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

BackgroundOsteoarthritis (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. MethodsIn 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 x 10(7) cells and were followed up for 18months. 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. ResultsSignificant improvement of all four clinical scores was observed within the first 6months (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. ConclusionsThe 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.This is the peer reviewed version of the paper : Spasovski, D., Spasovski, V., Baščarević, Z., Stojiljković, M., Vreća, M., Anđelković, M., & Pavlović, S. (2018). Intra-articular injection of autologous adipose-derived mesenchymal stem cells in the treatment of knee osteoarthritis. The Journal of Gene Medicine, 20(1), e3002. [https://doi.org/10.1002/jgm.3002

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 gt A and miR-146a rs2910164 C gt 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 gt 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.This is the peer reviewed version of the paper: Vreca, M., Andjelkovic, M., Tosic, N., Zekovic, A., Damjanov, N., Pavlovic, S., & Spasovski, V. (2018). Impact of alterations in X-linked IRAK1gene and miR-146a on susceptibility and clinical manifestations in patients with systemic sclerosis. Immunology Letters, 204, 1–8. [https://doi.org/10.1016/j.imlet.2018.10.002

Expression of miRNA-210 in human bone marrow-derived mesenchymal stromal cells under oxygen deprivation

A major limitation in the development of efficient clinical protocols for mesenchymal stromal cell (MStroC)-based tissue regeneration therapy is the low retention and survival of MStroC in injured tissue after therapeutic administration. Low oxygen concentration preconditioning (LOP) during ex vivo cultivation of MStroC, as a method for mimicking oxygenation in their physiological microenvironment, has been shown to be beneficial in clinical trials using MStroC. Introducing hypoxia-mimicking molecules into MStroC during cultivation could be an advantageous LOP strategy. MicroRNA (miRNA) drugs are good candidates for this approach. Analysis of the expression of miRNA-210 in human bone marrow-derived MStroC in conditions of acute and extended hypoxia (24 to 72 h) was performed using RT-qPCR methodology. HIF-1 alpha and HIF-2 alpha gene knockdown cell lines were generated using lentiviral transduction of short hairpin RNA (shRNA) in order to examine whether miRNA-210 expression is regulated by transcription factor HIF-1 and/or HIF-2. We detected a significant increase in miRNA-210 expression in hypoxic conditions at time points of 24, 48 and 72 h (p lt 0.05). Knocking down of HIF-1 alpha and HIF-2 alpha genes indicated involvement of both transcription factors in the elevation of miRNA-210 expression. These results point to miRNA-210 as a good candidate for a hypoxia-mimicking molecule in LOP strategy

Variants in vdr and nramp1 genes as susceptibility factors for tuberculosis in the population of Serbia

Tuberculosis (TB) is granulomatous diseases caused by Mycobacterium tuberculosis (MTB). TB is a highly infectious disease that primarily affects the lungs. One-third of human population is infected with MTB, therefore it is of utmost significance to determine the factors that influence the individual susceptibility to the disease. Host genetic factors have been recognized as essential for susceptibility to TB, since only 5% to 10% of infected individuals develop the disease. A number of candidate genes has been intensively studied, the most of which were connected with the function of macrophages, thus participating in immune response. Here we examined the gene variants of VDR (FokI) and NRAMP1 (INT4, D543N, 3'UTR) genes in aim to make the correlation between these genetic factors and risk of TB in Serbian patients. This study included 110 TB patients and 67 healthy controls. Pulmonary TB was diagnosed by clinical symptoms, radiological evidence of TB and bacteriological criteria (Culture-positive/smear-positive). Genotyping was performed using PCR-RFLP method. Our findings revealed significant prevalence of ff genotype and variant allele f of the FokI VDR gene variant in patients compared to control group. Based on the our results the carriers of ff genotype are five times more at risk to tuberculosis than carriers of FF and Ff genotype in our population. The results of analyzed SNPs in NRAMP1 gene showed no statistically significant difference in distribution of the gene variants between patient and control groups. Therefore, we could conclude that the genotype ff of the VDR gene is factor that strongly contribute to susceptibility to TB in Serbian population

Relevance of TNF-alpha, 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-alpha, 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-alpha 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-alpha and IL-6 gene expression. When clinical parameters were analyzed, increased levels of TNF-alpha mRNA were detected in patients with a longer duration of therapy ( gt 2 months) compared to those with a shorter therapy duration ( lt 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-alpha gene could be a marker for monitoring the clinical effect of the ant-tuberculosis drugs during therapy

Clinical case seminar - familial intracranial germinoma

Background: Intracranial germinomas (ICG) are uncommon brain neoplasms with extremely rare familial occurrence. Because ICG invades the hypothalamus and/or pituitary, endocrine dysfunction is one of the common determinants of these tumours. We present two brothers with a history of ICG. Patient 1 is a 25-year-old male who suffered from weakness of the right half of his body at the age of 18 years. Cranial MRI revealed a mass lesion in the left thalamus. He underwent neurosurgery, and the tumour was removed completely. Histopathological (HP) and immunohistochemical analyses verified the diagnosis of pure germinoma. He experienced complete remission of the tumour after radiation therapy. At the age of 22 years a diagnosis of isolated growth hormone deficiency (IGHD) was established and GH replacement was initiated. Molecular genetic analysis of the tumour tissue detected the mutation within exon 2 in KRAS gene. Patient 2 is a 20-year-old man who presented with diabetes insipidus at the age of 12 years. MRI detected tumour in the third ventricle and pineal region. After endoscopic tumour biopsy the HP diagnosis was pure germinoma. He received chemotherapy followed by radiotherapy and was treated with GH during childhood. At the age of 18 years GH replacement was reintroduced. A six-month follow-up during the subsequent two years in both brothers demonstrated the IGF1 normalisation with no MRI signs of tumour recurrence. Conclusion: To the best of our knowledge, so far only six reports have been published related to familial ICG. The presented two brothers are the first report of familial ICG case outside Japan. They have been treated successfully with GH therapy in adulthood

Opis przypadku — występujący rodzinnie wewnątrzczaszkowo germinoma

Background. Intracranial germinomas (ICG) are uncommon brain neoplasms with extremely rare familial occurance. Since ICG invades hypothalamus and/or pituitary, the endocrine dysfunction is one of the common determinants of these tumors. We presented two brothers with the history of ICG. Patient 1 is a 25-year-old male who had been suffering from the weakness of the right half of his body at the age of 18. Cranial MRI revealed mass lesion in the left thalamus. He underwent neurosurgery, tumor was removed completely. Histopathological (HP) and immunohistochemical analyses verified the diagnosis of pure germinoma. He experienced complete remission of the tumor after a radiation therapy. At the age of 22 the diagnosis of isolated growth hormone deficiency (IGHD) was established and GH replacement was initiated. Patient 2 is a 20-year old boy who was presented with diabetes insipidus at the age of 12. MRI detected tumor in the third ventricle and pineal region. After the endoscopic tumor biopsy the HP diagnosis was pure germinoma. He received chemotherapy followed by radiotherapy, and treated with GH during childhood. At the age of 18 GH replacement was reintroduced. A six month follow-up during the next two years in both brothers demonstrated the IGF1 normalization with no MRI signs of tumor recurrence. Conclusion. To the best of our knowledge so far, only six reports have been published related to familial ICG. The presented two brothers are the first report of familial ICG case outside of Japan. They are treated successfully with GH therapy in adult period. Wstęp: Rozrodczaki wewnątrzczaszkowe (intracranial germinomas, ICG) to rzadkie nowotwory mózgu, a szczególnie rzadko stwierdza się ich występowanie rodzinne. W związku z tym, że ICG zajmuje podwzgórze i/lub przysadkę mózgową, zaburzenia endokrynologiczne są jednym z najczęstszych wyznaczników obecności tych guzów. W pracy przedstawiono dwóch braci z ICG. Pacjent 1 to 25-letni mężczyzna, u którego w wieku 18 lat wystąpiło osłabienie mięśni po lewej stronie ciała. Badanie metodą rezonansu magnetycznego (MRI) czaszki ujawniło masę w lewym wzgórzu. Chorego poddano zabiegowi neurochirurgicznemu, podczas którego guz został całkowicie usunięty. Badania histopatologiczne i immunohistochemiczne potwierdziły rozpoznanie czystej postaci rozrodczaka. Po radioterapii nastąpiła całkowita remisja guza. W wieku 22 lat u chorego zdia­gnozowano izolowany niedobór hormonu wzrostu (isolated growth hormone deficiency, IGHD) i wdrożono terapię zastępczą hormonem wzrostu (growth hormone, GH). Genetyczna analiza molekularna tkanki guza wykazała mutację w eksonie 2 w genie KRAS. Pacjent 2 to 20-letni mężczyzna, u którego w wieku 12 lat stwierdzono moczówkę prostą. W badaniu MRI wykryto guz w okolicy trzeciej komory i szyszynki. Po ocenie histopatologicznej materiału pobranego za pomocą biopsji endoskopowej postawiono diagnozę czystego rozrod­czaka. U chorego zastosowano chemioterapię, a następnie radioterapię, a także podawano GH w okresie dzieciństwa. W wieku 18 lat u chorego wznowiono terapię GH. Sześciomiesięczna obserwacja obu braci w następnych 2 latach wykazała normalizację IGF1 przy braku objawów nawrotu guza w badaniu MRI. Wnioski: Według najlepszej wiedzy autorów dotychczas opublikowano 6 doniesień na temat rodzinnego występowania ICG. Przed­stawieni w niniejszej pracy bracia są pierwszym opisanym przypadkiem rodzinnego ICG poza Japonią. W okresie dorosłym chorzy są leczeni GH z dobrym skutkiem

Sources, background and enrichment of lead and other elements: Lower Guadiana River

The lower sector of the Guadiana River Basin, located in southern Portugal and Spain, hosts soils and plants with elevated Pb, Cu and As near former and current mining sites. Two geogenic and two anthropogenic sources of Pb were identified where elevated concentrations are mostly related to the occurrence of sulphide-rich ore deposits, Volcanic Sedimentary formations and mining. These were generally reflected by the multiple regression analysis (MRA) and confirmed by isotope determinations. Nevertheless, caution was needed in interpreting statistical and isotopic results; therefore the combination of both techniques was important. Elements, such as Ca, Na, Cu and As, show enrichment in soil developed on shale, sandstone and conglomerate of Upper Devonian age belonging to the Phyllite-Quartzite Group. Lead exhibits an enrichment in soil developed on felsic volcanic rocks from the Volcanic Sedimentary Complex (VSC) of Upper Devonian-Lower Carboniferous age, which has been identified by the relationship between topsoil median values of different lithologies and grand subsoil median values. In the same soil, Fe, As, Co, Ni and Cr are depleted. Translocation of Pb to the aerial parts of plants is insignificant in all three plant species studied and analysed (Cistus ladanifer L., Thymus vulgaris, Lavandula luisieri). High Pb concentrations in soil, where Cistus ladanifer L. developed, the only representative number of species analysed, do not correspond generally to elevated Pb contents in plants, except near mine sites, where lower pH of soil, increases Pb bioavailability. The different statistical methodologies combined with Pb isotopic studies were successfully applied in the identification of Pb sources in soil and Cistus ladanifer L. plant of the Lower sector of Guadiana River basin. Therefore, rocks, mineralisations, subsoil, topsoil and plant processes were successfully integrated to understand the migration of Pb into the food chai