Immunization with rP22 induces protective immunity against Schistosoma mansoni: Effects on granuloma down-modulation and cytokine production

AbstractSchistosomiasis remains a significant public health problem in tropical countries and it is recognized as the most important human helminth infection in terms of morbidity and mortality. Although the existing antischistosomal drugs are highly effective, they do not prevent against reinfection or granuloma formation. Therefore, vaccine strategies are essential for the control of schistosomiasis. Our group recently identified the recombinant (r) P22 protein, a component of the adult worm protein fraction PIII that has been shown to engender protective and immunomodulatory effects on murine schistosomiasis. A cDNA clone encoding rP22 was isolated from a Schistosoma mansoni adult worm cDNA library using anti-PIII rabbit serum; it exhibited complete identity with S. mansoni Sm21.7 EF-hand antigen. Confocal microscopy revealed that rP22 is a tegument protein localized on the surface of S. mansoni miracidia and adult worms. Mice immunized with rP22 exhibited a 51% and 22.5% decrease in adult worm burden and in hepatic eggs, respectively. Additionally, rP22 vaccine produced a reduction in 60% of liver granuloma size and 71% of fibrosis in mice, suggesting that rP22 might contribute to down-modulate granulomatous hypersensitivity to S. mansoni eggs. Protective immunity in mice was associated with high titers of rP22-specific IgG antibodies; elevated production of IFN-γ, TNF-α and IL-10; and a reduced level of IL-4. In conclusion, these findings indicate that rP22-based vaccines could be useful to elicit protection and reduce pathology associated to schistosomiasis

Inheritance of DNA Transferred from American Trypanosomes to Human Hosts

Interspecies DNA transfer is a major biological process leading to the accumulation of mutations inherited by sexual reproduction among eukaryotes. Lateral DNA transfer events and their inheritance has been challenging to document. In this study we modified a thermal asymmetric interlaced PCR by using additional targeted primers, along with Southern blots, fluorescence techniques, and bioinformatics, to identify lateral DNA transfer events from parasite to host. Instances of naturally occurring human infections by Trypanosoma cruzi are documented, where mitochondrial minicircles integrated mainly into retrotransposable LINE-1 of various chromosomes. The founders of five families show minicircle integrations that were transferred vertically to their progeny. Microhomology end-joining of 6 to 22 AC-rich nucleotide repeats in the minicircles and host DNA mediates foreign DNA integration. Heterogeneous minicircle sequences were distributed randomly among families, with diversity increasing due to subsequent rearrangement of inserted fragments. Mosaic recombination and hitchhiking on retrotransposition events to different loci were more prevalent in germ line as compared to somatic cells. Potential new genes, pseudogenes, and knockouts were identified. A pathway of minicircle integration and maintenance in the host genome is suggested. Thus, infection by T. cruzi has the unexpected consequence of increasing human genetic diversity, and Chagas disease may be a fortuitous share of negative selection. This demonstration of contemporary transfer of eukaryotic DNA to the human genome and its subsequent inheritance by descendants introduces a significant change in the scientific concept of evolutionary biology and medicine

Endothelial Differentiation of Human Stem Cells Seeded onto Electrospun Polyhydroxybutyrate/Polyhydroxybutyrate-Co-Hydroxyvalerate Fiber Mesh

Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules for inducing tissue regeneration. Despite its enormous potential, tissue engineering faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the delivery of nutrients to and removal of metabolites from the cells, which can lead to implant failure. In this sense, our investigation aims to develop a new strategy for enhancing vascularization in tissue engineering constructs. This study's aim was to establish a culture of human adipose tissue-derived stem cells (hASCs) to evaluate the biocompatibility of electrospun fiber mesh made of polyhydroxybutyrate (PHB) and its copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and to promote the differentiation of hASCs into the endothelial lineage. Fiber mesh was produced by blending 30% PHB with 70% PHB-HV and its physical characterization was conducted using scanning electron microscopy analysis (SEM). Using electrospinning, fiber mesh was obtained with diameters ranging 300 nm to 1.3 µm. To assess the biological performance, hASCs were extracted, cultured, characterized by flow cytometry, expanded and seeded onto electrospun PHB/PHB-HV fiber mesh. Various aspects of the cells were analyzed in vitro using SEM, MTT assay and Calcein-AM staining. The in vitro evaluation demonstrated good adhesion and a normal morphology of the hASCs. After 7, 14 and 21 days of seeding hASCs onto electrospun PHB/PHB-HV fiber mesh, the cells remained viable and proliferative. Moreover, when cultured with endothelial differentiation medium (i.e., medium containing VEGF and bFGF), the hASCs expressed endothelial markers such as VE-Cadherin and the vWF factor. Therefore, the electrospun PHB/PHB-HV fiber mesh appears to be a suitable material that can be used in combination with endothelial-differentiated cells to improve vascularization in engineered bone tissues

Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway

AbstractThe epidermal growth factor (EGF) transduces its actions via the EGF receptor (EGFR), which can traffic from the plasma membrane to either the cytoplasm or the nucleus. However, the mechanism by which EGFR reaches the nucleus is unclear. To investigate these questions, liver cells were analyzed by immunoblot of cell fractions, confocal immunofluorescence and real time confocal imaging. Cell fractionation studies showed that EGFR was detectable in the nucleus after EGF stimulation with a peak in nuclear receptor after 10min. Movement of EGFR to the nucleus was confirmed by confocal immunofluorescence and labeled EGF moved with the receptor to the nucleus. Small interference RNA (siRNA) was used to knockdown clathrin in order to assess the first endocytic steps of EGFR nuclear translocation in liver cells. A mutant dynamin (dynamin K44A) was also used to determine the pathways for this traffic. Movement of labeled EGF or EGFR to the nucleus depended upon dynamin and clathrin. This identifies the pathway that mediates the first steps for EGFR nuclear translocation in liver cells

New Developments of RNAi in <i>Paracoccidioides brasiliensis</i>: Prospects for High-Throughput, Genome-Wide, Functional Genomics

<div><p>Background</p><p>The Fungal Genome Initiative of the Broad Institute, in partnership with the <i>Paracoccidioides</i> research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1), Pb03 (PS2) and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for <i>P. brasiliensis</i>.</p><p>Methodology/Principal Findings</p><p>In this paper, we report <i>Agrobacterium tumefaciens</i> mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×10⁶ viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the <i>Streptoalloteichus hindustanus</i> bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in <i>P. brasiliensis</i>. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA) from a Gateway-adapted cassette (cALf) which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach.</p><p>Conclusions/Significance</p><p>We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future <i>Paracoccidioides</i> spp. functional genomics research.</p></div

Epifluorescence microscopy analysis of yeast cells expressing the bifunctional selective marker.

<p>Yeast cells carrying integrated cassettes under the transcriptional regulation of Prm_CBP1 (<b>A</b>), Prm_Act (<b>B</b>) or Prm_GP43 (<b>C</b>) were collected from late-log phase cultures and visualized through confocal microscopy. (D). Yeast cells carrying the integrated “Prm_CBP1::<i>Shble</i>::Ttr_GP43” expression cassette was used as negative control. Scale bar: 10 µm.</p

Cyclic adenosine monophosphate protects renal cell lines against amphotericin B toxicity in a PKA-independent manner.

Amphotericin B is the ??gold standard?? agent in the management of serious systemic fungal infections. However, this drug can cause nephrotoxicity, which contributes up to 25% of all acute kidney injuries in critically ill patients. Cyclic adenosine monophosphate can protect kidney cells from death due to injury or drug exposure in some cases. Hence, the objective of this work was to evaluate if cAMP could prevent cell death that occurs in renal cell lines subjected to AmB treatment and, if so, to assess the involvement of PKA in the transduction of this signal. Two different renal cell lines (LLC-PK1 and MDCK) were used in this study. MTT and flow cytometry assays showed increased cell survival when cells were exposed to cAMP in a PKA-independent manner, which was confirmed by western blot. This finding suggests that cAMP (db-cAMP) may prevent cell death caused by exposure to AmB. This is the first time this effect has been identified when renal cells are exposed to AmB?s nephrotoxic potential