Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34 ؉ CD38 ؊ CD90 ؉ Lin ؊ ) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.JAK ͉ signaling ͉ progenitors ͉ cell fate ͉ mutant allele
A single-step method for factor V Leiden genotyping is presented that uses rapid-cycle PCR and simultaneous fluorescence analysis with resonance energy transfer probes. A fragment of the factor V gene containing the mutation is amplified asymmetrically through use of a primer labeled with Cy5TM in the presence of a 3′-fluorescein-labeled probe that covers the mutation site. When the fluorescein probe is annealed to the extension product of the Cy5-labeled primer, the fluorophores are brought into close enough contact for resonance energy transfer to occur. As the temperature increases, the probe melts from its target, decreasing the resonance energy transfer. When the probe is complementary to the product strand, it melts at 65 °C; if the single-base mutation is present, the probe melts at 57 °C. Concurrent amplification and analysis from genomic DNA takes 20–45 min and requires no sample manipulation after the fluorescence thermal cycler is loaded.
SummarySafe and effective adjuvants for influenza vaccines that could increase both the levels of neutralizing antibody, including against drifted viral subtypes, and T-cell immunity would be a major advance in vaccine design. The JVRS-100 adjuvant, consisting of DOTIM/cholesterol cationic liposome-DNA complexes, is particularly promising for vaccines that require induction of high levels of antibody and T-cell immunity, including CD8 + cytotoxic T lymphocytes (CTL). Inclusion of protein antigens with JVRS-100 results in the induction of enhanced humoral and cell-mediated (i.e., CD4 + , and CD8 + T cells) immune responses. The JVRS-100 adjuvant combined with a split trivalent influenza vaccine (Fluzone ® -sanofi pasteur) elicited increased antibody and T-cell responses in mice and non-human primates compared to vaccination with Fluzone ® alone. Mice vaccinated with JVRS-100-Fluzone ® and challenged with antigenically drifted strains of H1N1 (PR/8/34) and influenza B (B/Lee/40) viruses had higher grade protection, as measured by attenuation of weight loss and increased survival, compared to recipients of unadjuvanted vaccine. The results indicate that the JVRS-100 adjuvant substantially increases immunogenicity and protection from drifted-strain challenge using an existing influenza vaccine.
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