Culture expanded primary chondrocytes have potent immunomodulatory properties and do not induce an allogeneic immune response.

OBJECTIVE: Allogeneic cell therapies, such as mesenchymal stromal cells (MSC), which have potent regenerative and anti-inflammatory potential are being investigated as a therapy for osteoarthritis (OA) and cartilage injury. Here we describe another potential source of regenerative and anti-inflammatory allogeneic cells, culture expanded primary chondrocytes (CEPC). In direct comparison to allogeneic MSC, we extensively assess the immunological interactions of CEPC in an allogeneic setting. METHODS: Chondrocytes were isolated from rat articular cartilage and cultured in normoxic or hypoxic conditions. In vitro co-culture assays with allogeneic lymphocytes and macrophages were used to assess the immunomodulatory capacities of the chondrocytes, followed by immune response analysis by flow cytometry, ELISA and qPCR. RESULTS: CEPC showed reduced induction of proliferation, activation and cytotoxic granzyme B expression in allogeneic T cells. Importantly, exposure to pro-inflammatory cytokines did not increase CEPC immunogenicity despite increases in MHC-I. Furthermore, CEPC had a potent ability to suppress allogeneic T cell proliferation, which was dependent on nitric oxide production. This suppression was contact independent in hypoxia cultured CEPC. Finally, chondrocytes were shown to have the capacity to modulate pro-inflammatory macrophage activity by reducing MHC-II expression and TNF-α secretion. CONCLUSION: These data indicate the potential use of allogeneic chondrocytes in OA and cartilage defects. The lack of evident immunogenicity, despite exposure to a pro-inflammatory environment, coupled with the immunomodulatory ability indicates that these cells have the potential to evade the host immune system and suppress inflammation, thus potentially facilitating the resolution of OA induced inflammation and cartilage regeneration.

Mesenchymal stem cell therapy promotes corneal allograft survival in rats by local and systemic immunomodulation.

Mesenchymal stem cells (MSCs) are being investigated extensively due to their ability to dampen immune responses. Here, we tested the ability of MSCs from three distinct sources to prolong rat corneal allograft survival. A fully allogeneic rat cornea transplant model (DA to LEW) was used. Recipient rats received 1 × 10(6) MSCs (syn [LEW], allo [DA] or third-party [Wistar Furth]) intravenously 7 days before transplantation and again on the day of transplantation (day 0). A high percentage of untreated and syn-MSC treated allografts were rejected (80% and 100%, respectively). Preactivation of syn-MSCs with interferon gamma also failed to prolong allograft survival. Conversely, corneal allograft survival was significantly prolonged in allo-MSC treated (90%) and third-party MSC treated (80%) allograft recipients. Flow cytometric analysis revealed less infiltrating natural killer T cells in corneas of both allo- and third-party MSC treated animals, coupled with a higher proportion of splenic CD4+Foxp3+ regulatory T cells, compared to controls. In the case of allo- and third-party MSCs, results from a delayed-type hypersensitivity assay clearly showed that hypo-responsiveness was specific for corneal donor-associated allo-antigens. Thus, allo- and third-party MSC treatment prolongs corneal allograft survival by suppressing peripheral immune responses and promoting an intragraft immunoregulatory milieu.

Role of lentivirus-mediated overexpression of programmed death-ligand 1 on corneal allograft survival.

To investigate the role of lentivirus-mediated overexpression of programmed death-ligand 1 (PD-L1) on rat corneal allograft survival. A fully allogeneic rat cornea transplant model was used for in vivo studies. Lentiviral (LV) vectors are efficient tools for ex vivo genetic modification of cultured corneas. LV vector encoding for PD-L1 (LV.PD-L1) and LV vector encoding for eGFP (LV.eGFP, as control) were constructed and tested. PD-L1 or eGFP expression was increased on corneal cells upon LV.PD-L1 and LV.eGFP transduction, respectively. Both allogeneic controls and allogeneic LV.eGFP transduced corneas were uniformly rejected (MST: 13.8 ± 1.7 days and 12.3 ± 1.9 days, respectively). In contrast, allogeneic LV.PD-L1 transduced corneas showed a high percentage (83%) of graft survival (MST > 30 days, n = 5, 15 days, n = 1). Graft opacity of PD-L1 transduced corneas was present but was significantly reduced compared to control or eGFP expressing corneas. Flow cytometric analysis revealed that percentages of CD3(+) CD8(+) CD161(+) and CD3(+) CD8(+) CD161(-) lymphocytes were decreased in animals receiving LV.PD-L1 transduced corneas compared to animals grafted with LV.eGFP transduced corneas. Moreover, reduced expression of proinflammatory cytokines (IFN-γ and IL-6) in PD-L1 transduced corneas compared to allogeneic controls was also observed. Local PD-L1 gene transfer in cultured corneas is a promising approach for the prolongation of corneal allograft survival and attenuation of graft rejection.

An anaesthetic pre-operative assessment clinic reduces pre-operative inpatient stay in patients requiring major vascular surgery.

BACKGROUND: Patients undergoing major vascular surgery (MVS) require extensive anaesthetic assessment. This can require extended pre-operative stays. AIMS: We investigated whether a newly established anaesthetic pre-operative assessment clinic (PAC) would reduce the pre-operative inpatient stay, avoid unnecessary investigations and facilitate day before surgery (DBS) admissions for patients undergoing MVS. PATIENT AND METHODS: One year following and preceding the establishment of the PAC the records of patients undergoing open or endovascular aortic aneurysm repair, carotid endarterectomy and infra-inguinal bypass were reviewed to measure pre-operative length of stay (LoS). RESULTS: Pre-operative LoS was significantly reduced in the study period (1.85 vs. 4.2 days, respectively, P < 0.0001). Only 12 out of 61 patients in 2007 were admitted on the DBS and this increased to 33 out of 63 patients (P = 0.0002). No procedure was cancelled for medical reasons. CONCLUSION: The PAC has facilitated accurate outpatient anaesthetic assessment for patients requiring MVS. The pre-operative in-patient stay has been significantly reduced.

A new theoretical approach to the investigation of the symmetry of protein oligomers with bifunctional reagents.

The use of bifunctional reagents to form cross-links between subunits in protein oligomers and subsequent disruption of noncovalent interactions with SDS allows comment upon the number of subunits and the symmetry in the original assembly. In existing treatments the number of equations needed to describe theoretically the proportions of all the cross-linked species that can be formed as a function of time in this way makes the analysis of the system unmanageable for proteins with more than four subunits. A method is presented that allows the required equations for any oligomer to be formulated as an algorithm suitable for solution by computer. Its application is illustrated with reference to experimental results obtained with two protein hexamers, Jasus hemocyanin and alpha-urease from jack bean.