Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates

We have previously reported the production of hepatitis C virus-like particles (HCV-LP) using a recombinant baculovirus containing the cDNA of the HCV structural proteins (core, E1, and E2). These particles resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice. Here we present evidence on the immunogenicity of HCV-LP and the effects of novel adjuvant systems in a nonhuman primate model, the baboon. Three groups of four baboons were immunized with HCV-LP, HCV-LP and adjuvant AS01B (monophosphoryl lipid A and QS21), or HCV-LP and the combination of AS01B and CpG oligodeoxynucleotides 10105. After four immunizations over an 8-month period, all animals developed HCV-specific humoral and cellular immune responses including antibodies to HCV structural proteins and gamma interferon(+) (IFN-gamma(+))CD4(+) and IFN-gamma(+)CD8(+) T-cell responses. The immunogenicity of HCV-LP was only marginally enhanced by the use of adjuvants. The overall HCV-specific immune responses were broad and long lasting. Our results suggest that HCV-LP is a potent immunogen to induce HCV-specific humoral and cellular immune responses in primates and may be a promising approach to develop novel preventive and therapeutic modalities.     

Role of BAG3 protein in leukemia cell survival and response to therapy

The ability of BAG3, a member of the BAG family of heat shock protein (Hsp) 70 - cochaperones, to sustain the survival of human primary B-CLL and ALL cells was recognized about nine years ago. Since then, the anti-apoptotic activity of BAG3 has been confirmed in other tumor types, where it has been shown to regulate the intracellular concentration and localization of apoptosis-regulating factors, including NF-κB-activating (IKKγ) and Bcl2-family (Bax) proteins. Furthermore, growing evidences support its role in lymphoid and myeloid leukemia response to therapy. Moreover in the last years, the contribution of BAG3 to autophagy, a process known to be involved in the pathogenesis and response to therapy of leukemia cells, has been disclosed, opening a new avenue for the interpretation of the role of this protein in leukemias' biology.     

The Plasminogen Activation System Modulates Differently Adipogenesis and Myogenesis of Embryonic Stem Cells

Regulation of the extracellular matrix (ECM) plays an important functional role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiation in vitro is unknown. We found that the PA system components are expressed at very low levels in undifferentiated ESCs and that upon differentiation uPA activity is detected mainly transiently, whereas tPA activity and PAI-1 protein are maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system display reduced adipogenic capacities after induction of the gene. Furthermore, the adipogenic differentiation capacities of PAI-1^−/− induced pluripotent stem cells (iPSCs) are augmented as compared to wt iPSCs. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms.     

Trichoderma spp. alleviate phytotoxicity in lettuce plants (Lactuca sativa L.) irrigated with arsenic-contaminated water

The influence of two strains of Trichoderma (T. harzianum strain T22 and T. atroviride strain P1) on the growth of lettuce plants (Lactuca sativa L.) irrigated with As-contaminated water, and their effect on the uptake and accumulation of the contaminant in the plant roots and leaves, were studied. Accumulation of this non-essential element occurred mainly into the root system and reduced both biomass development and net photosynthesis rate (while altering the plant P status). Plant growth-promoting fungi (PGPF) of both Trichoderma species alleviated, at least in part, the phytotoxicity of As, essentially by decreasing its accumulation in the tissues and enhancing plant growth, P status and net photosynthesis rate. Our results indicate that inoculation of lettuce with selected Trichoderma strains may be helpful, beside the classical biocontrol application, in alleviating abiotic stresses such as that caused by irrigation with As-contaminated water, and in reducing the concentration of this metalloid in the edible part of the plant.     

A MiRNA Signature for Defining Aggressive Phenotype and Prognosis in Gliomas

Gliomas represent a disparate group of tumours for which there are to date no cure. Thus, there is a recognized need for new diagnostic and therapeutic approaches based on increased understanding of their molecular nature. We performed the comparison of the microRNA (miRNA) profile of 8 WHO grade II gliomas and 24 higher grade tumours (2 WHO grade III and 22 glioblastomas) by using the Affymetrix GeneChip miRNA Array v. 1.0. A relative quantification method (RT-qPCR) with standard curve was used to confirm the 22 miRNA signature resulted by array analysis. The prognostic performances of the confirmed miRNAs were estimated on the Tumor Cancer Genome Atlas (TCGA) datasets. We identified 22 miRNAs distinguishing grade II gliomas from higher grade tumours. RT-qPCR confirmed the differential expression in the two patients'' groups for 13 out of the 22 miRNAs. The analysis of the Glioblastoma Multiforme (GBM) and Lower Grade Glioma (LGG) datasets from TCGA demonstrated the association with prognosis for 6 of those miRNAs. Moreover, in the GBM dataset miR-21 and miR-210 were predictors of worse prognosis in both univariable and multivariable Cox regression analyses (HR 1.19, p = 0.04, and HR 1.18, p = 0.029 respectively). Our results support a direct contribution of miRNAs to glioma cancerogenesis and suggest that miR-21 and miR-210 may play a role in the aggressive clinical behaviour of glioblastomas.     

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b₆f with the electron transfer proteins plastocyanin and cytochrome c₆. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c₆ from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c₆ were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge–charge interactions pre-orient cytochrome c₆ with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.     

Transduction of Cultured Oligodendrocytes from Normal and Twitcher Mice by a Retroviral Vector Containing Human Galactocerebrosidase (GALC) cDNA

Krabbe disease or globoid cell leukodystropy is a lysosomal disorder caused by a deficiency of galactocerebrosidase (GALC) activity. This results in defects in myelin that lead to severe symptoms and early death in most human patients and animals with this disease. With the cloning of the GALC gene and the availability of the mouse model, called twitcher, it was important to evaluate the effects of providing GALC via a retroviral vector to oligodendrocytes in culture. After differentiation, the untransduced cells from normal mice extended highly branched processes while those from the twitcher mice did not. oligodendrocytes in culture can be readily transduced to produce much higher than normal levels of GALC activity. Transduced normal and twitcher cells formed clusters when plated at high density. Transduction of twitcher oligodendrocytes plated at lower density, followed by differentiation, resulted in some cells having a completely normal appearance with highly branched processes. Other cells showed retraction and fragmentation. Perhaps over expression of GALC activity may be detrimental to oligodendrocytes. These studies demonstrate that the phenotype of twitcher oligodendrocytes can be corrected by providing GALC via gene transfer, and this could lead the way to future studies to treat this disease.