Median Nerve Shear Wave Elastography is Associated With the Neurophysiological Severity of Carpal Tunnel Syndrome.

OBJECTIVES: This study examines the associations between the median nerve (MN) shear wave elastography (SWE), the MN cross-sectional area (CSA), patient's symptoms, and the neurophysiological severity of carpal tunnel syndrome (CTS). The most appropriate site to perform SWE was also tested. METHODS: This prospective study comprised 86 wrists of 47 consecutive patients who volunteered for MN ultrasound after an electrodiagnostic study. The neurophysiological severity of CTS was assessed according to the results of a nerve conduction study (NCS). The MN CSA was measured at the carpal tunnel inlet (wCSA) and the forearm (fCSA). SWE was performed on the MN in a longitudinal orientation at the wrist crease (wSWE), at the forearm (fSWE), and within the carpal tunnel (tSWE). RESULTS: The wCSA and wSWE correlated positively with the neurophysiological severity of CTS (r = .619, P < .001; r = .582, P < .001, respectively). The optimal cut-off values to discriminate the groups with normal NCS and with findings indicating CTS were 10.5 mm2 for the wCSA and 4.12 m/s for the wSWE. With these cut-off values, wCSA had a sensitivity of 80% and specificity of 87% and wSWE a sensitivity of 88% and specificity of 76%. Neither tSWE nor fSWE correlated with the neurophysiological severity of CTS or differed between NCS negative and positive groups (P = .429, P = .736, respectively). CONCLUSION: Shear wave velocity in the MN at the carpal tunnel inlet increases in CTS and correlates to the neurophysiological CTS severity equivalently to CSA measured at the same site.

The regulation of TRPA1 expression and function by Th1 and Th2-type inflammation in human A549 lung epithelial cells.

BACKGROUND: Transient Receptor Potential Ankyrin 1 (TRPA1) is a cation channel that mediates pain, itch, cough, and neurogenic inflammation in response to pungent compounds such as acrolein in cigarette smoke. TRPA1 is also activated by endogenous factors and promotes inflammation in asthma models. We have recently shown that TRPA1 is upregulated by inflammatory cytokines in A549 human lung epithelial cells. Here, we explored the effects of Th1 and Th2-type inflammation on TRPA1. METHODS AND RESULTS: TRPA1 expression and function was studied in A549 human lung epithelial cells. To induce inflammation, the cells were exposed to a combination of cytokines TNF-α and IL-1ß; and to model Th1 or Th2-type responses, IFN-γ or IL-4/IL-13 was added, respectively. TRPA1 expression (measured by RT-PCR and Western blot) and function (assessed by Fluo-3AM intracellular calcium measurement) was enhanced under the influence of TNF-α + IL-1ß. IFN-γ further enhanced TRPA1 expression and function, whereas IL-4 and IL-13 suppressed them. The effects of IFN-γ and IL-4 on TRPA1 expression were reversed by the Janus kinase (JAK) inhibitors baricitinib and tofacitinib, and those of IL-4 also by the STAT6 inhibitor AS1517499. The glucocorticoid dexamethasone downregulated TRPA1 expression, whereas the PDE4 inhibitor rolipram had no effect. Under all conditions, TRPA1 blockade was found to reduce the production of LCN2 and CXCL6. CONCLUSIONS: TRPA1 expression and function in lung epithelial cells was upregulated under inflammatory conditions. IFN-γ further increased TRPA1 expression while IL-4 and IL-13 suppressed that in a JAK-STAT6 dependent manner which is novel. TRPA1 also modulated the expression of genes relevant to innate immunity and lung disease. We propose that the paradigm of Th1 and Th2 inflammation is a major determinant of TRPA1 expression and function, which should be considered when targeting TRPA1 for pharmacotherapy in inflammatory (lung) disease.

Human Osteoarthritic Chondrocytes Express Nineteen Different TRP-Genes-TRPA1 and TRPM8 as Potential Drug Targets.

Transient receptor potential (TRP) ion channels are expressed in neuronal and some non-neuronal cells and are involved particularly in pain and thermosensation. We previously showed that TRPA1 is functionally expressed in human osteoarthritic (OA) chondrocytes and mediates inflammation, cartilage degradation, and pain in monosodium-iodoacetate-induced experimental OA. In the present study, we explored the expression of TRP-channels in primary human OA chondrocytes and investigated whether drugs used in the treatment of OA, ibuprofen and glucocorticoids, have effects on TRP-channel expression. OA cartilage was obtained from knee replacement surgery and chondrocytes were isolated with enzyme digestion. NGS analysis showed the expression of 19 TRP-genes in OA chondrocytes, with TRPM7, TRPV4, TRPC1, and TRPM8 having the highest counts in unstimulated cells. These results were verified with RT-PCR in samples from a different group of patients. Interleukin-1ß (IL-1ß) significantly increased TRPA1 expression, while TRPM8 and TRPC1 expression was decreased, and TRPM7 and TRPV4 expression remained unaffected. Furthermore, dexamethasone attenuated the effect of IL-1ß on TRPA1 and TRPM8 expression. The TRPM8 and TRPA1 agonist menthol increased the expression of the cartilage-degrading enzymes MMP-1, MMP-3, and MMP-13 and the inflammatory factors iNOS and IL-6 in OA chondrocytes. In conclusion, human OA chondrocytes express 19 different TRP-genes, of which the significant TRPM8 expression is a novel finding. Dexamethasone attenuated IL-1ß-induced TRPA1 expression. Interestingly, the TRPM8 and TRPA1 agonist menthol increased MMP expression. These results support the concept of TRPA1 and TRMP8 as potential novel drug targets in arthritis.

Dexamethasone Attenuates the Expression of MMP-13 in Chondrocytes through MKP-1.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is upregulated in inflammation and reduces the activity of proinflammatory mitogen-activated protein kinases (MAP kinases) by dephosphorylation. MAP kinases are intracellular signaling pathways that mediate the cellular effects of proinflammatory cytokines. In the present study, we investigated the effects of the glucocorticoid dexamethasone on the expression of catabolic enzymes in chondrocytes and tested the hypothesis that these effects are mediated through MKP-1. Dexamethasone was found to significantly attenuate the expression of matrix metalloproteinase (MMP)-13 in human OA chondrocytes as well as in chondrocytes from MKP-1 WT mice, but not in chondrocytes from MKP-1 KO mice. Dexamethasone also increased the expression of MKP-1 in murine and human OA chondrocytes. Furthermore, p38 MAP kinase inhibitors significantly attenuated MMP-13 expression in human OA chondrocytes, while JNK MAP kinase inhibitors had no effect. The results indicate that the effect of dexamethasone on MMP-13 expression in chondrocytes was mediated by an MKP-1 and p38 MAP kinase-dependent manner. These findings, together with previous results, support the concept of MKP-1 as a protective factor in articular chondrocytes in inflammatory conditions and as a potential drug target to treat OA.

Large Intraneural Ganglion Cyst in the Peroneal Nerve.

A 23-year-old previously healthy male was referred to the clinical neurophysiology unit due to a relatively fast-onset paralysis of muscles of the anterior right leg. Electroneuromyography (ENMG) revealed a total denervation of the muscles innervated by the deep peroneal nerve, diminished sensory response of the superficial peroneal nerve, and partial denervation of the peroneus longus muscle. Ultrasound and magnetic resonance imaging (MRI) revealed a large fluid collection inside the common peroneal nerve, primarily suspected to be an intraneural ganglion cyst. The cyst was surgically excised, and the function of the muscles innervated by the peroneal nerve was recovering at the control ENMG 6 months later. We describe a case of a large intraneural ganglion cyst of the peroneal nerve in an otherwise healthy young male, diagnosis by ENMG, ultrasound, and MRI, as well as subsequent operative treatment. This report demonstrates the utility of nerve ultrasound in differentiating between different causes of peroneal nerve dysfunction.

Effects of Lingonberry (Vaccinium vitis-idaea L.) Supplementation on Hepatic Gene Expression in High-Fat Diet Fed Mice.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is growing worldwide in association with Western-style diet and increasing obesity. Lingonberry (Vaccinium vitis-idaea L.) is rich in polyphenols and has been shown to attenuate adverse metabolic changes in obese liver. This paper investigated the effects of lingonberry supplementation on hepatic gene expression in high-fat diet induced obesity in a mouse model. C57BL/6N male mice were fed for six weeks with either a high-fat (HF) or low-fat (LF) diet (46% and 10% energy from fat, respectively) or HF diet supplemented with air-dried lingonberry powder (HF + LGB). HF diet induced a major phenotypic change in the liver, predominantly affecting genes involved in inflammation and in glucose and lipid metabolism. Lingonberry supplementation prevented the effect of HF diet on an array of genes (in total on 263 genes) associated particularly with lipid or glucose metabolic process (such as Mogat1, Plin4, Igfbp2), inflammatory/immune response or cell migration (such as Lcn2, Saa1, Saa2, Cxcl14, Gcp1, S100a10) and cell cycle regulation (such as Cdkn1a, Tubb2a, Tubb6). The present results suggest that lingonberry supplementation prevents HF diet-induced adverse changes in the liver that are known to predispose the development of NAFLD and its comorbidities. The findings encourage carrying out human intervention trials to confirm the results, with the aim of recommending the use of lingonberries as a part of healthy diet against obesity and its hepatic and metabolic comorbidities.

Effects of ibuprofen on gene expression in chondrocytes from patients with osteoarthritis as determined by RNA-Seq.

Non-steroidal anti-inflammatory drugs are a widely used symptomatic treatment in osteoarthritis (OA), but their effects on cartilage remain controversial. We studied the effects of ibuprofen on gene expression in chondrocytes from patients with OA using RNA-Seq. Chondrocytes were isolated from cartilage samples of patients with OA undergoing knee replacement surgery, cultured with ibuprofen, and total mRNA was sequenced. Differentially expressed genes were identified with edgeR using pairwise comparisons. Functional analysis was performed using ingenuity pathway analysis (IPA). Ibuprofen did not induce statistically significant changes in chondrocyte transcriptome when the cells were cultured in the absence of added cytokines. In inflammatory conditions (when the cells were exposed to the OA-related cytokine interleukin (IL)-1ß), 51 genes were upregulated and 42 downregulated by ibuprofen with fold change >1.5 in either direction. The upregulated genes included anti-inflammatory factors and genes associated with cell adhesion, while several mediators of inflammation were among the downregulated genes. IPA analysis revealed ibuprofen having modulating effects on inflammation-related pathways such as integrin, IL-8, ERK/MAPK and cAMP-mediated signalling pathways. In conclusion, the effects of ibuprofen on primary OA chondrocyte transcriptome appear to be neutral in normal conditions, but ibuprofen may shift chondrocyte transcriptome towards anti-inflammatory phenotype in inflammatory environments.

Chondrocytes from Osteoarthritis Patients Adopt Distinct Phenotypes in Response to Central TH1/TH2/TH17 Cytokines.

Chronic low-grade inflammation plays a central role in the pathogenesis of osteoarthritis (OA), and several pro- and anti-inflammatory cytokines have been implicated to mediate and regulate this process. Out of these cytokines, particularly IFNγ, IL-1ß, IL-4 and IL-17 are associated with different phenotypes of T helper (TH) cells and macrophages, both examples of cells known for great phenotypic and functional heterogeneity. Chondrocytes also display various phenotypic changes during the course of arthritis. We set out to study the hypothesis of whether chondrocytes might adopt polarized phenotypes analogous to TH cells and macrophages. We studied the effects of IFNγ, IL-1ß, IL-4 and IL-17 on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were harvested from the cartilage of OA patients undergoing knee replacement surgery and then cultured with or without the cytokines for 24 h. Total RNA was isolated and sequenced, and GO (Gene Ontology) functional analysis was performed. We also separately investigated genes linked to OA in recent genome wide expression analysis (GWEA) studies. The expression of more than 2800 genes was significantly altered in chondrocytes treated with IL-1ß [in the C(IL-1ß) phenotype] with a fold change (FC) > 2.5 in either direction. These included a large number of genes associated with inflammation, cartilage degradation and attenuation of metabolic signaling. The profile of genes differentially affected by IFNγ (the C(IFNγ) phenotype) was relatively distinct from that of the C(IL-1ß) phenotype and included several genes associated with antigen processing and presentation. The IL-17-induced C(IL-17) phenotype was characterized by the induction of a more limited set of proinflammatory factors compared to C(IL-1ß) cells. The C(IL-4) phenotype induced by IL-4 displayed a differential expression of a rather small set of genes compared with control, primarily those associated with TGFß signaling and the regulation of inflammation. In conclusion, our results show that OA chondrocytes can adopt diverse phenotypes partly analogously to TH cells and macrophages. This phenotypic plasticity may play a role in the pathogenesis of arthritis and open new therapeutic avenues for the development of disease-modifying treatments for (osteo)arthritis.

Transient Receptor Potential Ankyrin 1 (TRPA1) Is Involved in Upregulating Interleukin-6 Expression in Osteoarthritic Chondrocyte Models.

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-bound ion channel found in neurons, where it mediates nociception and neurogenic inflammation. Recently, we have discovered that TRPA1 is also expressed in human osteoarthritic (OA) chondrocytes and downregulated by the anti-inflammatory drugs aurothiomalate and dexamethasone. We have also shown TRPA1 to mediate inflammation, pain, and cartilage degeneration in experimental osteoarthritis. In this study, we investigated the role of TRPA1 in joint inflammation, focusing on the pro-inflammatory cytokine interleukin-6 (IL-6). We utilized cartilage/chondrocytes from wild-type (WT) and TRPA1 knockout (KO) mice, along with primary chondrocytes from OA patients. The results show that TRPA1 regulates the synthesis of the OA-driving inflammatory cytokine IL-6 in chondrocytes. IL-6 was highly expressed in WT chondrocytes, and its expression, along with the expression of IL-6 family cytokines leukemia inhibitory factor (LIF) and IL-11, were significantly downregulated by TRPA1 deficiency. Furthermore, treatment with the TRPA1 antagonist significantly downregulated the expression of IL-6 in chondrocytes from WT mice and OA patients. The results suggest that TRPA1 is involved in the upregulation of IL-6 production in chondrocytes. These findings together with previous results on the expression and functions of TRPA1 in cellular and animal models point to the role of TRPA1 as a potential mediator and novel drug target in osteoarthritis.

Widespread regulation of gene expression by glucocorticoids in chondrocytes from patients with osteoarthritis as determined by RNA-Seq.

BACKGROUND: Intra-articular glucocorticoid (GC) injections are widely used as a symptomatic treatment for osteoarthritis (OA). However, there are also concerns about their potentially harmful effects, and their detailed effects on chondrocyte phenotype remain poorly understood. METHODS: We studied the effects of dexamethasone on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were isolated from the cartilage from OA patients undergoing knee replacement surgery and cultured with or without dexamethasone for 24 h. Total RNA was isolated and sequenced, and functional analysis was performed against the Gene Ontology (GO) database. Results for selected genes were confirmed with RT-PCR. We also investigated genes linked to OA in recent genome-wide expression analysis (GWEA) studies. RESULTS: Dexamethasone increased the expression of 480 and reduced that of 755 genes with a fold change (FC) 2.0 or greater. Several genes associated with inflammation and cartilage anabolism/catabolism as well as lipid and carbohydrate metabolism were among the most strongly affected genes. In the GO analysis, genes involved in the extracellular matrix organization, cell proliferation and adhesion, inflammation, and collagen synthesis were enriched among the significantly affected genes. In network analysis, NGF, PI3KR1, and VCAM1 were identified as central genes among those most strongly affected by dexamethasone. CONCLUSIONS: This is the first study investigating the genome-wide effects of GCs on the gene expression in OA chondrocytes. In addition to clear anti-inflammatory and anticatabolic effects, GCs affect lipid and glucose metabolism in chondrocytes, an observation that might be particularly important in the metabolic phenotype of OA.

Regulation of gene expression by MF63, a selective inhibitor of microsomal PGE synthase 1 (mPGES1) in human osteoarthritic chondrocytes.

BACKGROUND AND PURPOSE: mPGES1 catalyses the production of PGE2 , the most abundant prostanoid related to inflammation and pain in arthritis. mPGES1 is suggested to be a safer and more selective drug target in inflammatory conditions compared to the COX enzymes inhibited by NSAIDs. In the present study, we investigated the effects of the selective mPGES1 inhibitor MF63 on gene expression in primary human chondrocytes from patients with osteoarthritis (OA). EXPERIMENTAL APPROACH: Chondrocytes were isolated from articular cartilage obtained from osteoarthritis patients undergoing knee replacement surgery. The effects of MF63 were studied in the primary chondrocytes with RNA-sequencing based genome-wide expression analysis. The main results were confirmed with qRT-PCR and compared with the effects of the NSAID ibuprofen. Functional analysis was performed with the GO database and interactions between the genes were studied with STRING. KEY RESULTS: MF63 enhanced the expression of multiple metallothionein 1 (MT1) isoforms as well as endogenous antagonists of IL-1 and IL-36. The expression of IL-6, by contrast, was down-regulated. These genes were also essential in functional and interaction network analyses. The effects of MF63 were consistent in qRT-PCR analysis, whereas the effects of ibuprofen overlapped only partly with MF63. There were no evident findings of catabolic effects by MF63. CONCLUSION AND IMPLICATIONS: Metallothionein 1 has been suggested to have anti-inflammatory and protective effects in cartilage. Up-regulation of the antagonists of IL-1 superfamily and down-regulation of the pro-inflammatory cytokine IL-6 also support novel anti-inflammatory and possibly disease-modifying effects of mPGES1 inhibitors in arthritis.

Novel adipokine associated with OA: retinol binding protein 4 (RBP4) is produced by cartilage and is correlated with MMPs in osteoarthritis patients.

OBJECTIVE: Retinol binding protein 4 (RBP4) is a member of the lipocalin family and a vitamin A carrier in the blood. More recently, RBP4 has been described as an adipokine that is involved in insulin resistance and metabolic syndrome (MetS). As obesity, MetS and some adipokines contribute to the pathogenesis of osteoarthritis (OA), we investigated RBP4 in patients with OA. MATERIALS AND METHODS: Cartilage, synovial fluid and blood samples were collected from 100 OA patients undergoing total knee replacement surgery. Primary chondrocytes and cartilage tissue were cultured to measure the RBP4 expression. The concentrations of RBP4, other adipokines (adipsin, adiponectin, leptin and resistin) and biomarkers of OA (COMP, MMP-1, MMP-3 and YKL-40) were measured by immunoassay, and gene expression was measured by next-generation RNA sequencing. RESULTS: The OA cartilage samples released RBP4 into the culture medium, and the levels correlated positively with the expression of the adipokines adipsin, adiponectin, leptin and resistin. RBP4 was the most prominently expressed of these adipokines in the OA chondrocytes, and the expression of the RBP4 receptors STRA6 (stimulated by retinoic acid gene homologue 6) and TLR4 (Toll-like receptor 4) was also detected. Within the cartilage culture medium, RBP4 showed a positive correlation with MMP-1, MMP-3 and YKL-40. RBP4 was also present in the synovial fluid from the OA patients and correlated positively with the concentrations of RBP4 found in the plasma and the cartilage culture medium. Plasma RBP4 concentrations also showed a positive correlation with MMP-3 and adipsin. CONCLUSIONS: We show here, for the first time, that RBP4 is produced within OA joints and that it is associated with increased levels of adipokines and MMPs. The results suggest a role for RBP4 in the pathogenesis of OA and as a possible target for the disease-modifying drugs for the treatment of OA.

MKP-1 promotes anti-inflammatory M(IL-4/IL-13) macrophage phenotype and mediates the anti-inflammatory effects of glucocorticoids.

Macrophage polarization refers to the ability of these cells to adopt different functional phenotypes according to their environment. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is known to regulate the classical lipopolysaccharide (LPS)-induced pro-inflammatory macrophage activation and the inflammatory response. Here, we investigated the effects of MKP-1 on the anti-inflammatory and healing-promoting macrophage phenotype induced by cytokines IL-4 and IL-13 and examined the potential mediator role of MKP-1 in glucocorticoid effects on the two macrophage phenotypes. In MKP-1-deficient macrophages treated with IL-4 and IL-13 to induce the anti-inflammatory phenotype, the expression of phenotypic markers arginase 1, Ym-1 and FGF2 was reduced as compared to wild-type cells. In contrast, LPS-induced expression of the pro-inflammatory factors IL-6 and iNOS was significantly higher in MKP-1-deficient macrophages. Dexamethasone suppressed the pro-inflammatory phenotype and enhanced the anti-inflammatory phenotype. Interestingly, both of these glucocorticoid effects were attenuated in macrophages from MKP-1-deficient mice. Accordingly, dexamethasone increased MKP-1 expression in both LPS- and IL4+13-treated wild-type cells. In conclusion, the findings support MKP-1 as an endogenous mechanism able to shift macrophage activation from the classical pro-inflammatory state towards the anti-inflammatory and healing-promoting phenotype. In addition, MKP-1 was found to mediate the anti-inflammatory effects of dexamethasone in a dualistic manner: by suppressing the pro-inflammatory macrophage activation and by enhancing the healing-promoting macrophage phenotype.

Attenuating Effects of Nortrachelogenin on IL-4 and IL-13 Induced Alternative Macrophage Activation and on Bleomycin-Induced Dermal Fibrosis.

Excessive alternative macrophage activation contributes to fibrosis. We studied the effects of nortrachelogenin, the major lignan component of Pinus sylvestris knot extract, on alternative (M2) macrophage activation. J774 murine and THP-1 human macrophages were cultured with IL-4+IL-13 to induce alternative activation, together with the extract and its components. Effects of nortrachelogenin were also studied in bleomycin-induced murine dermal fibrosis model. Knot extract significantly decreased the expression of alternative activation markers-arginase 1 in murine macrophages (97.4 ± 1.3% inhibition at 30 µg/mL) and CCL13 and PDGF in human macrophages-as did nortrachelogenin (94.9 ± 2.4% inhibition of arginase 1 at 10 µM). Nortrachelogenin also decreased PPARγ expression but had no effect on STAT6 phosphorylation. In vivo, nortrachelogenin reduced bleomycin-induced increase in skin thickness as well as the expression of collagens COL1A1, COL1A2, and COL3A1 (all by >50%). In conclusion, nortrachelogenin suppressed IL-4+IL-13-induced alternative macrophage activation and ameliorated bleomycin-induced fibrosis, indicating therapeutic potential in fibrosing conditions.

Gene expression in adverse reaction to metal debris around metal-on-metal arthroplasty: An RNA-Seq-based study.

Joint replacement surgery is a standard treatment of advanced osteoarthritis (OA). Since 2000, cobalt-chromium (CoCr) metal-on-metal (MoM) implants were widely used in hip arthroplasties. Some patients developed "adverse reaction to metal debris" (ARMD) around the prosthesis, resulting in a need for revision surgery. In the present study, we addressed the pathogenesis of ARMD by genome-wide expression analysis. Pseudosynovial ARMD tissue was obtained from revision surgery of Articular Surface Replacement (ASR, DePuy, Warsaw, IN, USA) hip arthroplasties. Control tissue was 1) OA synovium from primary hip arthroplasties and 2) inflammatory pseudosynovial tissue from metal-on-plastic (MoP) implant revisions. In ARMD tissue, the expression of 1446 genes was significantly increased and that of 1881 decreased as compared to OA synovium. Genes associated with immune response, tissue development and certain leukocyte signaling pathways were enriched in the differently (FC > 2) expressed genes. The network analysis proposed PRKACB, CD2, CD52 and CD53 as the central regulators of the greatest (FC > 10) differences. When ARMD tissue was compared to MoP tissue, the expression of 16 genes was significantly higher and that of 21 lower. Many of these genes were associated with redox homeostasis, metal ion binding and transport, macrophage activation and apoptosis. Interestingly, genes central to myofibroblast (AEBP1 and DES) and osteoclast (CCL21, TREM2 and CKB) development were upregulated in the MoP tissue. In network analysis, IL8, NQO1, GSTT1 and HMOX1 were identified as potential central regulators of the changes. In conclusion, excessive amounts of CoCr debris produced by MoM hip implants induces in a group of patients a unique adverse reaction characterized with enhanced expression of genes associated with inflammation, redox homeostasis, metal ion binding and transport, macrophage activation and apoptosis.