Association between PYTPN22 rs2476601, VEGF rs833070, TNFAIP3 rs6920220 Polymorphisms and Risk for Rheumatoid Arthritis in Early Undifferentiated Arthritis Patients: A Pilot Study.

Background and Objectives: About 40% of early undifferentiated arthritis (UA) progresses to rheumatoid (RA) or other chronic arthritis. Novel diagnostic tools predicting the risk for this progression are needed to identify the patients who would benefit from early aggressive treatment. Evidence on the role of single-nucleotide polymorphisms (SNPs) in the development of RA has emerged. The aim of our study was to investigate the association between rs2476601, rs833070, and rs6920220 SNPs and UA progression to RA. Materials and Methods: Ninety-two UA patients were observed for 12 months. At study entry, demographic and clinical characteristics were recorded, musculoskeletal ultrasonography was performed, and blood samples were drawn to investigate levels of inflammatory markers, rheumatoid factor (RF), anti-citrullinated protein antibodies (anti-CCP)detect SNPs. After 12 months, UA outcomes were assessed, and patients were divided into two (RA and non-RA) groups. The association between the risk of progression to chronic inflammatory arthritis and analyzed SNPs was measured by computing odds ratios (OR). Results: After a 12-month follow-up, 27 (29.3%) patients developed RA, and 65 (70.7%) patients were assigned to the non-RA group. The arthritis of 21 patients (22.8%) from the non-RA group resolved completely, while the other 44 (47.2%) patients were diagnosed with another rheumatic inflammatory disease. The patients who developed RA had a significantly greater number of tender and swollen joints (p = 0.010 and p = 0.021 respectively) and were more frequently RF or anti-CCP (p < 0.001), and both RF and anti-CCP positive (p < 0.001) at the baseline as compared with the patients in the non-RA group. No significant association between rs2476601 (OR = 0.99, p = 0.98), rs833070 (OR = 1.0, p = 0.97), and rs6920220 (OR = 0.48, p = 0.13) polymorphisms and the risk of developing RA were found. Conclusions: No association between analyzed SNPs and a greater risk to progress from UA to RA was confirmed, although patients with rs6920220 AA + AG genotypes had fewer tender joints at the disease onset.

VEGF Profile in Early Undifferentiated Arthritis Cohort.

Background and Objectives: Early undifferentiated arthritis (UA) is a group of inflammatory joint diseases that are not classified under any specific rheumatic or connective tissue disorder and might evolve into chronic inflammatory arthritis or may be a self-limiting condition. Early recognition and treatment are crucial for the future course of the disease. Vascular endothelial growth factor (VEGF) is an angiogenic regulator that induces the growth of new capillary blood vessels, which are important in joint invasion and destruction during the progression of chronic inflammatory arthritis. The aim of this study was to assess VEGF levels associated with sociodemographic, clinical, laboratory, and ultrasound findings in the early UA patient cohort as well as to evaluate VEGF as a potential prognostic marker for arthritis outcomes. Materials and Methods: Seventy-six patients with inflammatory arthritis in at least one joint, with a duration of arthritis <12 months at the study entry that did not meet any rheumatic disease classification criteria, were enrolled after informed consent was obtained. Patient's sociodemographic, laboratory data, and clinical disease characteristics were recorded, VEGF levels were measured, and ultrasound (US) of tender and swollen joints was performed. Results: VEGF levels had positive correlation with conventional rheumatic disease activity and diagnostic markers: erythrocyte sedimentation rate (ESR), C−reactive protein (CRP), and rheumatoid factor (RF) (p < 0.05). RF-positive patients had higher VEGF values (p = 0.024). A statistically higher number of patients whose VEGF levels were below the median value presented with active infection (p = 0.046). In patients with a higher number of swollen joints, and a higher score of synovitis and power doppler (PD) seen on US, VEGF levels were statistically significantly higher. Patients who after 12-month follow-up developed rheumatoid arthritis (RA) had statistically higher VEGF levels at baseline compared with those who developed spondyloarthropathies (p = 0.028). Conclusions: This study demonstrated that VEGF levels significantly represented inflammatory processes that were present in the joints (number of swollen joints, synovitis, and PD changes) of the early UA cohort.

Lyme Disease among Patients at an Ambulatory Unit in a Highly Endemic Country: Lithuania.

Background and objectives: Lyme disease is the most common tick-borne infectious disease in Europe, caused by the spirocheta bacteria of Borrelia burgdorferi. Several genospecies of B. burgdorferi are pathogenic to humans. B. burgdorferi sensu stricto, which is prevalent in North America, causes reactive arthritis, whereas B. garinii and B. afzelii, common in Europe, can affect the skin, heart, or nervous system; it has been shown that the clinical symptoms of the disease may be very different. The objective of this study was to identify the baseline characteristics of Lyme disease and to elucidate the frequency of different Lyme disease syndromes in Lithuania. Materials and Methods: Patients who were diagnosed with Lyme disease during an ambulatory visit to the Center of Infectious Diseases, Vilnius University Santaros clinics, from 2014 to 2016, were enrolled in this study. A retrospective material analysis was conducted. Results: In total, 1005 patients were enrolled with the following prevalence of clinical syndromes: erythema migrans (EM), 945 (94.02%); Lyme arthritis, 32 (3.18%); neuroborreliosis, 23 (2.28%); Lyme carditis, 4 (0.39%); and acrodermatitis, 1 (0.09%). Erythema migrans was dominant among middle-aged women, with a rash appearing mainly on the lower extremities. Lyme arthritis mainly manifested among middle-aged women as an oligoarthritis, mostly affecting the knee joint. Neuroborreliosis was seen more often in middle-aged women than men and the main symptom was nervus facialis neuropathy. Lyme carditis, manifested as an atrioventricular block, with a male/female ratio of 3:1, and the median age was 51. Acrodermatitis was diagnosed in a 61-year-old woman, as a painful, red rash on the hand. Conclusions: According to the prevalence of B. garinii and B. afzelii in Europe, previously it was thought that Lyme disease presented as erythema migrans, and less frequently as neuroborreliosis; however, this study revealed that other syndromes may also be seen. In addition, we revealed that the longer it takes for erythema migrans to appear, the greater the likelihood of Lyme arthritis developing.

The Expression of Inflammasomes NLRP1 and NLRP3, Toll-Like Receptors, and Vitamin D Receptor in Synovial Fibroblasts From Patients With Different Types of Knee Arthritis.

Activated rheumatoid arthritis (RA) synovial fibroblasts (SFs) are among the most important cells promoting RA pathogenesis. They are considered active contributors to the initiation, progression, and perpetuation of the disease; therefore, early detection of RASF activation could advance contemporary diagnosis and adequate treatment of undifferentiated early inflammatory arthritis (EA). In this study, we investigated the expression of nucleotide-binding, oligomerization domain (NOD)-like receptor family, pyrin domain containing (NLRP)1, NLRP3 inflammasomes, Toll-like receptor (TLR)1, TLR2, TLR4, vitamin D receptor (VDR), and secretion of matrix metalloproteinases (MMPs) in SFs isolated from patients with RA, osteoarthritis (OA), EA, and control individuals (CN) after knee surgical intervention. C-reactive protein, general blood test, anticyclic citrullinated peptide (anti-CCP), rheumatoid factor (RF), and vitamin D (vitD) in patients' sera were performed. Cells were stimulated or not with 100 ng/ml tumor necrosis factor alpha (TNF-α) or/and 1 nM or/and 0.01 nM vitamin D3 for 72 h. The expression levels of NLRP1, NLRP3, TLR1, TLR2, TLR4, and VDR in all examined SFs were analyzed by quantitative real-time PCR (RT-qPCR). Additionally, the secretion of IL-1ß by SFs and MMPs were determined by ELISA and Luminex technology. The expression of NLRP3 was correlated with the levels of CRP, RF, and anti-CCP, suggesting its implication in SF inflammatory activation. In the TNF-α-stimulated SFs, a significantly lower expression of NLRP3 and TLR4 was observed in the RA group, compared with the other tested forms of arthritis. Moreover, upregulation of NLRP3 expression by TNF-α alone or in combination with vitD3 was observed, further indicating involvement of NLRP3 in the inflammatory responses of SFs. Secretion of IL-1ß was not detected in any sample, while TNF-α upregulated the levels of secreted MMP-1, MMP-7, MMP-8, MMP-12, and MMP-13 in all patient groups. Attenuating effects of vitD on the expression of NLRP3, TLR1, and TLR4 suggest potential protective effects of vitD on the inflammatory responses in SFs. However, longer studies may be needed to confirm or fully rule out the potential implication of vitD in SF activation in inflammatory arthritis. Both VDR and NLRP3 in the TNF-α-stimulated SFs negatively correlated with the age of patients, suggesting potential age-related changes in the local inflammatory responses.

Epidemiology of Lyme Disease in a Highly Endemic European Zone.

Background and objective: Lyme disease, also known as Lyme borreliosis (LB), is a tick-borne infectious disease caused by the spirochete bacteria Borrelia. The risk of infection depends on the geographical area, ecological factors, and human behavior. Clinical manifestations of Lyme borreliosis have a wide range, but the most frequent clinical symptom, which is also a diagnostic symptom, is a skin rash called erythema migrans (EM). The disease is very common worldwide. In Lithuania, the disease frequency is 99.9 cases per 100,000 population (Centre for Communicable Diseases and AIDS, Lithuania, 2017). The main aim of this study was to obtain the baseline characteristics of the disease regarding the infected Lithuanian population. Materials and Methods: We analyzed data from the Centre for Communicable Diseases and AIDS about all Lyme disease (A69.2) diagnosed patients over a three-year period (from 2014 to 2016) in Lithuania. Results: In 2014-2016, 7424 (crude incidence rate 85.4) cases with LB were diagnosed in Lithuania. Most of them (4633 (62.4%)) were identified in women. Older people were more likely to suffer from LB. Urban residents were 2.6 times more often affected that those living in villages. Tick bites were primarily observed in high season months, from May to September (90%), with the highest peak in July. There was a higher number of observed tick bites (p = 0.003) in the urban residents. Erythema migrans occurred in 75.6% LB cases, while other symptoms did not exceed a quarter of all LB cases. There were 7353 (99.6%) cases where LB was confirmed via clinical symptoms and/or laboratory tests. Also, 1720 (23.2%) patients were tested for LB immunoglobulins. Conclusions: This study found a high incidence of Lyme disease in Lithuania. We elucidated the baseline characteristics regarding the infected Lithuanian population which may ease medical clinicians' work on new Lyme diagnoses.

Validation of Lithuanian Arthroplasty Register Telephone Survey of 2769 Patients Operated for Total Knee Replacement.

Background and objectives: The aim of our study is to validate the registration of knee arthroplasty revisions in the Lithuanian Arthroplasty Register (LAR) and thus give an indication of the accuracy of the published revision rates. Materials and methods: A total of 4269 primary total knee arthroplasties (TKAs) registered in the LAR between 2013 and 2015 were included. Two years after surgery the patients were contacted by phone in order to inquire if they had been subject to revision. The information from the patients was then cross checked against what had been registered in the LAR, and in case of a revision not having been registered hospital charts were investigated. Thus, the patients were followed up with regarding revision and/or death until 2017. A true revision was defined as an addition, exchange, or removal of one or all components. Results: Out of 4269 primary TKAs, we managed to contact and interview 2769 patients. Nine small hospitals were not able to provide contact details (telephone numbers) for 533 patients (549 knees). Sixty-seven patients (67 knees) were deceased (data from the Lithuanian National Census Register) and a further 438 patients (565 knees) appeared to have a wrong or non-valid telephone number, leaving 3031 (3091 knees) patients being contacted. Of those, 262 patients (266 knees) refused to participate in the study which left 2769 responders (2825 knees). Sixty-one patients said that reoperation had been performed on the index knee within two years of their primary surgery. After checking with the clinics, 10 were surgical procedures on the knee but not true revisions by our criteria. Out of the 51 true revisions we found that 46 were registered to the LAR as revised, while five (9.8%) revisions were missing. Conclusions: We conclude that the Lithuanian Arthroplasty Register has a good completeness of registered revision TKAs as only 9.8% of revisions were missing.

Inadequate evaluation and management of suspected -infections after TKA surgery in Lithuania: a retrospective study of 2,769 patients with 2-year follow-up.

Background and purpose - The evidence-based algorithms for treatment of periprosthetic joint infection (PJI) recommend surgical intervention in combination with the use of systemic antibiotics. However, still it is not unusual to treat total knee arthroplasty (TKA) patients with suspected infection using only antibiotics. We investigated treatment pathways for TKA patients with suspected infection in Lithuania. Patients and methods - Of the 4,069 TKA patients (4,269 knees) registered in the Lithuanian Arthroplasty Register (2013-2015) 2,769 patients (2,825 knees) were interviewed 2 years after the surgery. The patients were asked if they had been subject to antibiotic treatment after the TKA surgery and/or if any additional surgical interventions on the operated knee had been performed. The number of patients treated with antibiotics due to problems in the operated knee was identified and cumulative revision rates (CRR) were calculated. Results - 180 (7%) patients of the total 2,769 reported that they had been prescribed antibiotics after the primary TKA; 132 of these patients (70%) said they had received antibiotics due to problems with the operated knee. The 2-year CRR after TKA in patients not treated with antibiotics was 0.7% (95% CI 0.4-1), as compared with 24% (95% CI 17-32) in those who had used antibiotics due to the problems in the operated knee for more than 1 week. Interpretation - In Lithuania there seems to be a lack of adherence to evidence-based treatment guidelines when infection is suspected after primary TKA.

The safety and efficacy of light emitting diodes-based ultraviolet A1 phototherapy in bleomycin-induced scleroderma in mice.

PURPOSE: To define the efficacy and safety of narrowband ultraviolet A1 (UVA1) for the treatment of dermal fibrosis in bleomycin-induced mouse model of scleroderma. MATERIALS AND METHODS: 42 DBA/2 strain mice were included in the study: healthy mice and mice with established scleroderma, treated with high or medium dose of UVA1. Non-treated groups served as control. The equipment emitting 365±5nm UVA1 radiation was used in the study. The average cumulative doses were 1200J/cm2 for high and 600J/cm2 for medium dose course. Histological analysis was performed for the evaluation of the dermal thickness and mast cells density. The expressions of p53 and Ki-67 proteins were assessed by immunohistochemical analyses. RESULTS: Skin thickness of mice with scleroderma, treated with high and medium dose of UVA1, were lower (272.9±113.2µm and 394±125.9µm, respectively) in comparison to the dermal thickness of non-treated animals (599±55.7µm). The dermal mast cells count in mice with scleroderma was reduced after high and medium dose treatment to 11±1.7 and 13±2.2, respectively, as compared to that in non-treated mice (23±3.0). No significant upregulation of p53 nor Ki-67 proteins was observed in the skin of healthy mice and mice with scleroderma after high- and medium-dose of UVA1. CONCLUSIONS: The results of this study indicate that 365nm UVA1 with the cumulative doses of 1200J/cm2 and 600J/cm2 is safe and effective for the dermal fibrosis treatment.

Paracrine Potential of the Human Adipose Tissue-Derived Stem Cells to Modulate Balance between Matrix Metalloproteinases and Their Inhibitors in the Osteoarthritic Cartilage In Vitro.

Adipose tissue represents an abundant source of stem cells. Along with anti-inflammatory effects, ASC secrete various factors that may modulate metabolism of extracellular matrix in osteoarthritic (OA) cartilage, suggesting that the presence of ASC could be advantageous for OA cartilage due to the recovery of homeostasis between matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs). To evaluate these effects, cartilage explants (CE) were cocultured with ASC for 3 and 7 days under stimulation with or without IL-1ß. The pattern of gene expression in CE was modified by ASC, including the upregulation of COL1A1 and COL3A1 and the downregulation of MMP13 and COL10A1. The production of MMP-1, MMP-3, and MMP-13 by ASC was not significant; moreover, cocultures with ASC reduced MMP-13 production in CE. In conclusion, active production of TIMP-1, TIMP-2, TIMP-3, IL-6, IL-8, and gelatinases MMP-2 and MMP-9 by ASC may be involved in the extracellular matrix remodelling, as indicated by the altered expression of collagens, the downregulated production of MMP-13, and the reduced chondrocyte apoptosis in the cocultured CE. These data suggest that ASC modulated homeostasis of MMPs/TIMPs in degenerated OA cartilage in vitro and might be favourable in case of the intra-articular application of ASC therapy for the treatment of OA.

The impact of high-dose narrowband ultraviolet A1 on dermal thickness, collagen and matrix-metalloproteinases in animal model of scleroderma.

OBJECTIVE: The main purpose of the present study was to define the impact of high-dose of 365±5nm ultraviolet A1 (UVA1) on dermal fibrosis in the pre-established, bleomycin-induced mouse model of scleroderma. METHODS: DBA/2 strain mice with the pre-established, bleomycin-induced scleroderma were irradiated with cumulative UVA1 dose of 1200J/cm2 and in parallel were challenged with prolonged administration of bleomycin. Non-treated groups served as the control. Light source emitting a narrow band UVA1 light of 365±5nm and 21mW/cm2 power density was used in the study. Histological analysis was performed for the evaluation of dermal thickness. The expressions of matrix-metalloproteinase-1 (MMP-1), matrix-metalloproteinase-3 (MMP-3), collagen types I and III were evaluated by immunohistochemical analyses. The Mann - Whitney U test was used for statistical analysis. RESULTS: Dermal thickness in mice injected with bleomycin during all the experiment (8weeks) and irradiated with UVA1 for the last 5weeks was significantly lower than that in mice challenged only with bleomycin for 8weeks (253.96±31.83µm and 497.43±57.83µm, respectively; P=0.002). The dermal thickness after phototherapy was lower as compared with the pre-existing fibrotic changes observed after 3weeks of bleomycin injections (253.96±31.83µm and 443.87±41.76µm, respectively; P=0.002). High-dose of UVA1 induced the 5.8- and 5.2-fold increase in MMP-1 and MMP-3 expressions, respectively, and the 1.2- and 1.4-fold decrease in collagen type I and collagen type III expressions in the pre-established, bleomycin-induced scleroderma model as compared to that in the control non-irradiated mice (P=0.002). CONCLUSIONS: Our study has demonstrated that a cumulative 365±5nm UVA1 radiation dosage of 1200J/cm2 not only prevents the progression of dermal fibrosis, but also induces a regression of pre-existing fibrotic changes.

Age, Sex, Body Mass Index, Education, and Social Support Influence Functional Results After Total Knee Arthroplasty.

INTRODUCTION: Total knee arthroplasty (TKA) is an effective treatment for knee osteoarthritis. Patient-reported outcome after TKA is influenced by multiple patient-related factors. The aim of this study was to prospectively evaluate preoperative patient-related factors and to compare the self-reported outcomes 1 year after TKA among groups differing by age, sex, body mass index (BMI), education, and social support level. METHODS: 314 patients, who underwent TKA in Vilnius Republican University Hospital between the end of 2012 and the middle of 2014, were included in a study. The preoperative and 12-month follow-up measurements were obtained using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and Short Form-12 (SF-12). Differences between patient groups according to gender, age, BMI, level of education, and level of social support were analyzed. RESULTS: At 12-month follow-up men demonstrated better results than women in WOMAC (P = .003) and SF-12 both domains (P < .05). Patients with a higher social support demonstrated higher scores in physical function according to SF-12 (P = .008). Better preoperative WOMAC and SF-12 scores were a predictor of better outcome 1 year after surgery. There was no difference in postoperative scores in different age, BMI, and education groups according to WOMAC and SF-12. CONCLUSION: There is no difference in self-reported functional outcome between patient groups differing in age, BMI, and education. Men and socially supported patients demonstrate better postoperative functional results 12 months after TKA. Better preoperative knee function and overall physical and mental function are predictors of better outcome 1 year after TKA. Age and obesity should not be limiting factors when considering who should receive this surgery.

Microcarrier culture enhances osteogenic potential of human periodontal ligament stromal cells.

Regeneration of periodontal tissue represents a major challenge to modern tissue engineering, since cell-based therapies require large amounts of periodontal ligament stromal cells (PLSC), which can be obtained only by in vitro expansion. Ideally, the period of the in vitro expansion should be optimized for the generation of large enough numbers of pre-specified progenitor cells ready to contribute to the restoration of periodontal tissues. In the present study, we used a commercially available, three-dimensional culturing platform and alginate microcarrier cell culture system for the propagation of human PLSCs, which were derived using the explant outgrowth method. Induction of osteogenic differentiation resulted in rapid and robust mineralization of the extracellular matrix in PLSCs grown on microcarriers, but not in PLSCs grown under standard culture conditions. Gene expression studies revealed upregulation of osteogenesis-related genes, BMP2, ALP, RUNX2, MSX2, cementum protein 23, bone sialoprotein, osteopontin and periostin, in undifferentiated and differentiating microcarrier cultures of PLSCs. In addition, the microcarrier culture enhanced the expression of ß-catenin, intermediate filament protein vimentin and focal adhesion proteins vinculin and paxillin. Our study shows that microcarrier culture allows rapid generation of large numbers of PLSCs pre-specified towards an osteogenic-like phenotype. This method may be useful for the development of new tissue engineering protocols for the reconstruction of periodontal tissues.

Influence of Immunogenicity on the Efficacy of Long-Term Treatment with TNF α Blockers in Rheumatoid Arthritis and Spondyloarthritis Patients.

OBJECTIVE: To analyze the clinical relevance of the levels of TNFα blockers and anti-drug antibodies (anti-drug Ab) in patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA) treated with adalimumab (ADA), etanercept (ETA), or infliximab (INF) for a prolonged period of time. METHODS: Clinical characteristics (disease activity, and adverse events), serum TNFα blockers, and anti-drug Ab levels were evaluated in 62 RA and 81 SpA patients treated with TNFα blockers for a median of 28 months. RESULTS: Anti-ADA Ab were detected in 1 (4.0%) and anti-INF Ab in 14 out of 57 (24.6%) RA and SpA patients. Patient with anti-ADA Ab and 57.1% patients with anti-INF Ab were considered nonresponders to treatment. Anti-ETA Ab were not found in any of 61 ETA treated patients. Anti-ADA and anti-INF Ab levels differ between responders and nonresponders (P > 0.05). Three (5.3%) patients with high serum anti-INF Ab levels developed infusion related reactions. Patients with anti-INF Ab more often required changing to another biologic drug (OR 11.43 (95% CI 1.08-120.93)) and treatment discontinuation (OR 9.28 (95% CI 1.64-52.52)). CONCLUSION: Patients not responding to treatment had higher serum anti-ADA and anti-INF Ab concentrations. Anti-INF Ab formation is related to increased risk of infusion related reactions, changing to another biologic drug, and treatment discontinuation.

Exosomes from dental pulp stem cells rescue human dopaminergic neurons from 6-hydroxy-dopamine-induced apoptosis.

BACKGROUND AIMS: Stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) have unique neurogenic properties that could be potentially exploited for therapeutic use. The importance of paracrine SHED signaling for neuro-regeneration has been recognized, but the exact mechanisms behind these effects are presently unknown. In the present study, we investigated the neuro-protective potential of exosomes and micro-vesicles derived from SHEDs on human dopaminergic neurons during oxidative stress-induced by 6-hydroxy-dopamine (6-OHDA). METHODS: ReNcell VM human neural stem cells were differentiated into dopaminergic neurons and treated with 100 µmol/L of 6-OHDA alone or in combination with exosomes or micro-vesicles purified by ultracentrifugation from SHEDs cultivated in serum-free medium under two conditions: in standard two-dimensional culture flasks or on laminin-coated micro-carriers in a bioreactor. Real-time monitoring of apoptosis was performed with the use of time-lapse confocal microscopy and the CellEvent Caspase-3/7 green detection reagent. RESULTS: Exosomes but not micro-vesicles derived from SHEDs grown on the laminin-coated three-dimensional alginate micro-carriers suppressed 6-OHDA-induced apoptosis in dopaminergic neurons by approximately 80% throughout the culture period. Strikingly, no such effects were observed for the exosomes derived from SHEDs grown under standard culture conditions. CONCLUSIONS: Our results suggest that exosomes derived from SHEDs are considered as new potential therapeutic tool in the treatment of Parkinson's disease.

Molecular mechanisms behind progressing chronic inflammatory dilated cardiomyopathy.

BACKGROUND: Inflammatory dilated cardiomyopathy (iDCM) is a common debilitating disease with poor prognosis that often leads to heart failure and may require heart transplantation. The aim of this study was to evaluate sera and biopsy samples from chronic iDCM patients, and to investigate molecular mechanism associated with left ventricular remodeling and disease progression in order to improve therapeutic intervention. METHODS: Patients were divided into inflammatory and non-inflammatory DCM groups according to the immunohistochemical expression of inflammatory infiltrates markers: T-lymphocytes (CD3), active-memory T lymphocyte (CD45Ro) and macrophages (CD68). The inflammation, apoptosis, necrosis and fibrosis were investigated by ELISA, chemiluminescent, immunohistochemical and histological assays. RESULTS: The pro-inflammatory cytokine IL-6 was significantly elevated in iDCM sera (3.3 vs. 10.98 µg/ml; P < 0.05). Sera levels of caspase-9, -8 and -3 had increased 6.24-, 3.1- and 3.62-fold, (P < 0.05) and only slightly (1.3-, 1.22- and 1.03-fold) in biopsies. Significant release of Hsp60 in sera (0.0419 vs. 0.36 ng/mg protein; P < 0.05) suggested a mechanistic involvement of mitochondria in cardiomyocyte apoptosis. The significant MMP9/TIMP1 upregulation in biopsies (0.1931 - 0.476, P < 0.05) and correlation with apoptosis markers show its involvement in initiation of cell death and ECM degradation. A slight activation of the extrinsic apoptotic pathway and the release of hsTnT might support the progression of chronic iDCM. CONCLUSIONS: Data of this study show that significant increase of IL-6, MMP9/TIMP1 and caspases-9, -8, -3 in sera corresponds to molecular mechanisms dominating in chronic iDCM myocardium. The initial apoptotic pathway was more activated by the intramyocardial inflammation and might be associated with extrinsic apoptotic pathway through the pro-apoptotic Bax. The activated intrinsic form of myocardial apoptosis, absence of necrosis and decreased fibrosis are most typical characteristics of chronic iDCM. Clinical use of anti-inflammatory drugs together with specific anti-apoptotic treatment might improve the efficiency of therapies against chronic iDCM before heart failure occurs.

Exosomes from Human Dental Pulp Stem Cells Suppress Carrageenan-Induced Acute Inflammation in Mice.

The primary goal of this study was to examine the effects of human dental pulp stem cell-derived exosomes on the carrageenan-induced acute inflammation in mice. Exosomes were purified by differential ultracentrifugation from the supernatants of stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) cultivated in serum-free medium. At 1 h post-carrageenan injection, exosomes derived from supernatants of 2 × 10(6) SHEDs were administered by intraplantar injection to BALB/c mice; 30 mg/kg of prednisolone and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. Edema was measured at 6, 24, and 48 h after carrageenan injection. For the in vivo imaging experiments, AngioSPARK750, Cat B 750 FAST, and MMPSense 750 FAST were administered into the mouse tail vein 2 h post-carrageenan injection. Fluorescence images were acquired at 6, 24, and 48 h after edema induction by IVIS Spectrum in vivo imaging system. Exosomes significantly reduced the carrageenan-induced edema at all the time points studied (by 39.5, 41.6, and 25.6% at 6, 24, and 48 h after injection, respectively), to similar levels seen with the positive control (prednisolone). In vivo imaging experiments revealed that, both exosomes and prednisolone suppress activities of cathepsin B and matrix metalloproteinases (MMPs) at the site of carrageenan-induced acute inflammation, showing more prominent effects of prednisolone at the early stages, while exosomes exerted their suppressive effects gradually and at later time points. Our study demonstrates for the first time that exosomes derived from human dental pulp stem cells suppress carrageenan-induced acute inflammation in mice.

Predictors of mortality in patients with rheumatoid arthritis in Lithuania: Data from a cohort study over 10 years.

BACKGROUND AND OBJECTIVE: Increased mortality and shorter survival among rheumatoid arthritis (RA) patients are recognized but not fully explained. This cohort study aimed to identify predictors of mortality among RA patients at a tertiary clinical setting. MATERIALS AND METHODS: Patients with RA were recruited during 1998-2003 and followed up until April 1, 2012, or death whichever happened first. Baseline variables included sociodemographic and disease characteristics, and comorbidities. Cox regression and hazard risk (HR) were computed to estimate risks for mortality. RESULTS: One hundred ninety-one patients were included into the study, 186 patients were eligible for the analysis and of these 131 patients (70.4%) completed the entire period of followed-up while 55 patients (29.6%) died. The average follow up period was equivalent to 9.24 year per person. A Cox regression model identified four major factors having an impact on survival. History of a stroke at baseline was identified as a major factor (HR=5.33; 95% CI, 2.13-13.32). Statistically significant risk factors were also age over 50 years (HR=4.59; 95% CI, 2.04-10.30); education less than 11 years (HR=3.3; 95% CI, 1.72-6.33) and angina pectoris (HR=1.98; 95% CI, 1.03-3.80). CONCLUSIONS: Higher age, lower education and cardiovascular comorbidities were identified as predictors of mortality in this prospective cohort study while disease-related variables were not independent predictors of mortality.

A New Experimental Model for Neuronal and Glial Differentiation Using Stem Cells Derived from Human Exfoliated Deciduous Teeth.

Stem cells isolated from human adult tissues represent a promising source for neural differentiation studies in vitro. We have isolated and characterized stem cells from human exfoliated deciduous teeth (SHEDs). These originate from the neural crest and therefore particularly suitable for induction of neural differentiation. We here established a novel three-stage protocol for neural differentiation of SHEDs cells. After adaptation to a serum-free and neurogenic environment, SHEDs were induced to differentiate. This resulted in the formation of stellate or bipolar round-shaped neuron-like cells with subpopulations expressing markers of sensory neurons (Brn3a, peripherin) and glia (myelin basic protein). Commercial PCR array analyses addressed the expression profiles of genes related to neurogenesis and cAMP/calcium signalling. We found distinct evidence for the upregulation of genes regulating the specification of sensory (MAF), sympathetic (midkine, pleitrophin) and dopaminergic (tyrosine hydroxylase, Nurr1) neurons and the differentiation and support of myelinating and non-myelinating Schwann cells (Krox24, Krox20, apolipoprotein E). Moreover, for genes controlling major developmental signalling pathways, there was upregulation of BMP (TGF ß-3, BMP2) and Notch (Notch 2, DLL1, HES1, HEY1, HEY2) in the differentiating SHEDs. SHEDs treated according to our new differentiation protocol gave rise to mixed neuronal/glial cell cultures, which opens new possibilities for in vitro studies of neuronal and glial specification and broadens the potential for the employment of such cells in experimental models and future treatment strategies.

Protective induction of Hsp70 in heat-stressed primary myoblasts: Involvement of MAPKs.

The involvement of extracellular signal-regulated kinases 1 and 2 (ERK1,2), stress kinase p38 and c-Jun NH2 -terminal kinases 1 and 2 (JNK1,2) on Hsp70-upregulation following mild heat shock, and resulting cell protection, was studied on rabbit primary myoblasts. Cells subjected to heat stress (42°C; 60 min) showed a significantly enhanced amount of heat-shock-induced protein 70 (Hsp70), correlating with sustained phosphorylation of MAP kinases ERK1,2, inhibition of p38 and JNK1,2 activation. Induced Hsp70 did not autocrinally suppress activation of transcription factor c-Jun, suggesting involvement of the latter in the protection of myoblasts following heat shock. The inhibition of stress kinases p38, JNK1,2, and MEK1,2 by SP600125, SB203580, and UO126, respectively, established the involvement of JNK1,2 and p38 as upstream, and ERK1,2 as downstream targets of Hsp70 induction. Moreover, the effect of the MEK1,2 inhibitor UO126 revealed a new pathway of c-Jun activation by ERK1,2 in myogenic heat-stressed stem cells. The presented data show that transient activation of JNK1, JNK2, and p38 is necessary for Hsp70 induction and ensuing cell protection. In conclusion, affecting myogenic stem cell protective mechanisms might be a useful strategy in improving stem cell survival and their expanded application in therapy.

A variety of mild stresses upregulate stanniocalcin-1 (STC-1) and induce mitohormesis in neural crest-derived cells.

We induced upregulation of stanniocalcin-1 (STC-1) by various mild and long lasting stresses and assayed its influence on mitochondrial membrane potential (MMP) in the neural crest-derived cell line Paju. The obtained data showed that starvation (24-96 h), exposure to 10nM TPA, and low concentrations (0.05-1 µM) of As2O3 significantly (3-5 times) upregulated Paju cell STC-1 RNA and stabilized the mitochondrial membrane potential (MMP). However, high concentrations of As2O3 (2.5-5.0 µM) increased intracellular ROS and free calcium levels and, consequently, suppressed STC-1 and MMP. The results show that cells preconditioned by various mild stresses expressed more STC-1 and their MMP were more resistant to a secondary exposure to As2O3 (2.5-5 µM, 96 h) demonstrating mitohormesis. We suggest that MMP deviation from control levels, to an extent innocuous to cell viability, is a general signal for STC-1-induction and MMP-protection. Our findings of Paju cell MMP-regulation may be of great importance for inventing new ways to prevent neurodegenerative diseases and unravel the mechanisms behind drug resistance.