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The specific anti-T lymphocyte immunosuppressive drugs cyclosporine A (CsA), FK-506 (FK) and rapamycin (RPM) have revolutionized the field of allograft transplantation and have recently been evaluated for use in autoimmune diseases. Effects of immunosuppressive drugs on adhesion molecule expression in the putative autoimmune conditions psoriasis, through the use of keratinocyte (KC) cell culture techniques, and alopecia areata were investigated. In the Dundee Experiment Bald Rat, a model of alopecia areata, hair regrowth was only apparent with CsA therapy and FK had a high toxicity profile. <I>In vivo</I> the observed cutaneous reduction of ICAM-1 and LFA-1 expression particularly around the hair bulbs was due to the effects of CsA and FK on the inflammatory cell infiltrate, especially since the associated cutaneous lymphocyte populations were reduced with drug. This is corroborated by the results obtained from <I>in vitro</I> culture of human KC, since cytokine-induced expression and release of ICAM-1 by KC was unaffected by CsA, RPM or FK treatment. CsA and RPM, but not FK, however, had an observable cytostatic effect on unstimulated and more especially, cytokine-stimulated KC proliferation and may, therefore, clinically inhibit KC hyperplasia, which is characteristic of psoriasis. This study shows that CsA, FK and RPM may inhibit either directly but more likely in an indirect manner, cellular adhesion molecule expression during immune reactions. This effect will lower the possible number of cellular interactions and furthermore, reduce intracellular co-signalling events necessary for cellular activation which accounts for the overall inhibition of cell-mediated immune responses by these novel immunosuppressive drugs.

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Background: Fresh osteochondral allograft (OCA) transplantation represents a biologic restoration technique as an alternative treatment option for larger osteochondral lesions of the talus (OLT). The purpose of this study was to evaluate midterm outcomes after OCA transplantation for the treatment of OLT. Methods: Nineteen patients (20 ankles) received partial unipolar OCA transplant for symptomatic OLT between January 1998 and October 2014. The mean age was 34.7 years, and 53% were male. The average graft size was 3.8 cm2. All patients had a minimum follow-up of 2 years. Outcomes included the American Academy of Orthopaedic Surgeons Foot and Ankle Module (AAOS-FAM), the Olerud-Molander Ankle Score (OMAS), and pain and satisfaction questionnaires. Failure of OCA was defined as conversion to arthrodesis or revision OCA transplantation. Results: Five of 20 ankles (25%) required further surgery, of which 3 (5%) were considered OCA failures (2 arthrodesis and 1 OCA revision). The mean time to failure was 3.5 (range, 0.9 to 6.7) years. Survivorship was 88.7% at 5 years and 81.3% at 10 years. The median follow-up of the 17 patients with grafts in situ was 9.7 years. The mean OMAS improved significantly from 40 points preoperatively to 71 points postoperatively (P < .05; range, 5 to 55). The mean postoperative AAOS-FAM core score was 81.5 ± 15 (range, 40.5 to 96.6). Fifteen of 17 patients responded to follow-up questions regarding their ankle; 14 patients reported less pain and better function, and 13 patients were satisfied with the results of the procedure. Conclusion: Our study of midterm results after OCA transplantations showed that this procedure was a reasonable treatment option for large OLT. Level of Evidence: Level IV, case series.

Background: Osteochondral allograft (OCA) transplantation is a useful treatment for posttraumatic ankle arthritis in young patients, but failure rates are high and reoperations are not uncommon. The aim of this study was to evaluate the outcomes of failed ankle OCA transplantation converted to ankle arthrodesis (AA) or total ankle arthroplasty (TAA). Methods: We evaluated 24 patients who underwent salvage procedures (13 AA and 11 TAA) after primary failed ankle OCA transplantation. Reoperations were assessed. Failure of the salvage procedure was defined as an additional surgery that required a revision AA/TAA or amputation. Evaluation among nonfailing ankles included the American Academy of Orthopaedic Surgeons Foot and Ankle Module (AAOS-FAM), pain, and satisfaction. Results: In the salvage AA cohort, 3 patients were classified as failures (2 revision AA and 1 amputation). The 10 nonfailing patients had a mean follow-up of 7.4 years. Eighty-eight percent were satisfied with the procedure, but 63% reported continued problems with their ankle (eg, pain, swelling, stiffness). Mean pain level was 1.9 and AAOS-FAM core score was 83±13. In the salvage TAA cohort, 2 patients were classified as failures (both revision TAA). The 9 nonfailing patients had a mean follow-up of 3.8 years. Fifty percent were satisfied with the procedure, but 40% reported continued problems with their ankle. The mean pain level was 1.3, and the median AAOS-FAM core score was 82±26. Conclusion: Revision and reoperation rates for salvage procedures following failed OCA transplantation of the ankle are higher compared to published data for primary AA and TAA procedures. However, we believe OCA transplantation can serve as an interim procedure for younger patients with advanced ankle joint disease who may not be ideal candidates for primary AA or TAA at the time of initial presentation. Level of Evidence: Level IV, case series.

Regulatory T cells (Tregs), lymphocytes that suppress immunological reactions, are of great interest for our comprehension of basic immunology and as a therapeutic agent to treat immune-mediated pathologies. Understanding the physiology of these cells will help to inform clinical strategies targeting Tregs. In order to study the homing of human Tregs, we utilised genetic engineering to drive expression of fluorescent protein in human Tregs, permitting in vivo cell tracking. We optimised a protocol for lentivirus-mediated transduction of human Tregs during in vitro expansion, to generate high yields of stably-engineered cells. After infusing labelled cells into a humanised mouse model of skin allotransplantation, we detected human Tregs within a human skin graft by PCR and visualised Tregs moving in the graft, in a live mouse, by two-photon microscopy. Through reverse genetic analyses, we explored molecular mechanisms that allow Tregs to respond adaptively to environmental cues. Neuropilin-1 (NRP1), a transmembrane co-receptor, has been implicated in the function of mouse Tregs. Tregs transduced with shRNA to knock down NRP1 were severely impaired in their capacity to suppress cell proliferation in vitro and to prolong allograft survival in a humanised mouse model. qRT-PCR analysis revealed that transcription the gene encoding the anti-inflammatory cytokine IL-10, and the autophagy-associated genes BECN1, COPS4 and MAP1LC3B, was significantly diminished in NRP1-deficient Tregs. We concluded that in human Tregs, NRP1 is necessary for suppressive function, most likely via regulation of NRP1-dependent regulation of cytokine production and metabolism. Having identified a molecular target via which Treg function might be potentiated, we explored methods to target such molecules for cell therapy applications. Tregs engineered to over-express IL-10, but not NRP1, exerted significantly enhanced suppression of cell proliferation in vitro. Thus, relatively straightforward genetic engineering, compatible with generation of therapeutic cell yields, could be exploited to improve the efficacy of Treg cellular therapy.

Osteoarthritis is one of the most common causes of disability in adults in America. It is a progressive and degenerative disease where the articular cartilage is broken down and lost from the surfaces of bones causing chronic pain and swelling in the joints, and currently has no cure. The most commonly osteoarthritis starts from a focal lesion on the cartilage surface, which will expand on the surface and downwards through the thickness of the tissue. The current gold standard for correcting cartilage focal lesions is the osteochondral autograft/allograft transplantation (OAT), which replaces the defect with a fresh osteochondral graft. The main limiting factor for using the OAT comes from the limited number of autograft and allografts that are available for implantation. To address the concern of graft availability, this study will look at the development of a porcine osteochondral xenograft (OCXG). The first aim of this research is to establish a decellularization protocol that will remove the antigens and cellular debris, which are the leading causes of graft rejection when implanting animal tissue in humans. The second aim of this study is restoring the mechanical strength of the OCXG that was lost during the decellularization process through crosslinking the tissue using genipin and epigallocatechin gallate (EGCG). The third aim is comparing the performance of the complete crosslinked OCXG at different degrees of crosslinking in a long-term goat animal model. The final aim is an alternative way to correct focal lesions through the development of an injectable collagen stabilizing treatment with genipin and punicalagin that will slow or stop the growth of a lesion and prevent osteoarthritis.