as a rehabilitation technique, extracorporeal shock wave therapy, intraarticular corticosteroids, intraarticular hyaluronan, intraarticular intravenous and oral hyaluronan, intraarticular and intramuscular polysulfated glycosaminoglycans, intramuscular pentosan polysulfate, avocado-soy as an oral joint supplement, intraarticular and intravenous Polyglycan ®, gene therapy with IL-1ra, autologous conditioned serum (Orthokine Equine ®), IRAP I, IRAP II and autologous conditioned serum, bone marrow-derived mesenchymal stem cells and stromal vascular fraction. Differences between betamethasone esters, triamcinolone acetonide and (Vetolag ®, Kenalog ®) and methylprednisolone (MPA) (Depo Medrol ®) have been demonstrated. Beneficial symptom modifying and disease-modifying effects with triamcinolone acetonide contrast with deleterious effects to the articular cartilage with MPA. Our gene therapy study with adeno-IL-1ra not only demonstrated the value of inhabiting IL-1 but also was found to be the most potent therapy that we have tested in the OA model.
Mesenchymal Stem (Stromal) Cell (MSC) Therapies have also been examined in the equine OA model. Superiority over stromal vascular fraction (SVF) has been demonstrated in the OA model and a clinical trial demonstrated value with intraarticular injection of twenty-million (BMSCs) 30 days after surgery for articular cartilage defects, damaged intraarticular soft tissue structures (primarily meniscus and meniscal-tibial ligaments) as well as progressive osteoarthritis. (2)Intraarticular administered BMSCs have also been shown valuable in the value of repair of experimental microfractured full-thickness articular cartilage defects in the medial femoral condyle of the horse (3) but, in contrast, unsatisfactory results with BMSCs implanted in fibrin/PRP into microfractured defects and the PRP/fibrin alone showing superior results. (4)
Equine Models of Articular Cartilage Repair have been developed in pre-clinical studies (5)A desire to validate microfracture in humans led to the development of the first model of articular cartilage repair in horses, simulating femorol tibial defects in humans. Microfracture was first studied in a 12- month study of full-thickness defects in the medial condale of the femur in which microfractured and non-microfractured defects were compared. (5) The use of microfracture showed a significant enhancement of the amount of repaired tissue both macroscopically and microscopically with improvement as well as increased Type II collagen levels with microfracture. We also showed significant upregulation of Type II collagen expression eight weeks after microfracture. In arguably the most pivotal study, we also clarified the absolute need to completely remove the calcified cartilage layer for maximal positive effects with microfracture. (7)The clinical use of microfracture in human patients has been the subject of a number of studies. The negative effects have been stressed by some including the occurrence of intralesional osteophytes and failure after two years but there are also long-term studies showing considerable benefit. The technique remains the standard of care for many orthopaedic surgeons. We have also done studies on further manipulation of endogenous healing with intraarticular AdIL-1ra/ AdIGF-1 gene therapy (8) as well as intraarticular BMSCs. (3)Different models of articular cartilage defects on the medial and lateral trochlear ridges have been very useful to show significant repair augmentation with a modified MACI ® (10)technique as well as evaluation of a one-step cartilage fragment implantation system (CAIS). All studies go out to twelve months and involve 9 months of athletic exercise to physically test repair to show as well.
Translational Medicine Institute: The evolution of building a translational research program has now resulted in the establishment of the Translational Medicine Institute at Colorado State University. We will continue to do everything we do for horses but with added partnerships and people, we are escalating our translational research in human musculoskeletal injury and disease.
References
References:
(1) McIlwraith, CW, Frisbie, DD, Kawcak, CE
The horse has a model of naturally occurring osteoarthritis Bone Joint Research
(2) Ferris DJ, Frisbie DD, Kisiday JD, McIlwraith CW, Hague BA, Major MD, Schneider RK, Zubrod CJ, Kawcak CE, Goodrich LR. Clinical outcome after intra-articular administration of bone marrow derived mesenchymal stem cells in 33 horses with stifle injury. Vet Surg 2014;43:255-265. doi: 10.1111/j.1532-950X.2014.12100.x.
(3) McIlwraith CW, Frisbie DD, Rodkey WG, Kisiday JD, Werpy NM, Kawcak CE, Steadman JR. Evaluation of intra-articular mesenchymal stem cells to augment healing of microfractured chondral defects. Arthroscopy 2011;27:1552-61. doi: 10.1016/j.arthro.2011.06.002.
(4) Goodrich LR, Chen AC, Werpy NM, Willams AA, Kisiday JD, Su AW, Cory E, Morely PS, McIlwraith CW, Sah RL, Chu CR. Addition of mesenchymal stem cells to autologous platelet-enhanced fibrin scaffolds in chondral defects. Does it enhance repair? J Bone Joint Surg 2016;98:23-34. http://dx.doi.org/10.2106/JBJS.0.00407.
(5) McIlwraith, C.W., Frisbie, D.D., Nixon, A.J
Equine models of articular cartilage repair, Cartilage 2011, 22, 317-326
(6) Frisbie DD, Trotter GW, Powers BE, Rodkey WG, Steadman JR, Howard RD, Park RD, McIlwraith CW. Arthroscopic subchondral bone plate microfracture technique augments healing of large osteochondral defects in the radial carpal bone and medial femoral condyle of horses. Vet Surg 1999;28:242-255
(7) Frisbie DD, Morisset S, Ho CP, Rodkey WG, Steadman JR, McIlwraith CW. Effects of calcified cartilage on healing of chondral defects treated with microfracture in horses. Am J Sports Med 2006;34:1824-1831
(8) Morisset S, Frisbie DD, Robbins PD, Nixon AJ, McIlwraith CW. IL-1Ra/IGF-1 gene therapy modulates repair of microfractured chondral defects. Clin Orthop Related Res 2007;462:221-228.
(9) Frisbie DD, Bowman SM, Calhoun HA, DiCarlo EF, Kawcak CE, McIlwraith CW. Evaluation of autologous chondrocyte concentration via a collagen membrane in equine articular defects – results at 12 and 18 months. Osteoarthritis Cartilage 2008;16:667-679.