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Rheumatoid arthritis is characterised by a progressive, intermittent inflammation at the synovial membrane, which ultimately leads to the destruction of the synovial joint. The synovial membrane as the joint capsule's inner layer is lined with fibroblast-like synoviocytes that are the key player supporting persistent arthritis leading to bone erosion and cartilage destruction. While microfluidic models that model molecular aspects of bone erosion between bone-derived cells and synoviocytes have been established, RA's synovial-chondral axis has not yet been realised using a microfluidic 3D model based on human patient in vitro cultures. Consequently, we established a chip-based three-dimensional tissue coculture model that simulates the reciprocal cross talk between individual synovial and chondral organoids. When co-cultivated with synovial organoids, we could demonstrate that chondral organoids induce a higher degree of cartilage physiology and architecture and show differential cytokine response compared to their respective monocultures highlighting the importance of reciprocal tissue-level cross talk in the modelling of arthritic diseases.

The theory of mental models allows modal truth values (‘possibly true and possibly false’) and counterfactual truth values (‘true but it could have been false’), which are outside standard logics. Naive individuals have the intuition that the disjunction, ‘You arrived at Preston or at Crewe,’ is true if you arrived at Preston, but their deliberations consider the status of the alternative destination. If this counterfactual possibility is false, e.g., the road to Crewe is blocked, they are likely to select a modal truth value (in Experiment 1), or a counterfactual truth value (in Experiment 2). The results are pertinent to claims that cognition cannot be algorithmic (as Gödel argued on the grounds that consistent formal systems cannot prove truths that mathematicians grasp about the arithmetic of natural numbers). Human thinking, however, differs from formal logics: it relies on semantic algorithms that can evaluate truth and falsity.

Hypothetical thinking is an extraordinary human ability that allows people to think beyond the facts of a situation to imagine alternative possibilities. I first consider how people understand factual conditionals, such as, ‘if there was an apple in the fruit bowl there was a banana’, then how they understand counterfactual conditionals, such as, ‘if Pearl had studied harder she would have passed the exam’, and then how they understand counterpossible conditionals, that is, conditionals about impossibilities, such as, ‘ if people were made of steel they would not bruise easily’. I discuss the theory that people understand conditionals by envisaging models of possibilities, and consider experimental evidence that corroborates its predictions for factual, counterfactual, and impossible conditionals.

Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Forkhead box O 3 (FOXO3) perturbations in immune cells are increasingly linked to RA pathogenesis. Here, we show that FOXO3 is distinctly inactivated/phosphorylated in the FLS of rheumatoid synovitis. In vitro, stimulation of FLS with tumor necrosis factor-alpha α (TNFα) induced a rapid and sustained inactivation of FOXO3. mRNA profiling revealed that the inactivation of FOXO3 is important for the sustained pro-inflammatory interferon response to TNFα (CXCL9, CXCL10, CXCL11, and TNFSF18). Mechanistically, our studies demonstrate that the inactivation of FOXO3 results from TNF-induced downregulation of phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1). Thus, we identified FOXO3 and its modulator PIK3IP1 as a critical regulatory circuit for the inflammatory response of the resident mesenchymal cells to TNFα and contribute insight into how the synovial tissue brings about chronic inflammation that is driven by TNFα. Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Here, the authors identified a previously unknown signaling circuit that contributes to tumor necrosis factor (TNF)-induced activation of FLS. The authors show that FOXO3 and its modulator PIK3IP1 are crucial for the TNF-driven interferon (IFN) response in RA-FLS.

Rheumatoid arthritis is a chronic, systemic joint disease in which an autoimmune response translates into an inflammatory attack resulting in joint damage, disability and decreased quality of life. Despite recent introduction of therapeutic agents such as anti-TNFα, even the best current therapies fail to achieve disease remission in most arthritis patients. Therefore, research into the mechanisms governing the destructive inflammatory process in rheumatoid arthritis is of great importance and may reveal novel strategies for the therapeutic interventions. To gain deeper insight into its pathogensis, we have developed for the first time a three-dimensional synovium-on-a-chip system in order to monitor the onset and progression of inflammatory synovial tissue responses. In our study, patient-derived primary synovial organoids are cultivated on a single chip platform containing embedded organic-photodetector arrays for over a week in the absence and presence of tumor-necrosis-factor. Using a label-free and non-invasive optical light-scatter biosensing strategy inflammation-induced 3D tissue-level architectural changes were already detected after two days. We demonstrate that the integration of complex human synovial organ cultures in a lab-on-a-chip provides reproducible and reliable information on how systemic stress factors affect synovial tissue architectures.

Surgical nerve transfers lead to superior regeneration and specific muscular changes for potentially improved prosthesis control., Selective nerve transfers surgically rewire motor neurons and are used in extremity reconstruction to restore muscle function or to facilitate intuitive prosthetic control. We investigated the neurophysiological effects of rewiring motor axons originating from spinal motor neuron pools into target muscles with lower innervation ratio in a rat model. Following reinnervation, the target muscle’s force regenerated almost completely, with the motor unit population increasing to 116% in functional and 172% in histological assessments with subsequently smaller muscle units. Muscle fiber type populations transformed into the donor nerve’s original muscles. We thus demonstrate that axons of alternative spinal origin can hyper-reinnervate target muscles without loss of muscle force regeneration, but with a donor-specific shift in muscle fiber type. These results explain the excellent clinical outcomes following nerve transfers in neuromuscular reconstruction. They indicate that reinnervated muscles can provide an accurate bioscreen to display neural information of lost body parts for high-fidelity prosthetic control.

Osteons are the end product of cortical bone remodeling. Changes in their mechanical properties may be an indicator of overall bone health. Aszenci and Bonucci first evaluated the compressive properties of individual osteons in 1968. However, these results have never been independently validated and technological advances allow for further investigation of osteon mechanics. In the present study, an experimental protocol was established such that osteons ~250 µm in diameter were successfully extracted from human cortical bone (males, ages 93 and 64) and loaded in compression. Both micro-computed tomography and second harmonic generation (SHG) microscopy imaging methods were incorporated into the protocol to improve the structural and compositional assessment of each osteon. Further, a high-resolution videography system was used to monitor the osteons during mechanical testing, permitting insight into deformation mechanisms in situ. The results show that the older donor osteons (male, age 93 (n = 13)) were stiffer (p = 0.04) and more highly mineralized (p = 0.03), while the younger donor osteons (male, 64 (n = 14)) exhibited more heterogeneity in the measured mechanical properties with the presence of both stiff and more compliant osteons. SHG intensity indicated predominantly longitudinal fiber alignment for all osteons while the videography data revealed three distinct failure modes and confirmed that whitening observed during yielding of macroscale bone specimens represents failure at the local level. The stiffness and strength of the younger donor osteons were heavily dependent on the gross structure of the osteon, while these properties were dominated by collagen orientation in the older donor osteons.

Introduction The synovium demonstrates a distinct cellular structure with a densely packed synovial lining layer that sits on top of a loosely organised sublining layer. In rheumatoid arthritis (RA), this tissue hosts the inflammatory reaction and undergoes drastic structural changes. We hypothesise that this inflammatory remodelling results in tissue dysfunction, thereby perpetuating the inflammatory process. To explore the relationship between tissue structure and function, we use a 3D human synovial organoid culture system to model healthy and diseased synovium. Methods FLS from RA patients were resuspended in Matrigel and cultured as 3D organoids for an extended period of time. This allows FLS to re-establish a synovial tissue-like structure. To mimic inflammation, 3D synovial organoids were challenged with TNF. A re-stimulation experiment was designed in which synovial organoids were exposed to TNF for 10 days, followed by a 3 day wash out phase. Thereafter, the organoids were re-stimulated with TNF. To prevent TNF-driven remodelling, Marimastat, a broad MMP inhibitor, was added during the first TNF stimulation. For selected experiments, synovial tissue-like structure was assessed by HE staining in paraffin embedded sections of synovial organoids. qPCR and RNA Seq were used in gene expression profiling. IL-6, IL-8 and MMP3 protein production was determined by ELISA. Results TNF-stimulated synovial organoids demonstrated lining layer hyperplasia and cellular condensation in the sublining layer. After TNF-stimulation, gene expression of MMP1, MMP3 and IL-6 and protein production of IL-6, IL-8 and MMP3 was upregulated. Upon re-stimulation of synovial organoids with TNF, protein levels of IL-6, IL-8 and MMP3 were significantly increased when compared to previous stimulations. In order to explore whether this effect is due to TNF-induced structural changes, tissue remodelling was blocked using Marimastat. IL-6 protein level was reduced in 21%. To further explore the increased TNF response upon re-stimulation, epigenetic mechanisms are currently being tested. Conclusions TNF stimulation has a direct effetc in synovial organoids function and structure. TNF-driven tissue remodelling is associated with an increased in IL-6, IL-8 and MMP3 expression response upon re-stimulation. Inhibition of TNF-induced remodelling partially prevents this effect. Additional epigenetic mechanisms may account for tissue memory. Disclosure of interest None declared

Introduction The synovium is primarily built by fibroblast-like-synoviocytes (FLS). In the healthy synovium FLS form a complex tissue network via long-distance intercellular connexions (nanotubes). Using a 3D synovial micromass culture system, our previous research demonstrated that FLS exchange cytoplasmic cargo, i.e. organelles like mitochondria, via transfer through direct cell-to-cell connexions. The adaptive synovial tissue response to inflammation (i.e. to TNFa) likely depends upon the concerted communication between FLS. Objectives To determine how the cellular organisation of the synovial tissue affects intercellular cargo exchange and how it changes under inflammatory conditions. Methods Human FLS were isolated from joint tissues after synovectomies; passaged FLS were used to generate 3D micromass cultures using Matrigel (BD). For ultrastructural 3D reconstruction ultrathin sections of fixed micromasses were cut with an ATUMtom for FE-SEM scanning, followed by alignment and reconstruction of individual cells with Fiji TrackEM. To compare unstimulated and TNF-stimulated micromass cultures, cells were dyed with Cell- and Mitotracker dyes (TF). Using confocal microscopy, micromass cultures were analysed for cellular organisation within the 3D sphere, cell volume of individual cells, cell-to-cell interconnectivity and cargo transfer between cells. Analyses of the 3D confocal imaging data were done in Bitplane Imaris. Results SEM 3D reconstruction of synovial micromass tissue revealed that FLS are compartmentalised, and thus, much larger and extended than expected. Thin membrane tubes not only connect different cells but also compartments of the same cell. Additionally, one nanotube may provide a link to several other cells and these connexions are in general separated by membranes. Treatment of micromasses with TNFa resulted in cellular condensation, reduced cell volume as well as diminished cellular connectivitiy when compared to unstimulated micromasses. In particular, the abundance of interconnecting nanotubes was significantly attenuated. The mitochondrial transfer rate increased as FLS clustered upon TNFa stimulation. Conclusions TNFa directs FLS cellular re-organisation that is associated with increased intercellular transfer of mitochondria. These studies may provide insight into the concerted cooperation of FLS that is likely critical for the adaptive synovial response to inflammation. Disclosure of interest None declared

Background Emerging evidence suggests that fibroblast-like synoviocytes (FLS) are key effector cells in rheumatoid arthritis (RA) and research into the mechanisms defining FLS activity in RA indicate that cytokines secreted by leukocytes play a crucial role. Nevertheless, the contribution of IFNγ, which is increased in rheumatoid synovitis, to the inflammatory synovial tissue reaction is not known. Objectives To explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodeling in RA, and to determine whether IFNγ-signaling controls the invasive potential of FLS. Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analyzed in migration-, spreading- and invasion assays. Signaling events relevant to cellular motility were defined by western blots. Baricitinb and siRNA pools were used to suppress Janus Kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration, pronounced actin and focal adhesion (FA) re-organization upon IFNγ stimulation. Since actin and FA dynamics and, thus, cell motility are integrated by the focal adhesion kinase (FAK), we next analyzed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated FAK activation by IFNγ. Correspondingly, IFNγ-inudced FAK activation and invasion of FLS was abrogated by the JAK-inhibitor baricitinib. Conclusions Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA. Disclosure of Interest None declared

Background The synovial membrane is a distinctly organised tissue structure. During the course of rheumatoid arthritis (RA), the synovium becomes hyperplastic and demonstrates thickening of the lining layer and cellular condensation at the sublining layer. Using a three-dimensional synovial organ culture system, we explore cellular mechanisms of synovial tissue formation and inflammatory remodelling. Materials and methods Fibroblast-like synoviocytes (FLS) derived from patients with RA were cultured in 3D micromasses. To mimic synovial inflammation, micromasses were challenged with TNF. For histological analyses, micromasses were embedded in paraffin, sections were stained with haematoxylin and eosin; Ki67 labelling was performed to identify proliferating cells. 3D confocal micrographs were analysed using Imaris Bitplane software. mRNA levels for various genes expressed in FLS were determined by qPCR. Results Synovial micromasses demonstrated thickening of the lining layer over time. When stimulated with TNF, hyperplasia of the lining layer and cellular aggregation at the sublining layer was observed. In order to identify the origin of cells contributing to the thickening of the lining layer, proliferation studies were conducted. Intriguingly, in the early phase of the culture period, the percentage of proliferating cells in the lining layer was higher when compared to the sublining layer. This proliferative activity, however, was no longer present in the late phase, after the lining layer was established. In the presence of TNF, an increased number of proliferating cells at the lining layer was maintained for an extended period of time, consistent with higher rates of cellular proliferation at the synovial lining in sections of RA synovial tissues when compared to OA. qPCR data indicate that MMP1, MMP3, and IL-6 are differentially expressed during the early phase and the mature phase of the culture period. By contrast, lubricin, cadherin-11, CCL20, and STAT1 were not differentially expressed. Conclusions The three-dimensional FLS micromass culture reveals spontaneous cellular organisation that strikingly resembles the lining/sublining architecture of the synovium. This process involves FLS proliferation as well as expression of genes that allow for tissue remodelling. In inflammatory conditions similar cellular programs are re-activated resulting in synovial lining hyperplasia and a pannus-like condensed mass of cells.

Social learning is considered one of the hallmarks of cognition. Observers learn from demonstrators that a particular behavior pattern leads to a specific consequence or outcome, which may be either positive or negative. In the last few years, social learning has been studied in a variety of taxa including birds and bony fish. To date, there are few studies demonstrating learning processes in cartilaginous fish. Our study shows that the cartilaginous fish freshwater stingrays (Potamotrygon falkneri) are capable of social learning and isolates the processes involved. Using a task that required animals to learn to remove a food reward from a tube, we found that observers needed significantly (P

Objectives The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

A taxonomy of the movement possibilities for any species, within the constraints of its neural and skeletal systems, should be one of the foundations of the study of its behaviour. Caribbean reef squid, Sepioteuthis sepioidea, appear to have many degrees of freedom in their movement as they live in a three-dimensional habitat and have no fixed skeleton but rather a muscular hydrostatic one. Within this apparent lack of constraints, there are regularities and patterns of common occurrences that allow this article to describe an ethogram of the movements, postures and positions of squid. Squid have a combination of bent, spread and twisted maintained postures of the eight arms and two tentacles that enhance camouflage. Their body–arm posture combinations are actively maintained in the water but also influenced by gravity. Positions related to conspecifics are stereotyped and important for communication. For locomotion, squid use a well-coordinated dual fin–jet locomotion system. This motor system uses tonic...

PURPOSE To explore the utility of multimodal microscopy as a noninvasive tool to assess corneal collagen cross-linking (CXL) efficacy, we investigated the correlation between riboflavin (RF) axial profile, second harmonic generation (SHG) imaging, and histological/biochemical changes of human corneas after RF-ultraviolet A (UVA)-catalyzed CXL. METHODS De-epithelialized human corneoscleral tissues were imaged by confocal and multiphoton microscopy to study RF tissue diffusion profile and SHG-based roughness index (Rq) after CXL. We installed 0.1% RF for 5, 10, and 20 minutes, respectively, followed by UVA irradiation, while dextran drug vehicle-treated corneas served as controls. Masson's trichrome staining and collagenase digestion assay were employed to assess ultrastructural modifications of collagen lamellae and bioenzymatic strength following RF-UVA CXL. RESULTS Stromal absorption of RF was significantly higher in 20 minutes compared with 5- and 10-minute drug instillations. The roughness index of SHG images was reduced after RF-UVA CXL at all RF instillation time points compared with dextran controls. Interestingly, correlation between axial profiles of RF dosage and Rq index was only observed in 10- and 20-minute RF instillations (R(2) = 0.13 and 0.28, respectively, all P < 0.05), but not in the 5-minute group. Masson's trichrome staining revealed collagen fibril compaction in cross-linked corneas in an RF dose-dependent manner. Collagenase digestion assay showed significantly increased biochemical strength by higher RF doses in cross-linked corneas. CONCLUSIONS Intrastromal RF distribution profiles correlated with histological and functional property changes in RF-UVA cross-linked corneas. A riboflavin-defined threshold further determined the sensitivity of SHG imaging as a noninvasive imaging modality to assess the efficacy of RF-UVA CXL.

Behavioural lateralization has been demonstrated in many species of vertebrates, but there has been scarce evidence for it in invertebrates. We have previously documented lateral asymmetry of eye use in individual octopuses. In the present study we investigated lateralization at the population level. Octopus eyes are on the sides of the head, and these animals prefer monocular to binocular vision. We determined preferential eye use by recording the time that the octopuses watched a stimulus outside the tank while holding on to the front glass of the tank. Thirteen of 25 subjects were highly significantly left-eyed, 10 highly significantly right-eyed, and two showed no preference. Individual octopuses had lateralized eye use but, unlike handedness in humans, eye preference in octopuses at the population level had no systematic bias towards left or right. This behavioural asymmetry follows an antisymmetrical distribution and could therefore be genetically or epigenetically determined. This study extends assessment of lateralization to the population level to include invertebrates in the discussion of the evolution of lateralization.

Senescence is a normal stage of an octopus's life cycle that often occurs before death. Some of the following symptoms typify it: lack of feeding, retraction of skin around the eyes, uncoordinated movement, increased undirected activity, and white unhealing lesions on the body. There is inter- and intraspecific variability. Senescence is not a disease or a result of disease, although diseases can also be a symptom of it. Both males and females go through a senescent stage before dying-the males after mating, the females while brooding eggs and after the eggs hatch. There are many aspects of octopus senescence that have not yet been studied. This study discusses the ecological implications of senescence.

The lateralization of sensory and motor functions has been recently demonstrated in various groups of vertebrates. We examined lateral asymmetry of eye use in Octopus vulgaris by behavioural methods. Octopus vulgaris uses monocular vision almost exclusively and can move its eyes independently. The amount of binocular vision is small because the eyes are on the sides of the head. We tested eight octopuses in two conditions (one with and one without moving stimuli) where the use of the eye for frontal vision could be determined unequivocally. Data were recorded on videotape. All animals showed a preference for one eye (five left, three right). There was no correlation between eye use and the animal's direction of movement. Pigmentation of the ventral side of the arms tended to be most intense on the side of the preferred eye and the body was most pigmented on the side of the eye currently in use. We found no sex differences for visual lateralization. Pigmentation of the ventral side of the arms was lighter in females than in males. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.

The recently identified epidermal growth factor-related peptide cripto-1 has been previously implicated in the development of the malignant phenotype. The identification of gene products that can act as prognostic markers in bladder cancer would be value in determining the management of this heterogeneous group of patients. This study examines cripto-1 expression in benign and malignant bladder using immunohistochemical techniques. The expression of cripto-1 protein in benign and malignant bladder was examined in 45 bladder tumours (Ta/T1 n = 26, T2 n = 5, T3/T4 n = 14) and six benign controls. All 45 tumours showed positive cytoplasmic staining for cripto-1, including areas of carcinoma in situ. None of the six benign controls showed any evidence of positive cripto-1 staining. Twenty-three (60 per cent) bladder tumours had areas of papillary tumour that showed strong positive staining for cripto-1 as opposed to six (29 per cent) sections of histologically normal urothelium adjacent to tumour (P < 0.05). There was no association between cripto-1 staining and tumour grade, stage, or clinical outcome. Cripto-1 protein appears to be specifically expressed in malignant and benign adjacent urothelium of patients with bladder cancer. Its clinical significance, however, remains to be determined.

While evidence implicates both the adaptive and innate immune system in rheumatoid arthritis (RA) pathogenesis, accumulating data indicate that the synovial tissue itself actively participates in the destructive inflammatory process. Specifically, resident fibroblast-like synoviocytes (FLS), together with macrophages, re-organise to form an aggressive cell mass, called pannus, which destroys the articular cartilage and the subchondral bone. The exact molecular mechanisms of synovial pannus formation, FLS expansion and invasion into adjacent tissues are not yet known. Our data strongly suggest that the T-cell derived cytokine IFNγ is involved in FLS-mediated joint destruction. Migration and invasion assays revealed increased migratory activity for IFNγ-stimulated FLS, when compared to unstimulated FLS. Further, biochemical studies showed that IFNγ promotes the migratory and invasive activity of FLS via Janus kinase 2 (JAK2) and the focal adhesion kinase (FAK), a kinase known to integrate focal adhesion turnover and thus, regulates cell migration. In detail, IFNγ stimulation of FLS distinctly resulted in the phosphorylation of FAK-Y925, a phospho-site that has recently been demonstrated to be required for FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated the IFNγ-induced activation of FAK. Correspondingly, baricitinib, a JAK inhibitor that is currently successfully probed in RA clinical trials, abrogated IFNγ-stimulated activation of FAK. In conclusion, our studies contribute insight into the synovial response to IFNγ and reveal JAK2 and FAK as potential targets for synoviocyte-mediated joint destruction in arthritis, especially in RA.

Background and objectives Fibroblast like Synoviocytes (FLS) are key for the formation of the synovial lining / sublining architecture via unique cadherin-11 mediated cell-to-cell adherens junctions. Binding to the juxta-membrane domain of the cytoplasmic tail of cadherins, p120 is indispensable for the stabilisation of cadherins on the cell surface. By contrast, unbound p120-catenin interferes with small Rho GTPases, thereby regulating actin cytoskeletal organisation as well as NF-κB pathway activity. Here we probe into the function of p120-catenin for synovial tissue formation. Methods Primary FLS were cultured from synovial tissue after synovectomy of RA classified patients. The knock down (KD) of p120-catenin was achieved by the expression of specific shRNA via a lentiviral vector. Immunoblotting was performed to verify decreased p120-catenin expression levels as well as the effects on the expression of cellular cadherin-11. 2D FLS cultures were analysed using phase contrast microscopy as well as confocal immunofluorescence imaging of actin filaments and cadherin-11 mediated cell-to-cell junctions. For the analysis of FLS capacity in forming synovial-like tissue, FLS were cultured in Matrigel® micromasses. Data quantification was done with ImageJ; for statistical analyses Graphpad Prism® was used. Results p120-catenin silenced FLS demonstrated an altered cellular phenotype regarding the size, shape and the cytoskeletal organisation when compared to control cells. In conventional 2D cultures, the surface area covered by KD cells was increased threefold (p Conclusion Although the immunological role of p120-catenin for FLS function in synovial inflammation (NF-kB activity) has yet to be shown, our findings strongly suggest the importance of p120-catenin for the formation of the normal synovial tissue.

Background The synovial membrane of diarthrodial joints is primarily composed of fibroblast-like synoviocytes (FLS) that form a complex tissue via discrete cell-to-cell connexions with wide intercellular matrix spaces. The distinct synovial tissue function that is crucial to joint homeostasis depends upon integrated activity of individual FLS. Using a in-vitro synovial organ culture system, we explore mechanisms of FLS cellular cooperation with special interest in the cell-to-cell transfer of mitochondria via interconnecting membrane nanotubes. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. Cells were cultured in spherical matrigel micromasses with an average size of 2 mm O. Data was acquired by confocal live cell imaging. Analysis of the resulting 4D movies was done with Imaris® software. Results To examine whether or not FLS transfer cytoplasmic cargo, we labelled 50% of FLS with red cell tracker dye and loaded the other 50% with green non-degradable microspheres. In a time series (8 days), we found that microspheres appear in red labelled cells. First evidence was found on Day 1 and over the course of the following days microspheres accumulated in red labelled cells with a transfer rate of 10% of newly affected cells/day. With special interest in the transfer of mitochondria, we repeated similar experiments with labelled mitochondria. We found that the transfer rate for these organelles is similar to the one for microspheres. Additionally, we were able to capture evidence that FLS indeed use their nanotube connexions for transfer of mitochondria. Conclusions Our experiments suggest transfer of cytoplasmic cargo, including organelles such as mitochondria, between FLS. These studies may provide insight into how synoviocytes orchestrate their activity. Further studies will demonstrate the significance of directed cargo exchange for cellular cooperation and the function of the normal as well as the diseased synovium.

Neutrophilic granulocytes and monocytes (granulomonocytic cells; GMC) drive the inflammatory process at the earliest stages of rheumatoid arthritis (RA). The migratory behavior and functional properties of GMC within the synovial tissue are, however, only incompletely characterized. Here we have analyzed GMC in the murine collagen-induced arthritis (CIA) model of RA using multi-photon real time in vivo microscopy together with ex vivo analysis of GMC in tissue sections. GMC were abundant as soon as clinical arthritis was apparent. GMC were motile and migrated randomly through the synovial tissue. In addition, we observed the frequent formation of cell clusters consisting of both neutrophilic granulocytes and monocytes that actively contributed to the inflammatory process of arthritis. Treatment of animals with a single dose of prednisolone reduced the mean velocity of cell migration and diminished the overall immigration of GMC. In summary, our study shows that the combined application of real time in vivo microscopy together with elaborate static post-mortem analysis of GMC enables the description of dynamic migratory characteristics of GMC together with their precise location in a complex anatomical environment. Moreover, this approach is sensitive enough to detect subtle therapeutic effects within a very short period of time.

Objective The binding of abatacept (CTLA-4Ig) to the B7 ligands CD80 and CD86 prevents the engagement of CD28 on T cells and thereby prevents effector T cell activation. In addition, a direct effect of CTLA-4Ig on antigen-presenting cells (APCs) could contribute to the therapeutic effect. To further elucidate the mechanism of CTLA-4Ig, we performed phenotype and functional analyses of APCs in patients with rheumatoid arthritis (RA) before and after the initiation of CTLA-4Ig therapy. Methods Peripheral blood mononuclear cells were analyzed before and at 2 and 4 weeks after the initiation of CTLA-4Ig therapy. Proportions of APCs were determined by flow cytometry. CD14+ monocytes were further analyzed for the expression of costimulatory and adhesion molecules and for their transendothelial migratory capacity in vitro. In addition, CD14+ monocytes from healthy controls were analyzed for their migratory and spreading capacity. Results Proportions and absolute numbers of monocytes were significantly increased in RA patients treated with CTLA-4Ig. The expression of several adhesion molecules was significantly diminished. In addition, monocytes displayed a significant reduction in their endothelial adhesion and transendothelial migratory capacity upon treatment with CTLA-4Ig. Likewise, isolated monocytes from healthy controls revealed a significant reduction in their migratory and spreading activity after preincubation with CTLA-4Ig or anti-CD80 and anti-CD86 antibodies. Conclusion We describe direct effects of CTLA-4Ig therapy on phenotype and functional characteristics of monocytes in RA patients that might interfere with the migration of monocytes to the synovial tissue. This additional mechanism of CTLA-4Ig might contribute to the beneficial effects of CTLA-4Ig treatment in RA patients.

SummaryOctopuses are intelligent, soft-bodied animals with keen senses that perform reliably in a variety of visual and tactile learning tasks [1–6]. However, researchers have found them disappointing in that they consistently fail in operant tasks that require them to combine central nervous system reward information with visual and peripheral knowledge of the location of their arms [6–8]. Wells [6] claimed that in order to filter and integrate an abundance of multisensory inputs that might inform the animal of the position of a single arm, octopuses would need an exceptional computing mechanism, and “There is no evidence that such a system exists in Octopus, or in any other soft bodied animal.” Recent electrophysiological experiments, which found no clear somatotopic organization in the higher motor centers, support this claim [9]. We developed a three-choice maze that required an octopus to use a single arm to reach a visually marked goal compartment. Using this operant task, we show for the first time that Octopus vulgaris is capable of guiding a single arm in a complex movement to a location. Thus, we claim that octopuses can combine peripheral arm location information with visual input to control goal-directed complex movements.Video Abstract

We describe for the first time fluorescent virus-like particles decorated with biologically active mono- and multisubunit immune receptors of choice and the basic application of such fluorosomes (FSs) to visualize and target immune receptor-ligand interactions. For that purpose, human embryonic kidney (HEK)-293 cells were stably transfected with Moloney murine leukemia virus (MoMLV) matrix protein (MA) GFP fusion constructs. To produce FSs, interleukins (ILs), IL-receptors (IL-Rs), and costimulatory molecules were fused to the glycosyl phosphatidyl inositol anchor acceptor sequence of CD16b and coexpressed along with MoMLV group-specific antigen-polymerase (gag-pol) in MA::GFP+ HEK-293 cells. We show that IL-2 decorated but not control-decorated FSs specifically identify normal and malignant IL-2 receptor-positive (IL-2R+) lymphocytes by flow cytometry. In addition to cytokines and costimulatory molecules, FSs were also successfully decorated with the heterotrimeric IL-2Rs, allowing identification of IL-2+ target cells. Specificity of binding was proven by complete inhibition with nonlabeled, soluble ligands. Moreover, IL-2R FSs efficiently neutralized soluble IL-2 and thus induced unresponsiveness of T cells receiving full activation stimuli via T-cell antigen receptor and CD28. FSs are technically simple, multivalent tools for assessing and blocking mono- and multisubunit immune receptor-ligand interactions with natural constituents in a plasma membrane context.—Kueng, H. J., Manta, C., Haiderer, D., Leb, V. M., Schmetterer, K. G., Neunkirchner, A., Byrne, R. A., Scheinecker, C., Steinberger, P., Seed, B., Pickl, W. F. Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor-ligand interactions.

Testing the cognitive abilities of cartilaginous fishes is important in understanding the evolutionary origins of cognitive functions in higher vertebrates. We used five South American fresh water stingrays (Potamotrygon castexi) in a learning and problem-solving task. A tube test apparatus was developed to provide a simple but sophisticated procedure for testing cognitive abilities of aquatic animals. All five subjects quickly learned to use water as a tool to extract food from the testing apparatus. The experimental protocol, which gave the animals the opportunity of correcting a wrong visual cue decision, resulted in four out of five subjects correcting an error rather than making an initial right choice. One of five subjects reached 100% correct trials in the visual discrimination task. The ability to use water as an agent to extract food from the testing apparatus is a first indication of tool use in batoid fishes. Performance in the instrumental task of retrieving food from a novel testing apparatus and the rapid learning in the subsequent discrimination/error correction task shows that cartilaginous fish can be used to study the origins of cognitive functions in the vertebrate lineage.

Background The synovium is primarily built by fibroblast-like synoviocytes (FLS) that are connected to one another via cellular processes, thus forming a continuous network of cells. Its multicellularity requires precise coordination between cells to bring forth specialised tissue functions critical to joint homeostasis. Cell-to-cell transfer of cytoplasmic cargo may provide a means for intercellular communication, thereby facilitating the concerted behaviour of FLS. Using a 3D in-vitro model of the synovium, we analysed FLS capacity for exchange of cytoplasmic content. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. Cells were cultured in spherical matrigel micromasses with an average size of 2 mm O. For confocal live cell imaging and transmission electron microscopy, FLS were loaded with fluorescent non-degradable microspheres and labelled with fluorescent membrane dyes. Analysis of the resulting 4D movies was done with Imaris® software. Results To examine intercellular cytoplasmatic transfer, we labelled 50% of FLS with red cell tracker dye and loaded the other 50% with green microspheres. In a time series (8 days), we found that microspheres do indeed appear in red labelled cells. First evidence was found on Day 1 and over the course of the following days microspheres accumulated in red labelled cells with a transfer rate of 10% of newly affected cells/day. A similar experiment in 2D demonstrated microsphere movement within interconnecting membrane nanotubes. Transfer rates for microspheres into red cells were identical. Transmission Electron Microscopy (TEM) identified extracellular vesicles (exosomes) in discrete regions of intimate cell-to-cell contact but also open interconnecting tubes, suggesting distinct modes for transfer. Conclusions These studies reveal transfer of cytoplasmic cargo between FLS and may provide insight into how the synovial tissue operates. Further studies will demonstrate the significance of directed cargo exchange for cellular cooperation and the function of the normal as well as the diseased synovium.

Background Monocytes (Mo) are among the first hematopoietic cells to migrate into the inflamed synovial tissue where they integrate into the synovial lining network of fibroblast-like synovial cells (FLS). Here we analyse the dependence of Mo – FLS interaction on proinflammatory cytokines and the effect of anti-IL-6 antibody (Ab) Tocilizumab using real time confocal/multiphoton microscopy of 3D micromass tissue cultures. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. CD14 + Mo were isolated from peripheral blood by magnetic bead sorting. For confocal imaging fluorescent labelled FLS and Mo were cultured in spherical extracellular matrix micromasses. Micromass cultures were set up in the presence of Tocilizumab or control Ab and stimulated with TNF-alpha. The resulting 4D live cell imaging movies were analysed with Imaris ® Software. Results The highest migratory activity of Mo was observed on days 4–7 of culture, upon the formation of a FLS lining layer around a 3D micromass, The presence of Tocilizumab in TNF-alpha stimulated cultures i) increased the proportion of migrating Mo from 12% in control cultures to 22%, ii) increased the maximum track length of migrating monocytes and iii) stimulated a fast oscillating movement pattern of Mo. Conclusions The 3D synovial tissue culture system allows for monitoring and analyses of subtle migration patterns of Mo in relation to the organised synovial lining architecture that depends on the presence of proinflammatory cytokines. Tocilizumab causes significant changes in the migratory behaviour of Mo that might lead to the release of Mo from the inflamed synovial tissue and contribute to the dissolution of inflammation. Ongoing experiments will address the molecular mechanism (s) of Mo – FLS interaction as well as the cellular source and sequence of cytokine production in order to identify potential targets for future therapeutic interventions in arthritis.

Studying play behavior in octopuses is an important step toward understanding the phylogenetic origins and function of play as well as the cognitive abilities of invertebrates. Fourteen Octopus vulgaris (7 subadults and 7 adults) were presented 2 Lego objects and 2 different food items on 7 consecutive days under 2 different levels of food deprivation. Nine subjects showed play-like behavior with the Lego objects. There was no significant difference in play-like behavior corresponding to food deprivation, age, and sex of the octopuses. The sequence of behaviors, from exploration to play-like behavior, had a significant influence on the establishment of play-like behavior, as it occurred mostly on Days 3-6 of the 7-day experiment. The pattern of development of play-like activities after a period of exploration and habituation in this study agrees with the hypothesis that object play follows object exploration. A homologous origin of this behavioral trait in vertebrates and invertebrates is highly unlikely, as the last common ancestor might not have had the cognitive capacity to possess this trait.

Octopus macropus and Octopus vulgaris have overlapping habitats and are exposed to similar temporal changes. Whereas the former species is described as nocturnal in the field, there are conflicting reports about the activity time of the latter one. To compare activity patterns, the authors tested both species in the laboratory. Octopuses were exposed to a light-dark cycle and held under constant dim light for 7 days each. O. macropus showed nocturnal and light-cued activity. According to casual observations, O. vulgaris started out nocturnal but had switched to mostly diurnal when the experiment began. Individual variation of its activity was found. The different activity patterns of 0. macropus and 0. vulgaris might reflect their lifestyles, the latter species being more generalist.

Previous behavioral studies in Octopus vulgaris revealed lateralization of eye use. In this study, the authors expanded the scope to investigate arm preferences. The octopus's generalist hunting lifestyle and the structure of their arms suggest that these animals have no need to designate specific arms for specific tasks. However, octopuses also show behaviors, like exploration, in which only single or small groups of arms are involved. Here the authors show that octopuses had a strong preference for anterior arm use to reach for and explore objects, which points toward a task division between anterior and posterior arms. Four out of 8 subjects also showed a lateral bias. In addition, octopuses had a preference for a specific arm to reach into a T maze to retrieve a food reward. These findings give evidence for limb-specialization in an animal whose 8 arms were believed to be equipotential.

This study aims to investigate the octopus' eye and arm coordination and raises the question if visual guidance determines choice of arm use. Octopuses possess eight seemingly identical arms but have recently been reported to show a preference as to which arm they use to initiate contact with objects. These animals also exhibit lateralized eye use, therefore, a connection between eye and arm preference seems possible. Seven Octopus vulgaris were observed during approach, contact initiation and exploration of plastic objects that were positioned on three different levels in the water column. The subjects most commonly used an arm to initiate contact with an object that was in a direct line between the eye used to look at the object, and the object itself. This indicates that choice of arm use is spatially rather opportunistic when depending on visual guidance. Additionally, first contact with an object was usually established by the central third of the arm and in arm contact sequences neighboring arms were the most likely to follow an arm already touching the object. Although results point towards strong eye/arm coordination, we did not find lateralized behavior in this experiment. Results are discussed from a neuro-anatomical, behavioral and ecological perspective.

Polymorphonuclear neutrophils (PMNs) participate in the initiation of rheumatoid arthritis (RA), but their precise role at the earliest stages of the synovial inflammatory response is only partially understood. We therefore analysed the initial steps of synovial inflammation in the mouse model of collagen-induced arthritis (CIA) with regard to the role of PMN. CIA was induced in C57/BL6 mice. Mice were tested for anti-CII serum antibodies by ELISA. Mice were killed on days 10 and 20 after the first immunisation before clinical …

Background Monocytes (Mo) are among the first hematopoietic cells to migrate into the inflamed synovial tissue of arthritic joints. Monocyte migration appears to be associated with the expanding synovial lining network of fibroblast-like synovial cells (FLS). Whether cognitive interaction between Mo and FLS is required for an orchestrated migratory behaviour has not been analysed so far. We analysed Mo migratory activity under inflammatory conditions in regard to cell-cell interactions with FLS. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. CD14+ Mo were isolated from peripheral blood by magnetic bead sorting. FLS and Mo were labeled with fluorescent membrane dyes and cultured in spherical extracellular matrix micromasses with an average size of 1.5 mm for up to two weeks. For stimulation experiments, micromasses were cultured in medium containing 10 ng/ml of tumor necrosis factor (TNF). At different time-points cell migration was monitored in individual micromasses by real-time confocal microscopy. Results Cell migration could be subdivided into three successive phases of cell movement. Phase I (day 1-3 of culture) was characterised by the formation of the synovial lining layer. Mo in close contact with FLS appeared sessile. On average 20% of Mo were in no apparent contact with FLS and displayed a mobile and seeking behaviour. During phase II (day 3-7) the majority of Mo remained sessile whereas a fraction of Mo displayed a directed cell movement with an impressive maximum speed of up to 15 mcm/min. In addition the formation of Mo cell clusters was observed. The rapid Mo migration finally ceased during phase III (day 7-14). The addition of TNF i) increased the frequency and size of Mo cell clusters during phase II two and ii) prolonged the mobility of Mo into phase III. Conclusion The 3D synovial tissue culture system allows to monitor and analysed subtle migration patterns of Mo in relation to the organised synovial lining architecture. Ongoing experiments address molecular mechanism(s) of Mo – FLS interaction in order to identify potential targets for future therapeutic intervention in arthritis.

Background Monocytes (Mo) are among the first hematopoietic cells to migrate into the inflamed synovial tissue of arthritic joints. Monocyte migration appears to be associated with the expanding synovial lining network of fibroblast-like synovial cells (FLS). Whether cognitive interaction between Mo and FLS is required for an orchestrated migratory behavior has not been analyzed so far. Objectives We analyzed Mo migratory activity under inflammatory conditions in regard to cell-cell interactions with FLS. Methods Human FLS were prepared from synovial tissues obtained as discarded specimens following joint arthroplasty. CD14 + Mo were isolated from peripheral blood by magnetic bead sorting. FLS and Mo were labeled with fluorescent membrane dyes and cultured in spherical extracellular matrix micromasses with an average size of 1.5 mm for up to two weeks. For stimulation experiments, micromasses were cultured in medium containing 10 ng/ml of tumor necrosis factor (TNF). At different time-points cell migration was monitored in individual micromasses by real-time confocal microscopy. Results Cell migration could be subdivided into three successive phases of cell movement. Phase I (day 1-3 of culture) was characterized by the formation of the synovial lining layer. Mo in close contact with FLS appeared sessile. On average 20% of Mo were in no apparent contact with FLS and displayed a mobile and seeking behavior. During phase II (day 3-7) already >95% of Mo were in contact with FLS. The majority of Mo remained sessile whereas a fraction of Mo displayed a directed cell movement with an impressive maximum speed of up to 15 mcm/min. In addition the formation of Mo cell clusters was observed. The rapid Mo migration finally ceased during phase III (day 7-14). The addition of TNF i) increased the frequency and size of Mo cell clusters during phase II two and ii) prolonged the mobility of Mo into phase III. Conclusions The 3D synovial tissue culture system allows to monitor and analyzed subtle migration patterns of Mo in relation to the organized synovial lining architecture. Ongoing experiments address molecular mechanism(s) of Mo – FLS interaction in order to identify potential targets for future therapeutic intervention in arthritis. Disclosure of Interest None Declared

In rheumatoid arthritis (RA), a systemic autoimmune response translates into an inflammatory attack on the synovium that yields the formation of an aggressive cell mass, called pannus, which invades into and destroys the articular cartilage. Cartilage destruction is primarily mediated by fibroblast-like synoviocytes (FLS). Among the pro-inflammatory mediators produced by infiltrating T cells, interferon γ (IFNγ) may contribute to FLS driven joint destruction. Of note, IFNγ elicits its effects via the JAK1/JAK2-Stat1 signaling pathway. To establish a role for IFNγ in the invasive potential of FLS, the authors used an in-vitro invasion assay (matrix-associated trans-epithelial resistance invasion-(MATRIN)-assay). Strikingly, FLS that were stimulated with IFNγ demonstrated a markedly increased invasive capacity when compared to un-stimulated FLS. Cell invasion involves several steps, including attachment to extracellular matrix (ECM), cell migration, and digestion of the ECM by proteases. As determined by qPCR, however, IFNγ did not up-regulate the expression of metalloproteinases (MMPs) by FLS. Therefore, the authors hypothesised that IFNγ directs FLS motility. Indeed, exposure of FLS to IFNγ resulted in their increased migratory activity as determined in Boyden chamber assays. Since cell motility is partly controlled by focal adhesion kinase (FAK), the authors next analysed whether or not IFNγ modulates FAK activity in FLS. Western blot analysis revealed that activation of the IFNγ-JAK1/2-Stat1 signaling cascade is associated with phosphorylation of FAK. As Stat1 deficient U3A cells similarly responded to IFNγ stimulation, activation of FAK was independent of Stat1. Instead, inhibition of JAK1/2 abrogated IFNγ induced activation of FAK in FLS, indicating that IFNγ regulates FAK activity through JAK1/2, but not Stat1. Importantly, inhibition of JAKs abrogated IFNγ induced invasive activity of FLS. These results confirm that JAK1/2 play a critical role in translating the signal induced by IFNγ to promote invasion in FLS. These studies suggest a role for IFNγ in the destructive mesenchymal tissue response to inflammation and may provide insight into FLS behavior and function in RA.

Background Granulomonocytic cells (GMC) drive the inflammatory process at the earliest stages of rheumatoid arthritis (RA). The migratory behavior and functional properties of GMC within the synovial tissue are, however, only incompletely understood. This tempted us to study GMC in the murine collagen induced arthritis (CIA) model of RA with the help of multi-photon real time in vivo microscopy together with the subsequent and sequential ex vivo analysis of GMC on tissue sections. Methods CIA was induced in LysM-EGFP C57BL/6 transgenic animals that carry the EGFP fluorescence protein under the lysozyme promoter. Individual joints were prepared by surgical microscopy in healthy control and in CIA subjects and EGFP + GMC were analysed by 2-photon laser microscopy over 2 h. One group of animals received one single dose (0.25 mg) of prednisolone intravenously before in vivo imaging. Afterwards, the animals were killed and cryo-, and paraffin sections were prepared for immunofluorescence and histomorphological analysis, respectively. Results GMC were barely detectable in healthy animals but were abundant in the synovial tissue as soon as clinical arthritis was apparent. GMC were motile and migrated randomly through the synovial tissue with a reduced mean velocity (2.75±1.17 µm/min) of as compared to healthy controls (3.11±1.51 µm/min; p high neutrophilic granulocytes and EGFP low monocytes. In addition EGFPl low F4/80 + TRAP + osteoclast precursor cells were occasionally observed at the synovial-bone junction and areas of bone erosions.Prednisolone treatment reduced the mean velocity of cell migration (2.19±1.06 µm/min; p Conclusion The combined application of real time in vivo microscopy together with elaborate static postmortem analysis of GMC enabled the description of dynamic migratory characteristics of GMC together with their precise allocation in a complex anatomical environment. Moreover, this approach was found sensitive enough to detect subtle therapeutic effects within a very short period of time.

Rheumatoid arthritis (RA) is the most common chronic inflammatory joint disease. In RA, a systemic autoimmune response translates into an inflammatory attack on the synovium that yields the formation of an aggressive cell mass called pannus which attaches to, encroaches over and destroys the articular cartilage. Cartilage destruction is mediated by fibroblast-like synoviocytes (FLS) which are the prevailing cell type of the …