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Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase (CP) in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis (BC), characterized by aberrant activation of [beta]-catenin within granulocyte-macrophage progenitors (GMP). A major barrier to predicting and inhibiting blast crisis transformation has been the identification of mechanisms driving [beta]-catenin activation. Here we show that BC CML myeloid progenitors, in particular GMP, serially transplant leukemia in immunocompromised mice and thus ate enriched for leukemia stem cells (LSC). Notably, cDNA sequencing of Wnt/[beta]-catenin pathway regulatory genes, including adenomatous polyposis coli, GSK3[beta], axin 1, [beta]-catenin, lymphoid enhancer factor-1, cyclin D1, and c-myc, revealed a novel in-frame splice deletion of the GSK3[beta] kinase domain in the GMP of BC samples that was not detectable by sequencing in blasts or normal progenitors. Moreover, BC CML progenitors with misspliced GSK3[beta] have enhanced [beta]-catenin expression as well as serial engraftment potential while reintroduction of full-length GSK3[beta] reduces both in vitro replating and leukemic engraftment. We propose that CP CML is initiated by BCR-ABL expression in an HSC clone but that progression to BC may include missplicing of GSK3[beta] in GMP LSC, enabling unphosphorylated [beta]-catenin to participate in LSC self-renewal. Missplicing of GSK3[beta] represents a unique mechanism for the emergence of BC CML LSC and might provide a novel diagnostic and therapeutic target. blast crisis chronic myeloid leukemia | wnt pathway | xenograft | self-renewal | cancer stem cells

Varicella-zoster virus (VZV) causes varicella, establishes latency in sensory ganglia, and reactivates as herpes zoster. Human dorsal root ganglia (DRGs) xenografts in immunodeficient mice provide a model for evaluating VZV neuropathogenesis. Our investigation of the role of glycoprotein I (gI), which is dispensable in vitro, examines the functions of a VZV gene product during infection of human neural cells in vivo. Whereas intact recombinant Oka (rOka) initiated a short replicative phase followed by persistence in DRGs, the gI deletion mutant, rOka[DELTA]gI, showed prolonged replication with no transition to persistence up to 70 days after infection. Only a few varicella-zoster nucleocapsids and cytoplasmic virions were observed in neurons, and the major VZV glycoprotein, gE, was retained in the rough endoplasmic reticulum in the absence of gI. VZV neurotropism was not disrupted when DRG xenografts were infected with rOka mutants lacking gI promoter elements that bind cellular transactivators, specificity factor 1 (Sp1) and upstream stimulatory factor (USF). Because gI is essential and Sp1 and USF contribute to VZV pathogenesis in skin and T cells in vivo, these DRG experiments indicate that the genetic requirements for VZV infection are less stringent in neural cells in vivo. The observations demonstrate that gI is important for VZV neurotropism and suggest that a strategy to reduce neurovirulence by deleting gI could prolong active infection in human DRGs. herpesvirus | latency | neurotropism | neuropathogenesis | herpes

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 (CD3[4.sup.+]CD3[8.sup.-]CD9[0.sup.+]Li[n.sup.-]) 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

The placenta is the principal metabolic, respiratory, excretory, and endocrine organ for the first 9 months of fetal life. Its role in fetal and maternal physiology is remarkably diverse. Because of the central role that the placenta has in fetal and maternal physiology and development, the possibility that variation in placental gene expression patterns might be linked to important abnormalities in maternal or fetal health, or even variations in later life, warrants investigation. As an initial step, we used DNA microarrays to analyze gene expression patterns in 72 samples of amnion, chorion, umbilical cord, and sections of villus parenchyma from 19 human placentas from successful full-term pregnancies. The umbilical cord, chorion, amnion, and villus parenchyma samples were readily distinguished by differences in their global gene-expression patterns, many of which seemed to be related to physiology and histology. Differentially expressed genes have roles that include placental trophoblast secretion, signal transduction, metabolism, immune regulation, cell adhesion, and structure. We found interindividual differences in expression patterns in villus parenchyma and systematic differences between the maternal, fetal, and intermediate layers. A group of genes that was expressed in both the maternal and fetal villus parenchyma sections of placenta included genes that may be associated with preeclampsia. We identified sets of genes whose expression in placenta was significantly correlated with the sex of the fetus. This study provides a rich and diverse picture of the molecular variation in the placenta from healthy pregnancies. microarray | pregnancy | transcriptome | preeclampsia

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory ganglia. VZV reactivation results in herpes zoster. We developed a model using human dorsal root ganglion (DRG) xenografts in severe combined immunodeficient (SCID) mice to investigate VZV infection of differentiated neurons and satellite cells in vivo. DRG engrafted under the kidney capsule and contained neurons and satellite cells within a typical DRG architecture. VZV clinical isolates infected the neurons within DRG. At 14 days postinfection, VZ virions were detected by electron microscopy in neuronal cell nuclei and cytoplasm but not in satellite cells. The VZV genome copy number was 7.1 x [10.sup.7] to 8.0 x [10.sup.8] copies per [10.sup.5] cells, and infectious virus was recovered. This initial phase of viral replication was followed within 4-8 weeks by a transition to VZV latency, characterized by the absence of infectious virus release, the cessation of virion assembly, and a reduction in VZV genome copies to 3.7 x [10.sup.5] to 4.7 x [10.sup.6] per [10.sup.5] cells. VZV persistence in DRG was achieved without any requirement for VZV-specific adaptive immunity and was associated with continued transcription of the ORF63 regulatory gene. The live attenuated varicella vaccine virus exhibited the same pattern of short-term replication, persistence of viral DNA, and prominent ORF63 transcription as the clinical isolates. VZV-infected T cells transferred virus from the circulation into DRG, suggesting that VZV lymphotropism facilitates its neurotropism. DRG xenografts may be useful for investigating neuropathogenic mechanisms of other human viruses. herpesvirus latency | neurotropism | varicella vaccine

The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10-5, with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies. [ABSTRACT FROM AUTHOR]