Siriwan Ongchai

Evaluation of serum chondroitin sulfate and hyaluronan: biomarkers for osteoarthritis in canine hip dysplasia

Hip dysplasia (HD) is one of the most important bone and joint diseases in dogs. Making the radiographic diagnosis is sometime possible when the disease has markedly progressed. Chondroitin sulfate (CS) and hyaluronan (HA) are the most important cartilage biomolecules that are elevated in the serum taken from dogs with osteoarthritis. The serum CS and HA can be detected by an ELISA technique, with using monoclonal antibodies against CS epitope 3B3 and WF6 and the HA chain as the primary antibodies. The aim of this study was to compare the levels of serum CS (both epitopes) and HA in non-HD and HD dogs. All 123 dogs were categorized into 2 groups. The non-HD group was composed of 98 healthy dogs, while the HD group was comprised of 25 HD dogs. Blood samples were collected for analyzing the serum CS and HA levels with using the ELISA technique. The results showed that the average serum level of the CS epitope WF6 in the HD group (2,594 ± 3,036.10 ng/ml) was significantly higher than that in the non-HD group (465 ± 208.97 ng/ml) (p < 0.01) while the epitope 3B3 in the HD group (105 ± 100.05 ng/ml) was significantly lower than that in the non-HD group (136 ± 142.03 ng/ml) (p < 0.05). The amount of serum HA in the HD group (134.74 ± 59.71 ng/ml) was lower than that in the non HD group (245.45 ± 97.84 ng/ml) (p < 0.05). The results indicate that the serum CS and HA levels might be used as biomarkers for osteoarthritis in HD dogs.

Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E2 synthesis in human gingival fibroblasts

BACKGROUND AND OBJECTIVEnOral epithelial cells express three antimicrobial peptide human beta-defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in non-immune cells, such as human gingival fibroblasts.nnnMATERIAL AND METHODSnCultured fibroblasts were treated with different concentrations of hBD-1, -2, -3 or interleukin-1 beta, as a positive control, for various times, in the presence or absence of NS-398, a specific COX-2 inhibitor. The levels of COX-1 and COX-2 mRNA expression were analyzed using RT-PCR and real-time PCR. Whole cell lysates were analyzed for COX-1 and COX-2 protein expression by western blotting. Cell-free culture supernatants were assayed for PGE(2) levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs.nnnRESULTSnTen and 40 microg/mL of hBD-3 up-regulated COX-2 mRNA and protein expression, consistent with COX-2 up-regulation by interleukin-1 beta, whereas hBD-1 and hBD-2 did not. However, COX-1 mRNA and protein were constitutively expressed. The time-course study revealed that hBD-3 up-regulated COX-2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX-2 up-regulation, 10 and 40 microg/mL of hBD-3 significantly increased PGE(2) levels in cell-free culture supernatants (p < 0.05), and this was inhibited by NS-398 in a dose-dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells.nnnCONCLUSIONnThese findings indicate that epithelial human beta-defensin-3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.

Chondroitin sulfate epitope (WF6) and hyaluronic acid as serum markers of cartilage degeneration in patients following anterior cruciate ligament injury

Serum chondroitin sulfate epitope (WF6) and hyaluronic acid (HA) levels were determined to be of clinical relevance to an anterior cruciate ligament (ACL) injury. This cross-sectional study recruited participants from two distinct groups. Group A was comprised of 74 healthy controls, and group B consisted of 33 ACL injury patients. Serum samples were taken and assayed by a competitive immunoassay with monoclonal antibody WF6. Serum HA was also determined by an ELISA-based assay using biotinylated HA-binding proteins. Both groups A and B shared similar values of age, body mass index, white blood cell count and percentage of polymorphonuclear cells. ESR levels were also shown to be within normal limits. The serum WF6 epitope levels of group B were significantly higher than those of group A, whereas serum HA levels were not different between the two groups. The serum WF6 epitope level is more sensitive to changes in articular cartilage due to a non-inflammatory instability condition than the serum HA level, and should prove to be one of the most promising assays for early post-traumatic arthritis detection.

Authenticity analyses of Phyllanthus amarus using barcoding coupled with HRM analysis to control its quality for medicinal plant product

The Phyllanthus genus, a plant used in traditional Thai medicine, has according to several pharmacopeias hepatoprotective properties. Not only is the anatomical morphology of these species relatively similar but they also share the Thai common names Look-Tai-Bai (ลูกใต้ใบ) and Yah-Tai-Bai (หญ้าใต้ใบ), which might cause confusion for laypersons. This study attempted to develop a method for accurate identification of Phyllanthus species, especially Phyllanthus amarus, and to detect contaminants in P. amarus products by using DNA barcoding coupled with high resolution melting (HRM) analysis (bar-HRM). Two plastid loci (rbcL and trnL) were chosen for DNA barcoding to generate a suitable primer for distinguishing Phyllanthus species by HRM analysis. The five species of Phyllanthus were subjected to amplification for testing the specificity and discrimination power of the designed primers derived from rbcL and trnL regions. Sensitivity of the method (DNA barcoding conjugated with HRM) to detect adulterant in P. amarus samples was evaluated. The commercial P. amarus products obtained from a local market were authenticated. The primer pair derived from trnL DNA barcoding (PhylltrnL) had more specificity and power of discrimination for Phyllanthus species than that derived from rbcL DNA barcoding (PhyllrbcL). The result showed that Tm of P. amarus, Phyllanthus urinaria, Phyllanthus debilis, Phyllanthus airy-shawii, and Phyllanthus virgatus was 74.3±0.08, 73.04±0.07, 73.36±0.05, 72.21±0.06, 72.77±0.15°C, respectively. This method proved to be a very sensitive tool that can be used for rapid detection of contamination as low as 1% of other Phyllanthus species in P. amarus admixtures. All commercial products of P. amarus obtained from a local market in Thailand were found to contain pure raw materials of P. amarus without any substitution or contamination. Our results indicated that the use of DNA barcoding coupled with HRM was an efficient molecular tool for correct species identification. This molecular tool provides a noteworthy benefit for quality control of medicinal plants and industry plants for pharmacological prospects.

The effect of doxycycline on canine hip osteoarthritis: design of a 6-months clinical trial

Twenty-five dogs were included in a randomized, double-blind trial to assess the efficacy of doxycycline (DOX) orally administered twice a day at 4 mg/kg/day (n = 12) for the treatment of osteoarthritis of the hip. Chondroitin sulfate (CS; 525 mg/day) was used as a positive control (n = 13). Dogs were re-examined monthly for 6 months after initiation of treatment. The assessment protocol included clinical score, radiographic findings and serum osteoarthritis biomarkers. Dogs treated with DOX showed statistically significant improvements (p < 0.05) in lameness, joint mobility, pain on palpation, weight-bearing and overall score at 2, 6, 4, 4 and 4 months, respectively, after treatment. Biomarker levels of CS-WF6 epitope and hyaluronan were significantly increased and decreased (p < 0.05) at 2 and 3 months after treatment compared to pretreatment. These results showed that DOX had a positive therapeutic effect in dogs with osteoarthritis.

Effects of low molecular weight hyaluronan combined with carprofen on canine osteoarthritis articular chondrocytes and cartilage explants in vitro

Intra-articular injection with non-steroidal anti-inflammatory drugs (NSAIDs) is used to treat inflammatory joint disease, but the side effects of NSAIDs include chondrotoxicity. Hyaluronan has shown positive effects on chondrocytes by reducing apoptosis and increasing proteoglycan synthesis. The purposes of this study were to evaluate the effects of low molecular weight hyaluronan (low MW HA), carprofen 25xa0mg/ml, carprofen 12.5xa0mg/ml, and a combination of HA and carprofen on canine osteoarthritis (OA) articular chondrocytes and a cartilage explant model in terms of cell viability, extracellular matrix remaining, and gene expression after exposure. In chondrocyte culture, MTT assay was used to evaluate the chondrotoxicity of IC50 and IC80 of carprofen with HA. In cartilage explant culture, two kinds of extracellular matrix (uronic acid and collagen) remaining in cartilage were used to evaluate cartilage damage for 14xa0d after treatment. Expression of COL2A1, AGG, and MMP3 was used to evaluate the synthesis and degradation of the matrix for 7xa0d after treatment. In chondrocyte culture, low MW HA could preserve OA chondrocyte viability but could not reduce the chondrotoxicity level of carprofen (Pu2009<u20090.05). In explant culture, low MW HA combined with 12.5xa0mg/ml carprofen caused less destruction of uronic acid and collagen structure when compared with the control (Pu2009<u20090.05). Low MW HA caused high expression levels of COL2A1 and AGG in OA cartilage (Pu2009<u20090.05); HA combined with carprofen resulted in higher COL2A1 and AGG expression levels than carprofen alone.

Raised chondroitin sulphate WF6 epitope levels in gingival crevicular fluid in chronic periodontitis.

AIMnTo determine the levels of chondroitin sulphate (CS) WF6 epitope, recognized by WF6 monoclonal antibody, in gingival crevicular fluid (GCF) from different stages of periodontal disease and healthy periodontium, and to correlate those levels with clinical parameters.nnnMATERIAL AND METHODSnGCF samples, collected from 389 sites, were analysed for the WF6 epitope levels by the competitive enzyme-linked immunosorbent assay.nnnRESULTSnThe median WF6 epitope level was significantly higher in chronic periodontitis sites (n=185) than in healthy and gingivitis sites (n=204) (p<0.001), whereas the median levels did not significantly differ between healthy (n=65) and gingivitis sites (n=139). The median level in severe periodontitis sites (n=60) was significantly higher than that in moderate periodontitis sites (n=63) (p=0.019). Similarly, the median level in moderate periodontitis sites was significantly higher than that in slight periodontitis sites (n=62) (p=0.001). The WF6 epitope levels significantly correlated with probing depth (r=0.777, p=0.001) and loss of clinical attachment level (r=0.814, p=0.001).nnnCONCLUSIONnElevated CS WF6 epitope levels in GCF are associated with severity of periodontitis. The WF6 antibody may therefore be clinically applied to monitor disease severity and progression.

Hyaluronic acid : Additional biochemical marker in the diagnosis of biliary atresia

Background: The purpose of the present paper was to evaluate the value of biochemical markers, including conventional liver function tests, γ‐glutamyl transferase (GGT), and hyaluronic acid (HA), in the diagnosis of neonatal cholestasis.

Chondroprotective potential of bioactive compounds of Zingiber cassumunar Roxb. against cytokine-induced cartilage degradation in explant culture

The active compounds, cis-3-(2,4,5-trimethoxyphenyl)-4-{(E)-2,4,5-trimethoxystyryl}cyclohex-1-ene (Compound C) and (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-ol (Compound D), have been identified in hexane fraction ofxa0Zingiber cassumunarxa0Roxb., the medicinal plant which has been used for pain relief in arthritis including osteoarthritis (OA) and rheumatoid arthritis (RA). It was therefore interesting to investigate the chondroprotective activity of these compoundsxa0in vitro. Articular cartilage explants were cultured in the culture media containing 7 ng/ml of interleukin-1β (IL-1β), in the presence or absence of Compound C or Compound D at the concentration range of 1 to 100 μM. It was found that these compounds at concentrations of 10 and 100 μM significantly inhibited the IL-1β-induced cartilage degeneration by conserving the content of the cartilage matrix biomolecules such as collagen and uronic acid (UA) within the cartilage explants; and also resulted in the decline of releasing sulfated glycosaminoglycans and hyaluronic acid (HA) into the culture media. The increase in activities of matrix metalloproteinase-2 (MMP-2) and MMP-13 caused by IL-1β was significantly diminished by Compound C and Compound D. Additionally, the results showed no significant difference between the two active constituents and diacerein, an anti-arthritic agent used in OA, in those activities at a concentration of 100 μM. This is a pioneering evidence that indicate the potential chondroprotective property of thexa0Z. cassumunarxa0active compounds. n n xa0 n n Key words:xa0Zingiber cassumunar, chondroprotective activity, cartilage explant, rheumatoid arthritis, osteoarthritis, cis-3-(2,4,5-trimethoxyphenyl)-4-{(E)-2,4,5-trimethoxystyryl}cyclohex-1-ene, (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-ol.

Chondroitin sulphate (WF6 epitope) levels in peri-miniscrew implant crevicular fluid during orthodontic loading.

The aim of this study was to monitor changes in chondroitin sulphate (CS; WF6 epitope) levels in peri-miniscrew implant crevicular fluid (PMICF) during orthodontic loading. Ten patients (seven males and three females; aged 22.0 +/- 3.4 years), who required orthodontic treatment with extraction of all four premolar teeth, participated in the study. Twenty miniscrew implants (used as orthodontic anchorage) were placed, two in each patient, buccally and bilaterally in the alveolar bone between the roots of the maxillary posterior teeth. Sentalloy closed-coil springs (50 g) were used to load the miniscrew implants and to move the maxillary canines distally. During the unloaded period, PMICF samples were collected on days 1, 3, 5, and 7 after miniscrew implant placement and on days 14, 21, 28, and 35 during the loaded period. Clinical mobility assessments of the miniscrew implants were recorded at each visit. The competitive enzyme-linked immunosorbent assay with monoclonal antibody WF6 was used to detect CS (WF6 epitope) levels in the PMICF samples. The differences between the CS (WF6 epitope) levels during the unloaded and loaded periods were determined by a Mann-Whitney U-test. During the loaded period, two miniscrew implants were considered to have failed. The CS (WF6 epitope) levels during the unloaded period ranged from 0.00 to 758.03 ng/ml and those during the loaded period from 0.00 to 1025.11 ng/ml. Medians of CS (WF6 epitope) levels, around immobile miniscrew implants, between the unloaded and loaded periods were not significantly different (P = 0.07). CS (WF6 epitope) levels in PMICF can be detected and may be used as biomarkers for assessing alveolar bone remodelling around miniscrew implants during orthodontic loading.