Inflammation and joint destruction may be linked to the generation of cartilage metabolites of ADAMTS-5 through activation of toll-like receptors.

OBJECTIVE: Links between pain and joint degradation are poorly understood. We investigated the role of activation of Toll-like receptors (TLR) by cartilage metabolites in initiating and maintaining the inflammatory loop in OA causing joint destruction. METHODS: Synovial membrane explants (SMEs) were prepared from OA patients' synovial biopsies. SMEs were cultured for 10 days under following conditions: culture medium alone, OSM + TNFα, TLR2 agonist - Pam2CSK4, Pam3CSK4 or synthetic aggrecan 32-mer, TLR4 agonist - Lipid A. Release of pro-inflammatory and degradation biomarkers (acMMP3 and C3M) were measured by ELISA in conditioned media along with IL-6. Additionally, human cartilage was digested with ADAMTS-5, with or without the ADAMTS-5 inhibiting nanobody - M6495. Digested cartilage solution (DCS) and synthetic 32-mer were tested for TLR activation in SEAP based TLR reporter assay. RESULTS: Western blotting confirmed TLR2 and TLR4 in untreated OA synovial biopsies. TLR agonists showed an increase in release of biomarkers - acMMP3 and C3M in SME. Synthetic 32-mer showed no activation in the TLR reporter assay. ADAMTS-5 degraded cartilage fragments activated TLR2 in vitro. Adding M6495 - an anti-ADAMTS-5 inhibiting nanobody®, blocked ADAMTS-5-mediated DCS TLR2 activation. CONCLUSION: TLR2 is expressed in synovium of OA patients and their activation by synthetic ligands causes increased tissue turnover. ADAMTS-5-mediated cartilage degradation leads to release of aggrecan fragments which activates the TLR2 receptor in vitro. M6495 suppressed cartilage degradation by ADAMTS-5, limiting the activation of TLR2. In conclusion, pain and joint destruction may be linked to generation of ADAMTS-5 cartilage metabolites.

Aggrecanase degradation of type III collagen is associated with clinical knee pain.

There is a lack of biochemical markers for non-invasive and objective assessment of symptomatic osteoarthritis (OA). Aggrecanase activity has been shown to be associated with joint deterioration and symptomatic disease through the degradation of extracellular matrix proteins, such as type III collagen. Our study aimed to identify and develop a novel biomarker by measuring an aggrecanase-mediated type III collagen neoepitope, and correlate levels of this biomarker with OA joint pain. Mass spectrometric analysis of purified type III collagen, degraded by the aggrecanase A Disintigrin and Metalloproteinase with Thrombospondin motif (ADAMTS), revealed a fragment generated by ADAMTS-1, -4 and -8. A monoclonal antibody was raised against the neoepitope of this fragment (COL3-ADAMTS) and a competitive ELISA was developed and tested; using serum samples from a cross-sectional cohort of patients with different degrees of knee OA (n = 261). The COL3/ADAMTS ELISA was technically robust and specific for the ADAMTS-1, -4 and -8 generated neoepitope. COL3/ADAMTS was released form cytokine stimulated synovial cultures, indicating a biologic link between the marker and synovium. In OA patients, serum COL3/ADAMTS was independently associated with pain scores (rho = -0.13-0.17, p < 0.05). This association was associated significantly with the presence of radiographic OA. Together, these data indicate that COL3/ADAMTS could be a marker of early osteoarthritis and the underlining pathology.

Inflammation (or synovitis)-driven osteoarthritis: an opportunity for personalizing prognosis and treatment?

The disabling and painful disease osteoarthritis (OA) is the most common form of arthritis. Strong evidence suggests that a subpopulation of OA patients has a form of OA driven by inflammation. Consequently, understanding when inflammation is the driver of disease progression and which OA patients might benefit from anti-inflammatory treatment is a topic of intense research in the OA field. We have reviewed the current literature on OA, with an emphasis on inflammation in OA, biochemical markers of structural damage, and anti-inflammatory treatments for OA. The literature suggests that the OA patient population is diverse, consisting of several subpopulations, including one associated with inflammation. This inflammatory subpopulation may be identified by a combination of novel serological inflammatory biomarkers. Preliminary evidence from small clinical studies suggests that this subpopulation may benefit from anti-inflammatory treatment currently reserved for other inflammatory arthritides.

Osteoarthritis year in review 2015: soluble biomarkers and the BIPED criteria.

OBJECTIVE: To review and summarize biomarker data published from April 2014 to May 2015 to provide insight to the ongoing work in the field of osteoarthritis (OA). Furthermore, to summarize the BIPED criteria and set it in context of the medical needs of 2015. METHODS: PubMed was used as searching machine: Time period 2014/04/01-2015/05/01, MeSH term [Biomarker] AND [Osteoarthritis], Language; English, Full text available. Reviews were excluded. Only papers describing protein based biomarkers measured in human body fluids from OA patients were included. RESULTS: Biomarkers of joint tissue turnover, cytokines, chemokines and peptide arrays were measured in different cohorts and studies. Amongst those were previously tested biomarkers such as osteocalcin, Carboxy-terminal cross-linked fragment of type II collagen (CTX-II) and cartilage oligomeric matrix protein (COMP). A majority of the biomarker were classified as I, B or B biomarkers according to the BIPED criteria. Work is continuing on testing biomarkers in OA. There is still a huge, unmet medical need to identify, test, validate and qualify novel and well-known biomarkers. A pre-requisite for this is better characterization and classification of biomarkers to their needs, which may not be reached before higher understanding of OA phenotypes has been gained. In addition, we provide some references to some recent guidelines from Food and Drug Administration (FDA) and European Medicines Agency (EMA) on qualification and usage of biomarkers for drug development and personalized medicine, which may provide value to the field.

The airborne microflora of poultry houses.

Two experiments were conducted to compare effects of housing temperatures and bird density on the airborne microflora of poultry houses. Temperatures of 15.6 and 26.7 C were used with birds housed at densities of .42 or .84 m3 per bird. Air samples were taken using a New Brunswick STA 200 microbiological air sampler. Numbers of aerobic, anaerobic, coliform and lactic acid bacteria, and molds were determined by plate counts with numbers of Escherichia coli and Staphylococcus aureus determined by most probable numbers procedures. Microorganisms were identified by picking representative colonies from plates and inoculating into differential media for biochemical tests. Higher bird density (.42 m3/bird) resulted in greater numbers of airborne microorganisms in both experiments. Fifteen genera of bacteria were identified with two or more species identified for eight genera. Among the most commonly identified aerobic genera were Bacillus, Micrococcus, Proteus, Pseudomonas and Staphylococcus, while four species of Clostridia were the most frequently identified anaerobes. Nine genera of molds were identified with over one-half of all isolates being either Aspergillus or Penicillium. Microorganisms represented only a small fraction of the airborne particulate matter in the study.

Effect of pH on eggshell penetration by Salmonellae.

Experiments were conducted to study effects of pH on penetration of eggs by three species of Salmonella. Eggs having an average specific gravity of 1.078 were subjected to challenge by either S. typhimurium, S. st. paul, or S. derby. Challenge solutions ranged from pH 5.0 to 9.5 in .5 pH increments and contained an average of 7.5 x 10(3) Salmonella/ml. Egg temperature was 22 C and solution temperature 4.4 C when challenged. Tartaric acid (10%) or 1 N. NaOH were used to adjust solution prior to adding challenge organisms. Eggs were challenged for 3 min then allowed to dry and held at 22 C for 24 hr, after which they were opened aseptically. Salmonella penetration was determined by swabbing the inner shell membrane and incubating in selenite cystine and tetrathionate enrichment broths for 24 hr followed by plating on MacConkey and SS agars. Penetration rates for all three organisms were significantly less at pH 5.0 than at any higher pH tested. There was an increase in penetration from pH 5.5 to 7.0 for all species. Maximum penetration rates were 42% of eggs challenged at pH 7.5, 22% at pH 8.5, and 34% at pH 7.0 for S. typhimurium, S. derby, and S. st. paul, respectively. In no case was penetration of eggs at pH 9.0 significantly different from pH at maximum penetration of challenge eggs. Penetration by S. st paul, at pH 9.5 was significantly less (P less than .05) than at pH 7.0. Decalcification of the eggshell was less than .01%/min at pH 4.0. Shell losses at pH 3.5 and 3.0 were .03% and .33%/min, respectively.

Survival among patients with liver metastases from cancer of the colon and rectum.

Factors influencing spontaneous survival in 49 patients with liver metastases after cancer in colon/rectum were evaluated. In addition the same evaluation was performed in 12 patients treated with 5-Fluoro-uracil systemically of intraarterially in the hepatic artery. Alkaline phosphatases, elevated more than 4 times normal values, elevated serum alanine aminotransferase, or jaundice are all unfavorable prognostic signs in the spontaneous group. In the 5-Fluoro-uracil treated group only elevated serum dilirubin had the same unfavorable prognostic sign. Even though it seems to be an increased survival time in the 5-Fluoro-uracil treated group it is concluded that metastases to the liver from cancer in colon/rectum assume to be more or less resistent to 5-Fluoro-uracil.

Dietary and management procedures for development of consistent hemorrhagic symptoms in chicks.

Chicks hatched from S.C.W.L. pullets housed in laying cages and fed a diet without vitamin K were compared with chicks hatched from pullets fed a similar ration supplemented with vitamin K3 at 750 mcg./kg. of feed. Eggs were incubated after 4, 5 and 7 months vitamin K depletion. Chicks were fed a basal chick starter with no source of vitamin K or the basal supplemented with various levels of sulfaquinoxaline (SQ) (0.0125-0.050%) or with 750 mcg./kg. of vitamin K3. Blood clotting time was determined on chicks at the beginning and at weekly intervals during each experiment. Blood clotting time was significantly increased by a SQ feeding, but no hemorrhages developed in chicks hatched from hens fed the diet containing vitamin K. Day-old chicks hatched after 4 months vitamin K depletion showed elevated blood clotting times. There was no significant change in blood clotting time during the experiment for chicks fed the basal. Blood clotting time was significantly reduced in one week for chicks supplemented with vitamin K and increased for chicks fed 0.0375 or 0.050% SQ. These levels of SQ also resulted in 25 and 47.5% skin hemorrhages. Five months vitamin K depletion of breeder hens resulted in increased incidence of skin hemorrhages, but blood clotting times were similar to those at four months. Seven months vitamin K depletion of breeder hens resulted in increased blood clotting times as compared with 4 months and 100% incidence of hemorrhages in chicks fed rations containing 0.0375 or 0.050% SQ.