The MicroRNA-371 Family as Plasma Biomarkers for Monitoring Undifferentiated and Potentially Malignant Human Pluripotent Stem Cells in Teratoma Assays.

Predicting developmental potency and risk of posttransplantation tumor formation by human pluripotent stem cells (hPSCs) and their derivatives largely rely on classical histological analysis of teratomas. Here, we investigated whether an assay based on microRNAs (miRNA) in blood plasma is able to detect potentially malignant elements. Several hPSCs and human malignant germ cell tumor (hGCT) lines were investigated in vitro and in vivo after mouse xenografting. The multiple conventional hPSC lines generated mature teratomas, while xenografts from induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and hGCT lines contained undifferentiated and potentially malignant components. The presence of these elements was reflected in the mRNA and miRNA profiles of the xenografts with OCT3/4 mRNA and the miR-371 and miR-302 families readily detectable. miR-371 family members were also identified in mouse plasma faithfully reporting undifferentiated elements in the xenografts. This study demonstrated that undifferentiated and potentially malignant cells could be detected in vivo.

Mucosal delivery of a vectored RSV vaccine is safe and elicits protective immunity in rodents and nonhuman primates.

Respiratory Syncytial Virus (RSV) is a leading cause of severe respiratory disease in infants and the elderly. No vaccine is presently available to address this major unmet medical need. We generated a new genetic vaccine based on chimpanzee Adenovirus (PanAd3-RSV) and Modified Vaccinia Ankara RSV (MVA-RSV) encoding the F, N, and M2-1 proteins of RSV, for the induction of neutralizing antibodies and broad cellular immunity. Because RSV infection is restricted to the respiratory tract, we compared intranasal (IN) and intramuscular (M) administration for safety, immunogenicity, and efficacy in different species. A single IN or IM vaccination completely protected BALB/c mice and cotton rats against RSV replication in the lungs. However, only IN administration could prevent infection in the upper respiratory tract. IM vaccination with MVA-RSV also protected cotton rats from lower respiratory tract infection in the absence of detectable neutralizing antibodies. Heterologous prime boost with PanAd3-RSV and MVA-RSV elicited high neutralizing antibody titers and broad T-cell responses in nonhuman primates. In addition, animals primed in the nose developed mucosal IgA against the F protein. In conclusion, we have shown that our vectored RSV vaccine induces potent cellular and humoral responses in a primate model, providing strong support for clinical testing.

Fusion of HCV nonstructural antigen to MHC class II-associated invariant chain enhances T-cell responses induced by vectored vaccines in nonhuman primates.

Despite viral vectors being potent inducers of antigen-specific T cells, strategies to further improve their immunogenicity are actively pursued. Of the numerous approaches investigated, fusion of the encoded antigen to major histocompatibility complex class II-associated invariant chain (Ii) has been reported to enhance CD8(+) T-cell responses. We have previously shown that adenovirus vaccine encoding nonstructural (NS) hepatitis C virus (HCV) proteins induces potent T-cell responses in humans. However, even higher T-cell responses might be required to achieve efficacy against different HCV genotypes or therapeutic effect in chronically infected HCV patients. In this study, we assessed fusion of the HCV NS antigen to murine and human Ii expressed by the chimpanzee adenovirus vector ChAd3 or recombinant modified vaccinia Ankara in mice and nonhuman primates (NHPs). A dramatic increase was observed in outbred mice in which vaccination with ChAd3 expressing the fusion antigen resulted in a 10-fold increase in interferon-γ(+) CD8(+) T cells. In NHPs, CD8(+) T-cell responses were enhanced and accelerated with vectors encoding the Ii-fused antigen. These data show for the first time that the enhancement induced by vector vaccines encoding li-fused antigen was not species specific and can be translated from mice to NHPs, opening the way for testing in humans.

Structure and evolution of vertebrate aldehyde oxidases: from gene duplication to gene suppression.

Aldehyde oxidases (AOXs) and xanthine dehydrogenases (XDHs) belong to the family of molybdo-flavoenzymes. Although AOXs are not identifiable in fungi, these enzymes are represented in certain protists and the majority of plants and vertebrates. The physiological functions and substrates of AOXs are unknown. Nevertheless, AOXs are major drug metabolizing enzymes, oxidizing a wide range of aromatic aldehydes and heterocyclic compounds of medical/toxicological importance. Using genome sequencing data, we predict the structures of AOX genes and pseudogenes, reconstructing their evolution. Fishes are the most primitive organisms with an AOX gene (AOXα), originating from the duplication of an ancestral XDH. Further evolution of fishes resulted in the duplication of AOXα into AOXß and successive pseudogenization of AOXα. AOXß is maintained in amphibians and it is the likely precursors of reptilian, avian, and mammalian AOX1. Amphibian AOXγ is a duplication of AOXß and the likely ancestor of reptilian and avian AOX2, which, in turn, gave rise to mammalian AOX3L1. Subsequent gene duplications generated the two mammalian genes, AOX3 and AOX4. The evolution of mammalian AOX genes is dominated by pseudogenization and deletion events. Our analysis is relevant from a structural point of view, as it provides information on the residues characterizing the three domains of each mammalian AOX isoenzyme. We cloned the cDNAs encoding the AOX proteins of guinea pig and cynomolgus monkeys, two unique species as to the evolution of this enzyme family. We identify chimeric RNAs from the human AOX3 and AOX3L1 pseudogenes with potential to encode a novel microRNA.

Simian immunodeficiency virus-Vpx for improving integrase defective lentiviral vector-based vaccines.

BACKGROUND: Integrase defective lentiviral vectors (IDLV) represent a promising delivery system for immunization purposes. Human dendritic cells (DC) are the main cell types mediating the immune response and are readily transduced by IDLV, allowing effective triggering of in vitro expansion of antigen-specific primed CD8+ T cells. However, IDLV expression in transduced DC is at lower levels than those of the integrase (IN) competent counterpart, thus requiring further improvement of IDLV for future use in the clinic. RESULTS: In this paper we show that the addition of simian immunodeficiency (SIV)-Vpx protein in the vector preparation greatly improves transduction of human and simian DC, but not of murine DC, thus increasing the ability of transduced DC to act as functional antigen presenting cells, in the absence of integrated vector sequences. Importantly, the presence of SIV-Vpx allows for using lower dose of input IDLV during in vitro transduction, thus further improving the IDLV safety profile. CONCLUSIONS: These results have significant implications for the development of IDLV-based vaccines.

Neuronal apoptosis in Alzheimer's disease: the role of age-related mitochondrial metabolic competence.

To assess the role of the mitochondrial metabolic competence (MMC) in the development of age-related changes, we measured the levels of immunohistochemically stained (IH) mitochondrial- and nuclear-encoded subunits of cytochrome oxidase (COX II and COX IV, respectively) and compared these data with mRNA in situ hybridization (ISH) of the same subunits and with cytochemically evidenced COX activity in the temporal (TC) and frontal (FC) cortex of adult and late-adult monkeys. Quantitative cytochemistry of COX activity was performed by calculating the ratio (R) of the area of the cytochemical precipitate to the area of the respective organelle. Although ISH studies showed reduced gene expression of both subunits in FC of late-adult monkeys, no significant age-related difference was found either in TC or FC when considering the IH data. R was significantly increased in FC of late-adult animals, and a quartile distribution of the mitochondrial area showed that R is higher in the FC of older animals independent of the organelle size. The assessment of COX genetic and phenotypic parameters reliably reports on MMC because this enzyme is the terminal complex of the electron transport chain. Taken together, the present IH, ISH, and R findings suggest that, with advancing age, compensating mechanisms are activated to preserve the mitochondrial functional metabolic capacities. Although significant mitochondrial defects are currently reported to occur in Alzheimer's disease pathogenesis, our data document that MMC is actively involved in the physiological rearrangement of the age-related neuronal network and may provide substantial metabolic support for the energy demand of neuronal apoptosis.

Selective decline of the metabolic competence of oversized synaptic mitochondria in the old monkey cerebellum.

The morphofunctional features of synaptic mitochondria, positive to the activity of cytochrome oxidase (COX), were investigated in the cerebellar cortex of adult and old monkeys (Macaca fascicularis) to assess the potential age-related changes in the energy metabolism occurring at the neuronal synaptic compartment. The following mitochondrial ultrastructural parameters-numeric density (Nv), volume density (Vv), average volume (V), and average length (Fmax)-were measured by computer-assisted morphometric methods. The ratio (R) area of the COX cytochemical precipitate/area of the mitochondrion was semi-automatically calculated and considered as an estimation of the mitochondrial metabolic competence (MMC), that is, the capacity of single organelles to provide adequate amounts of adenosinetriphosphate. No age-related significant differences were found in any of the ultrastructural parameters taken into account, whereas a significant decrease of R was observed in old animals. In these animals, the quartile distribution of the COX-positive organelles, according to their respective cross-sectional area, showed no significant difference of R when comparing small (I quartile), medium-sized (II quartile), and large (III quartile) mitochondria, while a significant decrease of R was evident in oversized mitochondria (IV quartile). Although our data document an age-related preservation of the morphological features of COX-positive mitochondria in the monkey cerebellum, the significant decrease of R in old animals needs to be considered from the functional standpoint. Since COX is the terminal enzyme of the mitochondrial respiratory chain, the estimation of its activity is regarded as a reliable MMC index; thus our findings, by matching preferential cytochemistry and morphometry, support the hypothesis that the specific functional impairment of enlarged synaptic mitochondria may seriously affect information processing and cell-to-cell communication at synaptic junctional areas with aging.

Alterations of synaptic turnover rate in aging may trigger senile plaque formation and neurodegeneration.

The changes of synaptic ultrastructure were investigated by morphometry in the frontal (FC) and temporal (TC) cortex of adult and aged monkeys, to assess the potential role of age-related synaptic deterioration in neurodegeneration. The average synaptic size (S), the synaptic numeric density (Nv: number of synapses/microm(3) of tissue), the synaptic surface density (Sv: overall area of synaptic junctional zones/microm(3) of tissue), and the number of synapses/neuron (Syn/Neur) were calculated. In FC, significant differences of Nv and Sv due to age were not revealed, while the S value was significantly increased in the aged animals. In TC, Sv did not change in relation to age, whereas Nv was significantly decreased and S significantly increased in aged monkeys. A percent distribution of S showed that the fraction of enlarged synapses (>0.20 microm(2)) was higher in TC than in FC, regardless of the age of the animals (21.3% versus 16.9% in adult and 33.9% versus 26.0% in aged monkeys, respectively). In aged animals, Syn/Neur was not significantly decreased in TC and not significantly increased in FC (4.4%). The above morphometric parameters account for the ongoing rearrangements of synaptic ultrastructure, reacting to the environmental stimuli. Our findings provide evidence of an age-related decline of synaptic plasticity in the brain of aged monkeys that is statistically significant in TC. According to current literature data on synaptic structural dynamics, this decay may represent an early and subtle alteration able to trigger the development of senile plaques and neurodegenerative events.

Preservation of mitochondrial volume homeostasis at the early stages of age-related synaptic deterioration.

A morphometric study on synaptic mitochondria was performed in the frontal (FC) and temporal (TC) cortex of adult and aged monkeys to seek ultrastructural alterations due to age. The overall volume covered by mitochondria (volume density: Vv), the number of mitochondria/microm(3) of tissue (numeric density: Nv), the average mitochondrial size (average volume: V), and the average mitochondrial shape (average length: Fmax) were calculated. Either in FC and TC, no significant age-related differences were revealed for any of the above-mentioned morphometric parameters. Namely, in FC of aged monkeys, a decrease of Vv (2%) and Nv (6%) was observed, whereas V and Fmax were increased by 5% and 2%, respectively. In TC of aged animals, both Vv and Nv increased by 7%, V decreased by 2%, and Fmax increased by 1%. The above morphometric parameters account for changes in single aspects of mitochondrial ultrastructure; nonetheless, when considered together per experimental group, they provide information regarding the structural rearrangements occurring on discrete populations of organelles. Considering these assumptions, the present findings document a preservation of the mitochondrial volume homeostasis in the brain of aged monkeys. Because our data from a previous investigation on the same animals showed early signs of synaptic deterioration in FC and TC during aging, this seems to be in contrast with the results of the present study. However, the clear age-related preservation of the mitochondrial potential for structural dynamics may be interpreted as a reactive response to early signs of synaptic deterioration.

Synaptic and mitochondrial morphometry provides structural correlates of successful brain aging.

Average synaptic size (S), synaptic numeric density (Nv) and surface density (Sv), average mitochondrial volume, mitochondrial numeric density, and mitochondrial volume density were measured by morphometry in the frontal (FC) and temporal (TC) cortex from adult and old monkeys (Macaca fascicularis). In relation to aging, Sv did not change, while Nv was significantly decreased in TC, but not in FC. S was significantly increased in FC and TC. No significant difference due to age was found with regard to mitochondrial ultrastructure. Considering the functional significance of the above parameters, their substantial age-related constancy suggests that they may reasonably represent structural correlates of successful brain aging.

Synaptic pathology in the brain cortex of old monkeys as an early alteration in senile plaque formation.

Synaptic numeric density (Nv), average size (area: S), surface density (Sv) and number of synapses/neurone (Syn/Neur) were morphometrically measured in frontal (FC) and temporal (TC) cortex of adult and old monkeys. Sv was constant, a clear age-related trend to decrease by Nv and increase by S were observed in both areas investigated. Syn/Neur significantly decreased in TC of aged animals (-21.1%), whereas FC showed a not significant reduction (-2.6%). The present data support the hypothesis of an increased sensitivity to deterioration of TC synapses in aged monkeys, which might constitute a predisposing condition to the development of senile plaques.

Drug-induced expansion and differentiation of V gamma 9V delta 2 T cells in vivo: the role of exogenous IL-2.

Human Vgamma9Vdelta2 T cells recognize nonpeptidic Ags generated by the 1-deoxy-d-xylulose 5-phosphate (many eubacteria, algae, plants, and Apicomplexa) and mevalonate (eukaryotes, archaebacteria, and certain eubacteria) pathways of isoprenoid synthesis. The potent Vgamma9Vdelta2 T cell reactivity 1) against certain cancer cells or 2) induced by infectious agents indicates that therapeutic augmentations of Vgamma9Vdelta2 T cell activities may be clinically beneficial. The functional characteristics of Vgamma9Vdelta2 T cells from Macaca fascicularis (cynomolgus monkey) are very similar to those from Homo sapiens. We have found that the i.v. administration of nitrogen-containing bisphosphonate or pyrophosphomonoester drugs into cynomolgus monkeys combined with s.c. low-dose (6 x 10(5) U/animal) IL-2 induces a large pool of CD27+ and CD27- effector/memory T cells in the peripheral blood of treated animals. The administration of these drugs in the absence of IL-2 is substantially less effective, indicating the importance of additional exogenous costimuli. Shortly after the costimulatory IL-2 treatment, only gammadelta (but not alphabeta) T cells expressed the CD69 activation marker, indicating that Vgamma9Vdelta2 T lymphocytes are more responsive to low-dose IL-2 than alphabeta T cells. Up to 100-fold increases in the numbers of peripheral blood Vgamma9Vdelta2 T cells were observed in animals receiving the gammadelta stimulatory drug plus IL-2. Moreover, the expanded Vgamma9Vdelta2 T cells were potent Th1 effectors capable of releasing large amounts of IFN-gamma. These results may be relevant for designing novel (or modifying current) immunotherapeutic trials with nitrogen-containing bisphosphonate or pyrophosphomonoester drugs.

In vivo DNA gene electro-transfer: a systematic analysis of different electrical parameters.

BACKGROUND: Intramuscular plasmid injection followed by electroporation is an efficient method for gene therapy or vaccination. Several protocols have been described that give good transduction levels with several reporter genes. METHODS: In this work we have explored the efficiency of gene delivery upon variation of the different electrical parameters such as pulse length frequency and voltage monitoring both on short- and long-term protein production. RESULTS: Having defined the best performing parameters, we have designed a short electric treatment that gives good levels of plasmid-encoded protein in different species such as mice, rabbits and monkeys.

Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates.

BACKGROUND: Anemia due to impaired erythropoietin (EPO) production is associated with kidney failure. Recombinant proteins are commonly administered to alleviate the symptoms of this dysfunction, whereas gene therapy approaches envisaging the delivery of EPO genes have been tried in animal models in order to achieve stable and long-lasting EPO protein production. Naked DNA intramuscular injection is a safe approach for gene delivery; however, transduction levels show high inter-individual variability in rodents and very poor efficiency in non-human primates. Transduction can be improved in several animal models by application of electric pulses after DNA injection. METHODS: We have designed a modified EPO gene version by changing the EPO leader sequence and optimizing the gene codon usage. This modified gene was electro-injected into mice, rabbits and cynomolgus monkeys to test for protein production and biological effect. CONCLUSIONS: The modified EPO gene yields higher levels of circulating transgene product and a more significant biological effect than the wild-type gene in all the species tested, thus showing great potential in clinically developable gene therapy approaches for EPO delivery.