Characterization of Nanohybridosomes from Lipids and Spruce Homogenate Containing Extracellular Vesicles.

Introduction: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered. Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet-visual spectroscopy, interferometric light microscopy and liquid chromatography-mass spectrometry. Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes. Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible.

The Role of Autophagy and Apoptosis in Affected Skin and Lungs in Patients with Systemic Sclerosis.

Systemic sclerosis (SSc) is a complex autoimmune inflammatory disorder with multiple organ involvement. Skin changes present the hallmark of SSc and coincide with poor prognosis. Interstitial lung diseases (ILD) are the most widely reported complications in SSc patients and the primary cause of death. It has been proposed that the processes of autophagy and apoptosis could play a significant role in the pathogenesis and clinical course of different autoimmune diseases, and accordingly in SSc. In this manuscript, we review the current knowledge of autophagy and apoptosis processes in the skin and lungs of patients with SSc. Profiling of markers involved in these processes in skin cells can be useful to recognize the stage of fibrosis and can be used in the clinical stratification of patients. Furthermore, the knowledge of the molecular mechanisms underlying these processes enables the repurposing of already known drugs and the development of new biological therapeutics that aim to reverse fibrosis by promoting apoptosis and regulate autophagy in personalized treatment approach. In SSc-ILD patients, the molecular signature of the lung tissues of each patient could be a distinctive criterion in order to establish the correct lung pattern, which directly impacts the course and prognosis of the disease. In this case, resolving the role of tissue-specific markers, which could be detected in the circulation using sensitive molecular methods, would be an important step toward development of non-invasive diagnostic procedures that enable early and precise diagnosis and preventing the high mortality of this rare disease.

Can Pharmacogenetic Variants in TPMT, MTHFR and SLCO1B1 Genes Be Used as Potential Markers of Outcome Prediction in Systemic Sclerosis Patients?

Systemic sclerosis (SSc) is a rare connective tissue disorder with highest morbidity and mortality among rheumatologic diseases. Disease progression is highly heterogeneous between patients, implying a strong need for individualization of therapy. Four pharmacogenetic variants, namely TPMT rs1800460, TPMT rs1142345, MTHFR rs1801133 and SLCO1B1 rs4149056 were tested for association with severe disease outcomes in 102 patients with SSc from Serbia treated either with immunosuppressants azathioprine (AZA) and methotrexate (MTX) or with other types of medications. Genotyping was performed using PCR-RFLP and direct Sanger sequencing. R software was used for statistical analysis and development of polygenic risk score (PRS) model. Association was found between MTHFR rs1801133 and higher risk for elevated systolic pressure in all patients except those prescribed with MTX, and higher risk for kidney insufficiency in patients prescribed with other types of drugs. In patients treated with MTX, variant SLCO1B1 rs4149056 was protective against kidney insufficiency. For patients receiving MTX a trend was shown for having a higher PRS rank and elevated systolic pressure. Our results open a door wide for more extensive research on pharmacogenomics markers in patients with SSc. Altogether, pharmacogenomics markers could predict the outcome of patients with SSc and help in prevention of adverse drug reactions.

Small Cellular Particles from European Spruce Needle Homogenate.

Small cellular particles (SCPs) are being considered for their role in cell-to-cell communication. We harvested and characterized SCPs from spruce needle homogenate. SCPs were isolated by differential ultracentrifugation. They were imaged by scanning electron microscope (SEM) and cryogenic transmission electron microscope (cryo TEM), assessed for their number density and hydrodynamic diameter by interferometric light microscopy (ILM) and flow cytometry (FCM), total phenolic content (TPC) by UV-vis spectroscopy, and terpene content by gas chromatography-mass spectrometry (GC-MS). The supernatant after ultracentrifugation at 50,000× g contained bilayer-enclosed vesicles whereas in the isolate we observed small particles of other types and only a few vesicles. The number density of cell-sized particles (CSPs) (larger than 2 µm) and meso-sized particles (MSPs) (cca 400 nm-2 µm) was about four orders of magnitude lower than the number density of SCPs (sized below 500 nm). The average hydrodynamic diameter of SCPs measured in 10,029 SCPs was 161 ± 133 nm. TCP decreased considerably due to 5-day aging. Volatile terpenoid content was found in the pellet after 300× g. The above results indicate that spruce needle homogenate is a source of vesicles to be explored for potential delivery use.

Molecular Biomarkers in Perthes Disease: A Review.

BACKGROUND: Perthes disease is a juvenile form of osteonecrosis of the femoral head that affects children under the age of 15. One hundred years after its discovery, some light has been shed on its etiology and the biological factors relevant to its etiology and disease severity. METHODS: The aim of this study was to summarize the literature findings on the biological factors relevant to the pathogenesis of Perthes disease, their diagnostic and clinical significance, and their therapeutic potential. A special focus on candidate genes as susceptibility factors and factors relevant to clinical severity was made, where studies reporting clinical or preclinical results were considered as the inclusion criteria. PubMed databases were searched by two independent researchers. Sixty-eight articles were included in this review. Results on the factors relevant to vascular involvement and inflammatory molecules indicated as factors that contribute to impaired bone remodeling have been summarized. Moreover, several candidate genes relevant to an active phase of the disease have been suggested as possible biological therapeutic targets. CONCLUSIONS: Delineation of molecular biomarkers that underlie the pathophysiological process of Perthes disease can allow for the provision of earlier and more accurate diagnoses of the disease and more precise follow-ups and treatment in the early phases of the disease.

Autologous Platelet and Extracellular Vesicle-Rich Plasma as Therapeutic Fluid: A Review.

The preparation of autologous platelet and extracellular vesicle-rich plasma (PVRP) has been explored in many medical fields with the aim to benefit from its healing potential. In parallel, efforts are being invested to understand the function and dynamics of PVRP that is complex in its composition and interactions. Some clinical evidence reveals beneficial effects of PVRP, while some report that there were no effects. To optimize the preparation methods, functions and mechanisms of PVRP, its constituents should be better understood. With the intention to promote further studies of autologous therapeutic PVRP, we performed a review on some topics regarding PVRP composition, harvesting, assessment and preservation, and also on clinical experience following PVRP application in humans and animals. Besides the acknowledged actions of platelets, leukocytes and different molecules, we focus on extracellular vesicles that were found abundant in PVRP.

Assessment of Small Cellular Particles from Four Different Natural Sources and Liposomes by Interferometric Light Microscopy.

Small particles in natural sources are a subject of interest for their potential role in intercellular, inter-organism, and inter-species interactions, but their harvesting and assessment present a challenge due to their small size and transient identity. We applied a recently developed interferometric light microscopy (ILM) to assess the number density and hydrodynamic radius (Rh) of isolated small cellular particles (SCPs) from blood preparations (plasma and washed erythrocytes) (B), spruce needle homogenate (S), suspension of flagellae of microalgae Tetraselmis chuii (T), conditioned culture media of microalgae Phaeodactylum tricornutum (P), and liposomes (L). The aliquots were also assessed by flow cytometry (FCM), dynamic light scattering (DLS), ultraviolet-visible spectrometry (UV-vis), and imaging by cryogenic transmission electron microscopy (cryo-TEM). In Rh, ILM showed agreement with DLS within the measurement error in 10 out of 13 samples and was the only method used here that yielded particle density. Cryo-TEM revealed that representative SCPs from Tetraselmis chuii flagella (T) did not have a globular shape, so the interpretation by Rh of the batch methods was biased. Cryo-TEM showed the presence of thin filaments in isolates from Phaeodactylum tricornutum conditioned culture media (P), which provides an explanation for the considerably larger Rh obtained by batch methods than the sizes of particles observed by cryo-TEM images. ILM proved convenient for assessment of number density and Rh of SCPs in blood preparations (e.g., plasma); therefore, its use in population and clinical studies is indicated.

Crosstalk between Glycogen-Selective Autophagy, Autophagy and Apoptosis as a Road towards Modifier Gene Discovery and New Therapeutic Strategies for Glycogen Storage Diseases.

Glycogen storage diseases (GSDs) are rare metabolic monogenic disorders characterized by an excessive accumulation of glycogen in the cell. However, monogenic disorders are not simple regarding genotype-phenotype correlation. Genes outside the major disease-causing locus could have modulatory effect on GSDs, and thus explain the genotype-phenotype inconsistencies observed in these patients. Nowadays, when the sequencing of all clinically relevant genes, whole human exomes, and even whole human genomes is fast, easily available and affordable, we have a scientific obligation to holistically analyze data and draw smarter connections between genotype and phenotype. Recently, the importance of glycogen-selective autophagy for the pathophysiology of disorders of glycogen metabolism have been described. Therefore, in this manuscript, we review the potential role of genes involved in glycogen-selective autophagy as modifiers of GSDs. Given the small number of genes associated with glycogen-selective autophagy, we also include genes, transcription factors, and non-coding RNAs involved in autophagy. A cross-link with apoptosis is addressed. All these genes could be analyzed in GSD patients with unusual discrepancies between genotype and phenotype in order to discover genetic variants potentially modifying their phenotype. The discovery of modifier genes related to glycogen-selective autophagy and autophagy will start a new chapter in understanding of GSDs and enable the usage of autophagy-inducing drugs for the treatment of this group of rare-disease patients.

Association between active pulmonary tuberculosis and miRNA-146a: A preliminary study from Serbia.

INTRODUCTION: Tuberculosis (TB) continues to be a significant public health problem. The role of small non-coding RNAs, such as microRNAs (miRNAs), was investigated extensively in Mycobacterium tuberculosis (MTB) infection as well as in a variety of other pathophysiological processes in recent years. It was found that miRNAs act as regulators of both early reaction to MTB infection and in process of adaptation of the host immune cells during latent course of the disease. Molecule miRNA-146a is expressed exclusively in immune cells and it has the most prominent role in modulation of innate immunity. METHODOLOGY: We investigated the level of expression of miRNA-146a using an RT-qPCR technique in peripheral blood mononuclear cells of 44 patients with active pulmonary TB and 17 healthy individuals. We also analyzed the significance of miRNA-146a rs2910164 SNV for expression profile of miRNA-146a, in order to investigate potential usage of miRNA-146a as a biomarker for TB. RESULTS: There was statistically significant decrease of expression of miRNA-146a in TB group compared to control group. When gender cohorts were analyzed, the expression levels in TB male and TB female subgroup were significantly lower than the expression levels in the same gender control subgroups. CONCLUSIONS: Our results indicate that miRNA-146a plays a significant role in the pathogenesis of TB, suggesting that miRNA-146a could be used as a biomarker for active pulmonary TB.

Case Report: Successful Therapy of Spontaneously Occurring Canine Degenerative Lumbosacral Stenosis Using Autologous Adipose Tissue-Derived Mesenchymal Stem Cells.

The management of degenerative lumbosacral stenosis (DLSS) in dogs usually requires aggressive, costly surgical treatments that may themselves present complications, while do not fully resolve the symptoms of the disease. In this study, the dog diagnosed with severe DLSS, with hind limb paresis, was treated using a new and least invasive treatment. Cultured autologous adipose tissue-derived mesenchymal stem cells (AT-MSCs) were injected bilaterally at the level of L7-S1, in the vicinity of the external aperture of the intervertebral foramen of DLSS patient. In the previously described treatments of spontaneous intervertebral disc degeneration in dogs, intradiscal injections of MSCs did not lead to positive effects. Here, we report a marked improvement in clinical outcome measures related to the ability of a dog to walk and trot, which were expressed by a numeric rating scale based on a veterinary assessment questionnaire. The improved status persisted throughout the observed time course of 4.5 years after the AT-MSC transplantation. To the best of our knowledge, this is the first case of successful therapy, with long-term positive effect, of spontaneously occurring canine DLSS using presented treatment that, we believe, represents a contribution to current knowledge in this field and may shape both animal and human DLSS treatment options.

Genes and metabolic pathway of sarcoidosis: identification of key players and risk modifiers.

INTRODUCTION: Sarcoidosis is a rare multisystem granulomatous disease with unknown etiology. The interplay of vitamin D deficiency and genetic polymorphisms in genes coding for the proteins relevant for metabolism of vitamin D is an important, but unexplored area. The aim of this study was to investigate the association between single nucleotide polymorphisms (SNPs) in CYP2R1 (rs10741657), CYP27B1 (rs10877012), DBP (rs7041; rs4588), and VDR (rs2228570) genes and sarcoidosis, as well as the association between these SNPs and 25(OH)D levels in sarcoidosis patients. MATERIAL AND METHODS: For that purpose we genotyped 86 sarcoidosis patients and 50 healthy controls using the PCR-RFLP method. RESULTS: Subjects carrying the CC genotype of CYP27B1 rs10877012 have 10 times lower odds of suffering from sarcoidosis. Moreover, DBP rs4588 AA genotype was shown to be a susceptibility factor, where carriers of this genotype had eight times higher odds for developing sarcoidosis. In addition, the A allele of the DBP gene (rs4588) was associated with lower levels of 25(OH)D in sarcoidosis patients. CONCLUSIONS: These results suggest that patients with vitamin D deficiency should be regularly tested for genetic modifiers that are related to sarcoidosis in order to prevent development of serious forms of sarcoidosis.

Relevance of TNF-α, IL-6 and IRAK1 gene expression for assessing disease severity and therapy effects in tuberculosis patients.

INTRODUCTION: Tuberculosis (TBC) is a contagious chronic respiratory disease which despite the known cause, Mycobacterium tuberculosis (Mtb), and many decades of successful therapy, remains one of the leading global health problems. Immune responses against Mtb infection involve both of types of immunity, but cellular immunity, in which certain cytokines and Th1 cells play a key role, is crucial. A better understanding of the functions of the cytokine network involved in the state and progression of TBC could identify specific molecular markers for monitoring of disease activity as well as therapy outcomes in TBC patients. METHODOLOGY: We investigated expression of TNF-α, IL-6 and IRAK1 genes using an RT-qPCR technique in peripheral blood mononuclear cells of 33 TBC patients and 10 healthy individuals. RESULTS: Comparison between TBC patients and healthy individuals revealed statistically significant differences for all analyzed genes. The levels of expression of TNF-α and IL-6 mRNA were higher, while the level of IRAK1 mRNA was lower in the TBC group compared to controls. Moreover, a strong positive correlation was observed between TNF-α and IL-6 gene expression. When clinical parameters were analyzed, increased levels of TNF-α mRNA were detected in patients with a longer duration of therapy (>2 months) compared to those with a shorter therapy duration (< 2 months), and in patients without anemia. CONCLUSIONS: Our results indicate that the inflammatory genes we examined play a crucial role in the pathogenesis of tuberculosis, and that the expression of the TNF-α gene could be a marker for monitoring the clinical effect of the ant-tuberculosis drugs during therapy.

Impact of alterations in X-linked IRAK1gene and miR-146a on susceptibility and clinical manifestations in patients with systemic sclerosis.

Systemic sclerosis (SSc) is a heterogeneous multisystem autoimmune disease with unknown etiology. Numerous studies have indicated that the disease heterogeneity implies various genetic abnormalities. Considering that SSc is characterized by a strong sex bias and that the position of IRAK1 gene is on the X chromosome, we assume that variations in IRAK1 gene could explain female predominance of SSc. It was previously described that miR-146a has a role in 'fine-tuning' regulation of the TLR/NF-kB signaling pathway through down-regulation of IRAK1 gene. The aim of the present study was to analyze both variants and expression level of IRAK1 and miR-146a genes in terms of susceptibility to SSc and clinical presentation of SSc patients. We analyzed variants IRAK1 rs3027898 C > A and miR-146a rs2910164 C > G in 102 SSc patients and 66 healthy subjects. Genotyping was performed by Sanger sequencing. Expression level of IRAK1 mRNA and miR-146a in PBMCs was performed in subset of 50 patients and 13 healthy controls by RT-qPCR. Our results showed that there was no association between IRAK1 rs3027898 and the risk of SSc in women. However, the analysis of genotype distribution of the mir-146a rs2910164 C > G variant indicated that CC genotype shows strong association with lung fibrosis and active form of the disease. When expression level of IRAK1 gene was analyzed, we detected significant downregulation of IRAK1 mRNA in SSc patients compared to controls, as well as in male compared to female patients, in patients with ACAs autoantibodies and in patients with severe skin involvement. Regarding the expression level of miR-146a, we have found significantly reduced expression in SSc patients, in patients with skin involvement and in male SSc patients. The results from this study indicate that expression of IRAK1 gene could explain phenotypic heterogeneity of SSc and may be involved in the pathogenesis of SSc due to its differential expression in certain subgroups. Our results also suggested that miR-146a rs2910164 CC genotype may be predisposing factor for development lung fibrosis and more progressive form of SSc. Results from relative expression analysis of miR-146a demonstrated that changes in the level of this miRNA may have an impact on development and clinical course of SSc.

Genomic profiling supports the diagnosis of primary ciliary dyskinesia and reveals novel candidate genes and genetic variants.

Primary ciliary dyskinesia (PCD) is a rare inherited autosomal recessive or X-linked disorder that mainly affects lungs. Dysfunction of respiratory cilia causes symptoms such as chronic rhinosinusitis, coughing, rhinitis, conductive hearing loss and recurrent lung infections with bronchiectasis. It is now well known that pathogenic genetic changes lead to ciliary dysfunction. Here we report usage of clinical-exome based NGS approach in order to reveal underlying genetic causes in cohort of 21 patient with diagnosis of PCD. By detecting 18 (12 novel) potentially pathogenic genetic variants, we established the genetic cause of 11 (9 unrelated) patients. Genetic variants were detected in six PCD disease-causing genes, as well as in SPAG16 and SPAG17 genes, that were not detected in PCD patients so far, but were related to some symptoms of PCD. The most frequently mutated gene in our cohort was DNAH5 (27.77%). Identified variants were in homozygous, compound heterozygous and trans-heterozygous state. For detailed characterization of one novel homozygous genetic variant in DNAI1 gene (c. 947_948insG, p. Thr318TyrfsTer11), RT-qPCR and Western Blot analysis were performed. Molecular diagnostic approach applied in this study enables analysis of 29 PCD disease-causing and related genes. It resulted in mutation detection rate of 50% and enabled discovery of twelve novel mutations and pointed two possible novel PCD candidate genes.

The use of canine mesenchymal stem cells for the autologous treatment of osteoarthritis.

Mesenchymal stem cells (MSCs) hold enormous potential for cell-based therapy in the treatment of various diseases, particularly those which currently cannot be cured and result in poor outcomes or invasive surgery. Here we present results of the application of autologous, culture-expanded, adipose tissue (AT)-derived MSCs for the osteoarthritis (OA) treatment of 10 canine patients. The stemness of isolated cells has been confirmed by their ability to differentiate into osteo- and chondrocytic lineages. The clinical effect of a single injection of ATMSCs into the symptomatic joints was evaluated by a veterinarian for five disabilities characteristic of OA at 30, 60 and 90 days after treatment, which has been designated as the initial evaluation period. Functional outcomes for all analysed characteristics improved significantly at the end of this evaluation compared with the baseline. In addition, for 5 of these 10 patients, an extended follow-up study was performed from 1 to 4 years after the initial evaluation period. We detected long-lasting positive effects on two out of five analysed characteristics. The results demonstrate that the use of autologous AT-MSCs is a successful approach to canine OA therapy.

Association between the -174 C/G polymorphism in the interleukin-6 (IL-6) gene and gastrointestinal involvement in patients with systemic sclerosis.

Genetic predisposition to systemic sclerosis (SSc) has still not been fully revealed. Interleukin-6 (IL-6) is a mediator of T cell proliferation and fibrotic events in SSc. Polymorphisms in the IL-6 are found to be important in susceptibility to development of SSc. We aimed to assess the frequency of -174 C/G of IL-6 gene polymorphism in SSc patients and healthy controls, as well as correlation with disease manifestations. In the case-control study, 102 patients with SSc and 93 controls were included. PCR-RFLP method was performed for genotyping promotor variants -174 C/G of IL-6 gene. The expression level of IL-6 was determined by qRT-PCR on subset of 50 patients and 13 healthy controls with different IL-6 genotypes. We used UCLA GIT 2.0 questionnaire to assess gastrointestinal involvement in SSc patients. The expression level of IL-6 gene was significantly higher in patients with SSc in comparison with healthy controls (p < 0.05). Carriers of C-allele of IL-6 gene compared to those with G allele, showed higher expression of IL-6 gene (95.8 vs. 41.2, p < 0.05), higher GIT total score (0.85 vs. 0.5, p < 0.05) and higher distension scale score (1.4 ± 0.9 vs. 0.78 ± 0.8, p = 0.05). No significant differences in genotype distribution and allele frequency were observed between patients and controls. The expression of IL6 gene varies significantly during the course of SSc. The IL-6 gene variant -174 C/G (presence of C-allele) is associated with higher IL-6 gene expression and greater GIT impairment in patients with SSc.

Expression of TLR7, TLR9, JAK2, and STAT3 genes in peripheral blood mononuclear cells from patients with systemic sclerosis.

Systemic sclerosis (SSc) is a rare, chronic, multisystem autoimmune disease clinically characterized by progressive fibrosis of the skin and internal organs. The basic mechanism appears to involve endothelial cell injury, overproduction of extracellular matrix proteins, and aberrant immune activation. So far, there have been a few attempts to find genetic biomarkers for monitoring disease activity or for correlation with certain symptoms. In order to reveal reliable biomarkers, we analyzed the expression of four genes representing three important signaling pathways, TLR7, TLR9, and JAK2-STAT3. Using RT-qPCR technique, we analyzed the expression of TLR7, TLR9, JAK2, and STAT3 genes in peripheral blood mononuclear cells of 50 SSc patients and 13 healthy individuals. We detected significant upregulation of TLR7 gene expression in a group of SSc patients compared to non-SSc group. Receiver operating characteristic (ROC) analysis showed that TLR7 expression efficiently discriminates SSc cases from healthy individuals. High TLR7 expression positively correlated with the late form of disease, active SSc, and the presence of digital ulcers. Decreased levels of TLR9 and JAK2 mRNA were found in the patient's cohort in comparison to non-SSc individuals, but showed no correlation with specific clinical outcomes. The expression level of the STAT3 gene did not differ between the analyzed groups. This is the first study on the expression of TLR7, TLR9, and STAT3 genes in SSc patients. Our results show that TLR7, TLR9, and JAK2 genes are potential biomarkers for SSc. The results obtained in this study could contribute to better classification, monitoring, and outcome prediction of patients with SSc based on genetics.

Intra-articular injection of autologous adipose-derived mesenchymal stem cells in the treatment of knee osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a chronic degenerative joint disease and is considered to be the fourth leading cause of disability and the second cause of inability to work in men. Recently, adipose-derived mesenchymal stem cells (AD-MSCs) came into focus for regenerative medicine as a promising tool for the treatment of OA. The administration of stem cells into impaired joints results in pain relief and improves quality of life, accompanied by restoration of hyaline articular cartilage. METHODS: In the present study, nine patients (including two patients with bilateral symptoms) diagnosed with osteoarthritis (International Knee Documentation grade B in 5 and grade D in six knees) were treated using a single injection of AD-MSCs at a concentration of 0.5-1.0 × 107 cells and were followed up for 18 months. During follow-up, all the cases were evaluated clinically by Knee Society score (KSS), Hospital for Special Surgery knee score (HSS-KS), Tegner-Lysholm (T-L) score and visual analogue scale (VAS) of pain, as well as by plain radiography and by magnetic resonance imaging visualization with 2D Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score assessment. RESULTS: Significant improvement of all four clinical scores was observed within the first 6 months (KSS for 41.4 points, HSS-KS for 33.9 points, T-L score for 44.8 points, VAS of pain from 54.5 to 9.3) and improvement persisted throughout the rest of the follow-up. MOCART score showed significant cartilage restoration (from 43 ± 7.2 to 63 ± 17.1), whereas radiography showed neither improvement, nor further joint degeneration. CONCLUSIONS: The results obtained in the present study provide good basis for prospective randomized controlled clinical trials with respect to the use of AD-MSCs in the treatment of osteoarthritis.

New PAH gene promoter KLF1 and 3'-region C/EBPalpha motifs influence transcription in vitro.

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50 % in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60 % of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

Predictive genetic markers of coagulation, inflammation and apoptosis in Perthes disease­Serbian experience.

UNLABELLED: Perthes disease is one of the most common forms of pediatric femoral head osteonecrosis with an unknown etiology. Coagulation factors were the first genetic factors suspected to have a role in the pathogenesis of this disease, but studies showed inconsistent results. It is described that inflammation is present during early stages of Perthes disease, but its genetic aspect has not been studied extensively. Little is known regarding the status of apoptotic factors during the repair process that leads to the occurrence of hip deformity in patients. Therefore, the aim of this study was to analyze major mediators involved in coagulation, inflammation, and apoptotic processes as possible causative factors of Perthes disease. The study cohort consisted of 37 patients. Gene variants of TNF-α, FV, FII, and MTHFR genes were determined by PCR-RFLP, while IL-3 and PAI-1 were genotyped by direct sequencing. The expression level of Bax, Bcl-2, Bcl2L12, Fas and FasL was analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) technique. Our results showed a significantly increased level of expression of pro-apoptotic factor Bax along with significantly higher Bax/Bcl-2 ratio in the patient group. CONCLUSION: The results presented indicate that apoptosis could be one of the factors contributing to the lack of balanced bone remodeling process in Perthes patients.