Eicosapentaenoic acid modulates CyA-induced proinflammatory cytokine over-expression in osteoblastic cells in vitro.

Several adverse outcomes are reported in subjects undergoing long term Cyclosporin A (CyA) treatment. Severe osteopenia has been described in clinical and experimental reports, while beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) on bone metabolism are recognized. In the present study we investigated the effects of n-3 versus n-6 PUFAs on osteoblastic cells treated with CyA, evaluating the expression of interleukin (IL)-1ß, interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in two different experimental protocols and the production of IL-6, IL-1ß, and tumor necrosis factor alpha (TNFalpha) in cells challenged simultaneously with CyA and eicosapentaenoic acid (EPA) for 48h. IL-1ß and IL-6 up-regulation, induced by CyA, was counteracted by the addition of EPA in both protocols; on the contrary, arachidonic acid (AA) magnified CyA the effects. COX-2 and iNOS levels were not modified by CyA treatment. These in vitro results, that substantiate clinical reports of CyA-induced osteopenia, demonstrate a beneficial effect of EPA on CyA-altered cytokine profile, opening new perspectives in the non-pharmacological management of adverse outcomes in CyA-treated patients.

Immunogenetic aspects of erosive osteoarthritis of the hand in patients from northern Italy.

OBJECTIVES: To compare the distribution of human leucocyte antigen (HLA) class I and II alleles in patients with erosive hand osteoarthritis (EHOA) to that of patients with non-erosive hand OA (non-EHOA) and in healthy Italian Bone Marrow Donors (IBMDs), in order to evaluate possible immunogenetic associations with EHOA. In the EHOA group we also sought possible associations between HLA alleles and disease severity. METHODS: Ninety-four patients with EHOA (82 women, 12 men; mean age 61.4 ± 8.45 years) and 37 with non-EHOA (28 women, nine men; mean age 59.21 ± 9.07 years) were studied. Disease severity was measured by the number of clinically active joints (NCAJ) and by the radiographic score (RS) using the Kallman scale. HLA typing was undertaken for A, B, C, and DRB1 loci; HLA-DRB1* genotyping was determined using polymerase chain reaction (PCR) with sequence-specific primers. Frequencies were compared with those of the healthy IBMDs. RESULTS: The alleles found more frequently in EHOA patients than in non-EHOA patients and healthy controls were: A23, A26, and A29; B38, B44, and HLA DRB1*01 and *07. The RS was more severe in the EHOA compared to the non-EHOA group (63.60 ± 23.14 vs. 34.34 ± 20.24, p < 0.001). Within the EHOA group, HLA-DRB1*07 was associated with a higher RS (67.36 ± 23 vs. 64.5 ± 18.5, p = 0.029). CONCLUSION: In this study of North Italian patients affected with EHOA, the HLA-DRB1*07 allele was found to be associated with both the development and greater severity of the disease.

Influence of glucosamine sulphate on oxidative stress in human osteoarthritic chondrocytes: effects on HO-1, p22(Phox) and iNOS expression.

OBJECTIVE: Reactive oxygen species (ROS) are major determinants in the alteration of articular cartilage. Among protective cellular mechanisms, the inducible isoform of haem oxygenase (HO-1) plays a particularly relevant role. On the other hand, the enzymatic activity of the Nicotinamide adenine dinucleotide phosphate (NADPH) system could contribute to the generation of ROS. Glucosamine sulphate (GS) is one of the drugs used in the treatment of osteoarthritis; however, its mechanism of action is still largely unknown. The aim of the present study was to investigate the effects of GS on primary human chondrocytes in vitro, in particular with regard to HO-1, p22(Phox) (a subunit of NADPH complex) and inducible nitric oxide synthase (iNOS) expression. METHODS: Primary human chondrocytes were treated with different concentrations of GS; gene expression of HO-1, p22(Phox) and iNOS was assessed by the reverse transcriptase-polymerase chain reaction method. In a separate set of experiments, the cells were stimulated with human recombinant interleukin (IL)-1beta and simultaneously treated with GS. Moreover, HO-1 protein and total nitrite production were evaluated. RESULTS: HO-1 gene expression was up-regulated (+40% with respect to the controls, P < 0.001) by 10 mmol/l GS at 24 h, while p22(Phox) gene expression was down-regulated by 10 mmol/l GS with a maximum inhibitory effect observed after 48 h treatment. IL-1beta stimulation induced expression of iNOS reverted by 1 and 10 mmol/l GS. Moreover, HO-1 gene expression was down-regulated by IL-1beta and 10 mmol/l GS restored baseline values. These data were confirmed by evaluating HO-1 protein level and nitrite production. CONCLUSIONS: The influence of GS on oxidative stress observed in this study discloses a possible new mechanism of action and seems to be in keeping with a potential protective effect on chondrocyte population.

Synoviocyte infection with adeno-associated virus (AAV) is neutralized by human synovial fluid from arthritis patients and depends on AAV serotype.

Intraarticular gene transfer with adeno-associated viral (AAV) vectors may allow efficient therapeutic transgene expression within the joint in diseases such as rheumatoid arthritis (RA), allowing high expression of the protein within the joint, preventing both systemic diffusion and side effects. However, humans demonstrate antibodies against AAV, which can influence gene transfer. To better understand critical obstacles to intraarticular gene therapy with AAV, we have previously shown that synovial fluid (SF) contains IgG to AAV that neutralizes chondrocyte infection in vitro. Our objective was therefore to compare neutralization exerted by SF from RA patients for four different AAV serotypes (AAV serotypes 1, 2, 5, and 8) on human primary synoviocytes. Serotype 2 infected synoviocytes most efficiently followed, in decreasing order, by serotypes 1, 5, and 8. SF from all patients partially inhibited infection of synoviocytes by at least one of the four serotypes. Infection with serotypes 1 and 2 was the most inhibited by SF, whereas inhibition was weak for serotypes 5 and 8. Last, we have shown that inhibition of AAV1/interleukin (IL)-4 infection of synoviocytes by SF could be reversed by increasing the number of AAV1/IL-4 particles, with a dose-dependent effect. We conclude that the most infectious AAV serotypes (1 and 2) in synoviocytes are also the serotypes most neutralized by SF. Thus, serotype 5 seems to demonstrate the best infection efficiency:immunogenicity ratio for local use in articular diseases. These data may be useful for tailoring intraarticular AAV-mediated gene therapy to individual patients.

YKL-40 in human lumbar herniated disc and its relationships with nitric oxide and cyclooxygenase-2.

UNLABELLED: The basic pathophysiology of intervertebral disc degeneration and low back pain remains unclear. It has been hypo-thesized a role of biochemical mediators of inflammation and tissue degradation in intervertebral disc degeneration and herniation. Chitinase 3-like protein 1 (YKL-40) is a glycoprotein mainly secreted by chondrocytes which has been proposed as a possible marker of inflammation and/or cartilage alterations. OBJECTIVE: To investigate the YKL-40 presence in human lumbar disc tissue culture and its possible relationships with some substances relevant in inflammation such as cyclooxygenase-2 (COX-2) and nitric oxide (NO). PATIENTS AND METHODS: We analyzed lumbar discs from 19 patients who underwent surgery for lumbar disc herniation at L4-L5 or L5-S1 levels. The specimens were cultured and incubated for 72 hours. At the end of incubation, the supernatants were assayed for presence and concentration of YKL-40, COX-2 and NO. RESULTS: YKL-40 was detectable in all the samples analyzed. Mean (+/-SD) concentration was 1.54+/-1.29 ng/ml/mg compared to dry weight. COX-2 and NO levels were 25.25+/-11.42 pg/ml/mg and 1.3+/-1.8 microM/mgx10(-2), respectively. A correlation was found between YKL-40 and COX-2 (r=0.579, p<0.05) and YKL-40 and NO (r=0.509, p<0.05). CONCLUSION: To our knowledge, this is the first report demonstrating YKL-40 release by intervertebral disc culture. It may contribute to better clarify the role of this protein in the pathophysiology of discal degeneration and inflammation as confirmed by its relationships with COX-2 and NO in disc tissue culture.

[The enthesopathy of vitamin D-resistant osteomalacia in adults]. / La polientesopatia dell'osteomalacia vitamina D resistente dell'adulto.

A case of an adult patient with vitamin D-resistant osteomalacia or X-linked hypophosphatemic osteomalacia (XLH) with diffuse calcification of entheses is reported. XLH is the most frequent cause of rickets in developed countries. It is characterized by an impaired renal transport of the phosphate and mutation of PFEX (phosphate regulating gene, with homologies to endopeptidase on the X-chromosome). In childhood, the classic clinical presentation includes short stature and bow leg. While at this age the main radiographic features are characterised by rickets, in adult life they are dominated by a generalised calcific enthesopathy. Concerning the pathogenesis of the enthesopathic lesions of XLH, no convincing hypothesis has yet been made. As in our patient, the extension and the severity of enthesopathy seems not related to the severity of the biochemical changes nor to the treatment with calcitriol. The calcified enthesopathy is an integral part of XLH and it is possible that it is found in adult because many years are necessary to produce it.

[Frequency of the bleeding risk in patients receiving warfarin submitted to arthrocentesis of the knee]. / Frequenza del rischio emorragico nei pazienti in trattamento con warfarin sottoposti ad artrocentesi del ginocchio.

OBJECTIVE: To evaluate the potential bleeding risks of arthrocentesis in patients with different arthropathies and taking oral anticoagulants. MATERIALS AND METHODS: Fifteen consecutive patients, 8 males and 7 females, treated with anticoagulant therapy for atrial fibrillation (9 pts), deep venous thrombosis (4 pts) and replacement of cardiac valves (3 pts) were submitted to arthrocentesis for synovial fluid effusion due to different arthropathies. In all patients INR was