Identification of gene expression biomarkers to predict clinical response to methotrexate in patients with rheumatoid arthritis.

OBJECTIVES: To identify biomarkers at the gene expression level to predict response to methotrexate (MTX) in patients with rheumatoid arthritis (RA). METHODS: MTX-naïve patients with RA were started on MTX and followed up over three months. The disease activity score 28 (DAS28) was used to classify patients into responders and non-responders. Genome-wide gene expression analysis was performed in CD4 + and CD14 + mononuclear cells sampled from whole blood at baseline to identify differentially expressed genes in responders versus non-responders. Gene selection methods and prediction modelling obtained the most relevant differentially expressed genes. A logistic regression prediction model was subsequently constructed and validated via bootstrapping. The area under the receiver operating characteristic (AUC) curve was calculated to judge model quality. RESULTS: Seventy-nine patients with RA (53.4 ± 13.9 years, 74.7% females) were enrolled, and 70 finished the study with a documented treatment EULAR response (77.1% responders). Forty-six differentially expressed genes were found. The most promising genes were KRTAP4-11, LOC101927584, and PECAM1 in CD4 + cells and PSMD5 and ID1 in CD14 + cells. The final prediction model using these genes reached an AUC of 90%; the validation set's AUC was 82%. CONCLUSIONS: Our prediction model constructed via genome-wide gene expression analysis in CD4 + and CD14 + mononuclear cells yielded excellent predictions. Our findings necessitate confirmation in other cohorts of MTX-naïve RA patients. Especially if used in conjunction with previously identified clinical and laboratory (bio)markers, our results could help predict response to MTX in RA to guide treatment decisions. Key Points • Patients with rheumatoid arthritis may or may not respond to treatment with methotrexate, which is the recommended first-line drug in guidelines around the world. • In non-responders, valuable time is lost until second-line treatments are started. • This study aimed at predicting response to methotrexate by identifying differentially expressed genes from peripheral blood samples. • The final prediction model yielded excellent prognostic values, but validation in other cohorts is necessary to corroborate these findings.

Inhibition of Complex I of the Respiratory Chain, but Not Complex III, Attenuates Degranulation and Cytokine Secretion in Human Skin Mast Cells.

The mechanisms of mast cell (MC) degranulation and MC-driven skin symptoms are well-described. In contrast, data about the role of mitochondrial respiration for immune functions of human skin MCs are lacking. Oxygen consumption rate (OCR) in primary human skin MCs during IgE-mediated activation in the absence of glucose was examined using a metabolic flux analyzer. Effects of the inhibition of mitochondrial complex I (by rotenone A) and III (by myxothiazol) on degranulation and cytokine secretion (IL-4, IL-5, IL-6, IL-13, TNF-α, and GM-CSF) were explored by the ß-hexosaminidase release assay and multiplex ELISA. IgE-mediated activation rapidly increased the mitochondrial OCR and extracellular acidification; the contribution of non-mitochondrial oxygen consumption remained unchanged at lower levels. Both myxothiazol and rotenone A reduced OCR, the mitochondrial parameters, and extracellular acidification; however, myxothiazol did not affect degranulation and cytokine secretion. In contrast, degranulation and the secretion of IL-6, IL-13, TNF-α, and GM-CSF were reduced by rotenone A, whereas the secretion of IL-4 and IL-5 was not significantly affected. The inhibitors did not affect cell viability. Our results highlight the important role played by mitochondrial respiration in primary human skin MCs and allow for a conclusion on a hierarchy of their effector functions. Drugs targeting specific pathways in mitochondria may provide future options to control MC-driven skin symptoms.

Age-related increase of mitochondrial content in human memory CD4+ T cells contributes to ROS-mediated increased expression of proinflammatory cytokines.

Cellular metabolism modulates effector functions in human CD4+ T (Th) cells by providing energy and building blocks. Conversely, cellular metabolic responses are modulated by various influences, e.g., age. Thus, we hypothesized that metabolic reprogramming in human Th cells during aging modulates effector functions and contributes to "inflammaging", an aging-related, chronic, sterile, low-grade inflammatory state characterized by specific proinflammatory cytokines. Analyzing the metabolic response of human naive and memory Th cells from young and aged individuals, we observed that memory Th cells exhibit higher glycolytic and mitochondrial fluxes than naive Th cells. In contrast, the metabolism of the latter was not affected by donor age. Memory Th cells from aged donors showed a higher respiratory capacity, mitochondrial content, and intracellular ROS production than those from young donors without altering glucose uptake and cellular ATP levels, which finally resulted in higher secreted amounts of proinflammatory cytokines, e.g., IFN-γ, IP-10 from memory Th cells taken from aged donors after TCR-stimulation which were sensitive to ROS inhibition. These findings suggest that metabolic reprogramming in human memory Th cells during aging results in an increased expression of proinflammatory cytokines through enhanced ROS production, which may contribute to the pathogenesis of inflammaging.

Metabolic reprogramming of synovial fibroblasts in osteoarthritis by inhibition of pathologically overexpressed pyruvate dehydrogenase kinases.

Osteoarthritis (OA) is the most common degenerative joint disease and a major cause of age-related disability worldwide, mainly due to pain, the disease's main symptom. Although OA was initially classified as a non-inflammatory joint disease, recent attention has been drawn to the importance of synovitis and fibroblast-like synoviocytes (FLS) in the pathogenesis of OA. FLS can be divided into two major populations: thymus cell antigen 1 (THY1)- FLS are currently classified as quiescent cells and assumed to destroy bone and cartilage, whereas THY1+ FLS are invasively proliferative cells that drive synovitis. Both THY1- and THY1+ FLS share many characteristics with fibroblast-like progenitors - mesenchymal stromal cells (MSC). However, it remains unclear whether synovitis-induced metabolic changes exist in FLS from OA patients and whether metabolic differences may provide a mechanistic basis for the identification of approaches to precisely convert the pathologically proliferative synovitis-driven FLS phenotype into a healthy one. To identify novel pathological mechanisms of the perpetuation and manifestation of OA, we analyzed metabolic, proteomic, and functional characteristics of THY1+ FLS from patients with OA. Proteome data and pathway analysis revealed that an elevated expression of pyruvate dehydrogenase kinase (PDK) 3 was characteristic of proliferative THY1+ FLS from patients with OA. These FLS also had the highest podoplanin (PDPN) expression and localized to the sublining but also the lining layer in OA synovium in contrast to the synovium of ligament trauma patients. Inhibition of PDKs reprogrammed metabolism from glycolysis towards oxidative phosphorylation and reduced FLS proliferation and inflammatory cytokine secretion. This study provides new mechanistic insights into the importance of FLS metabolism in the pathogenesis of OA. Given the selective overexpression of PDK3 in OA synovium and its restricted distribution in synovial tissue from ligament trauma patients and MSC, PDKs may represent attractive selective metabolic targets for OA treatment. Moreover, targeting PDKs does not affect cells in a homeostatic, oxidative state. Our data provide an evidence-based rationale for the idea that inhibition of PDKs could restore the healthy THY1+ FLS phenotype. This approach may mitigate the progression of OA and thereby fundamentally change the clinical management of OA from the treatment of symptoms to addressing causes.

MIF does only marginally enhance the pro-regenerative capacities of DFO in a mouse-osteotomy-model of compromised bone healing conditions.

The initial phase of fracture healing is crucial for the success of bone regeneration and is characterized by an inflammatory milieu and low oxygen tension (hypoxia). Negative interference with or prolongation of this fine-tuned initiation phase will ultimately lead to a delayed or incomplete healing such as non-unions which then requires an effective and gentle therapeutic intervention. Common reasons include a dysregulated immune response, immunosuppression or a failure in cellular adaptation to the inflammatory hypoxic milieu of the fracture gap and a reduction in vascularizing capacity by environmental noxious agents (e.g. rheumatoid arthritis or smoking). The hypoxia-inducible factor (HIF)-1α is responsible for the cellular adaptation to hypoxia, activating angiogenesis and supporting cell attraction and migration to the fracture gap. Here, we hypothesized that stabilizing HIF-1α could be a cost-effective and low-risk prevention strategy for fracture healing disorders. Therefore, we combined a well-known HIF-stabilizer - deferoxamine (DFO) - and a less known HIF-enhancer - macrophage migration inhibitory factor (MIF) - to synergistically induce improved fracture healing. Stabilization of HIF-1α enhanced calcification and osteogenic differentiation of MSCs in vitro. In vivo, only the application of DFO without MIF during the initial healing phase increased callus mineralization and vessel formation in a preclinical mouse-osteotomy-model modified to display a compromised healing. Although we did not find a synergistically effect of MIF when added to DFO, our findings provide additional support for a preventive strategy towards bone healing disorders in patients with a higher risk by accelerating fracture healing using DFO to stabilize HIF-1α.

Production of IL-6 and Phagocytosis Are the Most Resilient Immune Functions in Metabolically Compromised Human Monocytes.

At sites of inflammation, monocytes carry out specific immune functions while facing challenging metabolic restrictions. Here, we investigated the potential of human monocytes to adapt to conditions of gradually inhibited oxidative phosphorylation (OXPHOS) under glucose free conditions. We used myxothiazol, an inhibitor of mitochondrial respiration, to adjust two different levels of decreased mitochondrial ATP production. At these levels, and compared to uninhibited OXPHOS, we assessed phagocytosis, production of reactive oxygen species (ROS) through NADPH oxidase (NOX), expression of surface activation markers CD16, CD80, CD11b, HLA-DR, and production of the inflammatory cytokines IL-1ß, IL-6 and TNF-α in human monocytes. We found phagocytosis and the production of IL-6 to be least sensitive to metabolic restrictions while surface expression of CD11b, HLA-DR, production of TNF-α, IL-1ß and production of ROS through NOX were most compromised by inhibition of OXPHOS in the absence of glucose. Our data demonstrate a short-term hierarchy of immune functions in human monocytes, which represents novel knowledge potentially leading to the development of new therapeutics in monocyte-mediated inflammatory diseases.

Fracture Healing Research-Shift towards In Vitro Modeling?

Fractures are one of the most frequently occurring traumatic events worldwide. Approximately 10% of fractures lead to bone healing disorders, resulting in strain for affected patients and enormous costs for society. In order to shed light into underlying mechanisms of bone regeneration (habitual or disturbed), and to develop new therapeutic strategies, various in vivo, ex vivo and in vitro models can be applied. Undeniably, in vivo models include the systemic and biological situation. However, transferability towards the human patient along with ethical concerns regarding in vivo models have to be considered. Fostered by enormous technical improvements, such as bioreactors, on-a-chip-technologies and bone tissue engineering, sophisticated in vitro models are of rising interest. These models offer the possibility to use human cells from individual donors, complex cell systems and 3D models, therefore bridging the transferability gap, providing a platform for the introduction of personalized precision medicine and finally sparing animals. Facing diverse processes during fracture healing and thus various scientific opportunities, the reliability of results oftentimes depends on the choice of an appropriate model. Hence, we here focus on categorizing available models with respect to the requirements of the scientific approach.

Functional Scaffold-Free Bone Equivalents Induce Osteogenic and Angiogenic Processes in a Human In Vitro Fracture Hematoma Model.

After trauma, the formed fracture hematoma within the fracture gap contains all the important components (immune/stem cells, mediators) to initiate bone regeneration immediately. Thus, it is of great importance but also the most susceptible to negative influences. To study the interaction between bone and immune cells within the fracture gap, up-to-date in vitro systems should be capable of recapitulating cellular and humoral interactions and the physicochemical microenvironment (eg, hypoxia). Here, we first developed and characterized scaffold-free bone-like constructs (SFBCs), which were produced from bone marrow-derived mesenchymal stromal cells (MSCs) using a macroscale mesenchymal condensation approach. SFBCs revealed permeating mineralization characterized by increased bone volume (µCT, histology) and expression of osteogenic markers (RUNX2, SPP1, RANKL). Fracture hematoma (FH) models, consisting of human peripheral blood (immune cells) mixed with MSCs, were co-cultivated with SFBCs under hypoxic conditions. As a result, FH models revealed an increased expression of osteogenic (RUNX2, SPP1), angiogenic (MMP2, VEGF), HIF-related (LDHA, PGK1), and inflammatory (IL6, IL8) markers after 12 and 48 hours co-cultivation. Osteogenic and angiogenic gene expression of the FH indicate the osteoinductive potential and, thus, the biological functionality of the SFBCs. IL-6, IL-8, GM-CSF, and MIP-1ß were detectable within the supernatant after 24 and 48 hours of co-cultivation. To confirm the responsiveness of our model to modifying substances (eg, therapeutics), we used deferoxamine (DFO), which is well known to induce a cellular hypoxic adaptation response. Indeed, DFO particularly increased hypoxia-adaptive, osteogenic, and angiogenic processes within the FH models but had little effect on the SFBCs, indicating different response dynamics within the co-cultivation system. Therefore, based on our data, we have successfully modeled processes within the initial fracture healing phase in vitro and concluded that the cross-talk between bone and immune cells in the initial fracture healing phase is of particular importance for preclinical studies. © 2021 American Society for Bone and Mineral Research (ASBMR).

A Human Osteochondral Tissue Model Mimicking Cytokine-Induced Key Features of Arthritis In Vitro.

Adequate tissue engineered models are required to further understand the (patho)physiological mechanism involved in the destructive processes of cartilage and subchondral bone during rheumatoid arthritis (RA). Therefore, we developed a human in vitro 3D osteochondral tissue model (OTM), mimicking cytokine-induced cellular and matrix-related changes leading to cartilage degradation and bone destruction in order to ultimately provide a preclinical drug screening tool. To this end, the OTM was engineered by co-cultivation of mesenchymal stromal cell (MSC)-derived bone and cartilage components in a 3D environment. It was comprehensively characterized on cell, protein, and mRNA level. Stimulating the OTM with pro-inflammatory cytokines, relevant in RA (tumor necrosis factor α, interleukin-6, macrophage migration inhibitory factor), caused cell- and matrix-related changes, resulting in a significantly induced gene expression of lactate dehydrogenase A, interleukin-8 and tumor necrosis factor α in both, cartilage and bone, while the matrix metalloproteases 1 and 3 were only induced in cartilage. Finally, application of target-specific drugs prevented the induction of inflammation and matrix-degradation. Thus, we here provide evidence that our human in vitro 3D OTM mimics cytokine-induced cell- and matrix-related changes-key features of RA-and may serve as a preclinical tool for the evaluation of both new targets and potential drugs in a more translational setup.

The in vitro human fracture hematoma model - a tool for preclinical drug testing.

The aim of the study was to establish an in vitro fracture hematoma (FH) model that mimics the in vivo situation of the human fracture gap in order to assess drug efficacy and effectiveness for the treatment of fracture healing disorders. Human peripheral blood and mesenchymal stromal cells (MSCs) were coagulated to produce in vitro FH models, which were incubated in osteogenic medium under normoxia/hypoxia and analyzed for cell composition, gene expression and cytokine/chemokine secretion. To evaluate the model, we studied the impact of dexamethasone (impairing fracture healing) and deferoxamine (promoting fracture healing). Under hypoxic conditions, MSCs represented the predominant cell population, while the frequencies of leukocyte populations decreased. Marker gene expression of osteogenesis, angiogenesis, inflammation, migration and hypoxic adaptation increased significantly over time and compared to normoxia, while cytokine/chemokine secretion remained unchanged. Dexamethasone favored the frequency of immune cells compared to MSCs, suppressed osteogenic and pro-angiogenic gene expression, and enhanced the secretion of inflammatory cytokines. Conversely, deferoxamine favored the frequency of MSCs over that of immune cells and enhanced the expression of the osteogenic marker RUNX2 and markers of hypoxic adaptation. In summary, we demonstrate that hypoxia is an important factor for modeling the initial phase of fracture healing in vitro and that both fracture-healing disrupting and promoting substances can influence the in vitro model comparable to the in vivo situation. Therefore, we conclude that our model is able to mimic in part the human FH and could reduce the number of animal experiments in early preclinical studies.

Macroscale mesenchymal condensation to study cytokine-driven cellular and matrix-related changes during cartilage degradation.

Understanding the pathophysiological processes of cartilage degradation requires adequate model systems to develop therapeutic strategies towards osteoarthritis (OA). Although different in vitro or in vivo models have been described, further comprehensive approaches are needed to study specific disease aspects. This study aimed to combine in vitro and in silico modeling based on a tissue-engineering approach using mesenchymal condensation to mimic cytokine-induced cellular and matrix-related changes during cartilage degradation. Thus, scaffold-free cartilage-like constructs (SFCCs) were produced based on self-organization of mesenchymal stromal cells (mesenchymal condensation) and (i) characterized regarding their cellular and matrix composition or secondly (ii) treated with interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα) for 3 weeks to simulate OA-related matrix degradation. In addition, an existing mathematical model based on partial differential equations was optimized and transferred to the underlying settings to simulate the distribution of IL-1ß, type II collagen degradation and cell number reduction. By combining in vitro and in silico methods, we aimed to develop a valid, efficient alternative approach to examine and predict disease progression and effects of new therapeutics.

Impact of Janus Kinase Inhibition with Tofacitinib on Fundamental Processes of Bone Healing.

Both inflammatory diseases like rheumatoid arthritis (RA) and anti-inflammatory treatment of RA with glucocorticoids (GCs) or non-steroidal anti-inflammatory drugs (NSAIDs) negatively influence bone metabolism and fracture healing. Janus kinase (JAK) inhibition with tofacitinib has been demonstrated to act as a potent anti-inflammatory therapeutic agent in the treatment of RA, but its impact on the fundamental processes of bone regeneration is currently controversially discussed and at least in part elusive. Therefore, in this study, we aimed to examine the effects of tofacitinib on processes of bone healing focusing on recruitment of human mesenchymal stromal cells (hMSCs) into the inflammatory microenvironment of the fracture gap, chondrogenesis, osteogenesis and osteoclastogenesis. We performed our analyses under conditions of reduced oxygen availability in order to mimic the in vivo situation of the fracture gap most optimal. We demonstrate that tofacitinib dose-dependently promotes the recruitment of hMSCs under hypoxia but inhibits recruitment of hMSCs under normoxia. With regard to the chondrogenic differentiation of hMSCs, we demonstrate that tofacitinib does not inhibit survival at therapeutically relevant doses of 10-100 nM. Moreover, tofacitinib dose-dependently enhances osteogenic differentiation of hMSCs and reduces osteoclast differentiation and activity. We conclude from our data that tofacitinib may influence bone healing by promotion of hMSC recruitment into the hypoxic microenvironment of the fracture gap but does not interfere with the cartilaginous phase of the soft callus phase of fracture healing process. We assume that tofacitinib may promote bone formation and reduce bone resorption, which could in part explain the positive impact of tofacitinib on bone erosions in RA. Thus, we hypothesize that it will be unnecessary to stop this medication in case of fracture and suggest that positive effects on osteoporosis are likely.

Hypoxia and mesenchymal stromal cells as key drivers of initial fracture healing in an equine in vitro fracture hematoma model.

Fractures in horses-whether simple fractures with just one clean break, or incomplete greenstick with stress fractures, or complications such as shattered bones can all be either minimal or even catastrophic. Thus, improvement in fracture healing is a hallmark in equine orthopedics. The fracture healing process implements a complex sequence of events including the initial inflammatory phase removing damaged tissue, re-establishment of vessels and mesenchymal stromal cells, a soft and hard callus phase closing the fracture gap as well as the remodeling phase shaping the bone to a scar-free tissue. Detailed knowledge on processes in equine fracture healing in general and on the initial phase in particular is apparently very limited. Therefore, we generated equine in vitro fracture hematoma models (FH models) to study time-dependent changes in cell composition and RNA-expression for the most prominent cells in the FH model (immune cells, mesenchymal stromal cells) under conditions most closely adapted to the in vivo situation (hypoxia) by using flow cytometry and qPCR. In order to analyze the impact of mesenchymal stromal cells in greater detail, we also incubated blood clots without the addition of mesenchymal stromal cells under the same conditions as a control. We observed a superior survival capacity of mesenchymal stromal cells over immune cells within our FH model maintained under hypoxia. Furthermore, we demonstrate an upregulation of relevant angiogenic, osteogenic and hypoxia-induced markers within 48 h, a time well-known to be crucial for proper fracture healing.

Collagen I-based scaffolds negatively impact fracture healing in a mouse-osteotomy-model although used routinely in research and clinical application.

Although several biomaterials for bone regeneration have been developed in the last decades, clinical application of bone morphogenetic protein 2 is clinically only approved when applied on an absorbable bovine collagen I scaffold (ACS) (Helistat; ACS-H). In research, another ACS, namely Lyostypt (ACS-L) is frequently used as a scaffold in bone-linked studies. Nevertheless, until today, the influence of ACS alone on bone healing remains unknown. Unexpectedly, in vitro studies using ASC-H revealed a suppression of osteogenic differentiation and a significant reduction of cell vitality when compared to ASC-L. In mice, we observed a significant delay in bone healing when applying ACS-L in the fracture gap during femoral osteotomy. The results of our study show for the first time a negative influence of both ACS-H and ACS-L on bone formation demonstrating a substantial need for more sophisticated delivery systems for local stimulation of bone healing in both clinical application and research. STATEMENT OF SIGNIFICANCE: Our study provides evidence-based justification to promote the development and approval of more suitable and sophisticated delivery systems in bone healing research. Additionally, we stimulate researchers of the field to consider that the application of those scaffolds as a delivery system for new substances represents a delayed healing approach rather than a normal bone healing which could greatly impact the outcome of those studies and play a pivotal role in the translation to the clinics. Moreover, we provide impulses on underlying mechanism involving the roles of small-leucine rich proteoglycans (SLRP) for further detailed investigations.

CTLA-4 Mediates Inhibitory Function of Mesenchymal Stem/Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) are stem cells of the connective tissue, possess a plastic phenotype, and are able to differentiate into various tissues. Besides their role in tissue regeneration, MSCs perform additional functions as a modulator or inhibitor of immune responses. Due to their pleiotropic function, MSCs have also gained therapeutic importance for the treatment of autoimmune diseases and for improving fracture healing and cartilage regeneration. However, the therapeutic/immunomodulatory mode of action of MSCs is largely unknown. Here, we describe that MSCs express the inhibitory receptor CTLA-4 (cytotoxic T lymphocyte antigen 4). We show that depending on the environmental conditions, MSCs express different isoforms of CTLA-4 with the secreted isoform (sCTLA-4) being the most abundant under hypoxic conditions. Furthermore, we demonstrate that the immunosuppressive function of MSCs is mediated mainly by the secretion of CTLA-4. These findings open new ways for treatment when tissue regeneration/fracture healing is difficult.

Journey into Bone Models: A Review.

Bone is a complex tissue with a variety of functions, such as providing mechanical stability for locomotion, protection of the inner organs, mineral homeostasis and haematopoiesis. To fulfil these diverse roles in the human body, bone consists of a multitude of different cells and an extracellular matrix that is mechanically stable, yet flexible at the same time. Unlike most tissues, bone is under constant renewal facilitated by a coordinated interaction of bone-forming and bone-resorbing cells. It is thus challenging to recreate bone in its complexity in vitro and most current models rather focus on certain aspects of bone biology that are of relevance for the research question addressed. In addition, animal models are still regarded as the gold-standard in the context of bone biology and pathology, especially for the development of novel treatment strategies. However, species-specific differences impede the translation of findings from animal models to humans. The current review summarizes and discusses the latest developments in bone tissue engineering and organoid culture including suitable cell sources, extracellular matrices and microfluidic bioreactor systems. With available technology in mind, a best possible bone model will be hypothesized. Furthermore, the future need and application of such a complex model will be discussed.

Antibodies against chemokine receptors CXCR3 and CXCR4 predict progressive deterioration of lung function in patients with systemic sclerosis.

BACKGROUND: The chemokine receptors CXCR3 and CXCR4 are involved in the pathogenesis of fibrosis, a key feature of systemic sclerosis (SSc). It is hypothesized that immunoglobulin (Ig)G antibodies (abs) against these two receptors are present in patients with SSc and are associated with clinical findings. METHODS: Anti-CXCR3 and anti-CXCR4 ab levels were measured in 449 sera from 327 SSc patients and in 234 sera from healthy donors (HD) by enzyme-linked immunosorbent assay (ELISA). In SSc, ab levels were compared with clinical data in a cross-sectional and longitudinal setting. Protein expression of CXCR3 and CXCR4 on peripheral blood mononuclear cells (PBMCs) was analyzed in 17 SSc patients and 8 HD by flow cytometry. RESULTS: Anti-CXCR3 and anti-CXCR4 ab levels were different among SSc subgroups compared with HD and were highest in diffuse SSc patients. The ab levels strongly correlated with each other (r = 0.85). Patients with SSc-related interstitial lung disease (SSc-ILD) exhibited higher ab levels which negatively correlated with lung function parameters (e.g., r = -0.5 and r = -0.43 for predicted vital capacity, respectively). However, patients with deterioration of lung function showed lower anti-CXCR3/4 ab levels compared with those with stable disease. Frequencies and median fluorescence intensities (MFI) of CXCR3+ and CXCR4+ PBMCs were lower in SSc patients compared with HD and correlated with the severity of skin and lung fibrosis. They correlated with the severity of skin and lung fibrosis. CONCLUSIONS: Anti-CXCR3/4 abs and their corresponding receptors are linked with the severity of SSc-ILD. Antibody levels discriminate patients with stable or decreasing lung function and could be used for risk stratification.

Optimization of a nonviral transfection system to evaluate Cox-2 controlled interleukin-4 expression for osteoarthritis gene therapy in vitro.

BACKGROUND: Gene therapy appears to have the potential for achieving a long-term remedy for osteoarthritis (OA). However, there is a risk of adverse reactions, especially when using cytomegalovirus-controlled expression. To provide a safe application, we focused on the expression of therapeutic cytokines [e.g. interleukin (IL)-4] in a disease-responsive manner by use of the previously cloned Cox-2 promoter as 'genetic switch'. In the present study, we report the functionality of a controlled gene therapeutic system in an equine osteoarthritic cell model. METHODS: Different nonviral transfection reagents were tested for their efficiency on equine chondrocytes stimulated with equine IL-1ß or lipopolysaccharide to create an inflammatory environment. To optimize the transfection, we successfully redesigned the vector by excluding the internal ribosomal entry site (IRES). The functionality of our Cox-2 promoter construct with respect to expressing IL-4 was proven at the mRNA and protein levels and the anti-inflammatory potential of IL-4 was confirmed by analyzing the expression of IL-1ß, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-3 and tumor necrosis factor (TNF)-α using a quantitative polymerase chain reaction. RESULTS: Nonviral transfection reagents yielded transfection rates from 21% to 44% with control vectors with and without IRES, respectively. Stimulation of equine chondrocytes resulted in a 20-fold increase of mRNA expression of IL-1ß. Such exogenous stimulation of chondrocytes transfected with pNCox2-IL4 led to an increase of IL-4 mRNA expression, whereas expression of inflammatory mediators decreased. The timely link between these events confirms the anti-inflammatory potential of synthesized IL-4. CONCLUSIONS: We consider that this approach has significant potential for translation into a useful anti-inflammation therapy. Molecular tools such as the described therapeutic plasmid pave the way for a local-controlled, self-limiting gene therapy.