ABSTRACT
The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry. We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008). These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies.
Subject(s)
Apoptosis , Cytokines/biosynthesis , Tendinopathy/metabolism , Adult , Aged , Animals , Apoptosis/genetics , Cumulative Trauma Disorders/genetics , Cumulative Trauma Disorders/metabolism , Cumulative Trauma Disorders/pathology , Cytokines/genetics , Disease Models, Animal , Humans , Inflammation Mediators/metabolism , Male , Middle Aged , Oligonucleotide Array Sequence Analysis/methods , Pain Measurement/methods , Random Allocation , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction/methods , Rotator Cuff/metabolism , Rotator Cuff/pathology , Rotator Cuff Injuries , Tendinopathy/genetics , Tendinopathy/pathology , Up-Regulation , Young AdultABSTRACT
OBJECTIVE AND DESIGN: To determine if the addition of nitric oxide (NO) via nitroflurbiprofen (NO-flurbiprofen) would enhance rat Achilles tendon healing. MATERIALS AND METHODS: Sixty-five male Sprague-Dawley rats were randomly divided into NO-flurbiprofen, flurbiprofen and vehicle groups, given drugs or vehicle subcutaneously, and their right Achilles tendon divided. Histological assessment was carried out at day 5, 10, and 15 post-operation. Healing tendon biomechanical properties and hydroxyproline content were measured at day 10. RESULTS: The healing Achilles tendon from the NO-flurbiprofen and flurbiprofen groups showed a better organization of extracellular collagenous matrix than that from the vehicle group. Flurbiprofen and NO-flurbiprofen decreased healing tendon cross-sectional area by 30% and 20%. This reduction was accompanied by a decreased failure load in the flurbiprofen group, but not the NO-flurbiprofen group. NO-flubiprofen prevented the reduction of body weight gain observed in the flubiprofen group. CONCLUSION: Both flurbiprofen and NO-flurbiprofen promoted better collagen reorganization during tendon healing. NO-flurbiprofen further improved tendon healing by increasing tendon stress and reducing the side effects (body weight loss) of flurbiprofen. The enhanced tendon healing by NO-flurbiprofen is likely due to the release of NO from the compound.
Subject(s)
Achilles Tendon/injuries , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Flurbiprofen/analogs & derivatives , Flurbiprofen/therapeutic use , Nitric Oxide/therapeutic use , Wound Healing/drug effects , Achilles Tendon/metabolism , Achilles Tendon/pathology , Animals , Biomechanical Phenomena , Body Water/metabolism , Collagen/metabolism , Coloring Agents , Extracellular Space/metabolism , Male , Nitric Oxide/metabolism , Organ Size/drug effects , Rats , Rats, Sprague-Dawley , Weight Gain/drug effectsABSTRACT
OBJECTIVE: To determine if Staphylococcus aureus stimulates the L-arginine-nitric oxide (NO) synthase pathway in articular cartilage. METHODS: A heat-killed and sonicated (denatured) S. aureus preparation was added to cultures of bovine articular cartilage. NO production was measured as accumulated nitrite in the culture medium and by the NO synthase-dependent conversion of 3H-L-arginine to 3H-L-citrulline in cartilage homogenates. Inducible NO synthase (iNOS) messenger RNA (mRNA) expression was analyzed by Northern blot. Proteoglycan synthesis was measured by 35SO4 incorporation into glycosaminoglycan. RESULTS: Nitrite accumulation and 3H-L-citrulline formation in cartilage were elevated by denatured S. aureus (compared with unstimulated control cartilage) and inhibited by the NO synthase inhibitor N(G)-monomethyl-L-arginine. Northern blot analysis revealed increased iNOS mRNA expression in bovine chondrocytes in response to denatured S. aureus stimulation. Denatured S. aureus suppressed the accumulation of 35SO4-labeled macromolecules representing newly synthesized proteoglycans in bovine articular cartilage. The suppressed proteoglycan synthesis was due to the presence of NO. CONCLUSION: These findings support the hypothesis that a component of S. aureus can stimulate iNOS in articular cartilage, and that NO generated from this enzyme down-regulates cartilage matrix proteoglycan synthesis.
Subject(s)
Cartilage, Articular/drug effects , Nitric Oxide Synthase/metabolism , Staphylococcus aureus/physiology , Animals , Cartilage, Articular/enzymology , Cattle , Cell Extracts/pharmacology , Cells, Cultured , Enzyme Activation , In Vitro Techniques , Lipopolysaccharides/pharmacology , Nitric Oxide Synthase/drug effects , Nitric Oxide Synthase Type II , Proteoglycans/metabolism , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Sulfates/metabolism , Sulfur Radioisotopes , Teichoic Acids/pharmacologyABSTRACT
The ability of pH-sensitive liposomes and immunoliposomes to deliver synthetic antisense oligonucleotides (oligos) into human myeloid and lymphoid leukaemia cells was examined. The cellular uptake of an 18mer anti-myb oligonucleotide encapsulated in liposomes was from three- to five-fold higher than that of 32P-oligos alone. In addition, anti-CD32 or anti-CD2 immunoliposomes improved the delivery of oligos to leukaemic cells carrying the appropriate receptor for the specific antibody-linked immunoliposome. The uptake of oligos was twice that of the liposome or non-specific immunoliposome encapsulated oligos. These findings support the use of liposomes or immunoliposomes to deliver antisense oligos into human leukaemic cells.
Subject(s)
Immunoconjugates/administration & dosage , Leukemia/pathology , Liposomes , Neoplastic Stem Cells/metabolism , Oligonucleotides, Antisense/administration & dosage , Cholesterol , Drug Carriers , Glucosides , HL-60 Cells/metabolism , Humans , Hydrogen-Ion Concentration , Oleic Acid , Oligonucleotides, Antisense/chemical synthesis , Phosphatidylethanolamines , Tumor Cells, CulturedABSTRACT
A recently developed enzyme-linked immunosorbent assay (EIAZ, ELISA) using two murine monoclonal anti-erythropoietin antibodies was compared with a radioimmunoassay (RIA) and a commercial in-vitro bioassay, EPOS, for measuring serum erythropoietin (Epo) in humans. Specificity and validity for Epo-EIA and the other two assays were examined. The serum Epo in normal subjects was 18 +/- 12 mU/ml (mean +/- SD, n = 80) for EIA compared with 22.5 +/- 18.5 mU/ml (n = 20) for RIA and 136 +/- 132 mU/ml (n = 14) for the bioassay. The serum Epo concentrations in normals and patients were highly comparable between EIA and RIA for Epo (P less than 0.01, r = 0.95). Epo concentrations by the EIA for normal female and male subjects were 20.5 +/- 13 and 16.5 +/- 10 mU/ml, respectively. Epo levels in patients with secondary polycythaemia or autoimmune haemolytic anaemia were significantly higher than normal subjects by the three methods. Epo levels in patients with chronic renal failure were within the normal range. By the EPOS bioassay, the Epo concentrations of normals and patients with renal failure were significantly higher than expected (136 +/- 132 and 447 +/- 273, respectively). Due to its inherent design, the EPOS bioassay possibly measures bone marrow proliferative activity in response to other serum growth regulators besides erythropoietin and was found to be unsuitable for clinical assessment of Epo. We concluded that the new EIA and RIA were similarly sensitive, reliable and accurate for measurement of serum Epo. The EIA method has the advantage of being less time consuming, more convenient and avoids the use of a radioisotope.
