Role of TMEM100 in mechanically insensitive nociceptor un-silencing.

Mechanically silent nociceptors are sensory afferents that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli during inflammation. Using RNA-sequencing and quantitative RT-PCR we demonstrate that inflammation upregulates the expression of the transmembrane protein TMEM100 in silent nociceptors and electrophysiology revealed that over-expression of TMEM100 is required and sufficient to un-silence silent nociceptors in mice. Moreover, we show that mice lacking TMEM100 do not develop secondary mechanical hypersensitivity-i.e., pain hypersensitivity that spreads beyond the site of inflammation-during knee joint inflammation and that AAV-mediated overexpression of TMEM100 in articular afferents in the absence of inflammation is sufficient to induce mechanical hypersensitivity in remote skin regions without causing knee joint pain. Thus, our work identifies TMEM100 as a key regulator of silent nociceptor un-silencing and reveals a physiological role for this hitherto enigmatic afferent subclass in triggering spatially remote secondary mechanical hypersensitivity during inflammation.

Minimally invasive Oxford medial unicompartmental knee replacement in patients 50 years of age or younger.

INTRODUCTION: The aim of the present study was to assess clinical outcome and mid-term survivorship of mobile-bearing unicompartmental knee arthroplasty in patients 50 years of age or younger. METHODS: This study reports the results of 119 patients (130 knees) following mobile-bearing medial UKA. Primary indication was advanced osteoarthritis or avascular necrosis of the femoral condyle. The anterior cruciate ligament (ACL) as well as the collateral ligaments were functionally intact, the varus deformity was manually correctable and there was no evidence of osteoarthritis in the lateral compartment. Survivorship analysis was performed with different endpoints and clinical outcome was measured using the Oxford Knee Score (OKS), American Knee Society Score and Functional Score (AKSS-O, AKSS-F), range of motion (ROM), Tegner activity score, University of California Los Angeles score (UCLA) and visual analogue scale for pain (VAS). RESULTS: The survival rate was 96.6% at 6.5 years (95% CI 98.7-91.3%; number at risk: 56) and 91.7% (95% CI 96.7-80%; number at risk: 22) at 10 years for the endpoint device related revisions and 91.5% at 6.5 years (95% CI 95.4-84.5%; number at risk: 56) and 86.8% (95% CI 93-76.2%; number at risk: 22) at 10 years for the endpoint revision for any reason. Outcome scores, VAS and ROM showed significant improvements (p < 0.001). The mean OKS increased from 26.7 (standard deviation (sd): 7.2) preoperatively to 40.9 (sd: 7.6) at final follow-up, the mean AKSS-O from 48.3 (sd: 13.3) to 87.8 (sd: 14.4) and the mean ROM from 118° (sd: 16.7) to 125° (sd: 11.4). The radiological analysis revealed progression of degenerative changes in the lateral compartment in 39.6% of patients without affecting the functional outcome. CONCLUSIONS: Medial mobile-bearing UKA is a viable surgical treatment option in young patients with significant improvements in knee function and pain. Further follow-up is necessary to evaluate the long-term efficacy. LEVEL OF EVIDENCE: Retrospective cohort study, Level III.

CD8+ T Cells in OA Knee Joints Are Differentiated into Subsets Depending on OA Stage and Compartment.

Osteoarthritis (OA) is no longer considered a purely degenerative disease. OA is defined as a disease of the entire joint, in which inflammation occurs in various joint tissues. The overall aim of this study was to analyze the presence and polarization of CD8+ T cell subsets in OA knee joints, in relation to the OA stage and compartment (synovial fluid (SF), synovial membrane (SM,) peripheral blood (PB)). A quantitative flow analysis of CD8+ T cell subsets to compare the SF, SM, PB, was performed in patients with different stages of OA (early, unicondylar and bicondylar OA). Samples of the SF, SM and PB were harvested from a total of 55 patients at the time of surgery. Early OA was confirmed by independent surgeons intraoperatively. Uni- and bicondylar OA was confirmed and graded by two plane radiographs. Samples were analyzed by flow cytometry for surface markers, and cytokines by intracellular staining (ICS). CD8+ T cells were shown to be differentiated into pro-inflammatory IFN-γ producing Tc1 and IL-17A producing Tc17, as well as anti-inflammatory IL-4 producing Tc2. All CD8+ T cell subsets (Tc1, Tc17, and Tc2) were detected in both the SM and SF. The percentage of CD8+ T cell subsets of the total CD8+ T cell population was dependent on the OA stage and compartment. Compared with the peripheral blood (PB), the proportion of CD8+IFN-γ+ Tc1 and CD8+IL-17A+ Tc17 was significantly increased in OA SF. This was confirmed in our data for both early OA and end-stage OA. In the SM samples of end-stage OA patients, the proportion of CD8+IL-17A+ Tc17 was significantly increased compared to the PB. Comparing SF and SM samples of end-stage OA patients, the proportion of CD8+IFN-γ+ Tc1 was significantly increased in SF, whereas there were no differences concerning CD8+IL-4+ Tc2 and CD8+IL-17A+ Tc17. End-stage OA samples showed a significant increase of CD8+IL-4+ Tc2 in the SM for both unicondylar and bicondylar OA compared to early OA. CD8+ T cells infiltrating the SM and SF in OA knees are differentiated into IFN-γ-, IL-17A-, and IL-4-producing CD8+ T cell subsets (Tc1, Tc17, Tc2). This differentiation depends on the OA stage and OA compartment. Further investigation of CD8+ T cell subsets and their interaction with other inflammatory cells such as CD4+ T cells and macrophages may help to identify novel therapeutic anti-inflammatory strategies for containing OA progression.

Neuropathic pain caused by miswiring and abnormal end organ targeting.

Nerve injury leads to chronic pain and exaggerated sensitivity to gentle touch (allodynia) as well as a loss of sensation in the areas in which injured and non-injured nerves come together1-3. The mechanisms that disambiguate these mixed and paradoxical symptoms are unknown. Here we longitudinally and non-invasively imaged genetically labelled populations of fibres that sense noxious stimuli (nociceptors) and gentle touch (low-threshold afferents) peripherally in the skin for longer than 10 months after nerve injury, while simultaneously tracking pain-related behaviour in the same mice. Fully denervated areas of skin initially lost sensation, gradually recovered normal sensitivity and developed marked allodynia and aversion to gentle touch several months after injury. This reinnervation-induced neuropathic pain involved nociceptors that sprouted into denervated territories precisely reproducing the initial pattern of innervation, were guided by blood vessels and showed irregular terminal connectivity in the skin and lowered activation thresholds mimicking low-threshold afferents. By contrast, low-threshold afferents-which normally mediate touch sensation as well as allodynia in intact nerve territories after injury4-7-did not reinnervate, leading to an aberrant innervation of tactile end organs such as Meissner corpuscles with nociceptors alone. Genetic ablation of nociceptors fully abrogated reinnervation allodynia. Our results thus reveal the emergence of a form of chronic neuropathic pain that is driven by structural plasticity, abnormal terminal connectivity and malfunction of nociceptors during reinnervation, and provide a mechanistic framework for the paradoxical sensory manifestations that are observed clinically and can impose a heavy burden on patients.

Intrinsically disordered intracellular domains control key features of the mechanically-gated ion channel PIEZO2.

A central question in mechanobiology is how mechanical forces acting in or on cells are transmitted to mechanically-gated PIEZO channels that convert these forces into biochemical signals. Here we examined the role of the intracellular domains of PIEZO2, which account for 25% of the channel, and demonstrate that these domains fine-tune properties such as poking and stretch-sensitivity, velocity coding and single channel conductance. Moreover, we show that the intrinsically disordered linker between the transmembrane helices twelve and thirteen (IDR5) is required for the activation of PIEZO2 by cytoskeleton-transmitted forces. The deletion of IDR5 abolishes PIEZO2-mediated inhibition of neurite outgrowth, while it only partially affected its sensitivity to cell indentation and does not alter its stretch sensitivity. Thus, we propose that PIEZO2 is a polymodal mechanosensor that detects different types of mechanical stimuli via different force transmission pathways, which highlights the importance of utilizing multiple complementary assays when investigating PIEZO function.

Proinflammatory T cell polarization is already present in patients with early knee osteoarthritis.

BACKGROUND: Investigating the pathophysiological mechanisms of early osteoarthritis (OA) is of utmost interest since this stage holds the strongest promise for therapeutic interventions. The aims of this study were to analyze if synovial inflammation is already present in early OA and to characterize the involved cell populations, by investigating synovial fluid (SF) and synovial membrane (SM) of early OA patients for the presence and polarization status of CD4 T cells. METHODS: A quantitative analysis of CD4+ T cell infiltration in SF and SM compared to peripheral blood (PB) was performed in patients with early stages of OA. We further investigated intracellular staining (ICS), surface marker, and chemokine receptor expression profiles of CD4+ T cells in SF, SM, and PB, as well as cytokine expression in native SF and PB. Matched samples of SF, SM, and PB were harvested from 40 patients with early OA at the time of surgery. Early OA was confirmed by independent surgeons intraoperatively. Samples were analyzed by flow cytometry for surface markers and cytokines, which are preferentially expressed by distinct T cell subsets (Th1, Th2, Th17, regulatory T cells). Furthermore, we analyzed native SF and PB supernatants using MACSPlex for multiple cytokine expression profiles. RESULTS: SF and SM showed a distinct infiltration of CD4+ T lymphocytes, with significantly increased expression of chemokine receptors CXCR3/CCR5, cytokine IFN-γ (preferentially expressed by Th1 cells), and CD161 (preferentially expressed by IL-17 producing Th17 cells) compared to PB. Furthermore, the percentage of CD4+ T cells polarized to Treg was significantly increased in SM compared to SF and PB. No significant differences were observed for CCR3 and CCR4 (preferentially expressed by Th2 cells), although IL-4 values were significantly higher in SM and SF compared to PB. Cytokine analysis showed comparable results between PB and SF, with only IL-6 being significantly increased in SF. CONCLUSIONS: Early OA joints show already significant inflammation through CD4+ T cell infiltration, with predominant Th1 cell polarization. Inflammation seems to be driven by direct proinflammatory cell interaction. Cytokine signaling seems to be negligible at the site of inflammation in early OA, with only IL-6 being significantly increased in SF compared to PB.

Impact of Mononuclear Cell Infiltration on Chondrodestructive MMP/ADAMTS Production in Osteoarthritic Knee Joints-An Ex Vivo Study.

Progressive loss of joint function in osteoarthritis (OA) is driven by degenerative and inflammatory processes and their complex interaction. Decoding the link between degeneration and inflammation is one of the most exciting approaches in understanding OA pathophysiology and holds the promise to open new therapeutic avenues. The overarching goal of this project was to analyze the impact of mononuclear cells (MNC) on enzymatic chondrodestructive processes (MMP/ADAMTS) in OA. Synovial membrane (SM), articular cartilage (AC) and peripheral blood (PB) were obtained from a total of 21 patients with advanced knee OA who underwent arthroplastic surgery. In supernatants of native synovial cell cultures, T cell-depleted synovial cell cultures and macrophage-depleted synovial cell cultures, the concentrations of various metalloproteinases were examined by Enzyme Linked Immunosorbent Assay (ELISA). Furthermore, ELISA was used to analyze concentrations of metalloproteinases in supernatants of chondrocyte monocultures and chondrocyte co-cultures with CD4+CD127dim/- enriched peripheral blood mononuclear cells (PBMC), Treg depleted CD4+CD25-CD127dim/- enriched PBMC and CD4+CD25+CD127dim/- Treg. Compared to native synovial cell culture, T cell depletion led to significantly lower levels of MMP1, MMP3 and MMP-9 and macrophage depletion led to a significant decline of MMP1, MMP3, MMP9 and ADAMTS-5 concentration. Compared to T cell depletion, macrophage depletion resulted in a significantly stronger reduction of MMP1, MMP3, MMP9 and ADAMTS5. In chondrocyte co-culture with CD4+CD127dim/- enriched PBMC the concentration of MMP1 and ADAMTS5 was significantly increased compared to chondrocyte monoculture. No significant differences were found between chondrocyte monoculture and chondrocyte coculture with Treg as well as between co-culture with CD4+CD127dim/- enriched PBMC containing Treg and co-culture with Treg-depleted CD4+CD25-CD127dim/- enriched PBMC. In conclusion, our data suggests that both synovial macrophages and T cells have a catabolic potential by inducing the release of chondrodestructive metalloproteinases in OA synovium. This study also supports the hypothesis that MNC affect the release of metalloproteinases by chondrocytes and are hereby involved in the cartilageinduced chondrodestructive process. In this study no suppressive effect of Treg was shown.

Multidisciplinary Pain Management of Chronic Back Pain: Helpful Treatments from the Patients' Perspective.

Multidisciplinary pain management programs (MPMP) for patients suffering from chronic back pain include a variety of treatment modalities. The patients' perceived helpfulness of these treatment modalities remains unclear. The aims of this prospective observational cohort study were to assess (i) the patients' perceived helpfulness of different treatment modalities, (ii) the influence of sociodemographic characteristics on the patient's perspective and (iii) whether treatment outcomes are affected by helpfulness ratings. Treatment modalities of this three-week MPMP consisted of individual physiotherapy, group-based physiotherapy, relaxation therapy, aquatic therapy, back education, medical training therapy, biofeedback, psychological pain therapy and music therapy. The study comprised 395 patients. The main outcome was the patients' perceived treatment helpfulness at the end of the program measured by a self-reported questionnaire ranging from 1 (not at all helpful) to 6 (extremely helpful). Secondary outcomes were treatment effects on pain, pain related disability, functional ability and level of depressive symptoms measured by self-reported questionnaires (NRS, PDI, FFbH-R, ADS-L). A total of 276 patients (22-64 years, 57% female) were available for overall analysis. Multivariate-analysis-of-variance- (MANOVA-) related results revealed that perceived treatment helpfulness (range 1-6) differed significantly between treatment modalities: individual physiotherapy (M = 5.00), group-based physiotherapy (M = 4.87), relaxation therapy (M = 4.6), aquatic therapy (M = 4.54), back education (M = 4.43), medical training therapy (M = 3.38), biofeedback (M = 3.31), psychological pain therapy (M = 3.15), music therapy (M = 3.02). Pain, pain related disability and levels of depressive symptoms significantly improved after the program (p < 0.001) whereas functional ability decreased (p < 0.01). Significant correlations were found between helpfulness ratings and sociodemographic data indicating that perceived treatment helpfulness was influenced by patient-related factors. Importantly, the degree of pain-related improvements was affected by the patients' perceived treatment helpfulness. In conclusion, patients' perceived treatment helpfulness differs significantly between treatment modalities and corresponds to treatment outcome.

Long-Term Results of a Second-Generation, Small-Diameter, Metal-On-Metal Bearing in Primary Total Hip Arthroplasty at 14-year Follow-Up.

(1) Background: The objective of the present study was to review the clinical and radiological results of a small-head, MoM bearing in primary THA and to determine blood metal ion levels at long-term follow-up. (2) Methods: We retrospectively evaluated the clinical and radiological results of 284 small-diameter, MoM 28-mm Metasul THA at a mean follow-up of 14.5 years, and measured blood metal ion concentrations in 174 of these patients. (3) Results: After 14 years, survival free for revision due to any reason was 94%. Proximal femoral osteolysis was seen in 23% of hips, and MRI demonstrated ARMD in 27 of the 66 investigated hips (41%). Mean cobalt, chromium, and titanium ion concentrations were 0.82 µg/L (range 0.22-4.45), 1.51 µg/L (0.04-22.69), and 2.68 µg/L (0.26-19.56) in patients with unilateral THA, and 2.59 µg/L (0.43-24.75), 2.50 µg/L (0.26-16.75), and 3.76 µg/L (0.67-19.77), respectively in patients with bilateral THA. Twenty-nine percent of patients showed cobalt or chromium ion levels > 2 µg/L. (4) Conclusions: Despite good clinical long-term results, increased blood metal ion levels (cobalt or chromium > 2 µg/L) were found in approximately one-third of asymptomatic patients, and proximal femoral osteolysis and ARMD were frequently seen in this cohort. Blood metal ion analysis appears helpful in the long-term follow-up of these patients in order to identify individuals at risk. In accordance with contemporary consensus statements, symptomatic patients with elevated metal ion levels and/or progressive osteolysis should be considered for additional CT or MARS MRI to determine the extent of soft tissue affection prior to revision surgery. Further studies are necessary to investigate the clinical relevance of ARMD in asymptomatic patients with small-head, MoM THA.

Divergent Mononuclear Cell Participation and Cytokine Release Profiles Define Hip and Knee Osteoarthritis.

Osteoarthritis (OA) is a progressive joint disease driven by a blend of inflammatory and biomechanical processes. Studies using human samples to understand inflammatory mechanisms in OA frequently recruit OA patients with different affected joints, even though recent evidence indicates that OA is a heterogeneous disease which only culminates in a common end point. Differences in age of onset and the dynamics of disease progression suggest that different joints may represent different disease entities, thereby diluting the discovery potential in a combined analysis. We hypothesized that different OA joints may also differ in immunopathology within the synovium. To investigate this hypothesis, we profiled the immune cell contribution (flow cytometry) and cytokine release profiles (ELISA) in purified synovial membrane mononuclear cells from 50 patients undergoing either hip (n = 34) or knee (n = 16) replacement surgery. Unsupervised computational approaches were used for disease deconstruction. We found that hip and knee osteoarthritis are not identical in respect to the inflammatory processes that take place in the synovial membrane. Instead, we report that principally CD14+ macrophages are expanded fourfold in the synovial membrane of patients with knee OA compared to hip OA, with a trend to higher expression in CD8+ T cells, while CD4+ T cells, B cells, and NK cells were found at comparable quantities. Upon isolation and culture of cells from synovial membrane, isolates from hip OA released higher concentrations of Eotaxin (CCL11), G-CSF, GM-CSF, INF-γ, IP-10 (CXCL10), TNF-α, MIP-1α (CCL3), MIP-1ß (CCL4), IL-4, IL-10, IL-17, and lower concentrations of stem cell factor (SCF), thereby highlighting the difference in the nature of hip and knee osteoarthritis. Taken together, this study establishes hip and knee OA as immunologically distinct types of OA, and creates a resource of the cytokine expression landscape and mononuclear cell infiltration pattern of patients with hip and knee osteoarthritis.

Synovial Cytokines Significantly Correlate with Osteoarthritis-Related Knee Pain and Disability: Inflammatory Mediators of Potential Clinical Relevance.

The aim of this study was to identify inflammatory mediators of potential clinical relevance in synovial fluid (SF) samples of patients with knee osteoarthritis (OA). Therefore, radiographic OA severity, knee pain and function of 34 OA patients undergoing unicompartmental (UC) and bicompartmental (BC) knee arthroplasty were assessed prior to surgery and SF samples were analyzed for a broad variety of inflammatory mediators, including interleukins (ILs), interferons (IFNs), C-X-C motif ligand chemokines (CXCLs), and growth factors (nerve growth factor; NGF, vascular endothelial growth factor; VEGF, and stem cell growth factor ß; SCGF-ß) using multiplex assay. Significant differences were observed between the SF levels of different inflammatory markers. When compared to UC OA, significantly higher concentrations of IL-7, IL-8, IL-10, IL-12, IL-13, IFN-γ, VEGF and CXCL1 were detected in BC OA. Correlation analyses revealed significant associations between OA severity and IL-6, IL-8, IFN-γ, SCGF-ß, VEGF, CXCL1. Interestingly, increases in both anti- (IL-10, IL-13) and pro-inflammatory (IL-7, IL-12, IFN-γ) cytokines, as well as growth factors (SCGF-ß, VEGF), correlated significantly with the level of knee pain. Poorer knee function was associated with higher IL-6, IL-10, IL-12, IL-13, IL-18, ßNGF, SCGF-ß, VEGF and CXCL9 levels. In conclusion, this study provides an extensive profile of synovial inflammatory mediators in knee OA and identifies cytokines of potential clinical relevance. In fact, five of the mediators examined (IL-10, IL-12, IL-13, SCGF-ß, VEGF) significantly correlate with both knee pain and function.

Sensorimotor Activity Partially Ameliorates Pain and Reduces Nociceptive Fiber Density in the Chronically Injured Spinal Cord.

A large proportion of patients suffering from spinal cord injury (SCI) develop chronic central neuropathic pain. Previously, we and others have shown that sensorimotor training early after SCI can prevent the development of mechanical allodynia. To determine whether training initiated in the subchronic/chronic phase remains effective, correlates of below-level neuropathic pain were analyzed in the hindpaws 5-10 weeks after a moderate T11 contusion SCI (50 kDyn) in adult female C57BL/6 mice. In a comparison of SCI and sham mice 5 weeks post-injury, about 80% of injured animals developed mechanical hypersensitivity to light mechanical stimuli, whereas testing of noxious stimuli revealed hypo-responsiveness. Thermal sensitivity testing showed a decreased response latency after injury. Without intervention, mechanical and thermal hyper-responsiveness were evident until the end of the experiment (10 weeks). In contrast, treadmill training (2 × 15 min/day; 5 × /week) initiated 6 weeks post-injury resulted in partial amelioration of pain behavior and this effect remained stable. Analysis of calcitonin gene-related peptide (CGRP)-labeled fibers in lamina III-IV of the lumbar dorsal horn revealed an increase in labeling density after SCI. This was not due to changes in the number or size distribution of CGRP-labeled lumbar dorsal root ganglion neurons. Treadmill training reduced the CGRP-labeling density in the spinal cord of injured mice, whereas the density of non-peptidergic isolectin-B4 (IB4)+ fibers showed no changes in lamina IIi and a slight reduction of sparse IB4 labeling in laminae III-IV. Thus, sensorimotor activity initiated in the subchronic/chronic phase of SCI remains effective in ameliorating pain behavior and influencing structural changes of the nociceptive system.

Early-onset treadmill training reduces mechanical allodynia and modulates calcitonin gene-related peptide fiber density in lamina III/IV in a mouse model of spinal cord contusion injury.

Below-level central neuropathic pain (CNP) affects a large proportion of spinal cord injured individuals. To better define the dynamic changes of the spinal cord neural network contributing to the development of CNP after spinal cord injury (SCI), we characterized the morphological and behavioral correlates of CNP in female C57BL/6 mice after a moderate T11 contusion SCI (50 kdyn) and the influence of moderate physical activity. Compared with sham-operated animals, injured mice developed mechanical allodynia 2 weeks post injury when tested with small-diameter von Frey hair filaments (0.16 g and 0.4 g filament), but presented hyporesponsiveness to noxious mechanical stimuli (1.4 g filament). The mechano-sensory alterations lasted up to 35 days post injury, the longest time point examined. The response latency to heat stimuli already decreased significantly 10 days post injury reaching a plateau 2 weeks later. In contrast, injured mice developed remarkable hyposensitivity to cold stimuli. Animals that underwent moderate treadmill training (2 × 15 minutes; 5 d/wk) showed a significant reduction in the response rate to light mechanical stimuli as early as 6 days after training. Calcitonin gene-related peptide (CGRP) labeling in lamina III-IV of the dorsal horn revealed significant increases in CGRP-labeling density in injured animals compared with sham control animals. Importantly, treadmill training reduced CGRP-labeling density by about 50% (P < 0.01), partially reducing the injury-induced increases. Analysis of IB4-labeled nonpeptidergic sensory fibers revealed no differences between experimental groups. Abnormalities in temperature sensation were not influenced by physical activity. Thus, treadmill training partially resolves signs of below-level CNP after SCI and modulates the density of CGRP-labeled fibers.