Synovial Concentrations Research Articles

Cytokines and chemokines multiplex analysis in patients with low disease activity rheumatoid arthritis

Rheumatoid arthritis is a severe chronic autoimmune disorder that results from pathological activation of immune cells and altered cytokine/chemokine network. The aim of our study was to evaluate concentrations of chosen cytokines and chemokines in blood sera and synovial fluid samples isolated from low disease activity rheumatoid arthritis (RA) patients and osteoarthritis (OA) sufferers. Blood sera and synovial fluid samples have been obtained from 24 OA and 14 RA patients. Cytokines/chemokines levels have been determined using a Milliplex® Map 38-plex human cytokine/chemokine magnetic bead-based panel (Merck Millipore, Germany) and Luminex® MAGPIX® platform (Luminex USA). Low disease activity RA patients showed altered concentration of numerous cytokine/chemokine when compared to OA controls—they were characterized by, inter alia, increased: eotaxin/CCL11 (p = 0.037), GRO/CXCL1 (p = 0.037), IL-2 (p = 0.013), IL-4 (p = 0.017), IL-7 (p = 0.003), IL-8 (p = 0.0007) and GM-CSF (p = 0.037) serum levels, whilst MDC/CCL22 concentration was decreased in this group (p = 0.034). Eotaxin/CCL11 (p = 0.001), GRO/CXCL1 (p = 0.041), IL-10 (p = 0.003), GM-CSF (p = 0.01), IL-1RA (p = 0.0005) and VEGF (p = 0.01) concentrations in synovial fluid of RA females were also increased. Even with low disease activity score, RA patients exhibited increased concentrations of cytokines with pro- and anti-inflammatory activities, as well as numerous chemokines, growth factors and regulators of angiogenesis. Surprisingly, RA subjects also shown decreased concentration of CCL22 chemokine. The attempt to restore cytokine balance and tolerogenic environment is ineffective in RA sufferers even with good disease management. Distinguished factors could serve as possible indicators of disease progression even in low disease activity patients.

Synovial and Systemic Pharmacokinetics of Florfenicol and PK/PD Integration against Streptococcus suis in Pigs.

Florfenicol is a member of the phenicol group, a broad-spectrum antibacterial agent. It has been used for a long time in veterinary medicine, but there are some factors regarding its pharmacokinetic characteristics that have yet to be elucidated. The aim of our study was to describe the pharmacokinetic profile of florfenicol in synovial fluid and plasma of swine after intramuscular (i.m.) administration. In addition, the dosage regimen of treatment of arthritis caused by S. suis was computed for florfenicol using pharmacokinetic/pharmacodynamic (PK/PD) indices. As the first part of our investigation, the pharmacokinetic (PK) parameters of florfenicol were determined in the plasma and synovial fluid of six pigs. Following drug administration (15 mg/kgbw, intramuscularly), blood was drawn at the following times: 10, 20, 30, 40, 50 and 60 min, 2, 3, 4, 5, 6, 7, 8, 12, 24, 48 and 72 h; synovial fluid samples were taken after 1, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h. The concentration of florfenicol was determined by a validated liquid chromatography-mass spectrometry (LC-MS/MS) method via multiple reaction monitoring (MRM) modes. As the second part of our research, minimum inhibitory concentration (MIC) values of florfenicol were determined in 45 S. suis strains isolated from clinical samples collected in Hungary. Furthermore, a strain of S. suis serotype 2 (SS3) was selected, and killing-time curves of different florfenicol concentrations (0.5 µg/mL, 1 µg/mL and 2 µg/mL) were determined against this strain. Peak concentration of the florfenicol was 3.58 ± 1.51 µg/mL in plasma after 1.64 ± 1.74 h, while it was 2.73 ± 1.2 µg/mL in synovial fluid 3.4 ± 1.67 h after administration. The half-life in plasma was found to be 17.24 ± 9.35 h, while in synovial fluid it was 21.01 ± 13.19 h. The area under the curve (AUC24h) value was 54.66 ± 23.34 μg/mL·h for 24 h in plasma and 31.24 ± 6.82 μg/mL·h for 24 h in synovial fluid. The drug clearance scaled by bioavailability (Cl/F) in plasma and synovial fluid was 0.19 ± 0.08 L/h/kg and 0.29 ± 0.08 L/h/kg, respectively. The mean residence time (MRT) in plasma and synovial fluid was 24.0 ± 13.59 h and 27.39 ± 17.16 h, respectively. The steady-state volume of distribution (Vss) in plasma was calculated from Cl/F of 0.19 ± 0.08 L/h/kg, multiplied by MRT of 24.0 ± 13.59 h. For the PK/PD integration, average plasma and synovial fluid concentration of florfenicol was used in a steady-state condition. The obtained MIC50 value of the strains was 2.0 µg/mL, and MIC90 proved to be 16.0 µg/mL. PK/PD integration was performed considering AUC24h/MIC breakpoints that have already been described. This study is the first presentation of the pharmacokinetic behavior of florfenicol in swine synovia as well as a recommendation of extrapolated critical MICs of S. suis for therapeutic success in the treatment of S. suis arthritis in swine, but it should be noted that this requires a different dosage regimen to that used in authorized florfenicol formulations.

Нимесулид: преимущества, перспективы применения во время эпидемии коронавирусной инфекции

Objective of the Review: To discuss the specific properties of Nimesulide and its use in COVID-19. Key Points. A significant advantage of Nimesulide is a very rapid analgesic effect, due to rapid and meal-independent absorption in the gastrointestinal tract, almost complete binding to blood albumin, equally rapid achievement of the maximum concentration both in synovial fluid and blood. Now we have some data on the promising use of Nimesulide for COVID-19 management. Conclusion. An idea that Nimesulide should be among the medicinal products undergoing assessment for the use for COVID-19 management, seems rather rational. Although it is not clear whether such studies will be conducted and what their outcome will be, selection of Nimesulide as one of the primary drugs for pain management and inflammation therapy both by medical professionals and patients is not by chance; it is completely informed and justified. Keywords: Nimesulide, nonsteroidal anti-inflammatory drugs, COVID-19.

Effects of a Single Intra-Articular Injection of 2% Lidocaine or 0.5% Bupivacaine on Synovial Fluid Acute Phase Protein Concentrations in Healthy Horses

Effects of a Single Intra-Articular Injection of 2% Lidocaine or 0.5% Bupivacaine on Synovial Fluid Acute Phase Protein Concentrations in Healthy Horses

Detecting low blood concentrations in joints using T1 and T2 mapping at 1.5, 3, and 7 T: an in vitro study

BackgroundIntra-articular blood causes irreversible joint damage, whilst clinical differentiation between haemorrhagic joint effusion and other effusions can be challenging. An accurate non-invasive method for the detection of joint bleeds is lacking. The aims of this phantom study were to investigate whether magnetic resonance imaging (MRI) T1 and T2 mapping allows for differentiation between simple and haemorrhagic joint effusion and to determine the lowest blood concentration that can be detected.MethodsSolutions of synovial fluid with blood concentrations ranging from 0 to 100% were scanned at 1.5, 3, and 7 T. T1 maps were generated with an inversion recovery technique and T2 maps from multi spin-echo sequences. In both cases, the scan acquisition times were below 5 min. Regions of interest were manually drawn by two observers in the obtained T1 and T2 maps for each sample. The lowest detectable blood concentration was determined for all field strengths.ResultsAt all field strengths, T1 and T2 relaxation times decreased with higher blood concentrations. The lowest detectable blood concentrations using T1 mapping were 10% at 1.5 T, 25% at 3 T, and 50% at 7 T. For T2 mapping, the detection limits were 50%, 5%, and 25%, respectively.ConclusionsT1 and T2 mapping can detect different blood concentrations in synovial fluid in vitro at clinical field strengths. Especially, T2 measurements at 3 T showed to be highly sensitive. Short acquisition times would make these methods suitable for clinical use and therefore might be promising tools for accurate discrimination between simple and haemorrhagic joint effusion in vivo.

Oral prednisolone achieves measurable concentrations in equine synovial fluid within 3 hours of administration: Preliminary observations

SummaryBackgroundClinical and anecdotal evidence exists for the use of orally administered prednisolone in the treatment of osteoarthritis in human patients; however, prednisolone is not commonly prescribed by this route of administration in horses for the treatment of osteoarthritis.ObjectivesThis preliminary study tested the hypothesis that orally administered prednisolone could be detected in equine synovial fluid 3 h after administration.Study designPre/post study.MethodsSix horses were used in this study. Blood was drawn pre‐prednisolone administration to assure that all horses were prednisolone free. Three hours after being given 400 mg prednisolone per os, an additional blood sample was taken, as well as samples from the radiocarpal (RC) and tibiotarsal (TT) joints. Samples were frozen and sent to the laboratory for the determination of the presence of prednisolone using liquid chromatography tandem mass spectrometry.ResultsPrednisolone appeared in measurable concentrations in the blood (Mean = 92.655 ng/mL; SD = 54.95 ng/mL) and synovial fluid (RC Mean = 28.44 ng/mL; SD = 20.58 ng/mL and TT Mean = 20.33; SD = 15.67 ng/mL) of all horses when tested 3 h after administration.Main limitationsThis study did not test for any possible therapeutic effects of oral prednisolone in osteoarthritis. It did not attempt to establish a therapeutic dosage for the medication, nor did it attempt to establish the pharmacokinetics of the medication.ConclusionsOrally administered prednisolone appears in equine blood and synovial fluid at 3 h post‐administration. Since it appears in joint fluid at concentrations that have been shown to be therapeutic for other corticosteroids, orally administered prednisolone may have potential value as a therapeutic agent in the treatment of equine osteoarthritis.

Elevated APE1/Ref-1 Levels of Synovial Fluids in Patients with Rheumatoid Arthritis: Reflection of Disease Activity.

There is growing evidence that apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) regulates inflammatory responses. Rheumatoid arthritis (RA) is an autoimmune disease, which is characterized with synovitis and joint destruction. Therefore, this study was planned to investigate the relationship between APE1/Ref-1 and RA. Serum and synovial fluid (SF) were collected from 46 patients with RA, 45 patients with osteoarthritis (OA), and 30 healthy control (HC) patients. The concentration of APE1/Ref-1 in serum or SF was measured using the sandwich enzyme-linked immunosorbent assay (ELISA). The disease activity in RA patients was measured using the 28-joint disease activity score (DAS28). The serum APE1/Ref-1 levels in RA patients were significantly increased compared to HC and OA patients (0.44 ± 0.39 ng/mL for RA group vs. 0.19 ± 0.14 ng/mL for HC group, p < 0.05 and vs. 0.19 ± 0.11 ng/mL for OA group, p < 0.05). Likewise, the APE1/Ref-1 levels of SF in RA patients were also significantly increased compared to OA patients (0.68 ± 0.30 ng/mL for RA group vs. 0.31 ± 0.12 ng/mL for OA group, p < 0.001). The APE1/Ref-1 concentration in SF of RA patients was positively correlated with DAS28. Thus, APE1/Ref-1 may reflect the joint inflammation and be associated with disease activity in RA.

Low-Molecular-Weight Collagen Peptide Ameliorates Osteoarthritis Progression through Promoting Extracellular Matrix Synthesis by Chondrocytes in a Rabbit Anterior Cruciate Ligament Transection Model.

This study examined whether the oral administration of low-molecular-weight collagen peptide (LMCP) containing 3% Gly-Pro-Hyp with >15% tripeptide (Gly-X-Y) content could ameliorate osteoarthritis (OA) progression using a rabbit anterior cruciate ligament transection (ACLT) model of induced OA and chondrocytes isolated from a patient with OA. Oral LMCP administration (100 or 200 mg/kg/day) for 12 weeks ameliorated cartilage damage and reduced the loss of proteoglycan compared to the findings in the ACLT control group, resulting in dose-dependent (p < 0.05) improvements of the OARSI score in hematoxylin & eosin (H&E) and Safranin O staining. In microcomputed tomography analysis, LMCP also significantly (p < 0.05) suppressed the deterioration of the microstructure in tibial subchondral bone during OA progression. The elevation of IL-1βand IL-6 concentrations in synovial fluid following OA induction was dose-dependently (p < 0.05) reduced by LMCP treatment. Furthermore, immunohistochemistry illustrated that LMCP significantly (p < 0.05) upregulated type II collagen and downregulated matrix metalloproteinase-13 in cartilage tissue. Consistent with the in vivo results, LMCP significantly (p < 0.05) increased the mRNA expression of COL2A1 and ACAN in chondrocytes isolated from a patient with OA regardless of the conditions for IL-1βinduction. These findings suggest that LMCP has potential as a therapeutic treatment for OA that stimulates cartilage regeneration.

Effect of prophylactic administration of vitamin C in chickens with staphylococcal septic arthritis.

BackgroundSeptic arthritis (SA) due to Staphylococcus aureus is a major cause of lameness in poultry with improper response to antimicrobial therapy.ObjectivesThe study evaluates the effect of prophylactic administration of vitamin C on SA induced by methicillin resistant S. aureus in chickens.MethodsOne hundred and twenty chickens were randomly assigned into four groups: I. Negative control (NC), II. Positive control (PC) with SA induced at the age of 35 days by intra articular injection of S. aureus. III. Vehicle control (VC) and IV. Arthritic vitamin C‐treated (VitC) group (15 g/100 L of drinking water from day 25 to the end of the experiment). Samplings were performed on day 44 (sampling 1) and day 54 (sampling 2) of age.ResultsArthritic birds showed an obvious decrease in body weight with severe clinical arthritis and lameness which were not significantly affected by vitamin C administration at both samplings. Moreover, marked increase in serum malondialdehyde (MDA) concentration of the PC group was observed in sampling 1. Administration of vitamin C successfully reduced MDA concentration at both samplings. In sampling 2, birds in the VitC group showed significantly higher total antioxidant capacity (TAC) than NC birds (p < 0.05). Interleukin‐6 concentration in synovial fluid of chickens remained statistically similar among groups in both samplings, while histopathological changes were ameliorated in the VitC group in sampling 2.ConclusionsProphylactic administration of vitamin C especially for relatively longer period can ameliorate oxidative stress and histopathological changes due to staphylococcal arthritis in chickens, although it is not associated with a significant effect on clinical manifestations of the disease.

Comparison of tissue pharmacokinetics of esflurbiprofen plaster with flurbiprofen tablets in patients with knee osteoarthritis: A multicenter randomized controlled trial.

This open‐label, multicenter, prospective, randomized controlled trial aimed to determine the effectiveness of esflurbiprofen plaster (SFPP) and flurbiprofen tablets (FPTs) on knee osteoarthritis in patients scheduled for total knee arthroplasty by comparing the transfer of esflurbiprofen and flurbiprofen to tissues and fluids. Thirty‐eight patients were randomly assigned in a 1:1 ratio to receive SFPP or FPT. Both groups were then divided into four subgroups, according to whether they received the final dose of SFPP or FPT at 2, 7, 12, or 24 h before planned surgery. The primary endpoints were the esflurbiprofen concentrations in synovium, synovial fluid, and plasma. Areas under concentration–time curves (AUC0–24 h) of esflurbiprofen were calculated for each group. Pain was assessed using a numeric rating scale (NRS) 7 days before and immediately before surgery. The AUC0–24 h in the synovium were 4401.24 and 4862.70 ng·h/g in the SFPP and FPT groups, respectively. Maximum esflurbiprofen concentrations were observed in the synovium, synovial fluids, and plasma after SFPP application for 12 h. The NRS results indicated a long‐lasting effect of SFPP. The AUC of the synovial esflurbiprofen concentration of SFPP indicated that the SFPP is transferred to the synovium and synovial fluid in high concentration. The efficient deep‐tissue transfer of esflurbiprofen suggests that its pharmacokinetic characteristics differ from those of conventional topical NSAIDs. This study was prospectively registered in the Japan Registry of Clinical Trials (registration number: jRCTs031180228).

Effect of intravenous tiludronate disodium administration on the radiographic progression of osteoarthritis of the fetlock joint in Standardbred racehorses.

To compare the effects of tiludronate disodium and 3 other medical treatments on clinical and radiographic findings and biomarkers of disease progression in horses with osteoarthritis of the fetlock joint. 100 Standardbred racehorses with spontaneous traumatic injury of the fetlock joint. Horses were retrospectively grouped by whether they received tiludronate IV or triamcinolone acetonide and hyaluronan, polysulfated glycosaminoglycan, or interleukin-1 receptor antagonist protein intra-articularly. Data were collected on clinical, radiographic, and ultrasonographic findings and results for serum and synovial samples obtained before and 6 months after treatment. Lameness score, joint flexion test response, radiographic score, serum concentrations of tumor necrosis factor-α and carboxy-terminal telopeptides of collagen types I and II (CTX-I and II, respectively), and synovial fluid concentrations of interleukin-1β, prostaglandin E2, and CTX-II were compared among treatments. All treatments resulted in a significant improvement in lameness score and joint flexion test response at 6 months. In horses that received triamcinolone acetonide and hyaluronan, synovial fluid interleukin-1β, prostaglandin E2, and CTX-II concentrations decreased after treatment, suggesting this treatment inhibited progression of hyaline cartilage degeneration and inflammatory processes. Horses that received tiludronate were the only group that had a decrease in radiographic score and serum CTX-I concentration after treatment, supporting the effect of tiludronate on bone metabolism. Tiludronate treatment was also followed by increases in serum and synovial fluid concentrations of CTX-II, a marker of cartilage damage. Tiludronate appeared to inhibit the radiographic progression of osteoarthritis in high-motion joints of racehorses at 6 months after treatment by inhibiting subchondral bone remodeling. Whether this effect was associated with a worsening of progressive cartilage damage remains to be ascertained.

Preparation Technique Affects Recipient Immune Targeting of Autologous Mesenchymal Stem Cells.

Fetal bovine serum (FBS) is used for MSC preparation in pre-clinical animal models and veterinary applications, recently in US clinical trials, and for MSC products with current foreign market authorizations. The effect of anti-bovine titers, which are common in animals and humans, has not been investigated. In the equine model, where anti-bovine titers are universally high due to routine vaccination, we evaluated the recipient immune response to autologous MSCs prepared with and without FBS. Preparation of MSCs with FBS resulted in post injection inflammation and antibody mediated cytotoxicity of MSCs when compared to MSCs prepared without FBS. Importantly, synovial MSC concentrations were reduced and LPS induced pain was higher, when FBS was used to prepare MSCs, demonstrating reduced efficacy of FBS prepared MSCs. Fetal bovine serum should no longer be utilized for MSC preparation in pre-clinical study, clinical study, or veterinary applications. The use of FBS in previously reported studies, and in MSC therapeutics with current foreign market authorization, should be considered when interpreting results.

Disrupted homeostasis of synovial hyaluronic acid and its associations with synovial mast cell proteases of rheumatoid arthritis patients and collagen-induced arthritis rats.

Hyaluronic acid (HA) is the main component of the extracellular matrix (ECM) of joints, and it is important for a lubricating joint during body movement. Degradation is the main metabolic process of HA in vivo. Hyaluronidases (HAase) were known for HA degradation. The inflammation-induced HA rapid-metabolism can reduce HA viscosity and concentration in joints. Mast cells (MC) containing their specific proteases were found in synovium tissue. It is unclear if MC-proteases could be involved in HA degradation pathways. This study aims to explore the correlations between HA concentration vs mast cell proteases, or matrix metalloproteinase-2/9 (MMP-2/9) and to investigate the association of MC-specific proteases with disrupted synovial HA homeostasis in rheumatoid arthritis (RA) or collagen-induced arthritis rats. The synovial fluid samples from no-RA and RA patients were collected; the collagen-induced arthritis (CIA) rat model was established; HA concentration and the activities of MC-protease and MMP-2/9 in the samples were detected, and the correlations were analyzed. In vitro interaction experiment was carried out by mixing MC-proteases with HA to observe the degradation speed. The HA concentrations in synovial fluids were decreased in RA patients and CIA rats compared with those in no-RA subjects or normal rats respectively. The activities of mast cell proteases in synovial fluids were increased and positively correlated with MMP-9, but negatively correlated with HA concentrations. In vitro study, the addition of MC-chymase and tryptase promoted the speed in HA degradation. MC-proteases may influence HA degradation pathway.

Targeting Soluble Epoxide Hydrolase and Cyclooxygenases Enhance Joint Pain Control, Stimulate Collagen Synthesis, and Protect Chondrocytes From Cytokine-Induced Apoptosis.

Objective: To determine the symptomatic and disease-modifying capabilities of sEH and COX inhibitors during joint inflammation.Methods: Using a blinded, randomized, crossover experimental design, 6 adult healthy horses were injected with lipopolysaccharide (LPS; 3 μg) from E. coli in a radiocarpal joint and concurrently received the non-selective cyclooxygenase (COX) inhibitor phenylbutazone (2 mg/kg), the sEH inhibitor t-TUCB (1 mg/kg) or both (2 mg/kg phenylbutazone and 0.1, 0.3, and 1 mg/kg t-TUCB) intravenously. There were at least 30 days washout between treatments. Joint pain (assessed via inertial sensors and peak vertical forces), synovial fluid concentrations of prostanoids (PGE2, TxB2), cytokines (IL-1β, IL-6, TNF-α) and biomarkers of collagen synthesis (CPII) and degradation (C2C) were measured at pre-determined intervals over a 48-h period. The anti-apoptotic effect of COX and sEH inhibitors was determined via ELISA technique in primary equine chondrocytes incubated with TNF-α (10 ng/ml) for 24 h. Apoptosis was also determined in chondrocytes incubated with sEH-generated metabolites.Results: Combined COX and sEH inhibition produced significantly better control of joint pain, prostanoid responses, and collagen synthesis-degradation balance compared to each compound separately. When administered separately, pain control was superior with COX vs. sEH inhibition. Cytokine responses were not different during COX and/or sEH inhibition. In cultured chondrocytes, sEH inhibition alone or combined with COX inhibition, but not COX inhibition alone had significant anti-apoptotic effects. However, sEH-generated metabolites caused concentration-dependent apoptosis.Conclusions: Combined COX and sEH inhibition optimize pain control, attenuate loss of articular cartilage matrix during joint inflammation and cytokine-induced chondrocyte apoptosis.

Synovial Concentration of Trimethoprim-Sulphadiazine Following Regional Limb Perfusion in Standing Horses.

The aim of this study was to investigate the safety and pharmacokinetics of trimethoprim-sulphadiazine administered via intravenous regional limb perfusion (IVRLP) into the cephalic vein. According to the hypothesis, the drug could be administered without adverse effects and the synovial concentrations would remain above the minimum inhibitory concentration (MIC) for trimethoprim-sulphadiazine (0.5 and 9.5 µg/mL) for 24 h. Ten (n = 10) horses underwent cephalic vein IVRLP with an Esmarch tourniquet applied for 30 min. Four grams (4 g) of trimethoprim-sulphadiazine (TMP-SDZ) were diluted at 0.9% NaCl for a total volume of 100 mL. Synovial fluid and blood samples were obtained immediately before IVRLP and at 0.25, 0.5, 2, 6, 12 and 24 h after the initiation of IVRLP. Trimethoprim and sulphadiazine concentrations were determined using a method based on liquid chromatography/tandem mass spectrometry. The Cmax (peak drug concentration) values were 36 ± 31.1 and 275.3 ± 214.4 µg/mL (TMP and SDZ). The respective tmax (time to reach Cmax) values were 20 ± 7.8 and 26.4 ± 7.2 min. The initial synovial fluid concentrations were high but decreased quickly. No horse had synovial concentrations of trimethoprim-sulphadiazine above the MIC at 12 h. Severe vasculitis and pain shortly after IVRLP, lasting up to one week post-injection, occurred in five out of 10 horses. In conclusion, IVRLP with trimethoprim-sulphadiazine cannot be recommended due to the low concentrations of synovial fluid over time and the frequent severe adverse effects causing pain and discomfort in treated horses. Thus, in cases of septic synovitis with bacteria sensitive to trimethoprim-sulphadiazine, other routes of administration should be considered.

Nonlinear Mixed-Effect Pharmacokinetic Modeling and Distribution of Doxycycline in Healthy Female Donkeys after Multiple Intragastric Dosing-Preliminary Investigation.

Simple SummaryRational use of antibiotic is of most importance for human and animal health. Donkeys have differences in metabolism compared to horses, and drug dosage should not be extrapolated from horses’ data. Doxycycline, a common antibiotic used in equine medicine, has never been investigated in donkeys. The aim of this preliminary study was to describe the concentrations of doxycycline obtain in serum and other body tissue and fluid following its oral administration in donkeys at the recommended horse dosage. Doxycycline was administered to eight healthy, adult jennies. Serum, urine, synovial fluid and uterine tissue were collected. Doxycycline concentrations were measured with a commercial ELISA. A pharmacological model was used to analyze the serum concentration and calculate some pharmacological parameters. Results suggest that doxycycline is well absorbed following oral administration and calculated serum parameters suggest high tissue distribution. However, the concentration of doxycycline reached in all fluids and tissues analyzed would unlikely result in therapeutic concentration against common equine pathogens. Further investigations are warranted. This data can be used for designing future studies of doxycycline in donkeys. In the meantime, oral doxycycline at the horse dosage should not be consider a suitable treatment in donkeys until proven efficacious. Doxycycline (DXC) is a broad-spectrum antibacterial antimicrobial administered to horses for the treatment of bacterial infections which may also affect donkeys. Donkeys have a different metabolism than horses, leading to differences in the pharmacokinetics of drugs compared to horses. This study aimed to describe the population pharmacokinetics of DXC in donkeys. Five doses of DXC hyclate (10 mg/kg) were administered via a nasogastric tube, q12 h, to eight non-fasted, healthy, adult jennies. Serum, urine, synovial fluid and endometrium were collected for 72 h following the first administration. Doxycycline concentration was measured by competitive enzyme immunoassay. Serum concentrations versus time data were fitted simultaneously using the stochastic approximation expectation-maximization algorithm for nonlinear mixed effects. A one-compartment model with linear elimination and first-order absorption after intragastric administration, best described the available pharmacokinetic data. Final parameter estimates indicate that DXC has a high volume of distribution (108 L/kg) as well as high absorption (10.3 h−1) in donkeys. However, results suggest that oral DXC at 10 mg/kg q12 h in donkeys would not result in a therapeutic concentration in serum, urine, synovial fluid or endometrium by comparison to the minimum inhibitory concentration of common equine pathogens. Further studies are recommended to identify appropriate dosage and dosing intervals of oral DXC in donkeys.

RANKL expression in chondrocytes and its promotion by lymphotoxin-α in the course of cartilage destruction during rheumatoid arthritis.

We investigated the expression and localization of the receptor activator nuclear factor κB ligand (RANKL) in cartilage from patients with rheumatoid arthritis (RA) of relevance to cartilage degeneration. We also examined the role of exogenous lymphotoxin (LT)-α on RANKL expression in human chondrocytes and its effect on in vitro osteoclast differentiation. Cartilage and synovial fluid samples were obtained from 45 patients undergoing total joint replacement surgery or joint puncture, including 24 patients with osteoarthritis (OA) and 21 patients with RA. RANKL expression in articular cartilage was examined by immunohistochemistry. LT-α concentrations in synovial fluid were measured using an enzyme-linked immunosorbent assay (ELISA). Normal human chondrocytes were stimulated with LT-α, and the relative mRNA levels of RANKL, osteoprotegerin (OPG), matrix metalloproteinase-9, and vascular endothelial growth factor were examined by real-time polymerase chain reaction. Soluble RANKL protein in culture media was measured using ELISA, and membrane-bound RANKL protein in cells was examined by western blotting. Co-cultures of human chondrocytes with peripheral blood mononuclear cells (PBMCs) were stimulated with macrophage-colony stimulating factor and LT-α, and osteoclast differentiation was evaluated by staining for tartrate-resistant acid phosphatase. LT-α concentrations were higher in RA synovial fluid than in OA samples. The population of RANKL-positive chondrocytes of RA cartilage was higher than that of OA cartilage, and correlated with cartilage degeneration. Stimulation of cultured human chondrocytes by LT-α increased RANKL expression, the RANKL/OPG ratio, and angiogenic factors. Membrane-bound RANKL in chondrocytes was up-regulated after stimulation of LT-α, whereas soluble RANKL in culture medium did not increase. Co-cultures of human chondrocytes and PBMCs demonstrated that LT-α stimulated human chondrocytes to produce RANKL and induced osteoclastic differentiation of PBMCs. RANKL produced by chondrocytes may contribute to cartilage destruction during RA and LT-α could promote the expression of RANKL in human chondrocytes.

Effects of Normal Synovial Fluid and Interferon Gamma on Chondrogenic Capability and Immunomodulatory Potential Respectively on Equine Mesenchymal Stem Cells.

Synovial fluid contains cytokines, growth factors and resident mesenchymal stem cells (MSCs). The present study aimed to (1) determine the effects of autologous and allogeneic synovial fluid on viability, proliferation and chondrogenesis of equine bone marrow MSCs (BMMSCs) and (2) compare the immunomodulatory properties of equine synovial fluid MSCs (SFMSCs) and BMMSCs after stimulation with interferon gamma (INF-γ). To meet the first aim of the study, the proliferation and viability of MSCs were evaluated by MTS and calcein AM staining assays. To induce chondrogenesis, MSCs were cultured in a medium containing TGF-β1 or different concentrations of synovial fluid. To meet the second aim, SFMSCs and BMMSCs were stimulated with IFN-γ. The concentration of indoleamine-2,3-dioxygenase (IDO) and nitric oxide (NO) were examined. Our results show that MSCs cultured in autologous or allogeneic synovial fluid could maintain proliferation and viability activities. Synovial fluid affected chondrocyte differentiation significantly, as indicated by increased glycosaminoglycan contents, compared to the chondrogenic medium containing 5 ng/mL TGF-β1. After culturing with IFN-γ, the conditioned media of both BMMSCs and SFMSCs showed increased concentrations of IDO, but not NO. Stimulating MSCs with synovial fluid or IFN-γ could enhance chondrogenesis and anti-inflammatory activity, respectively, suggesting that the joint environment is suitable for chondrogenesis.

Evaluation of Intra-Articular Amikacin Administration in an Equine Non-inflammatory Joint Model to Identify Effective Bactericidal Concentrations While Minimizing Cytotoxicity.

Septic arthritis causes significant morbidity and mortality in veterinary and human clinical practice and is increasingly complicated by multidrug-resistant infections. Intra-articular (IA) antibiotic administration achieves high local drug concentrations but is considered off-label usage, and appropriate doses have not been defined. Using an equine joint model, we investigated the effects of amikacin injected at three different doses (500, 125, and 31.25 mg) on the immune and cartilage responses in tibiotarsal joints. Synovial fluid (SF) was sampled at multiple time points over 24 h, the cell counts determined, and amikacin concentrations measured by liquid chromatography-mass spectrometry. Cytokine concentrations and collagen degradation products in SF were measured by ELISA and multiplex immunoassays. The mean amikacin concentrations in SF were greater than or equal to the minimum inhibitory concentration (MIC) (0.004 mg/ml) for most common equine joint pathogens at all time points tested to 24 h for all three amikacin doses evaluated. The inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) increased significantly in SF in the highest amikacin dose group, despite the fact that increases in SF cell counts were not observed. Similarly, the biomarkers of cartilage type II collagen cleavage (C2C and C12C) were increased in SF following amikacin injection. Mechanistically, we further demonstrated using in vitro studies that chondrocytes and synoviocytes killed by exposure to amikacin underwent apoptotic cell death and were phagocytosed by macrophages in a non-inflammatory process resembling efferocytosis. Neutrophils and T cells were susceptible to amikacin cytotoxicity at clinically relevant doses, which may result in blunting of cellular inflammatory responses in SF and account for the lack of increase in total nucleated cell counts following amikacin injection. In summary, decisions on whether to inject cytotoxic antibiotics such as aminoglycosides intra-articularly and what doses to use should take into account the potential harm that antibiotics may cause and consider lower doses than those previously reported in equine practice.

In vivo pharmacokinetic evaluation of carprofen delivery from intra-articular nanoparticles in rabbits: A population modelling approach

Osteoarthritis is treated with COX or fosfolipase A2 inhibitors such as carprofen, a propionic acid NSAID. The enhancement of its action over the articular cartilage is mandatory to facilitate its therapeutic application. Drug uptake into the cartilage requires high synovial fluid concentrations, anticipating its rapid distribution towards bloodstream. Thus, intraarticular administration improves local targeting of the drug, lining with the site of action.A pharmacokinetic study in rabbits has been performed to evaluate carprofen nanoparticles after intraarticular administration. Pharmacokinetic analysis of plasma profiles through a modelling approach, has demonstrated the rapid distribution of drug outside of synovial chamber but mainly remaining in plasma. The data modelling has demonstrated the existence of two release-absorption processes when the nanoparticles are administered in the synovial space. Additionally, results are predictive of the PK profile of some other species such as cat, dogs or humans.