Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Intervertebral disc regeneration. Studies on stem cell niches and cell transplantation

Abstract Intervertebral disc regeneration Studies on stem cell niches and cell transplantation Helena Barreto Henriksson Gothenburg 2010 Low back pain is a common condition in the Western world and disc degeneration (DD) is considered a major cause. DD is characterized by dysfunctional cells and decreased matrix production. The aim of this thesis was to explore normal growth and regeneration in the intervertebral disc (IVD). Further, to test possibilities of cell therapy treatment for DDs. The methods used include in vitro- and in vivo experiments. In vitro methods were: monolayer, 3D cell cultures and explants models with human mesenchymal stem cells (hMSCs), articular chondrocytes and IVD cells. Cells/ tissues were analyzed for cell proliferation markers; BrdU, KI67, migration markers: β1-INTEGRIN, SNAIL-homolog-1 (SNAI1), SNAIL-homolog-2 (SLUG), progenitor/stem cell markers: STRO1, C-KIT, Notch1, CD105 and chondrogenic lineage markers: GDF5 and SOX9, matrix markers: COLLAGEN I and II, glycosaminoglycans, AGGRECAN by biochemical methods, flowcytometry, Real-time PCR and microscopy. Disc appearance was evaluated with MRI. Results from normal regeneration studies: a potential stem cell niche was identified in the IVD region lateral to the epihyseal plate and in the annulus fibrosus outer region, based on findings of label-retaining cells and presence of cells expressing stem cell/progenitor markers, in young and mature animals. Migrating cells expressing SNAI1, SLUG, β1-integrin and GDF5 and SOX9 around niches were observed. Results from the cell therapy experiments; In vitro analyses; 3D co-culture system of hMSC and IVD cells showed an increased COLLAGEN II production. In vivo: Xenotransplanted cells survived in vivo 6 months (porcine IVDs) and produced matrix in hydrogel/MSCs injected IVDs. Taken together, these findings illustrate a normal slow regeneration of the IVD, and that growth and regeneration is presumably supported by progenitor cells deriving from niches adjacent to the IVD. Further, that human IVD cells and MSCs interact positively on matrix production when co-cultured and the survival of transplanted cells in vivo support the possibility for cell therapy treatment of DD. These results encourage further studies to arrest IVD degeneration, by stimulation of regenerative mechanisms in situ or by cell therapy. ISBN 978-91-628-8147-

Pathological Effects of Cortisol on Intervertebral Disc Cells and Mesenchymal Stem Cells from Lower Back Pain Patients

In western countries, lower back pain (LBP) is one of the most common disorders, experienced by more than 80% of the population. Chronic LBP due to disc degeneration has been linked to ongoing inflammatory processes in the disc and endplates. Pain effects the body in different ways, inducing a general stress response in which the body responds by releasing the stress hormone cortisol. Little is known about the impact of pain-induced stress on the progression of disc degeneration. Thus, the effects of cortisol on disc cells (DCs) and human mesenchymal stem cells (hMSCs) were explored in vitro with the objective of investigating the repercussions of cortisol on these cell types involved in de- and regenerative mechanisms of the disc. DC and hMSC pellet cultures were exposed to cortisol at two concentrations (150 and 300 ng/mL) for 28 days to simulate pain-induced stress. Cell viability, histological staining, and GAG DNA, along with apo-ptotic assays were conducted. Detection of OCT4, SOX9, IL-1R, and CXCR2 expressions was performed by immunohistochemistry. With cortisol treatment, restricted cell proliferation and less GAG production in both DCs and hMSCs were observed. Suppression of the differentiation and immunomodulatory efficacy of hMSCs was also detected. Moreover, elevated expressions of IL-1R and CXCR2 were detected in both cell types. To conclude, constant exposure to cortisol even at a physiological level enhanced pathological cellular processes in both DCs and hMSCs, which further jeopardized chondrogenesis. This suggests that cortisol resulting from pain-induced stress is a contributing component of intervertebral disc degeneration and may negatively affect regenerative attempts of the disc

Determination of mechanical and rheological properties of a cell-loaded peptide gel during ECM production

The development of an injectable biomaterial that supports cell survival and maintains or promotes nucleus pulposus (NP) phenotype could aid delivery of cells to degenerated NPs causing low back pain. Mesenchymal cells were loaded and grown in a synthetic peptide gel, PuraMatrix \uae . Cells were observed within the gels over 0–28 days, and accumulation of glycosaminoglycans were detected by histological staining. The mechanical properties of the cell-loaded constructs, and the change of the mechanical properties were studied using stress relaxation of the gels under compression and confinement. The PuraMatrix \uae gel was shown to relax fast on compression indicating that the fluid could easily flow out of the gel, and thus indicating the presence of large pores/voids. The presence of these pores/voids was further supported by high mobility of dextran molecules, determined using fluorescence recovery after photo bleaching. The stress required to deform the cell-loaded constructs to a specific strain increases at day 21, at which point the presence of glycosaminoglycans within the cell-loaded constructs was also observed. The results provide evidence of changes in mechanical properties of the PuraMatrix \uae matrix upon excretion of the extracellular matrix by the cells

Biocompatibility and resorption of a radiopaque premixed calcium phosphate cement

Calcium phosphate cements (CPC) are used as bone void filler in various orthopedic indications; however, there are some major drawbacks regarding mixing, transfer, and injection of traditional CPC. By using glycerol as mixing liquid, a premixed calcium phosphate cement (pCPC), some of these difficulties can be overcome. In the treatment of vertebral fractures the handling characteristics need to be excellent including a high radio-opacity for optimal control during injection. The aim of this study is to evaluate a radiopaque pCPC regarding its resorption behavior and biocompatibility in vivo. pCPC and a water-based CPC were injected into a Ø 4-mm drilled femur defect in rabbits. The rabbits were sacrificed after 2 and 12 weeks. Cross sections of the defects were evaluated using histology, electron microscopy, and immunohistochemical analysis. Signs of inflammation were evaluated both locally and systemically. The results showed a higher bone formation in the pCPC compared to the water-based CPC after 2 weeks by expression of RUNX-2. After 12 weeks most of the cement had been resorbed in both groups. Both materials were considered to have a high biocompatibility since no marked immunological response was induced and extensive bone ingrowth was observed. The conclusion from the study was that pCPC with ZrO2 radiopacifier is a promising alternative regarding bone replacement material and may be suggested for treatment of, for example, vertebral fractures based on its high biocompatibility, fast bone ingrowth, and good handling properties. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012

Terminal complement complex formation is associated with intervertebral disc degeneration

Purpose!#!The complement system is a crucial part of innate immunity. Recent work demonstrated an unexpected contribution to tissue homeostasis and degeneration. This study investigated for the first time, in human disc tissues, the deposition profile of the complement activation product terminal complement complex (TCC), an inflammatory trigger and inducer of cell lysis, and its inhibitor CD59, and their correlation with the degree of disc degeneration (DD).!##!Methods!#!Disc biopsies were collected from patients diagnosed with DD (n = 39, age 63 ± 12) and adolescent idiopathic scoliosis (AIS, n = 10, age 17 ± 4) and compared with discs from healthy Young (n = 11, age 7 ± 7) and Elder (n = 10, age 65 ± 15) donors. Immunohistochemical detection of TCC and CD59 in nucleus pulposus (NP), annulus fibrosus (AF) and endplate (EP) was correlated with age, Pfirrmann grade and Modic changes.!##!Results!#!Higher percentage of TCC+ cells was detected in the NP and EP of DD compared to Elder (P < 0.05), and in the EP of Young versus Elder (P < 0.001). In DD, TCC deposition was positively correlated with Pfirrmann grade, but not with Modic changes, whereas for Young donors, a negative correlation was found with age, indicating TCC's involvement not only in DD, but also in early stages of skeletal development. Higher CD59 positivity was found in AIS and DD groups compared to Young (P < 0.05), and it was negatively correlated with the age of the patients.!##!Conclusion!#!TCC deposition positively correlated with the degree of disc degeneration. A functional relevance of TCC may exist in DD, representing a potential target for new therapeutics