Intracellular delivery of trehalose renders mesenchymal stromal cells viable and immunomodulatory competent after cryopreservation

Cytotechnology - Trehalose is a nontoxic disaccharide and a promising cryoprotection agent for medically applicable cells. In this study, the efficiency of combining trehalose with reversible...     

Strain typing with ISLpl1 in lactobacilli

Twenty-seven Lactobacillus plantarum ssp. plantarum, 11 Lactobacillus paraplantarum and five Lactobacillus casei-related strains, isolated from various autochthonous Serbian and Montenegro-fermented foods, were identified using phenotypical characterization and current PCR methods based on PCR of the recA gene or the 23S-5S rRNA gene intragenic spacer (IS) region. The strains were genotypically characterized by a new method based on the insertion sequence element ISLpl11 that grouped these lactobacilli into 10 IS-fingerprinting groups. Between six and 23 copies of the ISLpl1 were found in each strain and the ISLpl1-fingerprint groups correlated well with the origin of the strains. The method proved suitable for strain typing of lactic acid bacteria at the infraspecies level.     

Testing the potency of anti‐TNF‐α and anti‐IL‐1β drugs using spheroid cultures of human osteoarthritic chondrocytes and donor‐matched chondrogenically differentiated mesenchymal stem cells

Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor‐alpha (TNF‐α) and interleukin 1β (IL‐1β). New in vitro testing systems are needed to evaluate efficacies of new anti‐inflammatory biological drugs, ideally in a patient‐specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti‐inflammatory drugs, spheroids were exposed to TNF‐α, IL‐1β, or to supernatant containing secretome from activated macrophages (MCM). The anti‐inflammatory efficacies of anti‐TNF‐α biologicals adalimumab, infliximab, and etanercept, and the anti‐IL‐1β agent anakinra were assessed in short‐term microspheroid and long‐term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF‐α or IL‐1β. The differences in potency of anti‐TNF‐α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short‐term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti‐TNF‐α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045–1058, 2018     

The outcome of autologous chondrocyte transplantation treatment of cartilage lesions in the knee

Recent results of the clinical outcome of autologous chondrocyte transplantation (ACT) treatment in a group of 28 patients with focal femoral condyle cartilage lesions revealed a correlation trend with the quality of the in vitro cell culture matrix-protein synthesis. No impact of the patients' age and chondrocyte cryopreservation prior to implantation was observed. Further studies are needed to confirm the preliminary results.     

Structural studies of the capsular polysaccharide of Klebsiella type 52

The structure of the capsular polysaccharide from Klebsiella Type 52 has been investigated. Methylation analysis, characterization by gas-liquid chromatography-mass spectrometry of oligosaccharide derivatives obtained on partial hydrolysis of the methylated polysaccharide with acid, and specific degradation of the methylated polysaccharide by successive treatments with base and acid followed by characterization of the product, were the principal methods used. The polysaccharide is composed of hexasaccharide repeating-units containing D-glucuronic acid, D-galactose, and L-rhamnose, in the ratios 1:3:2. A structure for these units, disregarding the anomeric natures of the sugar residues, is proposed.     

Analysis of Glioblastoma Patients' Plasma Revealed the Presence of MicroRNAs with a Prognostic Impact on Survival and Those of Viral Origin

BackgroundGlioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions.ResultsWe identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients’ survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM.ConclusionThe GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients.     

Nucleus pulposus repair with cultured autologous elastic cartilage derived chondrocytes

Low back pain is one of the most common medical conditions in the Western world. Disc degeneration, an inevitable process of ageing, is one of the major causes of low back pain. Autologous chondrocyte transplantation (ACT) is an increasingly popular method of addressing pathological disorders of cartilage. The purpose of our study was to determine whether autologous chondrocytes from elastic cartilage could survive and synthesise a cartilage specific matrix in the intervertebral disc of rabbits. Sixteen lumbar intervertebral discs (IVD) of New Zealand White rabbits were analysed. In 6 IVD, the nucleus pulposus was evacuated and replaced with tissue engineered autologous chondrocytes from auricular cartilage. In the second group, only the nucleus pulposus was evacuated from 6 IVD, with no chondrocytes implantation. Four non-operated IVD were used as a control. Six months after the operation, the animals were euthanized and the IVD were analysed histologically. Autologous cartilage implants were well tolerated by the host for up to six months in vivo. There was only hyaline-like cartilage in the place of the nucleus pulposus. We could not detect any elastic fibres in the new cartilage matrix. In IVD from which only the nucleus pulposus was evacuated and no chondrocytes were implanted, just fibrous tissue was found instead of nucleus pulposus. The overall histological analysis of new cartilage produced after implantation in our study confirmed the hypothesis that ACT from auricular cartilage can be implanted into the IVD instead of the nucleus pulposus and that a significant percentage of implanted chondrocytes survive and produce hyaline-like cartilage.     

A "crossomics" study analysing variability of different components in peripheral blood of healthy caucasoid individuals

Background

Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable.

Methodology/Principal Findings

To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex “crossomics” analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4⁺T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset.

Conclusions/Significance

Our study shows that the intra-individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective.     

A Comparative Study of In Vitro Cytotoxic,Antioxidant, and Antimicrobial Activity of Pt(II), Zn(II), Cu(II), and Co(III) Complexes with N‐heteroaromatic Schiff Base (E)‐2‐[N′‐(1‐pyridin‐2‐yl‐ethylidene)hydrazino]acetate

In search for novel biologically active metal based compounds, an evaluation of in vitro cytotoxic, antioxidant, and antimicrobial activity of new Pt(II) complex and its Zn(II), Cu(II), and Co(III) analogues, with NNO tridentately coordinated N‐heteroaromatic Schiff base ligand (E)‐2‐[N′‐(1‐pyridin‐2‐yl‐ethylidene)hydrazino]acetate, was performed. Investigation of antioxidative properties showed that all of the compounds have strong radical scavenging potencies. The Zn(II) complex showed potent inhibition of DNA cleavage by hydroxyl radical. A cytotoxic action of investigated compounds was evaluated on cultures of human promyelocitic leukaemia (HL‐60), human glioma (U251), rat glioma (C6), and mouse melanoma (B16) cell lines. It was shown that binuclear pentacoordinated Zn(II) complex possesses a strong dose‐dependent cytotoxic activity, of the same order of magnitude as cisplatin on B16, C6, and U251 cells. Furthermore, Zn(II) complex causes oxidative stress‐induced apoptotic death of HL‐60 leukemic cells, associated with caspase activation, phosphatidylserine externalization, and DNA fragmentation.