K. Tóth

Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters

Numerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light-scattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease.

Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle) signatures in joint diseases.

Introduction Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. Methods In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. Results EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3+ and CD8+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p = 0.027 and p = 0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p = 0.009, after Bonferroni correction). Conclusions Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

The Effect of First Ray Shortening in the Development of Metatarsalgia in the Second through Fourth Rays after Metatarsal Osteotomy

Background: The aims of this study were to determine the severity of metatarsalgia of the second through fifth rays after shortening of the first ray for correction of hallux valgus deformity and patient satisfaction of the cosmetic results. Methods: Two hundred and forty metatarsal osteotomies (Wilson osteotomy as modified by Lindgren and Turan) were evaluated 4.19 ±1.29 years postoperatively. The procedure involved a slightly oblique subcapital osteotomy of the first metatarsal and fixation with one screw. Results: The average decrease in the hallux valgus angle was 26 ± 5 degrees, the 1–2 intermetatarsal angle was 8.4 ± 4 degrees, and the average shortening of the first metatarsal was 3.8 ± 1.8 mm. Positive correlations were found between metatarsalgia of the second through fourth rays and first ray shortening (p < 0.001 second ray, p < 0.001 third ray, and p < 0.001 fourth ray); there was no correlation between the fifth ray and first ray shortening. No correlation was found between a decrease in the hallux valgus angle or 1–2 intermetatarsal angle and metatarsalgia in the second through fifth rays. A positive correlation was detected between postoperative foot alignment and decrease in the hallux valgus (p < 0.001) and a negative correlation between postoperative foot alignment and first ray shortening (p < 0.01). No correlation was noted between postoperative foot alignment and the 1–2 intermetatarsal angle. Conclusion: Excessive shortening of the first metatarsal should be avoided to decrease the occurrence of postoperative transfer metatarsalgia. We found a greater patient satisfaction with foot alignment in patients who had a greater decrease in the hallux valgus angle and less shortening of the first ray.

Gene expression and activity of cartilage degrading glycosidases in human rheumatoid arthritis and osteoarthritis synovial fibroblasts

IntroductionSimilar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints.MethodsExpressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity.ResultsAccording to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFα, IL-1β and IL-17.ConclusionsAccording to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.

The antinociceptive effect of intrathecal kynurenic acid and its interaction with endomorphin-1 in rats

Kynurenic acid as an endogenous ligand antagonizes all types of ionotropic glutamate receptors, with preferential affinity for the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor. The purpose of the present study was to investigate the antinociceptive potency of continuously administered kynurenic acid on carrageenan-induced thermal hyperalgesia by means of a paw withdrawal test in awake rats. The possible interaction between kynurenic acid and the endogenous mu-opioid receptor agonist peptide, endomorphin-1, was examined in the same set-up. Kynurenic acid at the higher doses (1-4 microg/min) significantly decreased the thermal hyperalgesia and increased the paw withdrawal latencies on the non-inflamed side. These doses were also associated with motor impairment on both sides. Low doses of kynurenic acid (0.01-0.1 microg/min) potentiated, but did not prolong, the antinociceptive effect of endomorphin-1 (0.1-1 microg/min) on the inflamed side. There was no sign of motor impairment during the combined treatment. These findings demonstrate that the combination of low doses of these two endogenous ligands provides effective and well-controlled antinociception without side effects.

The significance of intrathecal catheter location in rats

Although chronic intrathecal catheterization is a widely used method in rats, few calibration experiments have been performed. In this study, we investigated the correlation between the side position of the catheter tip and the side differences observed in the motor and sensory disturbances after intrathecal administration of lidocaine to a large number of rats. The existence of a sensory block was determined by the paw withdrawal test. The motor impairment was assessed by observing the complete clubbing of the hindpaw and measuring the hindpaw grip strength. After experimental use, we established the position of the catheter tip. The catheter tips were variously located in all directions of the transverse plane in the rat spinal subarachnoid space. Lidocaine administration (100 or 500 microg/5 microL; n = 264 and 112, respectively) led to dose-dependent motor and sensory disturbances. The effect of 100 microg of lidocaine exhibited side differences; i.e., the extents of both motor (r = 0.77) and sensory (r = 0.60 and r = 0.67 for the right and the left side, respectively) disturbances correlated significantly with the location of the catheter tip. Our data have shown that detection of the paralytic and/or antinociceptive effect of small-dose lidocaine before planned experiments is a simple and reliable method for prediction of the location of the catheter tip. We suggest that the position of the catheter might cause side differences in the drug effect, especially if small doses of drugs are administered and their effects are investigated on both sides.

The recently identified hexosaminidase D enzyme substantially contributes to the elevated hexosaminidase activity in rheumatoid arthritis.

Since the 1970s, numerous reports have described elevated hexosaminidase activities in rheumatoid arthritis. However, due to the overlapping substrate specificities of different hexosaminidases, identification of the exact enzyme(s) responsible for the elevated activity remains incomplete. In this work we tested if the recently described enzyme, hexosaminidase D was expressed in human arthritic joints, and could contribute to the elevated hexosaminidase activity in rheumatoid arthritis. Thermostable β-d-N-acetyl-galactosaminidase (hexosaminidase D) activities were determined in synovial fluid samples, synovial membranes, synovial fibroblast cell strains and synovial fibroblast-derived extracellular vesicles of patients with rheumatoid arthritis and osteoarthritis using chromogenic substrates. Expression of the HEXDC gene was detected both in steady state and in TGF-β treated synovial fibroblasts by real time PCR. Strikingly, hexosaminidase D accounted for approximately 50% of the total β-N-acetyl-galactosaminidase activity in synovial membranes and synovial fibroblasts, and it was responsible for the vast majority of the β-d-N-acetyl-galactosaminidase activity in synovial fluid samples. TGF-β downregulated the expression of hexosaminidase D in synovial fibroblasts dose-dependently. Of note, significant activity of hexosaminidase D was also found in association with extracellular vesicles released by synovial fibroblasts. This first study that describes the expression and disease relevance of the HEXDC gene in humans demonstrates the expression of this novel enzyme within the joints, and suggests that its activity may significantly contribute to the overall local exoglycosidase activity.

Percutaneous decompression for the treatment of Mueller–Weiss syndrome

This report describes the case of a young athlete, who presented with a painful foot and was eventually diagnosed with early-stage Mueller–Weiss syndrome (spontaneous osteonecrosis of the navicular) by MRI. As non-operative management was unsuccessful, a percutaneous decompression of the navicular was performed. The patient made a full recovery and was able to return to her previous level of sporting activity. Subsequent imaging showed complete remodelling of the bony architecture of the affected navicular.

The Influence of the Length of the First Metatarsal on Transfer Metatarsalgia After Wu's Osteotomy

Background The aim of this study was to evaluate how changes in the length of the first metatarsal, hallux valgus angle (HVA), intermetatarsal 1–2 angle (IMA), plantar angulation and sesamoid position influence the severity of the postoperative 2–5 metatarsalgia and to determine patient satisfaction with the cosmetic outcome after Wus osteotomy. Materials and Methods A retrospective analysis of the clinical data and radiographs of 87 cases was performed at a mean followup time of 4.2 years after Wus subcapital cross osteotomies. Results The mean HVA decreased from 42 to 13 degrees, the mean IMA 1–2 from 22 to 10 degrees. The mean first metatarsal lengthening was 0.3 mm. A negative correlation was found between lengthening of the first metatarsal and metatarsalgia at rays 2 and 3. No such pattern was found between the fourth and fifth metatarsal. No correlation was found between the 2–5 metatarsalgia and the decrease in either the HVA or the IMA 1–2. A positive correlation was detected between the HVA decrease and the patients’ satisfaction with their postoperative foot alignment; there was no correlation between the perceived postoperative foot alignment and either the first metatarsal lengthening or the IMA 1–2 decrease. Conclusion Preservation of the length of the first metatarsal during osteotomy seems to prevent the postoperative transfer metatarsalgia on the second and third rays; however, it has no major influence on the satisfaction of the patients with their foot alignment. A greater correction of the HVA angle resulted in a higher level of satisfaction with the foot cosmesis.

Peripheral antinociceptive effect of 2-arachidonoyl-glycerol and its interaction with endomorphin-1 in arthritic rat ankle joints

1. Both cannabinoid and opioid receptors are localized at the peripheral level, and drugs acting on these receptors may produce antinociception after topical administration; however, the effect of endogenous ligands at these receptors is poorly understood. Our goal was to determine the antinociceptive potency of the endogenous cannabinoid 2‐arachidonoyl‐glycerol (2‐AG), and its interaction with endomorphin‐1 (EM1) at joint level in the rat inflammation model.