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Orthopaedic Proceedings
Vol. 97-B, Issue SUPP_16 | Pages 6 - 6
1 Dec 2015
Kostopoulou F Papathanasiou I Anastasopoulou L Aidarinis C Mourmoura E Malizos K Tsezou A
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Toll-like receptors (TLRs) are crucial components of the immune system that recognize microbial infection and trigger anti-microbial host defense responses. Gram positive bacteria are causative factors of bone infections, as they alter the balance of coordinated activities during bone remodeling, stimulating osteoclastogenesis. The aim of the study was to investigate whether genetic variation in TLR2 and TLR4 genes predisposes to bone infections’ susceptibility.

One hundred and twenty patients with bone infections (osteomyelitis) and 200 healthy controls were genotyped for two single nucleotide polymorphisms (SNPs), R753Q [A/G] in TLR2 gene and T399I [C/T] in TLR4 gene. DNA was extracted from whole blood and the above SNPs were typed with PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) method for genotype identification. All patients were infected by Gram-positive bacteria, predominantly Staphylococcus aureus. Statistical analysis was carried out using the chi-square test.

We observed a significantly increased frequency in patients carrying the GA genotype of TLR2 R753Q polymorphism compared to controls (p<0.05). We also found that the A allele was more common in patients than in controls. All individuals carrying the A allele were heterozygous for this variant, while homozygous mutant individuals were not detected in the patients and the control group. In contrast, we found that the TLR4 T399I [C/T] SNP was similarly distributed among the two groups (patients and controls). The mechanism through which TLR2 mediates its effect in bone infections is under investigation.

A significant difference was observed in the genotype frequency of TLR2 R753Q [A/G] polymorphism in patients, suggesting that genetic variability in TLR2 gene may be associated with susceptibility to osteomyelitis in response to bacterial invasion in the bone.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_III | Pages 336 - 336
1 Jul 2011
Tsezou A Poultsides L Papathanasiou I Malizos KN
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Septic arthritis induced by Staphylococcus aureus causes a rapid destruction of joint cartilage and periarticular bone. The mechanisms behind this phenomenon are not fully understood. Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria. TLR2 signaling can lead to the activation of NF-kB through myeloid differentiation factor 88 (MyD88) dependent pathway. The purpose of this study was to examine the catabolic role of TLR2 mediated by the NF-kB pathway in human septic arthritic chondrocytes.

Septic arthritic (SA) chondrocytes (n=7) and fibroblast-like synoviocytes (n=7) infected by gram-positive bacteria, mainly Staphylococcus aureus, as well as chondrocytes from healthy individuals (n=5) were used for this study.

The expression of TLR2 in septic articular cartilage and normal cartilage was analyzed by real time reverse transcription polymerase chain reaction as well western blot analysis. Production of matrix metalloproteinase MMP- 13 and IL-1b was evaluated by enzyme-linked immunosorbent assay. MyD88 protein expression levels and NF-kB activation were evalutated by western blot analysis. Downregulation of TLR2 expression was achieved after transfection with specific siRNA against TLR2 using liposomes.

We observed that TLR2 mRNA and protein expression was significantly up-regulated in septic arthritic cartilage. Also MMP-13 and IL-1b production were significantly increased in septic arthritic chondrocytes compared to normal. Blocking TLR2 in septic chondrocytes resulted in significant reduction of MyD88 and NF-kB protein levels as well as reduction in MMP-13 and IL-1b expression.

It could be suggested that stimulation of TLRs by microbial components may represent the initial signal promoting a pro-inflammatory environment that will enhance degeneration of articular cartilage and the surrounding synovial cells. Targeting NF-kB signalling pathway through TLR2 gene silencing may be of potential therapeutic value in treatment of joint diseases.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_III | Pages 360 - 360
1 Jul 2011
Tsezou A Papathanasiou I Orfanidou T Malizos K
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The Wnt/b-catenin signaling pathway participates in normal adult bone and cartilage biology and seems to be involved in cartilage degeneration and subsequent OA progression. The aim of this study was to investigate the activation of Wnt/b-catenin pathway in osteoarthritis and the role of LRP5, a coreceptor of Wnt/b-catenin pathway, in human osteoarhritic chondrocytes.

Human cartilage was obtained from 11 patients with primary osteoarthritis (OA) undergoing total knee and hip replacement surgery. Normal cartilage was obtained from 5 healthy individuals. b-catenin and LRP5 mRNA and protein levels were investigated using real time PCR and western blot analysis, respectively. Blocking LRP5 expression was performed using small interfering (siRNA) against LRP5 and subsequent MMP-13 mRNA and protein levels were evaluated by real time RCR and western blot analysis, respectively.

We confirmed the activation of Wnt/b-catenin pathway in osteoarthritis, as we observed significant upregulation of b-catenin mRNA and protein expression in osteoarthritic chondrocytes. We also observed that LRP5 mRNA and protein expression was significantly up-regulated in osteoarthritic cartilage compared to normal. Also, blocking LRP5 expression using siRNA against LRP5 resulted in a significant decrease in MMP-13 mRNA and protein expressions.

Our findings suggest that the upregulation of LRP5 mRNA and protein expression in osteoarthritic chondrocytes results in an increased activation of Wnt/b-catenin pathway in osteoarthritis. The observed reduction of MMP-13 expression after blocking LRP5 expression in osteoarthritic chondrocytes, suggests the involvement of LRP5 in the progression and pathogenesis of osteoarthritis.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_III | Pages 355 - 355
1 Jul 2011
Tsezou A Simopoulou T Kostopoulou F Malizos K
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The aim of the study was to investigate the expression of genes regulating cholesterol efflux in human chondrocytes and to study the effect of an LXR agonist on cholesterol efflux and lipid accumulation in osteoarthritic chondrocytes.

Human cartilage was obtained from 24 patients with primary osteoarthritis (OA) undergoing total knee replacement surgery. Normal cartilage was obtained from 8 individuals undergoing fracture repair surgery, with no history of joint disease. ATP-binding-cassette transporter A1(ABCA1), apolipoprotein A1 (ApoA1), and liver X receptors(LXRα and LXRβ) mRNA expression levels were evaluated using real-time PCR. The effect of the synthetic LXR agonist TO-901317 was studied after treatment of osteoarthritic chondrocytes and subsequent investigation of ABCA1 and ApoA1 mRNA expression levels. Cholesterol efflux was evaluated in osteoarthritic chondrocytes radiolabeled with [1,2(n)-3H] cholesterol after LXR treatment, while intracellular lipid accumulation was studied after Oil-red-O staining. Apoptosis was evaluated using flow cytometry.

ApoA1, ABCA1, LXRα and LXRβ mRNA expressions were significantly lower in osteoarthritic chondrocytes compared to normal. Treatment of osteoarthritic chondrocytes with the LXR agonist TO-901317 significantly increased ApoA1 and ABCA1 mRNA expression levels as well as cholesterol efflux, while it significantly reduced apoptosis. Additionally, osteoarthritic chondrocytes presented intracellular lipids deposits, while no deposits were found after treatment with TO-901317.

Our findings suggest that impaired expression of genes regulating cholesterol efflux may be a critical player in osteoarthritis, while the ability of the LXR agonist to facilitate cholesterol efflux and decrease apoptosis suggests that it may be a target for therapeutic intervention in osteoarthritis.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_III | Pages 354 - 355
1 Jul 2011
Tsezou A Orfanidou T Varytimidis S Malizos K
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Cartilage calcification induces the synthesis of degrading enzymes, such as matrix metalloproteinases (MMPs) and prostaglandin E2 leading to tissue degeneration. The aim of the study was to investigate the effect of vitamin D on the calcification process in osteoarthritic cartilage.

We evaluated the effect of vitamin D on klotho (KL), Fibroblast Growth Factor 23 (FGF23) and Fibroblast Growth Factor Receptor 1c (FGFR1c) mRNA and protein expression levels by real-time PCR and western blot analysis, respectively. Possible interactions between klotho and FGF23 on the receptor FGFR1c in normal chondrocytes were investigated using immunoprecipitation assay. The direct effect of 1,25 dihydroxyvitamin D3 (1,25D) on KL, FGF23 and FGFR1c promoter was also evaluated.

We found that FGF23 and FGFR1c mRNA expression levels were significantly increased in osteoarthritic chondrocytes compared to normal, while KL mRNA levels were decreased (p=0.001 for all genes). We showed that klotho-FGF23-FGFR1c form complexes in normal chondrocytes and confirmed the participation of klotho in the initiation of FGF23-FGFR1c signalling. Treatment of normal chondrocytes with 1,25D resulted in a significant dose and time dependent increase of FGF23 and FGFR1c mRNA levels and in an increase of KL mRNA levels in osteoarthritic chondrocytes compared to untreated (p=0.001). We revealed, for the fist time, the presence of conserved, canonical VDREs in the proximal promoters of KL, FGF23 and FGFR1c.

We propose a common regulatory scheme of mineral homeostasis and aging in osteoarthritic chondrocytes evidenced by the positive/negative feedback actions by KL, FGF23, FGFR1c and 1,25D, through binding of vitamin D receptor (VDR) on the promoters of the above mentioned genes.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_III | Pages 355 - 355
1 Jul 2011
Tsezou A Kostopoulou F Poultsides L Simopoulou T Malizos K
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Our study aimed to investigate the role of an HMG-CoA reductase inhibitor (atorvastatin) in human osteoarthritic chondrocytes and to test the in vivo effects of intra-articular injections of atorvastatin in a rabbit experimental osteoarthritis model.

Human articular osteoarthritic chondrocytes were cultured in the presence and absence of atorvastatin. mRNA and protein expression of MMP-13, COL2A1 and aggrecan were measured using real-time PCR and Western Blot analysis.

New Zealand rabbits (n=15) underwent bilateral anterior cruciate ligament transection (ACLT) to induce osteoarthritic degeneration and received intra-articular injections of atorvastatin and normal saline in the left and right knees respectively. The first injection was at the time of ACLT and injections were repeated every 3 days for 3 weeks. Data were obtained from macroscopic and histological evaluation as well as from gene expression analysis for COL2A1, aggrecan and MMP-13.

Incubation of the cultures with atorvastatin produced a decreasing effect in MMP-13 expression. Regarding aggrecan and COL2A1 expression a significant increase was observed.

Gross morphologic evaluation showed that the joints which received atorvastatin injections, showed minimal cartilage erosion, compared to the non-treated knees where the cartilage was markedly eroded, especially on the medial knee compartment. These results were supported by histological and gene expression analysis. The mRNA expression of MMP-13 was significantly reduced in the cartilage of the statin-treated knee joints, while the expression of COL2A1 and aggrecan was increased.

The clinical relevance of our results indicates a potential protective effect of atorvastatin on articular cartilage undergoing osteoarthritic degeneration.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_II | Pages 97 - 98
1 May 2011
Tsezou A Iliopoulos D Malizos K Simopoulou T
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Osteoarthritis is a complex joint disease in which all involved tissues play an important role in its onset and progression. It has been suggested that osteoarthritis is likely to be a systemic disease involving stromal cell differentiation and lipid metabolism while altered lipid metabolism has been implicated as a critical player in its pathogenesis.

As excessive accumulation of free cholesterol is toxic for the cells, the accumulation of lipids in chondrocytes may signify a causal relationship to development and/or progression of osteoarthritis; therefore we investigated the expression of genes regulating reverse cholesterol transport, as ABCA1, ApoA1, LXRa, LXR_, in human osteoarthritic chondrocytes. We also investigated the effect of an LXR agonist on ABCA1 and ApoA1 expression and, for the first time, on cholesterol effiux and lipid accumulation in osteoarthritic chondrocytes.

Articular cartilage samples were obtained from femoral condyles and tibial plateaus of patients with primary OA undergoing knee replacement surgery while normal cartilage was obtained from eight individuals undergoing fracture repair surgery, with no history of joint disease. Total cellular RNA was extracted from all samples and ABCA1, ApoA1, and LXRα and LXRβ mRNA and protein expression levels were evaluated using real-time PCR and Western blot analysis respectively.

The effect of the synthetic LXR agonist TO-901317 was studied after treatment of osteoarthritic chondrocytes and subsequent investigation of ABCA1 and ApoA1 mRNA expression levels. Cholesterol effiux was evaluated in osteoarthritic chondrocytes radiolabeled with [1,2(n)-3H] cholesterol after LXR treatment, while intracellular lipid accumulation was studied after Oil-red-O staining.

ApoA1 and ABCA1 mRNA levels were significantly lower in osteoarthritic cartilage compared to normal (p< 0.01 and p< 0.001 respectively). In addition, the two subtypes of the LXR, namely LXRα and LXRβ, mRNA levels were also found to be significantly lower in osteoarthritic cartilage (p< 0.05 and p< 0.01 respectively). The differential expression pattern of the cholesterol effiux genes between normal and osteoarthritic cartilage remained the same at the protein level as well. Treatment of osteoarthritic chondrocytes with the LXR agonist TO-901317 significantly increased ApoA1 and ABCA1 expression levels, as well as cholesterol effiux. Additionally, osteoarthritic chondrocytes presented intracellular lipids deposits, while no deposits were found after treatment with TO-901317.

Our findings suggest that impaired expression of genes regulating cholesterol effiux may be a critical player in osteoarthritis, while the ability of the LXR agonist to facilitate cholesterol effiux suggests that it may be a target for therapeutic intervention in osteoarthritis.


Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_II | Pages 315 - 315
1 May 2009
Tsezou A Poultsides L Satra M Malizos K
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It is known that wide variability exists among patients in the susceptibility to and outcome from infection. Polymorphisms in genes coding for proteins involved in the response to bacterial pathogens as tumor necrosis factor-alpha(TNF-a), interleukin (IL)-1alpha, IL-1beta, IL-1 receptor agonist, IL-6, IL-10 can influence the amount or function of the protein produced in response to bacterial stimuli. These genetic polymorphisms may influence the susceptibility to and outcome from infection.

The aim of the study was to investigate whether genetic variation in genes coding for components of the innate immune response might be a critical determinant of the inflammatory response and the risk for and outcome from severe bacterial infection in individuals with musculoskeletal infections. The relationship between single nucleotide polymorphisms (SNPs) in the above mentioned genes and susceptibility to infection was evaluated.

Forty patients with musculoskeletal infections hospitalised at the Orthopaedic Clinic of University Hospital of Larissa, as well as 80 healthy controls were included in the study. Genomic DNA was isolated from peripheral blood from all cases and controls and was extracted according to standard procedures. The following genes with their polymorphic positions were studied: IL 1α (IL 1α promoter −889), IL 1β (IL 1β promoter −511, pos. +3962), IL 1R (IL 1R pos. pst1 1970), IL 1RA (IL 1RA pos. mspa1 11100), IL 4Rα (IL 4Rα pos. +1902), IL 12 (IL 12 promoter −1188), TGF-β (TGF-β exon 1 codon 10, codon 25), TNF-α (TNF-α promoter −308, −238), IL 2 (IL 2 promoter −330, pos. +166), IL 4 (IL 4 promoter −1098, −590, −33), IL 6 (IL 6 promoter −174, pos. +nt 565) and IL 10 (IL 10 promoter −1082, −819, −592). Genotype distribution and allele frequencies in patients and controls were evaluated.

There was a significant difference in genotype and allele frequency of IL-1a (T/C −889) p=0.000 (CC, TC) between patients and the control group. Moreover, 2 SNPs of interleukin 4 [IL-4 (T/G −1098) p=0.000 (GG, GT) p=0.009 (TT) and IL-4 (T/C-590) p=0.000 (CC, CT) p=0.006 (TT)] showed significant genotypic and allelic differences between the two groups. Finally, 2 SNPs of interleukin 6 [IL-6 (G/C-174) p=0.000 (CC) p=0.014 (GG), IL-6 G/A nt565) p=0.000 (AA,GA,GG)] and TNF-a [(G/A-308) p=0.034 (AG)] showed significant differences in genotype and allele frequencies between patients and the control group.

We observed, for the first time, significant differences in genotype and allele frequencies of TNF-a (G/A-308), IL-1a (T/C -889), IL-4 (T/G -1098), IL-4 (T/C-590), IL-6 (G/C-174) and IL-6 G/A (nt565) in patients with musculoskeletal infections, a fact which points towards the involvement of cytokine gene polymorphisms in the pathogenesis of infection.