Location of the CD8 T Cell Epitope within the Antigenic Precursor Determines Immunogenicity and Protection against the Toxoplasma gondii Parasite

CD8 T cells protect the host from disease caused by intracellular pathogens, such as the Toxoplasma gondii (T. gondii) protozoan parasite. Despite the complexity of the T. gondii proteome, CD8 T cell responses are restricted to only a small number of peptide epitopes derived from a limited set of antigenic precursors. This phenomenon is known as immunodominance and is key to effective vaccine design. However, the mechanisms that determine the immunogenicity and immunodominance hierarchy of parasite antigens are not well understood.Here, using genetically modified parasites, we show that parasite burden is controlled by the immunodominant GRA6-specific CD8 T cell response but not by responses to the subdominant GRA4- and ROP7-derived epitopes. Remarkably, optimal processing and immunodominance were determined by the location of the peptide epitope at the C-terminus of the GRA6 antigenic precursor. In contrast, immunodominance could not be explained by the peptide affinity for the MHC I molecule or the frequency of T cell precursors in the naive animals. Our results reveal the molecular requirements for optimal presentation of an intracellular parasite antigen and for eliciting protective CD8 T cells.     

ATM-deficient human neural stem cells as an in vitro model system to study neurodegeneration

Loss of ATM kinase, a transducer of the DNA damage response and redox sensor, causes the neurodegenerative disorder ataxia-telangiectasia (A-T). While a great deal of progress has been made in elucidating the ATM-dependent DNA damage response (DDR) network, a key challenge remains in understanding the selective susceptibility of the nervous system to faulty DDR. Several factors appear implicated in the neurodegenerative phenotype in A-T, but which of them plays a crucial role remains unclear, especially since mouse models of A-T do not fully mirror the respective human syndrome. Therefore, a number of human neural stem cell (hNSC) systems have been developed to get an insight into the molecular mechanisms of neurodegeneration as consequence of ATM inactivation. Here we review the hNSC systems developed by us an others to model A-T.     

Telocytes express PDGFRα in the human gastrointestinal tract

Telocytes (TC), a cell population located in the connective tissue of many organs of humans and laboratory mammals, are characterized by a small cell body and extremely long and thin processes. Different TC subpopulations share unique ultrastructural features, but express different markers. In the gastrointestinal (GI) tract, cells with features of TC were seen to be CD34‐positive/c‐kit‐negative and several roles have been proposed for them. Other interstitial cell types with regulatory roles described in the gut are the c‐kit‐positive/CD34‐negative/platelet‐derived growth factor receptor α (PDGFRα)‐negative interstitial cells of Cajal (ICC) and the PDGFRα‐positive/c‐kit‐negative fibroblast‐like cells (FLC). As TC display the same features and locations of the PDGFRα‐positive cells, we investigated whether TC and PDGFRα‐positive cells could be the same cell type. PDGFRα/CD34, PDGFRα/c‐kit and CD34/c‐kit double immunolabelling was performed in full‐thickness specimens from human oesophagus, stomach and small and large intestines. All TC in the mucosa, submucosa and muscle coat were PDGFRα/CD34‐positive. TC formed a three‐dimensional network in the submucosa and in the interstitium between muscle layers, and an almost continuous layer at the submucosal borders of muscularis mucosae and circular muscle layer. Moreover, TC encircled muscle bundles, nerve structures, blood vessels, funds of gastric glands and intestinal crypts. Some TC were located within the muscle bundles, displaying the same location of ICC and running intermingled with them. ICC were c‐kit‐positive and CD34/PDGFRα‐negative. In conclusion, in the human GI tract the TC are PDGFRα‐positive and, therefore, might correspond to the FLC. We also hypothesize that in human gut, there are different TC subpopulations probably playing region‐specific roles.     

Identification of a Huperzine A-producing endophytic fungus from Phlegmariurus taxifolius

Molecular Biology Reports - Highly prized huperzine A (Hup A), a natural alkaloid formerly isolated from the Chinese medicinal plant Huperzia serrata, has been widely used for the treatment of...     

Staphylococci Isolated from Carriage Sites and Infected Sites of Dogs as a Reservoir of Multidrug Resistance and Methicillin Resistance

The aim of this study was to compare the spread of multidrug-resistant (MDR) and methicillin-resistant (MR) staphylococci in healthy dogs and in dogs with evident symptoms of infection. The samples from 172 healthy and 197 infected dogs were examined. The staphylococci were identified with conventional methods and by means of the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method (MboI). Susceptibility to 15 antibiotics from 10 different antimicrobial classes was tested. Resistance to methicillin was confirmed by the presence of Staphylococcus aureus mecA and S. sciuri mecA genes. Multidrug resistance was defined as resistance to three or more antimicrobial classes. The oral mucosa to be the most frequent site of staphylococcal colonization (55.8 %), followed by nasal cavity (44.2 %), and anus (32.6 %). The prevalence of MDR staphylococci in infected dogs was significantly higher than in the healthy animals (74/137 vs. 34/95, P = 0.006). The MR strains of S. pseudintermedius (2.9 %) originated solely from infected dogs. In contrast, the MR coagulase-negative strains (7.4 %) were isolated solely from healthy dogs. S. aureus strains originated from nasal swabs, MRSA strains were not isolated. MDR staphylococci and MR S. pseudintermedius are more common among infected dogs, but coagulase-negative staphylococci (mostly S. sciuri) seem to be a reservoir of methicillin resistance in healthy dogs.     

The Interplay Between NSAIDs and Candida albicans on the Gastrointestinal Tract of Guinea Pigs

Recent studies suggest that Candida albicans colonization is associated with several gastrointestinal inflammatory disorders and is also responsible for the delay in ulcer healing. No data are reported about the effects of C. albicans on the nonsteroidal anti-inflammatory drugs (NSAIDs)-induced necroinflammatory lesions. On the other hand, beneficial effects of NSAIDs regarding the colonization potential with C. albicans have been reported. Our aim was to investigate whether the association between NSAIDs and C. albicans could potentially induce necroinflammatory lesions in the guinea pigs gastric and enteral mucosa. Three interventional groups of 11 guinea pigs each were investigated after 5 days of receiving indomethacin, C. albicans or the association of both. C. albicans and necroinflammatory lesions were graded based on histological examinations. Statistical analysis used Mann–Whitney nonparametric test. NSAIDs did not significantly decrease C. albicans colonization grades on gastrointestinal mucosa. Administration of indomethacin subsequent to C. albicans determined significantly more severe necroinflammatory lesions compared to group that only received C. albicans. The association of NSAIDs and C. albicans did not cause significantly more severe degenerative or inflammatory lesions compared to the administration of only NSAIDs in this experimental model. Associations between NSAIDs and C. albicans caused significantly more severe necroinflammatory injuries than the lesions produced by C. albicans, without enhancing the mucosal injury or inflammation caused by NSAIDs.     

A tale of time and depth: intralacustrine radiation in endemic Gammarus species flock from the ancient Lake Ohrid

Tentatively dated, the Plio‐/Pleistocene origin of the ancient Lake Ohrid on the Balkan Peninsula makes it the oldest ancient lake in Europe. Given the surface area of the lake and the adjusted endemicity rate, it may be also defined as the most diverse of all the ancient lakes in the world. From all the animal groups endemic to this lake, gammarids are amongst the most scarcely known in terms of their diversity and phylogenetic relationships. Partial DNA sequences of two mitochondrial genes, cytochrome oxidase subunit I (cox1) and 16S ribosomal RNA (16S rRNA) of eight known endemic Gammarus species from the Lake Ohrid valley were analysed. Phylogenetic analyses showed that endemic Gammarus species comprise an ancient species flock, with Gammarus sketi from the feeder springs being their sister taxon outside the lake. Amongst the species inhabiting the lake, Gammarus solidus and Gammarus salemaai are morphologically and molecularly well defined. By contrast, Gammarus ochridensis, Gammarus parechiniformis, Gammarus lychnidensis, and Gammarus stankokaramani revealed high discrepancy between morphological and genetic data. None of these morphospecies form a monophyletic clade and a significant degree of apparent gene flow occurs between them. This could be caused by incomplete lineage sorting and/or hybridization events. Two novel mtDNA lineages were found within the lake, possibly constituting two new species (Gammarus sp. 1 and Gammarus sp. 2). Molecular clock analysis showed that the split between G. sketi and the Gammarus species flock from the lake occurred approximately 5–7 Mya, whereas within the flock there were at least two intralacustrine radiations: one estimated at 2–3 Mya and the second at less than 1 Mya. The first one could be associated with the origin of the lake and the second with the lake water‐level fluctuations during Pleistocene. © 2013 The Linnean Society of London     

The social and economic circumstances of mixed ethnicity children in the UK: findings from the Millennium Cohort Study

The number of people with a ‘mixed’ ethnicity heritage is growing in contemporary Britain. Research in this area has largely focused on implications for cultural and racialized identities, and little is known about associated economic and social factors. Data from the Millennium Cohort Study, a representative panel survey of children born in 2000–2001, are used to examine the circumstances of mixed ethnicity children in comparison with their non-mixed and white counterparts. Findings suggest a cultural location between ‘white’ and minority identities, and socio-economic advantage in comparison with non-mixed counterparts. For example, households of non-mixed white children had poorer economic profiles than households of both mixed white and mixed Indian children. This effect is associated with the presence of a white parent, and the factors underlying it are examined. Although the statistical approach used bypasses a consideration of the dynamics of identity, it provides important evidence on stratification and inequality, and the factors driving this.     

New Type of Outer Membrane Vesicle Produced by the Gram-Negative Bacterium Shewanella vesiculosa M7T: Implications for DNA Content

Outer membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacterium Shewanella vesiculosa M7^T has revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/μg OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacterium Shewanella vesiculosa M7^T that can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV).     

Genetic and molecular characterization of the human Osteosarcoma 3AB‐OS cancer stem cell line: A possible model for studying osteosarcoma origin and stemness

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second‐line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB‐OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB‐OS cells have hypertriploid karyotype with 71–82 chromosomes. By comparing 3AB‐OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan® Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let‐7/98 and miR‐29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets. J. Cell. Physiol. 228: 1189–1201, 2013. © 2012 Wiley Periodicals, Inc.