The Dutch version of the Forgotten Joint Score: test-retesting reliability and validation

The aim of this study was to translate the Forgotten Joint Score (FJS) into the Dutch language. This questionnaire was tested for internal consistency (Cronbach’s alpha) and test-retest reliability (intraclass correlation coefficients (ICC)). 159 patients were included in this study; 74 with a total hip arthroplasty (THA) and 85 with a total knee arthroplasty (TKA ). The FJS showed a high internal consistency (Cronbach’s alpha=0.957; ICC=0.943). The FJS showed a significant correlation (r=0.751) with the WOMAC and low ceiling effects (3.1%). This study proved the Dutch FJS to be highly discriminative in patients treated with a THA or TKA. This makes the FJS a reliable patient related outcome measurement, measuring a new dimension in arthroplasty: the ability to forget an artificial joint in everyday life

Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor

Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the cytosol. A plethora of human pathologies are associated with the altered expression or dysfunction of cell plasma membrane transport proteins, making them interesting therapeutic drug targets. However, the search for therapeutics is challenging, since many drug candidates targeting cell plasma membrane proteins fail in (pre)clinical testing due to inadequate selectivity, specificity, potency or stability. These latter characteristics are met by nanobodies, which potentially renders them eligible therapeutics targeting cell plasma membrane proteins. Therefore, a therapeutic nanobody-based strategy seems a valid approach to target and modulate the activity of cell plasma membrane transport proteins. This review paper focuses on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discusses several therapeutic nanobodies directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters

Mechanisms Underlying Connexin Hemichannel Activation in Disease

Gap junctions and connexin hemichannels mediate intercellular and extracellular communication, respectively. While gap junctions are seen as the “good guys” by controlling homeostasis, connexin hemichannels are considered as the “bad guys”, as their activation is associated with the onset and dissemination of disease. Open connexin hemichannels indeed mediate the transport of messengers between the cytosol and extracellular environment and, by doing so, fuel inflammation and cell death in a plethora of diseases. The present mini-review discusses the mechanisms involved in the activation of connexin hemichannels during pathology

Nanobody-based pannexin1 channel inhibitors reduce inflammation in acute liver injury

Background: The opening of pannexin1 channels is considered as a key event in inflammation. Pannexin1 channel-mediated release of adenosine triphosphate triggers inflammasome signaling and activation of immune cells. By doing so, pannexin1 channels play an important role in several inflammatory diseases. Although pannexin1 channel inhibition could represent a novel clinical strategy for treatment of inflammatory disorders, therapeutic pannexin1 channel targeting is impeded by the lack of specific, potent and/or in vivo-applicable inhibitors. The goal of this study is to generate nanobody-based inhibitors of pannexin1 channels. Results: Pannexin1-targeting nanobodies were developed as potential new pannexin1 channel inhibitors. We identified 3 cross-reactive nanobodies that showed affinity for both murine and human pannexin1 proteins. Flow cytometry experiments revealed binding capacities in the nanomolar range. Moreover, the pannexin1-targeting nanobodies were found to block pannexin1 channel-mediated release of adenosine triphosphate. The pannexin1-targeting nanobodies were also demonstrated to display anti-inflammatory effects in vitro through reduction of interleukin 1 beta amounts. This anti-inflammatory outcome was reproduced in vivo using a human-relevant mouse model of acute liver disease relying on acetaminophen overdosing. More specifically, the pannexin1-targeting nanobodies lowered serum levels of inflammatory cytokines and diminished liver damage. These effects were linked with alteration of the expression of several NLRP3 inflammasome components. Conclusions: This study introduced for the first time specific, potent and in vivo-applicable nanobody-based inhibitors of pannexin1 channels. As demonstrated for the case of liver disease, the pannexin1-targeting nanobodies hold great promise as anti-inflammatory agents, yet this should be further tested for extrahepatic inflammatory disorders. Moreover, the pannexin1-targeting nanobodies represent novel tools for fundamental research regarding the role of pannexin1 channels in pathological and physiological processes.</p

Non-canonical roles of connexins

Gap junctions mediate cellular communication and homeostasis by controlling the intercellular exchange of small and hydrophilic molecules and ions. Gap junction channels are formed by the docking of 2 hemichannels of adjacent cells, which in turn are composed of 6 connexin subunits. Connexin proteins as such can also control the cellular life cycle independent of their channel activities. This has been most demonstrated in the context of cell growth and cell death. Different mechanisms are involved mainly related to direct interaction with cell growth or cell death regulators, but also implying effects on the expression of cell growth and cell death regulators. The present paper focuses on these atypical roles of connexin proteins

Additional file 1 of Nanobody-based pannexin1 channel inhibitors reduce inflammation in acute liver injury

Additional file 1. Figure S1: Immunoblot analysis of Panx1 expression following transduction of DUBCA cells. a DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1). Protein levels of Panx1 were assessed by immunoblot analysis. Representative data of 3 independent experiments. b Panx1 protein levels were normalised against the total protein content and expressed as relative alteration compared to untransduced DUBCA cells. (n=3 independent experiments). All data was analysed by unpaired t-tests with Welch’s correction. Data were expressed as means ± S.D. Figure S2: Immunocytochemistry analysis of Panx1 expression following transduction of DUBCA cells. DUBCA cells were transduced with lentiviral vectors to express mouse Panx1 (mPanx1) or human Panx1 (hPanx1). DUBCA wild-type (WT), DUBCA mPanx1 and DUBCA hPanx1 cells were subjected to (1) immunocytochemistry analysis of Panx1 (red) with nuclear counterstaining (blue). Scale bar represents 500 µm (green). For (2) negative controls, the primary antibody directed against Panx1 was omitted. Representative data of 3 independent experiments. Figure S3: In vivo biodistribution of Panx1-targeting nanobodies. Nb1, Nb3, Nb9 and non-targeting Nb were radiolabeled with Technetium-99m (99mTc), and 5 µg of radiolabeled nanobody was injected intravenously in healthy adult mice. Biodistribution of nanobodies was determined by γ-counting of isolated organs and expressed as percentage of injected activity per gram of organ (n=3 animals per group). Data were expressed as means ± S.D. Figure S4: Analysis of necrosis in acetaminophen-overdosed mice. Adult mice were overdosed with acetaminophen (APAP) (300 mg/kg) or kept untreated (UTC). After 2 hours, some mice were additionally administered either nanobody (Nb1, Nb3, Nb9 or non-targeting Nb) (10 mg/kg) or N-acetylcysteine (NAC) (200 mg/kg). Sampling was performed 24 hours after APAP overdosing. The percentage of necrosis was determined by measuring areas of necrosis on 10 µm liver sections stained with hematoxylin and periodic acid Schiff base (H-PAS) (n=4 (UTC and APAP) or n=12 (Nb1, Nb3, Nb9, non-targeting Nb and NAC) animals per group). All data was analysed by parametric 1-way analysis of variance followed by post hoc tests with Bonferroni’s correction. Data were expressed as means ± S.D