MicroRNA signatures of iPSCs and endoderm-derived tissues

MicroRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression, play multiple roles in the cell, including cell fate specification. We have analyzed the differential expression of miRNAs during fibroblast reprogramming into induced pluripotent stem cells (iPSCs) and endoderm induction from iPSCs upon treatment with high concentrations of Activin-A. The reprogrammed iPSCs assumed an embryonic stem cell (ESC)-like miRNA signature, marked by the induction of pluripotency clusters miR-290–295 and miR-302/367 and conversely the downregulation of the let-7 family. On the other hand, endoderm induction in iPSCs resulted in the upregulation of 13 miRNAs. Given that the liver and the pancreas are common derivatives of the endoderm, analysis of the expression of these 13 upregulated miRNAs in hepatocytes and pancreatic islets revealed a tendency for these miRNAs to be expressed more in pancreatic islets than in hepatocytes. These observations provide insights into how differentiation may be guided more efficiently towards the endoderm and further into the liver or pancreas. Moreover, we also report novel miRNAs enriched for each of the cell types analyzed.     

Human adipose tissue is a source of multipotent stem cells

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.     

In Vivo Assessment of NS1-Truncated Influenza Virus with a Novel SLSYSINWRH Motif as a Self-Adjuvanting Live Attenuated Vaccine

Mutants of influenza virus that encode C-terminally truncated NS1 proteins (NS1-truncated mutants) characteristically induce high interferon responses. The dual activity of interferon in blocking virus replication and enhancing the development of adaptive immune responses makes these mutants promising as self-adjuvanting live-attenuated influenza vaccine (LAIV) candidates. Yet, among the NS1-truncated mutants, the length of NS1 is not directly correlated with the interferon-inducing efficiency, the level of attenuation, or effectiveness as LAIV. Using quantitative in vitro biologically active particle subpopulation analysis as a tool to identify potential LAIV candidates from a pool of NS1-truncated mutants, we previously predicted that a NS1-truncated mutant pc2, which was less effective as a LAIV in chickens, would be sufficiently effective as a LAIV in mammalian hosts. In this study, we confirmed that pc2 protected mice and pigs against heterologous virus challenge in terms of preventing clinical signs and reducing virus shedding. pc2 expresses a unique SLSYSINWRH motif at the C-terminus of its truncated NS1. Deletion of the SLSYSINWRH motif led to ~821-fold reduction in the peak yield of type I interferon induced in murine cells. Furthermore, replacement of the SLSYSINWRH motif with the wildtype MVKMDQAIMD sequence did not restore the interferon-inducing efficiency. The diminished interferon induction capacity in the absence of the SLSYSINWRH motif was similar to that observed in other mutants which are less effective LAIV candidates. Remarkably, pc2 induced 16-fold or more interferon in human lung and monkey kidney cells compared to the temperature-sensitive, cold-adapted Ann Arbor virus that is currently used as a master backbone for LAIVs such as FluMist. Although the mechanism by which the SLSYSINWRH motif regulates the vaccine properties of pc2 has not been elucidated, this motif has potential use in engineering self-adjuvanting NS1-truncated-based LAIVs.     

Cry Protein Crystals: A Novel Platform for Protein Delivery

Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.     

Epigenetic activation of SOX11 in lymphoid neoplasms by histone modifications

Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications.     

A second generation of double mutant cholera toxin adjuvants: enhanced immunity without intracellular trafficking

Nasal application of native cholera toxin (nCT) as a mucosal adjuvant has potential toxicity for the CNS through binding to GM1 gangliosides in the olfactory nerves. Although mutants of cholera toxin (mCTs) have been developed that show mucosal adjuvant activity without toxicity, it still remains unclear whether these mCTs will induce CNS damage. To help overcome these concerns, in this study we created new double mutant CTs (dmCTs) that have two amino acid substitutions in the ADP-ribosyltransferase active center (E112K) and COOH-terminal KDEL (E112K/KDEV or E112K/KDGL). Confocal microscopic analysis showed that intracellular localization of dmCTs differed from that of mCTs and nCTs in intestinal epithelial T84 cells. Furthermore, both dmCTs exhibited very low toxicity in the Y1 cell assay and mouse ileal loop tests. When mucosal adjuvanticity was examined, both dmCTs induced enhanced OVA-specific immune responses in both mucosal and systemic lymphoid tissues. Interestingly, although both dmCT E112K/KDEV and dmCT E112K/KDGL showed high Th2-type and significant Th1-type cytokine responses by OVA-specific CD4+ T cells, dmCT E112K/KDEV exhibited significantly lower Th1-type cytokine responses than did nCT and dmCT E112K/KDGL. These results show that newly developed dmCTs retain strong biological adjuvant activity without CNS toxicity.     

Bacillus anthracis edema toxin acts as an adjuvant for mucosal immune responses to nasally administered vaccine antigens

Anthrax edema toxin (EdTx) is an AB-type toxin that binds to anthrax toxin receptors on target cells via the binding subunit, protective Ag (PA). Edema factor, the enzymatic A subunit of EdTx, is an adenylate cyclase. We found that nasal delivery of EdTx enhanced systemic immunity to nasally coadministered OVA and resulted in high OVA-specific plasma IgA and IgG (mainly IgG1 and IgG2b). The edema factor also enhanced immunity to the binding PA subunit itself and promoted high levels of plasma IgG and IgA responses as well as neutralizing PA Abs. Mice given OVA and EdTx also exhibited both PA- and OVA-specific IgA and IgG Ab responses in saliva as well as IgA Ab responses in vaginal washes. EdTx as adjuvant triggered OVA- and PA-specific + T cells which secreted IFN-gamma and selected Th2-type cytokines. The EdTx up-regulated costimulatory molecule expression by APCs but was less effective than cholera toxin for inducing IL-6 responses either by APCs in vitro or in nasal washes in vivo. Finally, nasally administered EdTx did not target CNS tissues and did not induce IL-1 mRNA responses in the nasopharyngeal-associated lymphoepithelial tissue or in the olfactory bulb epithelium. Thus, EdTx derivatives could represent an alternative to the ganglioside-binding enterotoxin adjuvants and provide new tools for inducing protective immunity to PA-based anthrax vaccines.     

Low temperature induces the delivery of mature and immature CFTR to the plasma membrane

Deletion of phenylalanine 508 (ΔF508) is the most prevalent disease-causing mutation resulting in retention of the immature CFTR in the endoplasmic reticulum. The most common strategy to induce the delivery of ΔF508-CFTR to the surface of cells is by reducing the incubation temperature (≈28 °C). Cell surface biotinylation of HEK293T cells grown at 37 °C for 48 h, confirmed the presence of mature wild-type CFTR, but not ΔF508-CFTR at the cell surface. On the other hand, cells incubated at 28 °C for 16 h showed both mature and immature ΔF508-CFTR at their surface. The trafficking of immature ΔF508-CFTR, but not mature ΔF508-CFTR, to the cell surface occurred at low temperature even upon addition of BFA, suggesting the involvement of a Golgi-independent pathway. These results suggest that low temperature induces the appearance of a mix population of mature and immature CFTR molecules at the plasma membrane through distinct pathways.