Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis

Currently, little is known about the in vivo human immune response against Staphylococcus aureus during a biofilm-associated infection, such as osteomyelitis, and how this relates to protein production in biofilms in vitro. Therefore, we characterized IgG responses in 10 patients with chronic osteomyelitis against 50 proteins of S. aureus, analyzed the presence of these proteins in biofilms of the infecting isolates on polystyrene (PS) and human bone in vitro, and explored the relation between in vivo and in vitro data. IgG levels against 15 different proteins were significantly increased in patients compared to healthy controls. Using a novel competitive Luminex-based assay, eight of these proteins [alpha toxin, Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FlipR), glucosaminidase, iron-responsive surface determinants A and H, the putative ABC transporter SACOL0688, staphylococcal complement inhibitor (SCIN), and serine–aspartate repeat-containing protein E (SdrE)] were also detected in a majority of the infecting isolates during biofilm formation in vitro. However, 4 other proteins were detected in only a minority of isolates in vitro while, vice versa, 7 proteins were detected in multiple isolates in vitro but not associated with significantly increased IgG levels in patients. Detection of proteins was largely confirmed using a transcriptomic approach. Our data provide further insights into potential therapeutic targets, such as for vaccination, to reduce S. aureus virulence and biofilm formation. At the same time, our data suggest that either in vitro or immunological in vivo data alone should be interpreted cautiously and that combined studies are necessary to identify potential targets

FK506 protects against articular cartilage collagenous extra-cellular matrix degradation

Objective: Osteoarthritis (OA) is a non-rheumatologic joint disease characterized by progressive degeneration of the cartilage extra-cellular matrix (ECM), enhanced subchondral bone remodeling, activation of synovial macrophages and osteophyte growth. Inhibition of calcineurin (Cn) activity through tacrolimus (FK506) in invitro monolayer chondrocytes exerts positive effects on ECM marker expression. This study therefore investigated the effects of FK506 on anabolic and catabolic markers of osteoarthritic chondrocytes in 2D and 3D invitro cultures, and its therapeutic effects in an invivo rat model of OA. Methods: Effects of high and low doses of FK506 on anabolic (QPCR/histochemistry) and catabolic (QPCR) markers were evaluated invitro on isolated (2D) and ECM-embedded chondrocytes (explants, 3D pellets). Severe cartilage damage was induced unilaterally in rat knees using papain injections in combination with a moderate running protocol. Twenty rats were treated with FK506 orally and compared to twenty untreated controls. Subchondral cortical and trabecular bone changes (longitudinal microCT) and macrophage activation (SPECT/CT) were measured. Articular cartilage was analyzed exvivo using contrast enhanced microCT and histology. Results: FK506 treatment of osteoarthritic chondrocytes invitro induced anabolic (mainly collagens) and reduced catabolic ECM marker expression. In line with this, FK506 treatment clearly protected ECM integrity invivo by markedly decreasing subchondral sclerosis, less development of subchondral pores, depletion of synovial macrophage activation and lower osteophyte growth. Conclusion: FK506 protected cartilage matrix integrity invitro and invivo. Additionally, FK506 treatment invivo reduced OA-like responses in different articular joint tissues and thereby makes Cn an interesting target for therapeutic intervention of OA

Real-time assessment of bone metabolism in small animal models for osteoarthritis using multi pinhole-SPECT/CT

SummaryObjectiveDestructive techniques such as histology and biochemical assays are still regarded the gold standard to study the effects of novel therapies or etiologic aspects of osteoarthritis in small animal models. These techniques are time-consuming and require many animals. Multi-pinhole single photon emission computed tomography (MPH-SPECT) is a relatively novel, high resolution imaging technique which enables assessment of biological processes in real-time and thus it might provide a good substitute for destructive assessment techniques.DesignFor this study, we assessed mono-iodoacetate (MIA) induced osteoarthritic knees in 18 rats. The animals were scanned using MPH-SPECT/CT and a diphosphonate labelled with 99m-technetium as the radioactive tracer to monitor subchondral bone turnover (bone-scan) at 2 (n = 18), 14 (n = 12) and 42 (n = 6) days after injection of MIA. At each time-point six animals were sacrificed and also assessed with high-resolution micro-computed tomography (μCT) and histology.ResultsAt 2 days after injection of MIA, the MPH-SPECT/CT already showed elevated bone turnover in the affected knee, whereas with histology and μCT we could not detect clear alterations at all this time-point. The increase in bone turnover induced by MIA was elevated further at 14 and 42 days after injection. At this time alterations on histology and μCT scanning also became visible.ConclusionsMPH-SPECT/CT proved to be a highly sensitive assessment technique for experimental osteoarthritis in small animal models, detecting real-time changes in bone turnover at a very early time point, which might make it a valuable technique to measure the direct effect of interventional strategies on osteoarthritis

In vivo pharmacokinetics of celecoxib loaded endcapped PCLA-PEG-PCLA thermogels in rats after subcutaneous administration

Injectable thermogels based on poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) (PCLA-PEG-PCLA) containing an acetyl- or propyl endcap and loaded with celecoxib were developed for local drug release. The aim of this study was to determine the effects of the composition of the celecoxib/PCLA-PEG-PCLA formulation on their in vivo drug release characteristics. Furthermore, we want to obtain insight into the in vitro-in vivo correlation. Different formulations were injected subcutaneously in rats and blood samples were taken for a period of 8 weeks. Celecoxib half-life in blood increased from 5 h for the bolus injection of celecoxib to more than 10 days for the slowest releasing gel formulation. Sustained release of celecoxib was obtained for at least 8 weeks after subcutaneous administration. The release period was prolonged from 3 to 6-8 weeks by increasing the injected volume from 100 to 500 µL, which also led to higher serum concentrations in time. Propyl endcapping of the polymer also led to a prolonged release compared to the acetyl endcapped polymer (49 versus 21 days) and at equal injected dose of the drug in lower serum concentrations. Increasing the celecoxib loading from 10 mg/mL to 50 mg/mL surprisingly led to prolonged release (28 versus 56 days) as well as higher serum concentrations per time point, even when corrected for the higher dose applied. The in vivo release was about twice as fast compared to the in vitro release for all formulations. Imaging of organs of mice, harvested 15 weeks after subcutaneous injection with polymer solution loaded with infrared-780 labelled dye showed no accumulation in any of these harvested organs except for traces in the kidneys, indicating renal clearance. Due to its simplicity and versatility, this drug delivery system has great potential for designing an injectable to locally treat osteoarthritis, and to enable tuning the gel to meet patient-specific needs

In vivo pharmacokinetics of celecoxib loaded endcapped PCLA-PEG-PCLA thermogels in rats after subcutaneous administration

Injectable thermogels based on poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) (PCLA-PEG-PCLA) containing an acetyl- or propyl endcap and loaded with celecoxib were developed for local drug release. The aim of this study was to determine the effects of the composition of the celecoxib/PCLA-PEG-PCLA formulation on their in vivo drug release characteristics. Furthermore, we want to obtain insight into the in vitro-in vivo correlation. Different formulations were injected subcutaneously in rats and blood samples were taken for a period of 8 weeks. Celecoxib half-life in blood increased from 5 h for the bolus injection of celecoxib to more than 10 days for the slowest releasing gel formulation. Sustained release of celecoxib was obtained for at least 8 weeks after subcutaneous administration. The release period was prolonged from 3 to 6–8 weeks by increasing the injected volume from 100 to 500 µL, which also led to higher serum concentrations in time. Propyl endcapping of the polymer also led to a prolonged release compared to the acetyl endcapped polymer (49 versus 21 days) and at equal injected dose of the drug in lower serum concentrations. Increasing the celecoxib loading from 10 mg/mL to 50 mg/mL surprisingly led to prolonged release (28 versus 56 days) as well as higher serum concentrations per time point, even when corrected for the higher dose applied. The in vivo release was about twice as fast compared to the in vitro release for all formulations. Imaging of organs of mice, harvested 15 weeks after subcutaneous injection with polymer solution loaded with infrared-780 labelled dye showed no accumulation in any of these harvested organs except for traces in the kidneys, indicating renal clearance. Due to its simplicity and versatility, this drug delivery system has great potential for designing an injectable to locally treat osteoarthritis, and to enable tuning the gel to meet patient-specific needs

In vivo pharmacokinetics of celecoxib loaded endcapped PCLA-PEG-PCLA thermogels in rats after subcutaneous administration

Injectable thermogels based on poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) (PCLA-PEG-PCLA) containing an acetyl- or propyl endcap and loaded with celecoxib were developed for local drug release. The aim of this study was to determine the effects of the composition of the celecoxib/PCLA-PEG-PCLA formulation on their in vivo drug release characteristics. Furthermore, we want to obtain insight into the in vitro-in vivo correlation. Different formulations were injected subcutaneously in rats and blood samples were taken for a period of 8 weeks. Celecoxib half-life in blood increased from 5 h for the bolus injection of celecoxib to more than 10 days for the slowest releasing gel formulation. Sustained release of celecoxib was obtained for at least 8 weeks after subcutaneous administration. The release period was prolonged from 3 to 6-8 weeks by increasing the injected volume from 100 to 500 µL, which also led to higher serum concentrations in time. Propyl endcapping of the polymer also led to a prolonged release compared to the acetyl endcapped polymer (49 versus 21 days) and at equal injected dose of the drug in lower serum concentrations. Increasing the celecoxib loading from 10 mg/mL to 50 mg/mL surprisingly led to prolonged release (28 versus 56 days) as well as higher serum concentrations per time point, even when corrected for the higher dose applied. The in vivo release was about twice as fast compared to the in vitro release for all formulations. Imaging of organs of mice, harvested 15 weeks after subcutaneous injection with polymer solution loaded with infrared-780 labelled dye showed no accumulation in any of these harvested organs except for traces in the kidneys, indicating renal clearance. Due to its simplicity and versatility, this drug delivery system has great potential for designing an injectable to locally treat osteoarthritis, and to enable tuning the gel to meet patient-specific needs

In vivo pharmacokinetics of celecoxib loaded endcapped PCLA-PEG-PCLA thermogels in rats after subcutaneous administration

Injectable thermogels based on poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) (PCLA-PEG-PCLA) containing an acetyl- or propyl endcap and loaded with celecoxib were developed for local drug release. The aim of this study was to determine the effects of the composition of the celecoxib/PCLA-PEG-PCLA formulation on their in vivo drug release characteristics. Furthermore, we want to obtain insight into the in vitro-in vivo correlation. Different formulations were injected subcutaneously in rats and blood samples were taken for a period of 8 weeks. Celecoxib half-life in blood increased from 5 h for the bolus injection of celecoxib to more than 10 days for the slowest releasing gel formulation. Sustained release of celecoxib was obtained for at least 8 weeks after subcutaneous administration. The release period was prolonged from 3 to 6-8 weeks by increasing the injected volume from 100 to 500 µL, which also led to higher serum concentrations in time. Propyl endcapping of the polymer also led to a prolonged release compared to the acetyl endcapped polymer (49 versus 21 days) and at equal injected dose of the drug in lower serum concentrations. Increasing the celecoxib loading from 10 mg/mL to 50 mg/mL surprisingly led to prolonged release (28 versus 56 days) as well as higher serum concentrations per time point, even when corrected for the higher dose applied. The in vivo release was about twice as fast compared to the in vitro release for all formulations. Imaging of organs of mice, harvested 15 weeks after subcutaneous injection with polymer solution loaded with infrared-780 labelled dye showed no accumulation in any of these harvested organs except for traces in the kidneys, indicating renal clearance. Due to its simplicity and versatility, this drug delivery system has great potential for designing an injectable to locally treat osteoarthritis, and to enable tuning the gel to meet patient-specific needs