N-glycome profiling of Bothrops jararaca newborn and adult venoms

Glycosylation is an important post-translational modification of snake venom proteins and contributes to venom proteome complexity. Many snake venom components are known to be glycosylated, however, very little is known about the carbohydrate structures present in venom glycoproteins. Previous studies showed that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and shift in animal size are associated with changes in the venom proteome of the snake Bothrops jararaca. In this study we explored the composition of the N-glycome released from newborn and adult B. jararaca venom proteins. We used an ion trap mass spectrometer (IT-MS) to disassemble glycan structures based on the use of several pathways of MS (MSn) and demonstrate the presence of some structural isomers in both newborn and adult venom B. jararaca N-glycans. The main N-glycans identified in both venoms are of the hybrid/complex type however some mannose-rich type structures were also detected. The N-glycan composition of newborn and adult venoms did not vary indicating that differences in the utilization of the N-glycosylation motif could be the explanation for the differences in the glycosylation levels indicated by the differential electrophoretic profiles previously reported for B. jararaca newborn and adult venoms.     

Methodologic issues in the measurement of interleukin-16 in clinical blood samples using immunoassays

Quantitation of interleukin-16 (IL-16) in clinical blood samples has strongly increased, since IL-16 appears to be involved in the pathogenesis of several inflammatory diseases. IL-16 is synthesized in the cell cytoplasm as precursor protein (pro-IL-16), which can be processed by caspase-3 into N-terminal (N-IL-16) and C-terminal (C-IL-16) fragments. C-IL-16 is described to be subsequently secreted. Using commercially available IL-16 ELISA, a pro-IL-16 ELISA and immunoprecipitation analysis, we investigated, whether type and handling of blood samples influence IL-16 quantitation and whether existing IL-16 ELISA are specific for C-IL-16. We observed that cell-rich plasma samples reflect falsely-elevated IL-16 concentrations due to cell contaminations. Interestingly, not C-IL-16, but pro-IL-16 represents the major IL-16 form in cell-rich plasma samples. Notably, commercially IL-16 ELISA could not distinguish between C-IL-16 and pro-IL-16. Thus, cell-rich plasma samples should not be used for IL-16 measurements and new methods are necessary for quantitation of C-IL-16 and pro-IL-16 uniquely.     

Loss of CCR7 Expression on CD56bright NK Cells Is Associated with a CD56dimCD16+ NK Cell-Like Phenotype and Correlates with HIV Viral Load

NK cells are pivotal sentinels of the innate immune system and distinct subpopulations in peripheral blood have been described. A number of studies addressed HIV-induced alterations of NK cell phenotype and functionality mainly focusing on CD56^dimCD16⁺ and CD56⁻CD16⁺ NK cells. However, the impact of HIV-infection on CD56^bright NK cells is less well understood. Here we report a rise of CD56^bright NK cells in HIV-infected individuals, which lack CCR7-expression and strongly correlate with HIV viral load. CCR7⁻CD56^bright NK cells were characterized by increased cytolytic potential, higher activation states and a more differentiated phenotype. These cells thus acquired a number of features of CD56^dimCD16⁺ NK cells. Furthermore, CD56^bright NK cells from HIV patients exhibited higher degranulation levels compared to uninfected individuals. Thus, chronic HIV-infection is associated with a phenotypic and functional shift of CD56^bright NK cells, which provides a novel aspect of HIV-associated pathogenesis within the NK cell compartment.     

Concordance of Gene Expression and Functional Correlation Patterns across the NCI-60 Cell Lines and the Cancer Genome Atlas Glioblastoma Samples

Background

The NCI-60 is a panel of 60 diverse human cancer cell lines used by the U.S. National Cancer Institute to screen compounds for anticancer activity. We recently clustered genes based on correlation of expression profiles across the NCI-60. Many of the resulting clusters were characterized by cancer-associated biological functions. The set of curated glioblastoma (GBM) gene expression data from the Cancer Genome Atlas (TCGA) initiative has recently become available. Thus, we are now able to determine which of the processes are robustly shared by both the immortalized cell lines and clinical cancers.

Results

Our central observation is that some sets of highly correlated genes in the NCI-60 expression data are also highly correlated in the GBM expression data. Furthermore, a “double fishing” strategy identified many sets of genes that show Pearson correlation ≥0.60 in both the NCI-60 and the GBM data sets relative to a given “bait” gene. The number of such gene sets far exceeds the number expected by chance.

Conclusion

Many of the gene-gene correlations found in the NCI-60 do not reflect just the conditions of cell lines in culture; rather, they reflect processes and gene networks that also function in vivo. A number of gene network correlations co-occur in the NCI-60 and GBM data sets, but there are others that occur only in NCI-60 or only in GBM. In sum, this analysis provides an additional perspective on both the utility and the limitations of the NCI-60 in furthering our understanding of cancers in vivo.     

Type I IFN-induced, NKT cell-mediated negative control of CD8 T cell priming by dendritic cells

We investigated the negative effect of type I IFN (IFN-I) on the priming of specific CD8 T cell immunity. Priming of murine CD8 T cells is down-modulated if Ag is codelivered with IFN-I-inducing polyinosinic:polycytidylic acid (pI/C) that induces (NK cell- and T/B cell-independent) acute changes in the composition and surface phenotype of dendritic cells (DC). In wild-type but not IFN-I receptor-deficient mice, pI/C reduces the plasmacytoid DC but expands the CD8(+) conventional DC (cDC) population and up-regulates surface expression of activation-associated (CD69, BST2), MHC (class I/II), costimulator (CD40, CD80/CD86), and coinhibitor (PD-L1/L2) molecules by cDC. Naive T cells are efficiently primed in vitro by IFN-I-stimulated CD8 cDC (the key APC involved in CD8 T cell priming) although these DC produced less IL-12 p40 and IL-6. pI/C (IFN-I)-mediated down modulation of CD8 T cell priming in vivo was not observed in NKT cell-deficient CD1d(-/-) mice. CD8 cDC from pI/C-treated mice inefficiently stimulated IFN-gamma, IL-4, and IL-2 responses of NKT cells. In vitro, CD8 cDC that had activated NKT cells in the presence of IFN-I primed CD8 T cells that produced less IFN-gamma but more IL-10. The described immunosuppressive effect of IFN-I thus involves an NKT cell-mediated change in the phenotype of CD8 cDC that favors priming of IL-10-producing CD8 T cells. In the presence of IFN-I, NKT cells hence impair the competence of CD8 cDC to prime proinflammatory CD8 T cell responses.     

Thrombospondin-1 binds to ApoER2 and VLDL receptor and functions in postnatal neuronal migration

Apolipoprotein E receptor 2 (ApoER2), very low-density lipoprotein receptor (VLDLR), and Dab1 are the main components of the Reelin signalling cascade. Reelin is the sole ligand defined so far in signalling through this pathway. Postnatal migration of neuronal precursors from the subventricular zone (SVZ) to the olfactory bulb (OB), however, depends on ApoER2 and Dab1, but functions independently of Reelin. Here, we show that thrombospondin-1 (THBS-1) is a novel physiological ligand for ApoER2 and VLDLR. THBS-1 is present in the SVZ and along the entire rostral migratory stream (RMS). It binds to ApoER2 and VLDLR and induces phosphorylation of Dab1. In contrast to Reelin, it does not induce Dab1 degradation or Akt phosphorylation, but stabilizes neuronal precursor chains derived from subventricular explants. Lack of THBS-1 results in anatomical abnormalities of the RMS and leads to a reduction of postnatal neuronal precursors entering the OB.     

Phosphoinositide 3-kinases gamma and delta, linkers of coordinate C5a receptor-Fcgamma receptor activation and immune complex-induced inflammation

Fcgamma receptors (FcgammaR) and the C5a receptor (C5aR) are key effectors of the acute inflammatory response to IgG immune complexes (IC). Their coordinated activation is critical in IC-induced diseases, although the significance of combined signaling by these two different receptor classes in tissue injury is unclear. Here we used the mouse model of the passive reverse lung Arthus reaction to define their requirements for distinct phosphoinositide 3-kinase (PI3K) activities in vivo. We show that genetic deletion of class IB PI3Kgamma abrogates C5aR signaling that is crucial for FcgammaR-mediated activation of lung macrophages. Thus, in PI3Kgamma(-/-) mice, IgG IC-induced FcgammaR regulation, cytokine release, and neutrophil recruitment were blunted. Notably, however, C5a production occurred normally in PI3Kgamma(-/-) mice but was impaired in PI3Kdelta(-/-) mice. Consequently, class IA PI3Kdelta deficiency caused resistance to acute IC lung injury. These results demonstrate that PI3Kgamma and PI3Kdelta coordinate the inflammatory effects of C5aR and FcgammaR and define PI3Kdelta as a novel and essential element of FcgammaR signaling in the generation of C5a in IC disease.     

Histological assessment of cellular half-life in tissues in vivo

The assessment of cellular half-life is of fundamental importance for cell biology and biomedicine. Here, we show that cellular half-life in tissues can be histologically measured under steady state conditions in vivo by analyzing the loss of 5-bromo-2′-deoxyuridine (BrdU)-labeled cells over time after withdrawal of long-term BrdU labeling. To achieve efficient continuous cell labeling, we implanted BrdU-containing subcutaneous slow-release pellets into 12-month-old male Fischer 344 rats, delivering BrdU at a dose of 75 mg/kg per day over 1 (n = 20) or 3 weeks (n = 20). Four to five rats each were killed directly after the labeling or 1, 3, and 7 weeks post-labeling. Cellular half-life after withdrawal of BrdU was analyzed by nonlinear regression analysis of the labeling index, using a model of one-phase exponential decay. We initially validated our technique in the duodenum, where we determined a half-life of 2.4 days for crypt cells. Next, we applied this method to other tissues, and found a half-life of 2.2 weeks for cardiac endothelial cells, and of 5–6 days for pancreatic duct cells. In conclusion, we believe that this novel approach is an important step forward in the histological assessment of cellular half-life.     

Differentiating N-linked glycan structural isomers in metastatic and nonmetastatic tumor cells using sequential mass spectrometry

In an effort to understand the role of molecular glycosylationin cancer a murine model has been used to characterize and fingerprintmalignancies in established cell lines that manifest all thehallmarks of metastatic disease: spontaneous development, localinvasion, intravasation, immune system survival, extravasation,and secondary tumor formation involving liver, kidney, spleen,lung, and brain. Using astrocyte cell controls, we comparedN-linked glycosylation from a nonmetastatic brain tumor cellline and two different metastatic brain tumor cells. Selectedions in each profile were disassembled by ion trap mass spectrometry(MSⁿ) which exhibited multiple structural differences betweeneach tissue. These unique structures were identified withinisomeric compositions as pendant nonreducing termini of di-and trisaccharide fragments, probably transparent to a tandemMS approach but distinctively not to sequential ion trap MSⁿdetection.