Mitochondrial Dysfunction and Oxidative Stress in Rheumatoid Arthritis.

Control of excessive mitochondrial oxidative stress could provide new targets for both preventive and therapeutic interventions in the treatment of chronic inflammation or any pathology that develops under an inflammatory scenario, such as rheumatoid arthritis (RA). Increasing evidence has demonstrated the role of mitochondrial alterations in autoimmune diseases mainly due to the interplay between metabolism and innate immunity, but also in the modulation of inflammatory response of resident cells, such as synoviocytes. Thus, mitochondrial dysfunction derived from several danger signals could activate tricarboxylic acid (TCA) disruption, thereby favoring a vicious cycle of oxidative/mitochondrial stress. Mitochondrial dysfunction can act through modulating innate immunity via redox-sensitive inflammatory pathways or direct activation of the inflammasome. Besides, mitochondria also have a central role in regulating cell death, which is deeply altered in RA. Additionally, multiple evidence suggests that pathological processes in RA can be shaped by epigenetic mechanisms and that in turn, mitochondria are involved in epigenetic regulation. Finally, we will discuss about the involvement of some dietary components in the onset and progression of RA.

Identification of Exosomal MicroRNA Signature by Liquid Biopsy in Hereditary Hemorrhagic Telangiectasia Patients.

Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant vascular dysplasia characterized by epistaxis, mucocutaneous telangiectases, and arteriovenous malformations (AVM) in the visceral organs. The diagnosis of HHT is based on clinical Curaçao criteria, which show limited sensitivity in children and young patients. Here, we carried out a liquid biopsy by which we isolated total RNA from plasma exosome samples. A cohort of 15 HHT type 1 patients, 15 HHT type 2 patients, and 10 healthy relatives were analyzed. Upon gene expression data processing and normalization, a statistical analysis was performed to explore similarities in microRNA expression patterns among samples and detect differentially expressed microRNAs between HHT samples and the control group. We found a disease-associated molecular fingerprint of 35 miRNAs over-represented in HHT vs. controls, with eight being specific for HHT1 and 11 for HHT2; we also found 30 under-represented, including nine distinct for HHT1 and nine for HHT2. The analysis of the receiver operating characteristic (ROC) curves showed that eight miRNAs had good (AUC > 75%) or excellent (AUC > 90%) diagnosis value for HHT and even for type HHT1 and HHT2. In addition, we identified the cellular origin of these miRNAs among the cell types involved in the vascular malformations. Interestingly, we found that only some of them were incorporated into exosomes, which suggests a key functional role of these exosomal miRNAs in the pathophysiology of HHT.

Accurate Prediction of Sensory Attributes of Cheese Using Near-Infrared Spectroscopy Based on Artificial Neural Network.

The acceptance of a food product by the consumer depends, as the most important factor, on its sensory properties. Therefore, it is clear that the food industry needs to know the perceptions of sensory attributes to know the acceptability of a product. There exist procedures that systematically allows measurement of these property perceptions that are performed by professional panels. However, systematic evaluations of attributes by these tasting panels, which avoid the subjective character for an individual taster, have a high economic, temporal and organizational cost. The process is only applied in a sampled way so that its result cannot be used on a sound and complete quality system. In this paper, we present a method that allows making use of a non-destructive measurement of physical-chemical properties of the target product to obtain an estimation of the sensory description given by QDA-based procedure. More concisely, we propose that through Artificial Neural Networks (ANNs), we will obtain a reliable prediction that will relate the near-infrared (NIR) spectrum of a complete set of cheese samples with a complete image of the sensory attributes that describe taste, texture, aspect, smell and other relevant sensations.

Human Cartilage Engineering in an In Vitro Repair Model Using Collagen Scaffolds and Mesenchymal Stromal Cells.

The purpose of this study was to investigate cartilage repair of in vitro lesion models using human bone marrow mesenchymal stromal cells (hBMSCs) with different collagen (Col) scaffolds. Lesions were made in human cartilage biopsies. Injured samples were pre-treated with interleukin 1ß (IL1ß) for 24 h; also, samples were not pre-treated. hBMSCs were seeded on different types of collagen scaffolds. The resulting constructs were placed into the lesions, and the biopsies were cultured for 2 months in chondrogenic medium. Using the modified ICRSII scale, neotissues from the different scaffolds showed ICRS II overall assessment scores ranging from 50% (fibrocartilage) to 100% (hyaline cartilage), except for the Col I +Col II +HS constructs (fibrocartilage/hyaline cartilage, 73%). Data showed that hBMSCs cultured only on Col I +Col II +HS scaffolds displayed a chondrocyte-like morphology and cartilage-like matrix close to native cartilage. Furthermore, IL1ß pre-treated biopsies decreased capacity for repair by hBMSCs and decreased levels of chondrogenic phenotype of human cartilage lesions.

The role of osmolarity adjusting agents in the regulation of encapsulated cell behavior to provide a safer and more predictable delivery of therapeutics.

Transplantation of cells within alginate microspheres has been extensively studied for sustained drug delivery. However, the lack of control over cell behavior represents a major concern regarding the efficacy and the safety of the therapy. Here, we demonstrated that when formulating the biosystem, an adequate selection of osmolarity adjusting agents significantly contributes to the regulation of cell responses. Our data showed that these agents interact in the capsule formation process, influencing the alginate crosslinking degree. Therefore, when selecting inert or electrolyte-based osmolarity adjusting agents to encapsulate D1 multipotent mesenchymal stromal cells (MSCs), alginate microcapsules with differing mechanical properties were obtained. Since mechanical forces acting on cells influence their behavior, contrasting cell responses were observed both, in vitro and in vivo. When employing mannitol as an inert osmolarity adjusting agent, microcapsules presented a more permissive matrix, allowing a tumoral-like behavior. This resulted in the formation of enormous cell-aggregates that presented necrotic cores and protruding peripheral cells, rendering the therapy unpredictable, dysfunctional, and unsafe. Conversely, the use of electrolyte osmolarity adjusting agents, including calcium or sodium, provided the capsule with a suitable crosslinking degree that established a tight control over cell proliferation and enabled an adequate therapeutic regimen in vivo. The crucial impact of these agents was confirmed when gene expression studies reported pivotal divergences not only in proliferative pathways, but also in genes involved in survival, migration, and differentiation. Altogether, our results prove osmolarity adjusting agents as an effective tool to regulate cell behavior and obtain safer and more predictable therapies.

Improved control over MSCs behavior within 3D matrices by using different cell loads in both in vitro and in vivo environments.

The combination of multipotent mesenchymal stromal cells (MSCs) and different biomaterials has led to enormous advances in cell-based therapies, among which cell microencapsulation technologies are included. In the present work, we have studied the influence of different cell densities on the behavior of erythropoietin (EPO)-secreting MSCs immobilized in alginate microcapsules for their use as drug delivery systems. In vitro studies showed a more sustained and controlled EPO-secretion in groups with higher cell densities, which may be related to a more balanced renewal of the encapsulated cells, while low and intermediate densities gave rise to a continuous increase of both the number of cells and the EPO secretion levels. However, in vivo studies depicted a completely different scenario. Here the higher levels of cell proliferation led to a rapid space saturation and oxygen depletion of the capsule core, which eventually resulted in implant failure for the highest cell loads. On the contrary, lower cell densities showed a longer lasting release with a steadily increasing secretion profile. In conclusion, these results demonstrate how the final outcome of a cell-based drug delivery system may be tuned by just modifying the initial cell load, always taking into account the surrounding microenvironment.

* Human Amniotic Mesenchymal Stromal Cells as Favorable Source for Cartilage Repair.

INTRODUCTION: Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. MATERIAL AND METHODS: A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. RESULTS: HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. CONCLUSION: HAM is a useful material for articular cartilage repair in vitro when used as scaffold. In combination with hAMSCs, HAM showed better potential for cartilage repair with similar reparation capacity than chondrocytes.

Hybrid Alginate-Protein-Coated Graphene Oxide Microcapsules Enhance the Functionality of Erythropoietin Secreting C2C12 Myoblasts.

The beneficial effect of combining alginate hydrogel with graphene oxide (GO) on microencapsulated C2C12-myoblast viability has recently been described. However, the commercially available GO lacks homogeneity in size, this parameter being of high relevance for the cell fate in two-dimensional studies. In three-dimensional applications the capacity of this material for binding different kinds of proteins can result in the reduction of de novo released protein that can effectively reach the vicinity of the microcapsules. Undoubtedly, this could be an important hurdle in its clinical use when combined with alginate-PLL microcapsules. Here, we demonstrate that the homogenization of GO nanoparticles is not a mandatory preparation step in order to get the best of this material upon cell microencapsulation. In fact, when the superficial area of these particles is increased, higher amounts of the therapeutic protein erythropoietin (EPO) are adsorbed on their surface. On the other hand, we have been able to improve even more the favorable effects of this graphene derivative on microencapsulated cell viability by forming a protein biocorona. These proteins block the potential binding sites of EPO and, therefore, enhance the amount of therapeutic drug that is released. Finally, we prove that these hybrid alginate-protein-coated GO-microcapsules are functional in vivo.

Brief Report: European Mitochondrial Haplogroups Impact on Liver Fibrosis Progression Among HCV and HIV/HCV-Coinfected Patients From Northwest Spain.

The impact of mitochondrial DNA haplogroups on the outcome of liver fibrosis was evaluated in 362 hepatitis C virus infection (HCV)-monoinfected and HIV/HCV-coinfected patients (147 and 215, respectively) in clinical follow-up at 2 reference hospitals in the Northwest of Spain. The mitochondrial DNA haplogroup H was the most prevalent (50.3%) in this population. The cluster Others and V were recognized as risk factors for the development of liver fibrosis while haplogroup H and HCV genotype 4 confer a lower risk. This information might be useful for prioritization of HCV treatment, especially for F0-F1 patients for whom there is no urgency for treatment.

Perioperative Standard Oral Nutrition Supplements Versus Immunonutrition in Patients Undergoing Colorectal Resection in an Enhanced Recovery (ERAS) Protocol: A Multicenter Randomized Clinical Trial (SONVI Study).

To compare immunonutrition versus standard high calorie nutrition in patients undergoing elective colorectal resection within an Enhanced Recovery After Surgery (ERAS) program.Despite progress in recent years in the surgical management of patients with colorectal cancer (ERAS programs), postoperative complications are frequent. Nutritional supplements enriched with immunonutrients have recently been introduced into clinical practice. However, the extent to which the combination of ERAS protocols and immunonutrition benefits patients undergoing colorectal cancer surgery is unknown.The SONVI study is a prospective, multicenter, randomized trial with 2 parallel treatment groups receiving either the study product (an immune-enhancing feed) or the control supplement (a hypercaloric hypernitrogenous supplement) for 7 days before colorectal resection and 5 days postoperatively.A total of 264 patients were randomized. At baseline, both groups were comparable in regards to age, sex, surgical risk, comorbidity, and analytical and nutritional parameters. The median length of the postoperative hospital stay was 5 days with no differences between the groups. A decrease in the total number of complications was observed in the immunonutrition group compared with the control group, primarily due to a significant decrease in infectious complications (23.8% vs. 10.7%, P = 0.0007). Of the infectious complications, wound infection differed significantly between the groups (16.4% vs. 5.7%, P = 0.0008). Other infectious complications were lower in the immunonutrition group but were not statistically significantly different.The implementation of ERAS protocols including immunonutrient-enriched supplements reduces the complications of patients undergoing colorectal resection.This study is registered with ClinicalTrial.gov: NCT02393976.

Proteomic Analysis of Connexin 43 Reveals Novel Interactors Related to Osteoarthritis.

We have previously reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells. Cell-to-cell communication occurs through connexin channels termed Gap Junction (GJ) channels, which achieve direct cellular communication by allowing the intercellular exchange of ions, small RNAs, nutrients, and second messengers. The Cx43 protein is overexpressed in several human diseases and inflammation processes and in articular cartilage from patients with osteoarthritis (OA). An increase in the level of Cx43 is known to alter gene expression, cell signaling, growth, and cell proliferation. The interaction of proteins with the C-terminal tail of connexin 43 (Cx43) directly modulates GJ-dependent and -independent functions. Here, we describe the isolation of Cx43 complexes using mild extraction conditions and immunoaffinity purification. Cx43 complexes were extracted from human primary articular chondrocytes isolated from healthy donors and patients with OA. The proteomic content of the native complexes was determined using LC-MS/MS, and protein associations with Cx43 were validated using Western blot and immunolocalization experiments. We identified >100 Cx43-associated proteins including previously uncharacterized proteins related to nucleolar functions, RNA transport, and translation. We also identified several proteins involved in human diseases, cartilage structure, and OA as novel functional Cx43 interactors, which emphasized the importance of Cx43 in the normal physiology and structural and functional integrity of chondrocytes and articular cartilage. Gene Ontology (GO) terms of the proteins identified in the OA samples showed an enrichment of Cx43-interactors related to cell adhesion, calmodulin binding, the nucleolus, and the cytoskeleton in OA samples compared with healthy samples. However, the mitochondrial proteins SOD2 and ATP5J2 were identified only in samples from healthy donors. The identification of Cx43 interactors will provide clues to the functions of Cx43 in human cells and its roles in the development of several diseases, including OA.

Multifunctional hydrogel-based scaffold for improving the functionality of encapsulated therapeutic cells and reducing inflammatory response.

Since the introduction of cell immunoisolation as an alternative to protect transplanted cells from host immune attack, much effort has been made to develop this technology into a realistic clinical proposal. Several promising approaches have been investigated to resolve the biotechnological and biosafety challenges related to cell microencapsulation. Here, a multifunctional hydrogel-based scaffold consisting of cell-loaded alginate-poly-l-lysine-alginate (APA) microcapsules and dexamethasone (DXM)-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres embedded in alginate hydrogel is developed and evaluated. Initially, the feasibility of using an alginate hydrogel for enclosing APA microcapsules was studied in a xenogeneic approach. In addition, the performance of the local release of DXM was addressed. The in vitro studies confirmed the correct adaptation of the enclosed cells to the scaffolds in terms of metabolic activity and viability. The posterior implantation of the hydrogel-based scaffolds containing cell-loaded microcapsules revealed that the hematocrit levels were maintained high and constant, and the pericapsular overgrowth was reduced in the DXM-treated rats for at least 2months. This multifunctional scaffold might have a synergistic effect: (1) providing a physical support for APA microcapsules, facilitating administration, ensuring retention and recuperation and preventing dissemination; and (2) reducing post-transplantation inflammation and foreign body reaction, thus prolonging the lifetime of the implant by the continuous and localized release of DXM.

Hydrogel-based scaffolds for enclosing encapsulated therapeutic cells.

Cell encapsulation technology holds promise for the sustained and controlled delivery of different therapeutic proteins. Alginate-poly-L-lysine-alginate (APA) microcapsules represent one of the most widely studied alginate-polycation microcapsules. On the basis of this technology, two types of hydrogel-based scaffolds have been developed and analyzed with the aim of improving the retention and the retrieval of erythropoietin (Epo) secreting cell-loaded microcapsules in the tissue where they are implanted. Furthermore, these hydrogels may help to reduce the post-transplant inflammation and pericapsular fibrotic overgrowth typically observed around capsules. The hydrogel-based scaffolds could be administered as implantable forms (preformed scaffolds) or injectable forms (in situ formed scaffolds). The in vitro studies confirmed the correct adaptation of the enclosed cells to the scaffolds in terms of viability and protein expression. The posterior implantation of the cell-loaded capsules containing hydrogel-based scaffolds in mice revealed that the hematocrit levels were maintained up to 80% for at least 2 months. The histological analysis of the explanted microcapsules performed at that point showed that pericapsular overgrowth was reduced when cell-loaded microcapsules were enclosed in the hydrogels scaffolds. Incorporating microencapsulated cells within hydrogel-based scaffolds may help to improve their administration protocol and retention while reducing post-transplantation inflammation.

The Splicing Factor SRSF1 as a Marker for Endothelial Senescence.

Aging is the major risk factor per se for the development of cardiovascular diseases. The senescence of the endothelial cells (ECs) that line the lumen of blood vessels is the cellular basis for these age-dependent vascular pathologies, including atherosclerosis and hypertension. During their lifespan, ECs may reach a stage of senescence by two different pathways; a replicative one derived from their preprogrammed finite number of cell divisions; and one induced by stress stimuli. Also, certain physiological stimuli, such as transforming growth factor-ß, are able to modulate cellular senescence. Currently, the cellular aging process is being widely studied to identify novel molecular markers whose changes correlate with senescence. This review focuses on the regulation of alternative splicing mediated by the serine-arginine splicing factor 1 (SRSF1, or ASF/SF2) during endothelial senescence, a process that is associated with a differential subcellular localization of SRSF1, which typically exhibits a scattered distribution throughout the cytoplasm. Based on its senescence-dependent involvement in alternative splicing, we postulate that SRSF1 is a key marker of EC senescence, regulating the expression of alternative isoforms of target genes such as endoglin (ENG), vascular endothelial growth factor A (VEGFA), tissue factor (T3), or lamin A (LMNA) that integrate in a common molecular senescence program.

[Descriptive study of the use of DMARD in patients with rheumatoid arthritis or persistent arthritis who start drug treatment in Spain (FIRST)]. / Estudio descriptivo de la utilización de los FAMES en los pacientes con artritis reumatoide o artritis persistente que inician tratamiento farmacológico en España. (ESTUDIO FIRST).

INTRODUCTION: Rheumatoid arthritis is clinically very heterogeneous and variable in its progression, and no one treatment works the same for all patients, as this will depend on the clinical course and specific situations. OBJECTIVE: To describe the treatment with DMARDs established for the first time in patients with rheumatoid arthritis (RA) or persistent arthritis (PA) in routine clinical practice in Spain. MATERIAL AND METHODS: Epidemiological, cross-sectional, uncontrolled, multicenter study in 15 regions of Spain during a period of five months (July to November 2006). We included patients of both genders, aged 18 years and diagnosed with RA according to ACR criteria or PA defined as any arthritis (oligoarthritis or polyarthritis) lasting ≥12 weeks, which would be given DMARD to treat their disease. RESULTS: 1079 patients were recruited, 915 analyzed (33% ♂/♀ 67%) meeting all the criteria required to be evaluated in the study. Mean age of patients was 54.6 (SD=15.4) years. The mean time from onset of symptoms until the 1st visit with the rheumatologist was 6.3 (11.3) months and the time from the 1st visit with the rheumatologist and the start of treatment was 4 (13.5) months. Of the patients tested, 96.7% was treated with at least one DMARD, 62.1% were given NSAIDs, corticosteroids to 59.2% and 3.8% biological therapy. In patients who received DMARDs, 90.3% received treatment with a single DMARD, 9.5% with 2 DMARDs and 0.2% with three DMARDs. In polytherapy, the DMARDs that are most often administered together were MTX + hydroxychloroquine (4.8%), MTX + leflunomide (2.0%) and MTX + sulfasalazine (1.5%). The most frequently used DMARD in monotherapy was MTX (81.3%), followed by leflunomide (4.1%) and hydroxychloroquine (3.2%). In 89.6%, the treatment of first choice was adequate according to the SER. CONCLUSION: The most common pattern of initial treatment of RA is MTX monotherapy. Treatment of RA by rheumatologists has been homogenized in recent years.

Estudio descriptivo de la utilización de los FAMES en los pacientes con artritis reumatoide o artritis persistente que inician tratamiento farmacológico en España. (ESTUDIO FIRST) / Descriptive study of the use of DMARD in patients with Rheumatoid arthritis or persistent arthritis who start drug treatment in Spain (FIRST)

Introducción. La artritis reumatoide es clínicamente muy heterogénea y variable en su evolución, lo que ocasiona que no se pueda detallar un mismo tratamiento para todos los pacientes, ya que éste va a depender del curso clínico y de situaciones concretas que se van a presentar a lo largo del mismo. Objetivo. Realizar una descripción del tratamiento con fármacos modificadores de la enfermedad (FAME) que se instauran por primera vez en pacientes con artritis reumatoide (AR) o artritis persistente (AP) en la práctica clínica habitual en España. Material y métodos. Estudio epidemiológico, transversal, no controlado, multicéntrico realizado en 15 comunidades autónomas de España durante un período de 5 meses (julio a noviembre del 2006). Se incluyeron pacientes de ambos sexos, mayores de 18 años y diagnosticados de AR según los criterios de la ACR o bien de AP definida como toda artritis (oligoartritis o poliartritis) ≥12 semanas de duración, a los que se les iba a administrar el primer FAME para tratar su enfermedad. Resultados. Se reclutaron 1.079 pacientes, pero finalmente, 915 (33% ♂/67% ♀) cumplieron todos los criterios exigidos para ser evaluados en el estudio. La edad media de los pacientes fue de 54,6 (DE=15,4) años. El tiempo medio desde la aparición de los síntomas hasta la 1.a visita con el reumatólogo fue de 6,3 (11,3) meses y el tiempo desde la 1.a visita con el reumatólogo y el inicio del tratamiento fue de 4 (13,5) meses. Del total de pacientes evaluados, al 96,7% se les instauró tratamiento con al menos un FAME, al 62,1% se les administraron AINE, al 59,2% corticoesteroides y al 3,8% una terapia biológica. En los pacientes que recibieron FAME, el 90,3% recibió tratamiento con un solo FAME, el 9,5% con 2 FAME y el 0,2% con 3 FAME. En politerapia, los FAME que más a menudo se administraron conjuntamente fueron MTX+Hidroxicloroquina (4,8%), MTX+Leflunomida (2,0%) y MTX+Sulfasalazina (1,5%). El FAME más frecuentemente utilizado en monoterapia fue el MTX (81,3%), seguido de la leflunomida (4,1%) y la hidroxicloroquina (3,2%) En el 89,6%, el tratamiento de primera elección fue el adecuado según las recomendaciones de la SER. Conclusión. La pauta de tratamiento de inicio de la AR más frecuente es el MTX en monoterapia. El tratamiento de la AR por los reumatólogos se ha homogeneizado en los últimos años(AU)

Introduction. Rheumatoid arthritis is clinically very heterogeneous and variable in its progression, and no one treatment works the same for all patients, as this will depend on the clinical course and specific situations. Objective. To describe the treatment with DMARDs established for the first time in patients with rheumatoid arthritis (RA) or persistent arthritis (PA) in routine clinical practice in Spain. Material and methods. Epidemiological, cross-sectional, uncontrolled, multicenter study in 15 regions of Spain during a period of five months (July to November 2006). We included patients of both genders, aged 18 years and diagnosed with RA according to ACR criteria or PA defined as any arthritis (oligoarthritis or polyarthritis) lasting ≥12 weeks, which would be given DMARD to treat their disease. Results. 1079 patients were recruited, 915 analyzed (33% ♂/♀ 67%) meeting all the criteria required to be evaluated in the study. Mean age of patients was 54.6 (SD=15.4) years. The mean time from onset of symptoms until the 1st visit with the rheumatologist was 6.3 (11.3) months and the time from the 1st visit with the rheumatologist and the start of treatment was 4 (13.5) months. Of the patients tested, 96.7% was treated with at least one DMARD, 62.1% were given NSAIDs, corticosteroids to 59.2% and 3.8% biological therapy. In patients who received DMARDs, 90.3% received treatment with a single DMARD, 9.5% with 2 DMARDs and 0.2% with three DMARDs. In polytherapy, the DMARDs that are most often administered together were MTX + hydroxychloroquine (4.8%), MTX + leflunomide (2.0%) and MTX + sulfasalazine (1.5%). The most frequently used DMARD in monotherapy was MTX (81.3%), followed by leflunomide (4.1%) and hydroxychloroquine (3.2%). In 89.6%, the treatment of first choice was adequate according to the SER. Conclusion. The most common pattern of initial treatment of RA is MTX monotherapy. Treatment of RA by rheumatologists has been homogenized in recent years(AU)

Umbilical cord as a mesenchymal stem cell source for treating joint pathologies.

Articular cartilage disorders and injuries often result in life-long chronic pain and compromised quality of life. Regrettably, the regeneration of articular cartilage is a continuing challenge for biomedical research. One of the most promising therapeutic approaches is cell-based tissue engineering, which provides a healthy population of cells to the injured site but requires differentiated chondrocytes from an uninjured site. The use of healthy chondrocytes has been found to have limitations. A promising alternative cell population is mesenchymal stem cells (MSCs), known to possess excellent proliferation potential and proven capability for differentiation into chondrocytes. The "immunosuppressive" property of human MSCs makes them an important candidate for allogeneic cell therapy. The use of allogeneic MSCs to repair large defects may prove to be an alternative to current autologous and allogeneic tissue-grafting procedures. An allogeneic cell-based approach would enable MSCs to be isolated from any donor, expanded and cryopreserved in allogeneic MSC banks, providing a readily available source of progenitors for cell replacement therapy. These possibilities have spawned the current exponential growth in stem cell research in pharmaceutical and biotechnology communities. Our objective in this review is to summarize the knowledge about MSCs from umbilical cord stroma and focus mainly on their applications for joint pathologies.

Quantification of cells expressing mesenchymal stem cell markers in healthy and osteoarthritic synovial membranes.

OBJECTIVE: To quantify cells expressing mesenchymal stem cell (MSC) markers in synovial membranes from human osteoarthritic (OA) and healthy joints. METHODS: Synovial membranes from OA and healthy joints were digested with collagenase and the isolated cells were cultured. Synovial membrane-derived cells were phenotypically characterized for differentiation experiments using flow cytometry to detect the expression of mesenchymal markers (CD29, CD44, CD73, CD90, CD105, CD117, CD166, and STRO-1) and hematopoietic markers (CD34 and CD45). Chondrogenesis was assessed by staining for proteoglycans and collagen type II, adipogenesis by using a stain for lipids, and osteogenesis by detecting calcium deposits. Coexpression of CD44, CD73, CD90, and CD105 was determined using immunofluorescence. RESULTS: Cells expressing MSC markers were diffusely distributed in OA synovial membranes; in healthy synovial membrane these cells were localized in the subintimal zone. More numerous MSC markers in OA synovial membranes were observed in cells also expressing the CD90 antigen. FACS analysis showed that more than 90% of OA synovial membrane-derived cells were positive for CD44, CD73, and CD90, and negative for CD34 and CD45. OA synovial membrane-derived cells were also positive for CD29 (85.23%), CD117 (72.35%), CD105 (45.5%), and STRO-1 (49.46%). Micropellet analyses showed that the culture of cells with transforming growth factor-ß3 stimulated proteoglycan and collagen type II synthesis. CONCLUSION: Synovial membranes from patients with OA contain more cells positive for CD44, CD90, and CD105 antigens than those from joints with undamaged cartilage.

Bone marrow cells immunomagnetically selected for CD271+ antigen promote in vitro the repair of articular cartilage defects.

OBJECTIVE: The purposes of this project were to quantify the cells expressing the mesenchymal stem cell (MSC) marker CD271 in synovial membranes from human osteoarthritic (OA) and healthy joints, and to determine if those CD271 cells were involved in spontaneous human cartilage repair and were beneficial for the repair of human articular cartilage defects. METHODS: The coexpression of CD44/CD271, CD90/CD271, and CD105/CD271 antigens was determined by immunofluorescence in OA and healthy synovial membranes and during spontaneous cartilage repair. Isolated MSCs from the bone marrow of four OA patients (mean age: 64 years) were magnetically separated into MSC CD271+ and MSC CD271- subsets. The separated cell subsets were then implanted into 2 mm focal defects of articular cartilage. These implants were cultured in chondrogenic differentiation medium supplemented with recombinant human transforming growth factor-beta3 for 8 weeks. The repair tissues were analyzed by histochemistry (hematoxylin-eosin and safranin O) and immunohistochemistry for collage types I and II. RESULTS: Cells expressing the CD271 antigen were diffusely distributed in OA synovial membranes and localized in the subintimal zone in healthy synovial membranes. The number of cells expressing MSC markers was higher in OA synovial membranes than in synovia from healthy joints, corresponding to the highest level of coexpression of CD90/CD271 antigens (9.8% vs. 2.6%). Spontaneous repair tissue showed more cells expressing the CD271 antigen (9.9% ± 4.0%). The highest levels of expression were found to be associated with CD44; 64% of positive CD271 cells coexpressed the CD44 antigen. In both implant cell types, the repair tissue morphology resembled articular cartilage, having an extracellular matrix with a hyaline aspect and numerous lacunae containing cells, and was immunopositive for collagen types I and II. Statistical analyses of the repair tissue demonstrated that the implantation of MSC CD271+ provided such benefits as a greater filling of the chondral defect and better integration between the repair tissue and native cartilage. Safranin O staining of repair tissue was negative in implants of MSC CD271- but more positive in implants with MSC CD271+. The overall histologic score for CD271- implants was 9.5 ± 0.89 and 12.19 ± 1.01 for CD271+ implants. CONCLUSIONS: Synovial membranes from OA patients contain more cells expressing CD271 antigen than those from healthy joints, and spontaneous cartilage repair tissue contains cells positive for CD271 antigen. These data suggest the involvement of CD271 antigen in spontaneous cartilage repair and indicate that the cell subset MSC CD271+ provides higher quality chondral repair than the CD271- subset.

Design of a composite drug delivery system to prolong functionality of cell-based scaffolds.

Cell encapsulation technology raises hopes in medicine and biotechnology. However, despite important advances in the field in the past three decades, several challenges associated with the biocompatibility are still remaining. In the present study, the effect of a temporary release of an anti-inflammatory agent on co-administered encapsulated allogeneic cells was investigated. The aim was to determine the biocompatibility and efficacy of the approach to prevent the inflammatory response. A composite delivery system comprised of alginate-poly-l-lysine-alginate (APA)-microencapsulated Epo-secreting myoblasts and dexamethasone (DXM)-releasing poly(lactic-co-glycolic acid) (PLGA) microspheres was implanted in the subcutaneous space of Balb/c mice for 45 days. The use of independently co-implanted DXM-loaded PLGA microspheres resulted in an improved functionality of the cell-based graft, evidenced by significantly higher hematocrit levels found in the cell-implanted groups by day 45, which was found to be more pronounced when higher cell-doses (100 µL) were employed. Moreover, no major host reaction was observed upon implantation of the systems, showing good biocompatibility and capability to partially avoid the inflammatory response, probably due to the immunosuppressive effects related to DXM. The findings of this study imply that DXM-loaded PLGA microspheres show promise as release systems to enhance biocompatibility and offer advantage in the development of long-lasting and effective implantable microencapsulated cells by generating a potential immunopriviledged local environment and an effective method to limit the structural ensheathing layer caused by inflammation.