Rheumatology and Metabolic Medicine

Rheumatology and Metabolic Medicine 

Members of the Research Team:

Dr Oksana Kehoe, Dr Alasdair Kay, Mr Andrei Stefan and Miss Grace Long (intercalated student) Professor Jim Middleton (Honorary Clinical Scientist)

Clinical Support: Dr Mark Garton (Lead), Dr Ayman Askari, Dr Roshan Amarasena, Dr Wolfgang Schaefer

Our team has continued to be active this year carrying out basic science work into understanding mechanisms of rheumatoid arthritis (RA) progression and possible treatments.

In collaboration with Dr Mark Garton, Dr Nicola Kuiper, Professor Nick Forsyth (Keele), Dr Paul Roach (Keele) our group was awarded £98,475 from the RJAH Hospital Charity to investigate the extracellular vesicles derived from mesenchymal stem cells and chondrocytes with potential applications in inflammatory arthritis.

We have been very fortunate to receive a grant from the Orthopaedic Institute ‘Hypoxic vs normoxic grown MSC as a potential cell therapy for Rheumatoid Arthritis to purchase a Russkin Baker Hypoxic workstation.

We have been publishing our findings in European Cells and Materials, Journal of Tissue Engineering and Regenerative Medicine and the International Journal of Experimental Pathology.

We have presented our work at several national and international meetings such as Tissue and Cell Engineering Society, Southampton, July 2015; 4th TERMIS World Congress, Boston (USA) September 2015, UK Mesenchymal Stem Cell meeting, Manchester, December 2015;  British Society for Matrix Biology, Chester, April 2015; Midlands Academy of Medical Sciences Research Festival, Leicester, April 2015 in addition to scientific meetings at RJAH.

 

The projects which we are currently working are explained in detail below.

 

Stem Cells as Therapeutic Agents in Arthritis

Alasdair Kay, Andrei Stefan, Jim Middleton (Bristol University), Oksana Kehoe

Funded by Dowager Countess Eleanor Peel Trust, Orthopaedic Institute and Oswestry Rheumatology Association

Alasdair continues to work on his project aimed at understanding the role of mesenchymal stem cells in our pre-clinical arthritis model. Alasdair has shown intra-articular injection of mesenchymal stem cells conditioned medium attenuates progression of inflammation and cartilage destruction in pre-clinical arthritis model. Conditioned medium injection alleviated RA symptoms through reduction of inflammation (joint swelling) at day 3 and improved histological scores for hyperplasia, synovial infiltrate, cartilage depletion and overall arthritis index for disease severity. Therapeutic effects were associated with induction of regulatory anti-inflammatory T cells in spleen (Figure 1).  

 

Figure 1.  Increased FoxP3 expression (marker of anti-inflammatory “good” T lymphocytes) assessed by flow cytometry. This on-going work could identify new therapeutic targets or novel treatment methods to further reduce joint swelling in clinical studies in arthritis patients.

 

Mesenchymal stromal cells and syndecans as therapeutic agents in inflammatory arthritis

Andrei Stefan, Alasdair Kay, Nick Forsyth, Oksana Kehoe    

Funded by Keele University School of Medicine, Oswestry Rheumatology Association and Orthopaedic Institute

Andrei continues to work on his part-time PhD project based on the role of syndecan-3 and mesenchymal stromal cells in arthritis. Syndecans are heparan sulphate proteoglycans expressed by endothelial cells of blood vessels and play a role in trafficking of inflammatory cells in an arthritis model.  Andrei has shown that in vitro characterisation of bone marrow derived MSCs is not altered by the knockout of SDC-3 gene with two key exceptions: CD105 surface marker and cellular complexity.

 

Figure 2. Trilineage differentiation potential of WT (top row) and SDC-3-/- (bottom row) mMSCs (x10). The osteogenic (A and B) and adipogenic (C and D)  potential was identified for both cell types by the positive red stain of the alkaline phosphatase activity and lipid vesicles formation respectively. The positive chondrogenic (E and F) staining is represented by the intense blue staining of the glycosaminoglycans in the pellet sections. Scale bar = 200 μm.

Pre-clinical testing of hypoxic MSC as a novel cell therapy for Rheumatoid Arthritis

Andrei Stefan, Alasdair Kay and Oksana Kehoe

Funded by Oswestry Rheumatology Association

With gratefully received funding from the Oswestry Rheumatology Association, we have embarked upon an extensive and thorough study of the effects of physiologically normal oxygen levels applied to the growth of mMSC for therapeutic application. The study will look at how key genes are altered in hypoxic growth; the changes these alterations induce in protein production; and how these proteins affect the moderation of inflammatory arthritis.

Anti-inflammatory effect of mesenchymal stem cells conditioned media in inflammatory arthritis

George Tyler (Keele University School of Medicine), Alasdair Kay and Oksana Kehoe

INSPIRE is coordinated by the Academy of Medical Sciences and supported by the Wellcome Trust. Mr George Tyler, a second year medical student from Keele University, won the INSPIRE Summer studentship and spent 5 weeks last summer in our laboratory, working together with Dr Alasdair Kay. George was studying the mechanism underpinning anti-inflammatory effects of mesenchymal stem cell conditioned medium in an arthritis model. His results were included into Alasdair’s oral presentation at 4th TERMIS World Congress, Boston (USA) September 2015.

Immunoregulatory effects of mesenchymal stem cells (MSCs) and mesenchymal stem cells conditioned medium (CM-MSC) in experimental arthritis

Alasdair Kay, Grace Long (intercalated student, Keele University MMedSci Program), Andrei Stefan and Oksana Kehoe

Funded by Keele University School of Medicine, Oswestry Rheumatology Association and Orthopaedic Institute

Grace is an intercalated fourth year medical student working with Dr Kay. She is studying immunoregulatory effects of stem cells and stem cell conditioned medium on activated T lymphocytes. RA is characterised by an elevated autoimmune response with an abnormal shift in the natural balance of pro- and anti-inflammatory T cell production. It is hypothesized that therapeutic MSC and MSC products moderate the immune reaction by restoring the normal balance of T cells and reducing the effects of an autoimmune response. We have investigated the proliferation of activated T cells in the presence of either MSC or CM-MSC, and identified the type of T cell produced in lymph nodes and spleens of mice with induced arthritis. An optimised method has been developed to measure dividing cells, with preliminary data showing a reduction in the numbers of times cells divide when in the presence of CM-MSC (Figure 3).

 

Figure 3. Proliferation cycles of T cells. 

T cells from healthy spleens and lymph nodes cultured for 7 days A) with no activation; B) just T cells; and in the presence of C) adherent MSC’s or D) conditioned medium. Red peaks represent negative controls, green peaks represent initial cell numbers, and white peaks are proliferative cycles. Without activation (A), T cells die and no cells are seen at day 7. Reduced proliferation cycles and smaller peaks are seen in T cells co-cultured with MSC (C).

To assess the potential for naïve T cells to shift towards pro- or anti-inflammatory cells, spleens and lymph nodes of animals with induced arthritis and treated with either MSC or CM-MSC were used to extract T cells. These cells are therefore responding to inflammation caused by arthritic conditions. The production of markers characteristic of pro-inflammatory (Th1 and Th17 cells) or anti-inflammatory (Th2 and regulatory T cells) are assessed. The production of anti-inflammatory cytokines was increased in both MSC and CM-MSC treatment conditions and the proportion of pro-inflammatory ‘bad’ Th17 cells was reduced following CM-MSC treatment.

These results suggest that cell therapy using MSC or MSC products may act therapeutically to restore the balance of pro- and anti-inflammatory T cells by reducing ‘bad’ Th17 cells alongside increasing ‘good’ cytokine expression. Together these findings suggest a therapeutic application of MSC in immunomodulation acting through direct signalling. Further work will focus on positive identification of the components present in conditioned medium that may be isolated to act as a therapeutic agent.

 

Metabolic Medicine and Bone

Bone Health Clinical Service, Charles Salt Laboratories.

The bone health service currently has six members of staff who between them cover 4.3 whole time equivalent jobs.  Two work part time in the clinical measurement side of the service as well as in the DXA (bone densitometry unit) and four members of staff work solely in the DXA unit. The service also has 3 part time consultants and a nurse who does intravenous treatments both at the RJAH and in outreach clinics. In the last financial year we saw 5220 patients for a bone density scan only, 4202 clinic patients, many of whom also had a scan and 439 patients received intravenous treatment for their bones in our day care unit on Sheldon ward. 

In the laboratory over 1780 patient samples were analysed for the marker uNTx, a long established marker of bone breakdown.  Bisphosphonates (e.g. Alendronate) are the first line drug treatment for patients with osteoporosis. Following the recommendation of a treatment holiday in patients on long-term bisphosphonates due to rare but serious side-effects, we are continuing to study the effect of a treatment holiday on bone turnover

Research in the Charles Salt Laboratories.

Our work on bone and lean tissue coeliac disease has continued in men who are a relatively understudied group. We showed that both upper limb and lower limb lean body mass was significantly low in men with newly diagnosed coeliac disease, and suggested that inflammation, as has been suggested in Crohn’s disease, might be an underlying cause1. In the same patients we found that Bone Mineral density (BMD) in both the spine and femoral neck was similar to values in control subjects. In the same subjects serum alkaline phosphatase and serum parathyroid hormone levels were slightly higher than in control subjects, but were within the normal range. Generally BMD is thought to be low in Coeliac disease due to secondary hyperparathyroidism.

Previous studies had concentrated on the distal forearm site. We have been investigating BMD in newly diagnosed male coeliac subjects at sites in the lower limb using a DXA method that we have previously described2. The lower limb can be divided into sites which have a large component of trabecular bone (metaphyseal regions) and those with a greater proportion of cortical bone (diaphyseal regions). BMD is significantly low in the metaphyseal regions but not in the diaphyseal regions and at neither diaphyseal nor metaphyseal region is there a significant relationship with parathyroid hormone levels3. If hyperparathyroidism is an important cause of bone loss in coeliac disease, the effect may be different according to oestrogen status. Parathyroid hormone levels tend to increase in the absence of oestrogen and the same hormone also antagonises the breakdown effect of parathyroid hormone on bone. We are currently studying bone mineral density, together with parathyroid hormone values and biochemical markers of bone breakdown in pre and post menopausal women with newly diagnosed coeliac disease compared with healthy control subjects in collaboration with the University of Chester.

  1.  Davie M, Evans S, Sharp C. Limb, muscle, and bone in coeliac disease. Aliment Pharmacol Ther. 2015;42:1332-1333.
  2. Haddaway MJ, Davie MW, Davies HL, Sharp CA. Application of sub-regional analysis to bone mineral density of the lower limb from whole body DXA scans. Physiol Meas. 2013 ;34:757-768.
  3. Davie MWJ, Evans SF, Sharp CA. Lower limb metaphyseal bone is lost in men with Coeliac disease and does not relate to parathyroid status. In preparation.