Indrikis Muiznieks

Embryonic Stem Cell Marker Expression Pattern in Human Mesenchymal Stem Cells Derived from Bone Marrow, Adipose Tissue, Heart and Dermis

Mesenchymal stem cells (MSCs) have been isolated from a variety of human tissues, e.g., bone marrow, adipose tissue, dermis, hair follicles, heart, liver, spleen, dental pulp. Due to their immunomodulatory and regenerative potential MSCs have shown promising results in preclinical and clinical studies for a variety of conditions, such as graft versus host disease (GvHD), Crohn’s disease, osteogenesis imperfecta, cartilage damage and myocardial infarction. MSC cultures are composed of heterogeneous cell populations. Complications in defining MSC arise from the fact that different laboratories have employed different tissue sources, extraction, and cultivation methods. Although cell-surface antigens of MSCs have been extensively explored, there is no conclusive evidence that unique stem cells markers are associated with these adult cells. Therefore the aim of this study was to examine expression of embryonic stem cell markers Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4 in adult mesenchymal stem cell populations derived from bone marrow, adipose tissue, dermis and heart. Furthermore, we tested whether human mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions. We found that bone marrow MSCs express embryonic stem cell markers Oct4, Nanog, alkaline phosphatase and SSEA-4, adipose tissue and dermis MSCs express Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4, whereas heart MSCs express Oct4, Nanog, SOX2 and SSEA-4. Our results also indicate that human adult mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions during early passages, as shown by distinct germ layer and embryonic stem cell marker expression patterns. Studies are now needed to determine the functional role of embryonic stem cell markers Oct4, Nanog and SOX2 in adult human MSCs.

Ultracentrifugal analysis of the quaternary structure of the raf represser from Escherichia coli

The raf repressor from Escherichia coli regulates the expression of the plasmid‐borne raf operon by switching between active and inactive conformational states. Ultracentrifugal analysis of the largely purified repressor proves the DNA‐free protein to undergo concentration‐dependent dissociation‐association. High‐speed sedimentation equilibria show that the 72 kDa dimer prevails under meniscus depletion conditions. At intracellular concentrations the 144 kDa dimer‐of‐dimers is the dominating species. It is suggested that the tetrameric structure of the raf repressor is involved in the recognition of the 18‐basepair operator DNA.

Protective effect of adenylate deaminase (from Penicillium lanoso-viride) against acute infections in mice

We examined the effects of the immunomodulator-adenylate deaminase (E.C. 3.5.4.6) from Penicillium lanoso-viride on experimental mice infections. Prophylactic intraperitoneal administration of adenylate deaminase (ADA) increased survival time and numbers of survivors after infection with Salmonella typhimurium, Pseudomonas aeruginosa and influenza A (H3N2) virus. Protection against influenza virus after intranasal ADA application was also observed. The influence of ADA was time and dose dependent. The most pronounced protection was obtained by administration of 3 U ADA/mice 24 h prior to infection. ADA had no antibiotic effect against these bacterial strains. Protective effects of ADA were studied in immunosuppressed mice under different regimes of treatment including cyclosporin A and trypan blue. The results indicated, that the protective effect of ADA is of a complex nature and probably depends on both T-cell and macrophage components.

Successive binding of raf repressor to adjacent raf operator sites in vitro

SummaryThe raf repressor negatively regulates the transcription of the raf operon which encodes functions required for the uptake and hydrolysis of raffinose in Escherichia coli. Overexpression of the repressor gene under lac promoter control led to the formation of inclusion bodies. These were partially purified by centrifugation, solubilized in 0.1 % SDS and reactivated by dilution. DNase I protection and gel retardation experiments demonstrated the specific binding of raf repressor to DNA fragments that contained the previously identified raf operator, an element comprising two 18 by palindromic nucleotide sequences that flank the −35 raf promoter box. By using DNA fragments with one, two, or four copies of the 18 by palindrome, these experiments revealed concentration dependent, successive occupation of all available binding sites by raf repressor. Melibiose released the repressor from the operator complexes, whereas raffinose and other α-galactosides did not, indicating that melibiose is the actual inducer in vivo. We suggest that successive occupation by repressor of two strategically located operator sites is a specific type of stepwise down-regulation of gene expression in response to repressor concentration.

Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A

BackgroundImportant knowledge about the role of vitamin A in vertebrate heart development has been obtained using the vitamin A-deficient avian in ovo model which enables the in vivo examination of very early stages of vertebrate heart morphogenesis. These studies have revealed the critical role of the vitamin A-active form, retinoic acid (RA) in the regulation of several developmental genes, including the important growth regulatory factor, transforming growth factor-beta2 (TGFβ2), involved in early events of heart morphogenesis. However, this in ovo model is not readily available for elucidating details of molecular mechanisms determining RA activity, thus limiting further examination of RA-regulated early heart morphogenesis. In order to obtain insights into RA-regulated gene expression during these early events, a reliable in vitro model is needed. Here we describe a cell culture that closely reproduces the in ovo observed regulatory effects of RA on TGFβ2 and on several developmental genes linked to TGFβ signaling during heart morphogenesis.ResultsWe have developed an avian heart forming region (HFR) cell based in vitro model that displays the characteristics associated with vertebrate early heart morphogenesis, i.e. the expression of Nkx2.5 and GATA4, the cardiogenesis genes, of vascular endothelial growth factor (VEGF-A), the vasculogenesis gene and of fibronectin (FN1), an essential component in building the heart, and the expression of the multifunctional genes TGFβ2 and neogenin (NEO). Importantly, we established that the HFR cell culture is a valid model to study RA-regulated molecular events during heart morphogenesis and that the expression of TGFβ2 as well as the expression of several TGFβ2-linked developmental genes is regulated by RA.ConclusionsOur findings reported here offer a biologically relevant experimental in vitro system for the elucidation of RA-regulated expression of TGFβ2 and other genes involved in vertebrate early cardiovascular morphogenesis.

Role of two operators in regulating the plasmid-borne raf operon of Escherichia coli

The plasmid-borne raf operon encodes functions required for the inducible uptake and utilization of raffinose in Escherichia coli K12. The expression of three structural genes for α-galactosidase (rafA), Raf permease (rajB) and sucrose hydrolase (rafD) is negatively controlled by the binding of RafR repressor (rafR) to two operator sites, O1 and O2, that flank the − 35 sequence of the raf promoter, PA. In vitro, O1 and 02 are occupied on increasing the concentration of RafR, without detectable preference for one site or the other or any indication of cooperative binding. Nucleotide substitutions at positions 3, 4 or 5 in an operator half-site prevented repressor binding, supporting a model that postulates specific interactions of these base pairs with the recognition helix of RafR. To study the role of each operator site, we have compared by gel shift analysis the binding of purified RafR repressor to DNA fragments containing the original 0102 configuration or mutant O1 or 02. When either one of the two operators was inactivated by site-directed mutagenesis, both 01 and 02 exhibited the same affinity for repressor and the same sensitivity to arrest of repressor binding by the natural inducer, melibiose. However, in the native 0102 configuration, simultaneous binding of RafR to both operators was sterically hindered, leading to a 13-fold decrease in the intrinsic affinity of an operator site for repressor, once the other site had been occupied. To assess the role of each operator in vivo, rafA was used as a reporter gene. A 1200-fold repression (100%) was exerted by RafR binding to the native O1O2 configuration, whereas 02 alone exerted 45% and 01 alone 6% repression of rafA transcription. The differential effects of 01 versus 02 on transcription (despite matching affinities of 01 and 02 for repressor) suggest that positioning of the O2-repressor complex between the — 35 and —10 signals is crucial for transcription control and that repressor binding to the upstream 01 serves to enhance this effect.

The comparison of knee osteoarthritis treatment with single-dose bone marrow-derived mononuclear cells vs. hyaluronic acid injections

OBJECTIVE The aim of this study was to compare treatment methods of the knee joint degenerative osteoarthritis, using autologous bone marrow-derived mononuclear cells and hyaluronic acid injections and observe prevalence of adverse effects in both groups. MATERIALS AND METHODS A prospective randomized controlled clinical trial was carried out. The analysis of pain and changes in osteoarthritis symptoms after a single intra-articular bone marrow-derived mononuclear cell injection into the knee joint in the Kellgren-Lawrence stage II-III osteoarthritis during the 12-month period were performed. The results were compared with the control group treated routinely by hyaluronic acid injections therapy. A therapy group of patients (n=28) received single bone marrow-derived mononuclear cell intra-articular injections. A control group of patients (n=28) was treated with a total of three sodium hyaluronate intra-articular injections each one performed a week apart. The clinical results were obtained using the Knee Osteoarthritis Outcome Score (KOOS) and the Knee Society Score (KSS) before and 3, 6, and 12 months after injection. RESULTS A statistically significant improvement was observed in the mononuclear cell group over the starting point in all scores. At the endpoint at month 12, the KOOS score improved significantly (P<0.05) on the pain subscale (+25.44), activity and daily living subscale (+21.36), quality of life subscale (+28.83), and total KOOS (+18.25). The KSS score also demonstrated a significant improvement on the symptoms subscale (+25.42) and the function subscale (+38.32) (P<0.001). The KOOS symptoms and sports subscales improved without statistical significance. The difference between the control group treated with hyaluronic acid versus the bone marrow-derived mononuclear cells group at time points 6 and 12 months demonstrated a statistically significant (P<0.05) superiority in the KOOS pain subscale over the hyaluronic acid group. In both groups serious adverse effects were not observed. CONCLUSIONS The intra-articular injection of bone marrow-derived mononuclear cells is a safe manipulation with no side effects during the 12-month period. This treatment provides statistically significant clinical improvement between the starting point and 1, 3, 6, and 12 months after. When compared to hyaluronic acid treatment, better pain relief in the long-term period of mononuclear cell group was observed.

Microbiological characterization and sterilization-induced changes in the profile of the hydrophobic organic substances in Latvian balneological peat

The abundance and predominant groups of bacteria, filamentous fungi and yeasts have been studied by culture-dependent microbiological methods in peat probes obtained in two Latvian balneotherapy spa sites, Kemeri and Baldone. Unsterilized peat samples from both the sites contained 5.7–8.1 log bacterial colony-forming units (CFU) and 3.0–5.3 log fungal CFU per gram of dry peat. Isolated species belonged to Alpha-, Beta-, and Gamma-Proteobacteria, Actinobacteria, Clostridia, Bacilli and Flavobacteria as well as to filamentous fungi and yeasts. The composition of microbial population of the peat from both sites shared just four micro-organism groups (Bacillus mycoides, Burkholderia cepacia, Streptomyces spp. and Trichoderma spp.) within totally 36 groups identified. No pathogenic bacteria or fungi and no faecal pollution indicators were recovered. Decimal reduction doses for micro-organisms in peat samples and radiation sterilization doses of peat for the gamma and electron beam radiation were determined. The highest radiation resistance was observed for B. mycoides and Aureobasidium sp. Gamma-sitosterol was the most abundant hydrophobic organic compound in both peats according to GC–MS data. All the sterilization procedures increased concentration of alkanes, alcohols, and ketones and decreased the amount of fatty acids. Heat sterilization proved to be more preserving for the peat sterols than the radiation sterilization. It is concluded that the heat and radiation sterilization methods induce different changes of the profile of hydrophobic organic compounds of balneological peats, what may lead to different therapeutic effects at their application.

Particular characteristics of soil microbial communities in forest stands infected with Heterobasidion parviporum and Armillaria spp.

We compared soil microbial communities of forests infected with Heterobasidion parviporum and Armillaria spp. with soils of healthy forests using conventional plating and molecular methods. Plate counts from the soils of the infected forests reflected a significant decrease of the number of cultivable filamentous fungi (CFF) and a slight decrease of the total number of cultivable microorganisms. The diversity of CFF was reduced in the stands infected with H. parviporum. In the stands infected with Armillaria spp. the diversity of CFF and relative abundance of cultivable Trichoderma spp. was even higher than in healthy forest stands. Quantitative PCR revealed increased concentrations of total fungal DNA and Trichoderma spp. DNA in the soil of H. parviporum infected stands. In Armillaria spp. infected stands the total concentration of fungal DNA was decreased, but relative amount of Trichoderma spp. DNA was increased. No significant differences in the species diversity of fungi in the soil were found by ARDRA.

Treatment of Knee Osteoarthritis with Bone Marrow–Derived Mononuclear Cell Injection: 12-Month Follow-up

Objectives To evaluate the main symptoms of knee osteoarthritis (OA) and tissue structure changes after a single dose bone marrow–derived mononuclear cell (BM MNC) intra articular injection. Case series study. Patients with knee OA Kellgren Lawrence (K-L) grade II and III received 1 injection of BM MNC. The clinical results were analyzed with the Knee injury and Osteoarthritis Outcome Score (KOOS) and Knee Society Score (KSS) before, 3, 6, and 12 months after injection. Radiological evaluation was performed with a calibrated x-ray and the magnetic resonance (MR) imaging before and 6 to 7 months postinjection. Results A total of 34 knees were treated with BM MNC injections. Mean (±SD) age of patient group was 53.96 ± 14.15 years; there were 16 males, 16 females, KL grade II, 16; KL grade III, 18. The average injected count of BM MNCs was 45.56 ± 34.94 × 106 cells. At the endpoint of 12 months 65% of patients still had minimal perceptible clinical improvement of the KOOS total score. The mean improvement of KOOS total score was +15.3 and of the KSS knee score was +21.45 and the function subscale +27.08 (P < 0.05) points. The Whole Organ Magnetic Resonance Imaging Score (WORMS) improved from 44.31 to 42.93 points (P < 0.05). No adverse effects after the BM-MNC injection were observed. Conclusions The single dose BM MNC partially reduces clinical signs of the knee osteoarthritis stage II/III and in some cases, decreases degenerative changes in the joint building tissue over 12-month period.