Project description:Loss of immune function and an increased incidence of myeloid leukemia are two of the most clinically significant consequences of aging of the hematopoietic system. To better understand the mechanisms underlying hematopoietic aging, we evaluated the cell intrinsic functional and molecular properties of highly purified long-term hematopoietic stem cells (LT-HSCs) from young and old mice. We found that LT-HSC aging was accompanied by cell autonomous changes, including increased stem cell self-renewal, differential capacity to generate committed myeloid and lymphoid progenitors, and diminished lymphoid potential. Expression profiling revealed that LT-HSC aging was accompanied by the systemic down-regulation of genes mediating lymphoid specification and function and up-regulation of genes involved in specifying myeloid fate and function. Moreover, LT-HSCs from old mice expressed elevated levels of many genes involved in leukemic transformation. These data support a model in which age-dependent alterations in gene expression at the stem cell level presage downstream developmental potential and thereby contribute to age-dependent immune decline, and perhaps also to the increased incidence of leukemia in the elderly. 3 old mice and 5 young mice were assayed
Project description:ObjectivesTo expand the knowledge about the immunological consequences of cladribine (CLAD), a pulsed immune reconstitution therapy approved for active multiple sclerosis (MS), beyond the known short-term effects on peripheral immune cell subsets.MethodsIn this study, we characterized depletion and restitution kinetics as well as cytokine profiles of peripheral immune cell subsets in 18 patients with MS following treatment with oral CLAD. The methods involved blood collection prior to CLAD and every three months over a period of 24 months, and extensive characterization of various immune cells subsets by multiparametric flow cytometry.ResultsWe found a selectivity of CLAD towards central memory T cells and memory B cells and detected a hyper-repopulation of maturing B cells. Counts of classical (-65%) and various nonclassical TH17 cells (-84% to -87%) were markedly reduced 24 months after treatment start, and were comparable with depletion rates of class-switched memory B-cell phenotypes (-87% to -95%). The nadir of TH cells was more pronounced in the second treatment year. We observed a proportional surge of CD20 T-cell subsets and an expansion of regulatory T, B and NK cells. Natural killer T cells (NKT) were only depleted in year two and did not recover.InterpretationPeripheral immune cell profiling revealed more differentiated insights into the immunological effects of CLAD. While some immune cell subsets expanded, we also observed additive depleting effects after the second treatment course. Further studies are required to elucidate whether these changes are paramount for the consistent and prolonged disease-modifying effect of CLAD.
Project description:MicroRNAs are important negative regulators of protein coding gene expression, and have been studied intensively over the last few years. To this purpose, different measurement platforms to determine their RNA abundance levels in biological samples have been developed. In this study, we have systematically compared 12 commercially available microRNA expression platforms by measuring an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples, and synthetic spikes from homologous microRNA family members. We developed novel quality metrics in order to objectively assess platform performance of very different technologies such as small RNA sequencing, RT-qPCR and (microarray) hybridization. We assessed reproducibility, sensitivity, quantitative performance, and specificity. The results indicate that each method has its strengths and weaknesses, which helps guiding informed selection of a quantitative microRNA gene expression platform in function of particular study goals.
Project description:Abstract: The aim of the study was to analyze exhaled breath condensate (EBC) proteome changes due to the effects of spaceflight factors. All the protocols were approved at the Institute of Biomedical Problems – Russian Federation State Scientific Research Center in the frame of «Protocon» experiment. EBC samples were collected by commercial devices RTube one month before flight (background) at the Yu. A. Gagarin Research and Test Cosmonaut Training Center, immediately after landing of the landing modules in the field (R0), and on the seventh day after landing as a part of medical examination (R+7). Semi-quantitative label-free proteomic analysis of 13 EBC samples collected from 5 Russian cosmonauts before and after long-term (169–199 days) spaceflights were performed and resulted in 164 different proteins. The highest number of proteins was detected in EBC after landing (R0). Pathways enrichment analysis via GO database large group of proteins that take part in keratinization processes (CASP14, DSG1, DSP, JUP, and etc). Nine proteins were KRT2, KRT9, KRT1, KRT10, KRT14, DCD, KRT6C, KRT6A, KRT5 were detected in all groups (background, R0, R+1). A two-sample Welch’s t-test identified the significant changing of KRT2 and KRT9 levels after landing. Enrichment analysis via KEGG database revealed significant participation (2,07E-06) of detected proteins in pathogenic E. coli infection (ACTG1, TUBA1C, TUBA4A, TUBB, TUBB8, YWHAZ). Presumably, presents of this proteins can associated with changing of cosmonauts’ microbial composition. Thus EBC can be used for noninvasive monitoring of health status and respiratory tract pathologies during the spaceflight. The obtained data are important for the development of medicine in extreme situations.
Project description:In order to determine whether dis-regulation of a genetic pathway could explain the increased apoptosis of parp-2-/- double positive thymocytes, the gene expression profiles in double positive thymocytes derived from wild-type and parp-2-/- mice were analysed using Affymetrix oligonucleotide chips (mouse genome 430 2.0).
Project description:One of the most intriguing puzzles in biology is the degree to which evolution is repeatable. The repeatability of evolution, or parallel evolution, has been studied in a variety of model systems, but has rarely been investigated with clinically relevant viruses. To investigate parallel evolution of HIV-1, we passaged two replicate HIV-1 populations for almost 1 year in each of two human T-cell lines. For each of the four evolution lines, we determined the genetic composition of the viral population at nine time points by deep sequencing the entire genome. Mutations that were carried by the majority of the viral population accumulated continuously over 1 year in each evolution line. Many majority mutations appeared in more than one evolution line, that is, our experiments showed an extreme degree of parallel evolution. In one of the evolution lines, 62% of the majority mutations also occur in another line. The parallelism impairs our ability to reconstruct the evolutionary history by phylogenetic methods. We show that one can infer the correct phylogenetic topology by including minority mutations in our analysis. We also find that mutation diversity at the beginning of the experiment is predictive of the frequency of majority mutations at the end of the experiment.