Exosomes surf on filopodia to enter cells at endocytic hot spots,traffic within endosomes,and are targeted to the ER

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.     

The Contribution of Non-Conventional T Cells and NK Cells in the Mycobacterial-Specific IFNγ Response in Bacille Calmette-Guérin (BCG)-Immunized Infants

Background

The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest.

Methods

Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI).

Results

In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age.

Conclusion

In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.     

Manual Function in Cebus apella. Digital Mobility,Preshaping, and Endurance in Repetitive Grasping

Manual dexterity varies across species of primates in accord with hand morphology and degree of fine motor control of the digits. Platyrrhine monkeys achieve less direct opposition between thumb and index finger than that of catarrhine primates, and many of them typically whole-hand grip. However, tufted capuchins (Cebus apella), exhibit a degree of independent control of the digits and effective thumb–forefinger opposition. We report how capuchins prehended small objects, with particular attention to the form of sequential fine movements of the fingers, choice of hand, and differences between the two hands in the temporal properties of reaching and grasping. We compare these actions across tasks with differing demands for fine motor control. For tasks that required all the digits to flex in synchrony, capuchins displayed smooth, fast, and efficient reach-to-grasp movements and a higher endurance than for tasks requiring more complex digital coordination. These latter tasks induced a slightly differentiated preshaping of the hand when approaching the objects, indicating preparation for grasping in advance of contact with the object. A right-hand preponderance for complex digital coordination was evident. The monkeys coordinated their fingers rather poorly at the substrate, and they took longer to achieve control of the objects when complex coordination was required than when simultaneous flexion was sufficient. We conclude that precise finger coordination is more effortful and less well coordinated, and the coordination is less lateralized, in capuchins than in catarrhine primates.     

Time-resolved fluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR/HER2 dimers: a new method to evaluate the efficiency of targeted therapy using monoclonal antibodies

In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.     

Adaptation of Legionella pneumophila to the host environment: role of protein secretion, effectors and eukaryotic-like proteins

The intracellular pathogen Legionella pneumophila has evolved sophisticated mechanisms that enable it to subvert host functions, enter, survive and replicate in amoebae or alveolar macrophages, and to finally evade these hosts. Protozoa are essential for the growth of Legionella and the interaction with amoeba seems to be the driving force in the evolution of its pathogenicity. This is reflected in the genome of this pathogen, which encodes a high number and variety of eukaryotic-like proteins that are able to interfere in the various steps of the infectious cycle by mimicking functions of eukaryotic proteins. Central to the pathogenicity of L. pneumophila are the many secretion systems delivering these and other effectors to the host cell. Recent studies have highlighted the multi-functional role of these factors secreted by L. pneumophila, in host-pathogen interactions.     

Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents Abeta(25-35)-induced reduction in BPRP in PC12 cells

Several lines of evidence support that beta-amyloid (Abeta)-induced neurotoxicity is mediated through the generation of reactive oxygen species (ROS) and elevation of intracellular calcium. Salvianolic acid B (Sal B), the major and most active anti-oxidant from Salvia miltiorrhiza, protects diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the effects of Sal B against beta-amyloid peptide 25-35 (Abeta(25-35))-induced neurotoxicity, focused mainly on the neurotoxic effects of Abeta(25-35) and the neuroprotective effects of Sal B on the expression of brain-pancreas relative protein (BPRP), which is a new protein and mainly expressed in brain and pancreas. Following exposure of PC12 cells to 20 microM Abeta(25-35), a marked reduction in the expression of BPRP was observed, accompanied with decreased cell viability and increased cell apoptosis, as well as increased ROS production and calcium influx. Treatment of the PC12 cells with Sal B significantly reversed the expression of BPRP and cell viability while it decreased ROS production and intracellular calcium. These data indicate that Abeta(25-35) decreases the expression of BPRP via enhanced formation of intracellular ROS and increased intracellular calcium, and that Sal B, as an anti-oxidant, protects against Abeta(25-35)-induced reduction in expression of BPRP through its effects on suppressing the production of ROS, calcium flux, and apoptosis. However, the role(s) of BPRP in AD and the definite mechanisms by which Sal B protects against Abeta(25-35)-induced reduction in the expression of BPRP require further study.     

Effect of amino acid substitutions on the candidacidal activity of LFampin 265-284

LFampin 265-284, derived from bovine lactoferrin, has broad-spectrum antimicrobial activity against the yeast Candida albicans and several Gram-positive and Gram-negative bacteria. A glycine substitution scan was used to identify residues that are important for its candidacidal activity. Each single substitution of a positively charged residue led to considerable reduction in candidacidal activity, for each residue to a different extent. Substitution within the helix-facilitating N-terminal sequence DLIW had less severe effect; substitution of Ile and Trp led to a somewhat reduced potency. No substantial effects were found on the propensity to adopt a helical structure or to bind to C. albicans cells.     

A New 34-Kilodalton Isoform of Human Fibroblast Growth Factor 2 Is Cap Dependently Synthesized by Using a Non-AUG Start Codon and Behaves as a Survival Factor

Four isoforms of human fibroblast growth factor 2 (FGF-2) result from alternative initiations of translation at three CUG start codons and one AUG start codon. Here we characterize a new 34-kDa FGF-2 isoform whose expression is initiated at a fifth initiation codon. This 34-kDa FGF-2 was identified in HeLa cells by using an N-terminal directed antibody. Its initiation codon was identified by site-directed mutagenesis as being a CUG codon located at 86 nucleotides (nt) from the FGF-2 mRNA 5′ end. Both in vitro translation and COS-7 cell transfection using bicistronic RNAs demonstrated that the 34-kDa FGF-2 was exclusively expressed in a cap-dependent manner. This contrasted with the expression of the other FGF-2 isoforms of 18, 22, 22.5, and 24 kDa, which is controlled by an internal ribosome entry site (IRES). Strikingly, expression of the other FGF-2 isoforms became partly cap dependent in vitro in the presence of the 5,823-nt-long 3′ untranslated region of FGF-2 mRNA. Thus, the FGF-2 mRNA can be translated both by cap-dependent and IRES-driven mechanisms, the balance between these two mechanisms modulating the ratio of the different FGF-2 isoforms. The function of the new FGF-2 was also investigated. We found that the 34-kDa FGF-2, in contrast to the other isoforms, permitted NIH 3T3 cell survival in low-serum conditions. A new arginine-rich nuclear localization sequence (NLS) in the N-terminal region of the 34-kDa FGF-2 was characterized and found to be similar to the NLS of human immunodeficiency virus type 1 Rev protein. These data suggest that the function of the 34-kDa FGF-2 is mediated by nuclear targets.