text:
The molecular and genetic factors induced by human T-lymphotropic virus type-1 (HTLV-1) that initiate adult T-cell leukemia/lymphoma (ATLL) remain unclear, in part from the lack of an animal model that accurately recapitulates leukemogenesis. HTLV-1–infected humanized nonobese diabetic severe combined immunodeficiency (HU-NOD/SCID) mice were generated by inoculation of NOD/SCID mice with CD34+ hematopoietic progenitor and stem cells (CD34+ HP/HSCs) infected ex vivo with HTLV-1. HTLV-1-HU-NOD/SCID mice exclusively developed CD4+ T-cell lymphomas with characteristics similar to ATLL and elevated proliferation of infected human stem cells (CD34+CD38−) in the bone marrow were observed in mice developing malignancies. Purified CD34+ HP/HSCs from HTLV-1–infected patient peripheral blood mononuclear cells revealed proviral integrations suggesting viral infection of human bone marrow–derived stem cells. NOD/SCID mice reconstituted with CD34+ HP/HSCs transduced with a lentivirus vector expressing the HTLV-1 oncoprotein (Tax1) also developed CD4+ lymphomas. The recapitulation of a CD4+ T-cell lymphoma in HU-NOD/SCID mice suggests that HSCs provide a viral reservoir in vivo and act as cellular targets for cell transformation in humans. This animal model of ATLL will provide an important tool for the identification of molecular and cellular events that control the initiation and progression of the lymphoma and potential therapeutic targets to block tumor development.
Human T-lymphotropic virus type-1 (HTLV-1) has been linked to the development of adult T-cell leukemia/lymphoma (ATLL), a monoclonal malignancy of mature CD4+ T cells, which often express the activation marker CD25. It is estimated that approximately 15 to 20 million patients are infected with HTLV-1 worldwide, and although a majority of infected patients remain clinically asymptomatic, approximately 2% to 6% will develop some form of ATLL.1 Infection rates for HTLV-1 vary between 0.1% and 30% within endemic populations and occur more sporadically among at-risk groups from nonendemic regions, such as urban areas in the United States and Europe.2⇓–4
HTLV-1 typically is transmitted in endemic areas from mother to child through breastfeeding,5 but it also can be transmitted by exposure to infected blood or through the sharing of needles among intravenous drug users.6 Sexual transmission is less efficient but an important mode of transmission.7 The development of ATLL generally manifests in patients after a prolonged latency period (20-40 years).8⇓–10 The chronic-stage ATLL is characterized by leukocytosis, whereas the acute stage is characterized by multicentric lymphomatous masses, lymphoadenopathy, and hepatosplenomegaly.11⇓–13 Diagnostic criteria of ATLL include demonstration of clonally integrated HTLV-1 in neoplastic lymphocytes.14,15
Identification of primary target cells that harbor HTLV-1 infection in infants and characterization of factors that promote virally induced transformation are difficult to identify primarily because of the extremely long clinical latent period to ATLL development and the lack of appropriate animal models that accurately recapitulate leukemogenesis.16 Inoculation of nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice and NOD/SCID IL-2γ chainnull (NSG) mice with human CD34+ hematopoietic progenitor/stem cells (HP/HSCs) results in robust and extensive human hematopoiesis, including the maturation of human monocyte/macrophages and B and T lymphocytes, and sustained maintenance and expansion of human stem cells in the murine bone marrow (BM).17⇓–19
The ability of humanized SCID mice (HU-NOD/SCID and HU-NSG) to support maturation of all human hematopoietic lineages facilitates the evaluation of the effects of viral infection and gene transduction on human hematopoiesis in vivo. We previously demonstrated that HTLV-1 efficiently infects human CD34+ HP/HSCs ex vivo and that integrated proviral sequences persist in cells throughout maturation and differentiation in vitro.20⇓⇓–23 Herein, we report that HTLV-1 infection and Tax1 transduction of CD34+ HP/HSCs results in highly reproducible induction of CD4+ human T-cell lymphoma with clinical and pathologic features of ATLL in HU-SCID mice. In parallel, we provide data that CD34+ HP/HSCs from HTLV-1–infected patients contain proviral integrations, suggesting a role of human BM-derived stem cells in leukemogenesis. This animal model of ATLL will be useful to determine early factors that initiate or block the development of HTLV-1–associated lymphoma.
Ex vivo infection of CD34+ cells with HTLV-1 and generation of HU-NOD/SCID mice
Fetal liver (FL)–derived CD34+ HP/HSCs were freshly isolated without cryopreservation and infected with HTLV-1 or transduced with Tax1 or Tax1-lentiviral vectors (LV) as described previously.22⇓–24 CD34+ HPCs (5 × 106) were injected into sublethally irradiated (350 rads) 4- to 6-week-old NOD/SCIDpkrdc mice (The Jackson Laboratory) intravenously through the tail vein, as previously described25⇓–27 to generate HTLV-1-HU-NOD/SCID or Tax1-HU-NOD/SCID mice. HTLV-1–infected or mock CD34+ HPCs (2 × 105) were injected into sublethally irradiated (25 rads) 1- to 2-day-old NSG mice (The Jackson Laboratory) intrahepatically to generate HTLV-1-HU-NSG and mock HU-NSG mice as described previously.18 Mice were housed under specific pathogen–free conditions. Mice were killed after the administration of isoflurane and tissue collected under aseptic condition for flow cytometric, polymerase chain reaction (PCR), and histopathologic analysis.
Flow cytometry
The flow cytometric analysis was performed as described previously.20,27 In brief, cells from various murine organs were resuspended in phosphate-buffered saline with 2% human serum (Jackson ImmunoResearch Laboratories) and 2% mouse serum (Invitrogen). For surface antigen detection, the cells were stained with a combination of 5 μL each of murine anti–human CD4-fluorescein isothiocyanate, CD3-PE, CD8-Pe-CY7, CD19-APC-CY7, CD45-APC, CD25-APC, CD34-PE,CD38-APC, and CD14-PE-Cy7 (BD Biosciences) and rabbit anti–mouse CD45-peridinin chlorophyll protein complex (BD Biosciences) antibody at 4°C for 20 minutes. For detection of intracellular HTLV-1 p19gag, the cells were permeabilized/fixed by the use of Cytofix/Cytoperm solution (BD Biosciences) as per manufacturer's protocol and then incubated with mouse anti–HTLV-1 p19gag antibody (ZeptoMetrix; 3 μL/106 cells) for 30 minutes at 4°C, washed twice with phosphate-buffered saline, and then incubated with fluorescein isothiocyanate- or CY5-conjugated goat anti–mouse IgG monoclonal antibody (1 μL/106 cells; Dako) for 30 minutes in dark at 4°C. Then, 1 × 104 viable and/or HTLV-1 p19gag+ cells were acquired with the use of an LSR II flow cytometer (BD Biosciences). Acquired samples were analyzed with the use of the FlowJo 8.3 program (TreeStar Inc).
Histopathologic analysis
Tissue specimens were fixed directly in neutral buffered formalin (Sigma-Aldrich) embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) solution. In addition, tumor cells derived from NOD/SCID engrafted intraperitoneally with lymphomas from HTLV-1-HU-SCID/NOD and Tax1-HU-SCID/NOD mice were fixed in Cytospun (Sigma-Aldrich) solution and stained with the Giemsa stain. Embedded sections also were stained with anti–human CD45 antibody. All slides were examined under a light microscope, and 40× and 100× photomicrographs were generated with the use of a Nikon camera. Tissue samples were fixed in 10% neutral buffered formalin, conventionally processed, and embedded in paraffin. Three micron sections were obtained and mounted on charged slides. A representative section from each block was stained for H&E. Immunohistochemistry was performed on the BONDX automated stainer (Leica Microsystems). All aspects of the staining, including deparaffinization and epitope retrieval, are included in the protocols. The detection protocol was modified to include a 10-minute incubation of Rodent Block M (Biocare Medical) to reduce any endogenous mouse immunoglobulin activity. Mouse monoclonal anti–human antibodies were obtained (Leica Microsystems) and included CD45 (X16/99), CD20 (MJ1), and CD3 (LN10). Negative controls were performed by elimination of the primary antibody to address nonspecific staining of the detection system. Immunodetection was performed by the use of Bond Polymer Refine Detection (Leica Microsystems) with horseradish peroxidase as the visualization enzyme and DAB as the substrate chromogen. Slides were counterstained with hematoxylin, dehydrated through graded alcohol, cleared, and mounted with synthetic mounting media.
Serial transplantation of tumor cells into NOD/SCID mice
All animal studies were approved by the SUNY Upstate Medical University Committee for the Humane Use of Animals. Cells were harvested from the spleen and mesenteric lymph nodes of the primary HTLV-1–infected mice or from LV-Tax1–transduced mice and resuspended in serum-free Iscove modified Dulbecco medium (Invitrogen). Cells (5 × 106) were then injected intraperitoneally into naive NOD/SCID mice. Mice were killed when they displayed extended abdomens or evidence of ascites, and flow cytometric and histopathologic analyses were performed as described.
Real-time PCR detection of proviral sequences in patient peripheral blood mononuclear cell samples
Peripheral blood from HTLV-1– and HTLV-2–infected donors was collected after informed consent in accordance with the Declaration of Helsinki, under a research protocol approved by the Medical College of Wisconsin or the University of Miami Institutional Review Board. Cryopreserved peripheral blood mononuclear cell specimens were then shipped without unique identifiers to the Upstate Medical University of New York for the proposed molecular analysis. Quantitative PCR was performed with QuantiTect SYBR Green (QIAGEN) and primers specific for the HTLV-1 proviral genes gag (forward: 5′-CCTTCGTAGAACGCCTCAAC-3′ and reverse: 5′-ACAAGCCCGCAACATATCTC-3′), env (forward: 5′-TGGGAGCAAGGAGGATTATG-3′ and reverse: 5′-CGACAAGGGTGATTCCAGTT-3′), and tax (forward: 5′-TGTTTAGAGACTGTGTACAAGGCG-3′ and reverse: 5′-GTTGTATGAGTGATTGGCGGGGTAA-3′). The DNA products in each sample were adjusted for the total input DNA as monitored by the human β-globin gene detected with huBG240 primers (forward: 5′-GCCCTGGGCAGGTTGGTATCAAGGT-3′ and reverse: 5′-TGAGCCTTCACCTTAGGGTTGCCCA-3′). After an initial denaturation of 95°C for 15 minutes, followed by a 50-cycle PCR (94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, and 80°C for 30 seconds) with detection points at 72°C and 80°C, the reaction was concluded with a 0.5°C melt-curve from 60°C to 95°C with use of the IQ-5 iCycler (Bio-Rad Laboratories). Standard curve analysis was performed with the IQ-5 program, with R2 values of at least 0.990.
T-cell receptor rearrangement analysis
DNA from lymphoma samples were analyzed for combinatorial diversity of the human β-chain of the T-cell receptor (TCR) by multiplex PCR analysis (ImmunID Technologies).
Statistical analysis
Statistical analyses were performed with the Student t test.
Development of CD4+ T-cell lymphoma in HTLV-1-HU-NOD/SCID mice
To determine the effects of HTLV-1 infection on hematopoiesis in vivo, human FL-derived CD34+ HP/HSCs were infected with HTLV-1 and then inoculated intravenously into NOD/SCID mice to reconstitute hematopoiesis. A high percentage (43%) of HTLV-1-HU-NOD/SCID mice developed lymphoma after an average latency period of approximately 17 weeks (Table 1). Reconstitution of neonatal NSG mice with HTLV-1–infected CD34+ FL cells similarly resulted in the manifestation of CD4+ T-cell lymphomas. Mice exhibited multicentric lymphoma with masses in the mesenteric lymph nodes (MLNs), liver, and in the spleen, resulting in splenomegaly (Table 1; Figure 1). HU-NOD/SCID mice inoculated with uninfected donor-matched CD34+ cells ("mock") did not develop malignancies or lesions. Gross and histopathologic examination of affected mice revealed that presence of highly infiltrative lymphomas in the MLNs and spleen of infected mice that expressed human CD45 by immunohistochemistry (Figure 1F, G, and I). Infiltration of human lymphoma cells (CD45+) was also detected in the lungs, liver, kidney, pancreas, small intestine, and the BM in HTLV-1-HU-NOD/SCID mice (Figure 1K-R).
Lymphoma cells from HTLV-1-HU-NOD/SCID mice were CD4+/CD8−, coexpressed HTLV-1 viral p19gag envelope, and occasionally expressed CD25+, demonstrating that a mature T-cell lineage was the predominant phenotype among the infected lymphoma cells derived from these mice (Figure 2).28 Leukemic cells were detected in the peripheral blood, and Giemsa-stained tumor cells displayed an abnormal cellular morphology (Figure 2Q-R). HTLV-1–infected mice demonstrated CD4+/CD8− cells in the MLN and spleen, in contrast to the predominantly CD19+ B cells in the spleens of mock-infected HU-NOD/SCID mice as previously reported (Figure 2E-H).25,26 Cells recovered from the murine thymus of HTLV-1–infected mice demonstrated the presence of CD4+/CD8− human T cells in contrast to mock mice, which predominantly displayed CD4−/CD8− cells (Figure 2P).
Collectively, cells coexpressing CD45+/CD4+/p19gag+ were the predominant human cell population in the BM, thymus, spleen, and the MLN of HTLV-1-HU-NOD/SCID mice. Most HTLV-1-HU-NOD/SCID mice, which did not develop disease, failed to demonstrate engraftment of CD34+ cells, suggesting that the incidence of lymphomagenesis by HTLV-1 may be significantly greater than calculated. The slightly elevated incidence of lymphomagenesis in NSG mice most likely reflects the greater engraftment rate of CD34+ HP/HSCs in this mouse strain in contrast to NOD/SCID mice (P.B. and G.F., unpublished observations, December 2009). This finding demonstrates that HTLV-1 infection of CD34+ HP/HSCs induces the development of a mature CD4+ T-cell lymphoma within a relatively short time frame (12-20 weeks after reconstitution) with histopathologic, immunologic, and clinical characteristic features of ATLL in HU-NOD/SCID and HU-NSG mice. Furthermore, hyperproliferation of HTLV-1–infected stem cells in the BM suggests that infection of stem cells may be a predeterminant of lymphomagenesis (Figure 3). Intrahepatic injection of neonatal NSG mice with HTLV-1–infected CD34+ HP/HSCs also induced lymphoma and infected mice demonstrated a dramatic shift toward CD4+/CD8− T-cell maturation in comparison with mock-infected HU-NSG mice reconstituted with tissue-matched CD34+ cells (Figures 4–5).
To determine whether the lymphoproliferations from the HTLV-1-HU-NOD/SCID were fully transformed and tumorigenic, cells from the MLN and the spleen were pooled and inoculated intraperitoneally into naive NOD/SCID mice. Secondary recipient mice developed CD4+ lymphomas in the peritoneal cavity and in the MLN, and engrafted tumor cells expressed HTLV-1 p19gag (Figure 6). HTLV-1–induced lymphomas showed primarily oligoclonal patterns of T-cell proliferation as determined by TCR rearrangement patterns (Table 2). Serially transplanted lymphomas developed into monoclonal proliferations in secondary recipient mice for 81% of the HTLV-1–induced lymphomas (21/26 lymphoma cell lines), and these secondary lymphomas demonstrated infiltration into the spleen, kidney, and small intestine in these mice (Figure 6H-K).
LVs expressing Tax1 induce T-cell lymphomas
LVs, which coexpress HTLV-1 tax and green fluorescent protein (GFP; pHR'CMV-Tax1/GFP), have previously been described22⇓–24 and were used to transduce CD34+ HP/HSCs ex vivo before inoculation into NOD/SCID mice. Tax1-HU-NOD/SCID mice developed CD4+ malignancies with a slightly accelerated kinetics in comparison with HTLV-1-HU-NOD/SCID mice (Table 1; Figure 2). Some of the primary Tax1-generated lymphomas demonstrated monoclonality by TCR rearrangement patterns (Table 2). Tax1-induced lymphomas expressed GFP and demonstrated tissue distribution patterns similar to HTLV-1–induced lymphomas (Figure 2). HU-NOD/SCID mice reconstituted with CD34+ cells transduced with LVs encoding the Tax1 gene in the anti-sense orientation did not show any pathology (data not shown). This finding demonstrates that constitutive expression of Tax1 in CD34+ cells results in CD4+ lymphomas, which develop more rapidly into monoclonal proliferations, in contrast to HTLV-1–induced lymphomas. In addition, 100% (4/4 lymphoma cell lines) of Tax1-induced lymphomas engrafted and formed malignancies after intraperitoneal inoculation into naive NOD/SCID mice.
HTLV-1 infection induces hyperproliferation of HSCs in BM
We hypothesize that HTLV-1 infects CD34+ HP/HSCs and that the BM milieu allows productive expansion of HTLV-infected HSCs in vivo. HTLV-1-HU-NOD/SCID mice demonstrated 2- to 3-fold greater levels of infected CD34+/CD38− HSCs in the BM in comparison with HU-NOD/SCID mice generated from the same donor CD34+ cells (Figure 3). This finding demonstrates that HTLV-1 infection induces expansion of infected stem cell subpopulations in the BM and that the BM milieu may provide a suitable environment conducive for establishment of an HTLV-1–infected leukemic stem cell (ILSC).
HTLV-1 proviral sequences in CD34+ cells from patients
To determine whether HTLV-1 infection of CD34+ HP/HSCs is biologically relevant and detected in patient-derived CD34+ HP/HSCs, CD34+ and CD34− cells were purified from peripheral blood lymphocytes of human T-lymphotropic virus type I–associated myelopathy/tropical spastic paraparesis and HTLV-1–seropositive patients by fluorescence-activated cell sorting and subjected to real-time PCR by the use of primers specific for HTLV genes (Table 3). HTLV-1 proviral sequences were detected by PCR for all patient samples, and proviral loads were relatively elevated in purified CD34+ cells in comparison with CD34− cells, suggesting that HP/HSCs preferentially harbor HTLV proviral sequences in patients.
We describe the development of a novel humanized mouse model that recapitulates ATLL development and speculate that HTLV-1 infection of human CD34+ HP/HSCs contributes to the initiation of lymphomagenesis and the manifestation of this malignant disease. HTLV-1 infection of key target HP/HSCs in infants infected at birth has been proposed to be a predisposing factor in the development of leukemia/lymphoma.14,20,23,29,30 Allogeneic BM transplantation involving HTLV-1–infected stem cell donors has been associated with virus transmission,31 implicating HP/HSCs as harboring viral infection. Reconstitution of hematopoiesis in NOD/SCID mice with the use of HTLV-1–infected or Tax1-transduced human CD34+ HP/HSCs reproducibly recapitulates mature T-cell lymphomas with characteristic genetic, immunologic, histologic, pathologic, and clinical features of ATLL. It is notable that mature CD4+ T-cell lymphomas are established in Tax1-HU-NOD/SCID mice with the use of an LV that is not exclusively targeted to the human T-cell lineage. Recent reports of a Tax-transgenic mouse model demonstrates in vivo transformation of immature and mature T cells32,33 and a role for cancer stem cells in the generation of Tax-induced T-cell malignancy.30 It is noteworthy that HTLV-1 viral gene expression and Tax1 expression in lymphomas induced in HU-SCID mice are not reflective of the silencing of viral gene expression demonstrated by primary ATLL cells from patients. It is also important to note that the lymphomas initiate and progress in the absence of any major immune selective mechanisms in the HU-SCID mouse and that it has not yet been determined whether hallmarks of ATLL, such as hypercalcemia and constitutive NF-κB activation, are recapitulated in the HU-SCID mouse model. Moreover, the short time frame in the manifestation of these lymphomas in this small animal model could suggest that the progression to malignancy in HU-SCID mice may ultimately fail to reflect secondary mutations or immune evasion mechanisms that may be involved in the full progression to ATLL in human patients. Despite these shortcomings, this chimeric human-mouse model will be an important tool to systematically recapitulate ATLL disease and dissect HTLV-1 determinants of pathogenesis and oncogenesis.
Previous attempts to directly infect mature human T cells in the thymus-liver conjoint organ in HU-SCID mice with HTLV-1 failed to induce malignancy.21 This finding suggests that the combination of the appropriate immature hematopoietic target cells in the BM and the immunologically defective environment of the SCID mouse are both required for the manifestation of the ATLL phenotype. Our data demonstrate that infection of CD34+ HP/HSCs is a prerequisite to T-cell lymphoma development in HTLV-1-HU-SCID mice. CD34+ HP/HSCs represent a heterogeneous cell population encompassing early and committed multipotent progenitor cells, noncommitted differentiating cells and stem cells.34 Although Melkus et al35 failed to detect human T-cell development in humanized NOD/SCID mice, our ability to detect human T-cell maturation in NOD/SCID mice receiving freshly isolated FL-derived CD34+ cells is in agreement with other reports19,26 and suggests FL-derived CD34+ HP/HSCs are more pluripotent in robustly reconstituting lymphopoiesis.
HTLV-1 infection of CD34+ HP/HSCs dramatically skews hematopoiesis toward T-cell development, presumably at the expense of other hematopoietic lineages. It has recently been shown that HTLV-1 infection in HP/HSCs results in a perturbation of miRNA expression, resulting in the skewing of hematopoiesis toward T-cell lineages.36 Alternatively, the ability of HTLV-1–transformed T cells to preferentially proliferate in vivo may overshadow normal hematopoietic patterns. Clearly, HTLV-1 infection of HP/HSCs plays a pivotal role in the initiation and accelerated progression of malignancy during the course of HTLV-1 disease, and the immunodeficient environment in the SCID mouse allows for the rapid manifestation of a T-cell malignancy. It is interesting that HTLV-1 infection primarily induced oligoclonal T-cell proliferations in contrast to the predominantly monoclonal T-cell malignancies induced by LV-Tax1 expression. This finding could imply that transformation by HTLV-1 infection in HU-NOD/SCID mice occurs across a broader number of infected CD34+ cells or that numerous infected CD34+ HP/HSCs can progress into a T-cell malignancy. Alternatively, overexpression of Tax1 by the CMV promoter by LV may be a relatively strong selective event driving the expansion of a monoclonal, dominant transformed T-cell clone in comparison with HTLV-1–mediated transformation. The ability of the LV-Tax vector to induce lymphomagenesis relatively rapidly in comparison with HTLV-1 infection also may reflect the different dynamics involving transduction of stem cells with a LV in comparison with infection with HTLV-1. Clearly, evaluation of stages of transformation in vivo and quantitative analyses of Tax1 levels will be critical in deciphering the kinetics and mechanics of in vivo transformation by HTLV-1 infection and Tax1 expression.
HTLV-1 proviral DNA has previously been detected in cord blood samples, generally a rich source of HSCs.37 Our results demonstrate that CD34+ cells from patients consistently harbor HTLV-1 proviral DNA at relatively high levels in comparison with other hematopoietic lineages. It was previously reported that HTLV-1 proviral sequences were not detected in CD34+ BM cells from ATLL patients.38 Although this observation appears to be in discordance with our results, we speculate that ATLL patient BM-derived CD34+ cells are not truly representative of early acute stages of HTLV-1 infection in stem cells in neonates or pediatric cases but, rather, represent an end-stage disease process that may not recapitulate the BM environment of an early-stage infection. Analysis of BM samples for pediatric HTLV-1 infections would be essential in confirming the hypothesis that HTLV-1 infection enters and is sequestered in the CD34+ cells in the BM.
Manifestation of ATLL in patients generally occurs decades after infection, and it is most likely that HTLV-1 latently infects BM stem cells that are sequestered from immunologic surveillance. It is conceivable that initiation of the leukemogenesis in HSCs involves the generation of an ILSC that eventually manifests into the monoclonal ATLL malignancy. Clearly, the expansion of HTLV-1–infected CD34+/CD38− HSCs in the BM of HTLV-1-HU-NOD/SCID mice is consistent with the fact that infection increases the absolute numbers of retrovirally infected stem cells in the milieu of the BM environment, although it should be noted that this has not yet been demonstrated in human pediatric patients. To definitively establish whether HTLV-1 induces generation of ILSCs, we are currently testing whether BM-derived CD34+ cells purified from HTLV-1-HU-NOD/SCID mice are capable of re-establishing ATLL after transplantation.
The HU-NOD/SCID mouse model of ATLL provides a novel tool for characterizing the role of proviral integration sites and somatic mutations associated with induction of lymphoid malignancies and will also allow evaluation of the contribution of the HTLV-1 auxiliary genes in viral pathogenesis. Moreover, this model will allow preclinical therapeutic evaluation studies to be evaluated for a fatal malignancy that lacks effective treatment regimens.
Contribution: P.B., A.T., and L.C. performed the majority of experiments involving generation of lentivirus vectors, infection of stem cells, and reconstitution of SCID mice; P.B., L.C., and M.S. performed flow cytometric analyses and PCR analysis of DNA samples recovered from mice; M.D.L. performed histologic analyses; J.C.R. and W.H. analyzed tumor cells for TCR rearrangement patterns; M.A.B. provided peripheral blood lymphocytes patient samples; and G.F. supervised the experiments, interpreted the data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Gerold Feuer, Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NY 13210; e-mail: feuerg{at}upstate.edu.
This work was supported by grants from the National Institutes of Health (CA124595 to G.F. and P01 CA100730 to M.D.L.) and by the Empire State Stem Cell Fund through New York State Department of Health Contract (NYSTEM #C023059 and #N08G-127) to G.F.
This article is dedicated to the memory of William Harrington Jr, MD.
The opinions expressed here are solely those of the authors and do not necessarily reflect those of the Empire State Stem Cell Board, the New York State Department of Health, or the State of New York.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
- Submitted October 5, 2009.
- Accepted January 9, 2010.
- © 2010 by The American Society of Hematology


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Students work together to launch their experiment to space
Students get the opportunity to work with an astronaut in hopes of having their experiment sent to the International Space Station.
Students get the opportunity to work with an astronaut in hopes of having their experiment sent to the International Space Station.
"Consumers want to know what's in the products they use."
The Georgetown City Council voted 4 to 3 against allowing dogs on restaurant patios.
The suspect demanded from the teller without showing a weapon.
An audit shows the city's compensation division did not closely review monthly error reports, which also would have identified errors.
Fourteen-month-old Daisy Lynn Torres died March 29 after receiving dental treatment at Austin Children's Dentistry.
"I've never been in jail before so I don't know what to expect."
James Cloudt, 18, was pronounced dead at the scene. Police say an open alcoholic beverage was found in the car.
The new South Terminal at Austin-Bergstrom-International Airport is expected to open in 2017.
After further investigation, authorities discovered a similar incident involving him and a 16-year-old from a decade ago.
The Department of State Health Services says the mother recently traveled from Latin America, where she was likely infected.
She is being held on a total of $110,000 bonds and will be arraigned in Connecticut on Wednesday.
Frances Scott joined us in the studio with ideas on replacing the floors in your home.
"He looked like a dog inside the car just jumping back and forth from front to back."
"If that's gonna happen to somebody, he's probably the only one who would be able to come out of that as minimally scathed as he has."
All it takes is one distracted driver failing to move up to the intersection when the light is green causing the signal to turn red prematur…
The victim was at a bus stop in southeast Austin when a man robbed him at gunpoint.
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Translate a letter from English to Vietnamese
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Thomas Sawyer
- Registered on: 09/07/2008
- Last connection: 08/03/2016
- Assigned issues: 1
- Reported issues: 113
- 04:54 AM Ruby trunk Feature #9704: Refinements as files instead of modules
- Has any more thought been given to this? I had a recent request to have Ruby Facets to work as refinements, but I hes...
- 04:07 AM Ruby trunk Feature #12374: SingletonClass
- So...
Object..singleton_class.instance_of?(SingletonClass) #=> true
- 03:55 AM Ruby trunk Feature #12637: Unified and consistent method naming for safe and dangerous methods
- It would more interesting if there were a way to declare an object mutable or not, then when immutable destructive me...
- 03:23 PM Ruby trunk Feature #12360 (Rejected): More useful return values from bang methods
- It would be nice if bang methods returned results that where of some additional use. Presently most just return the e...
- 12:07 AM Ruby trunk Feature #11537: Introduce "Safe navigation operator"
- Philip Claren wrote:
> Thomas Sawyer wrote:
> > In this way is more an extension of the ternary operator -- the ini...
- 12:01 AM Ruby trunk Feature #11537: Introduce "Safe navigation operator"
- Laurentiu Macovei wrote:
> The same discussion happens to be on TypeScript and ES6 worlds.
> Using `..` instead of ...
- 05:10 AM Ruby trunk Feature #11537: Introduce "Safe navigation operator"
- Yukihiro Matsumoto wrote:
> I like the idea. My remaining concern is ".?" is too similar to "?." which is chosen by ...
- 06:49 PM Ruby trunk Bug #10924: String#b
- That's the irony. One of the reasons I choose #b is because I thought for sure that would almost certainly never beco...
- 11:57 PM Ruby trunk Bug #10924: String#b
- Robert A. Heiler wrote:
> I think that, the name aside, this would be a wonderful example for different
> namespace...
- 11:49 PM Ruby trunk Bug #10924: String#b
- Benoit Daloze wrote:
> Note #b is more #binary than #ascii.
That at least make a little more sense out of the nam...
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Pakistani Jewish Vote?
The Pakistani Election Commission lists approximately 800 voters as Jewish although there are virtually no Jews in the country, according to the country's The News website contributor Dr. Abdul Majeed Abid.
He facetiously asked, "We can only wait to see which way the 'Jewish vote' goes, if it goes anywhere."
He noted that no Jews are running for public office and no political party has tried to woo the supposed Jewish voters, leaving open the question why the Elections Commission bothers to list Jews as a minority.Tzvi Ben-Gedalyahu
About the Author: Tzvi Ben Gedalyahu is a graduate in journalism and economics from The George Washington University. He has worked as a cub reporter in rural Virginia and as senior copy editor for major Canadian metropolitan dailies. Tzvi wrote for Arutz Sheva for several years before joining the Jewish Press.
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1999 Daewoo Lanos SE 2dr Hatchback
I still have it and driving it. It a great car I turned in to a racer. So bsd they don`t sell it more in Puerto Rico. I had to do some changes because most of its part don`t sell it no more. Most of its part had to buy by the net to e-bay. Or get the from other cars at the Yunkers.
Features are good but I've change soon to make it look like a fast and furious car it still run fast.
I suggest if you want to make it more great make it an all wheel drive with turbo from factory. I will like to send pictures of it let you guys see what I did to it. I had it for more than ten years it still look new.
1999 Daewoo Lanos SX 2dr Hatchback
I have had it for two years. No major repairs. Only one minor repair. But the minor one of a hose. My mechanic said it was hard to get a hose He said luckily this was a minor repair but what happen if it becomes major. He said for this car parts are going to get hard and hard to find. This is the only reason why i am even considering selling it. It is such a reliably car only if you take care of routine maintenance, even then it cuts you a little slack. This is the best car and the least expensive car i have ever purchased, EVER. Don't worry about breaking down i can get in and go just about anywhere. Can park just about anywhere
Zips around Gas is great I'm 5' and its very accessible for a small person like me!
I have had a lot of fun with this car. It has saved me a lot of money in gas and oil. Great commuter car no problem.I think if you do the suggested maintenance you will have no problems. Most expense was I had the timing belt changed as suggested at 50,000 miles. With these engines that is a must. Just maintain the car as the book suggests.
Great gas mileage.
I would not recommend buying this car at all. It lasted 1 year. It's had a ton of maintenance problems. Many of the problems seem to be electrical. So far we've had to fix the transmission, replace the battery, and the car dies as soon as I put gas in it. The parts for the car are also cheap so everything will be falling apart within the first 3-5 months as I noticed. The windows get stuck when you try to roll them up. The sun roof doesn't work. It's very hard to keep clean. I thought this would be a first good car. I would definitely not recommend buying this or any Korean car.
1999 Daewoo Lanos SE 4dr Sedan
I am so sick of problems with this car I can't even begin to tell my story but let just say out of the six years I have owned this junk I have only been able to drive this car for 3 years because I can't locate simple parts. BEWARE! I haven't put any mileage on this car in 3 years so I don't even know my annual mileage.
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Find the best selection of new and used trumpets at all the best prices to keep you playing for hours with these easy to use websites for buying trumpets online.
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After blowing your load, the girl spreads your penis hole open and sticks her tongue in to get the rest out.
Hey John, your mom gave me a valve job last night and it was awesome.
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Questioning a fellow male if he would fuck a female they are checking out.
Q: Hey dude, drop the elbow?
A: Hell yeah! From the top rope!
A: Hell yeah! From the top rope!
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My grandpa just got a new dog and had his caregiver take pictures of him to send to me pic.twitter.com/qVwxgNmM7v— Jess (@JessAmante4) March 6, 2016
Grandads are all the rage online at the moment, with a charming elderly man going online again after his granddaughter posted a heartwarming image of him.
Jess Amante shared an image of her grandfather and his tiny new dog Penny, and it's been a hit, garnering 81,000 likes - and more than 40,000 retweets.
Hot on the heels of Sad Papaw who cooked for his grandkids and only one showed up, Jess' 90-year-old grandpa has been praised for his adorable photographs, with many congratulating him for taking on a new pet.
Read more: Sad grandpa 'Papaw' is star of massive party as hundreds attend cookout
"My grandpa just got a new dog and had his caregiver take pictures of him to send to me," she posted online.
My father in law (age 90) just adopted a sweet dog! He is already in love with his sweet Penny girlPosted by Debbie Amante on Sunday, 6 March 2016
Amante's mother, Debbie, also shared the sweet photos on Facebook.
Twitter was awash with love for the nonagenarian, with some posting images of themselves with running mascara after they've obviously had a cry.
@JessAmante4 @BRITROUGES pic.twitter.com/s9gkjyxOjV— dais (@kissingjohnson) March 7, 2016
@JessAmante4 pic.twitter.com/n7OhINaK6R— ˗ˏˋnaiˎˊ˗ (@nAAAYEomi) March 7, 2016
Big Earl posting: "That's so adorable I'm gonna get emotional," while Finna added: "Why is this one of the most heart warming things ever."
Jess was overwhelmed with the response, tweeting: "Gotta tell my grandpa that he's now Twitter famous."


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Sorry, this item sold. Have karleenlykken make something just for you, or try these other items:
This is a sterling silver necklace link connector. It measures approx. 22mm in diameter.
Sterling Silver Necklace Link Connector
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Home > Eyeglasses > Calvin Klein > CK536 (039)
CK 536 is available in 4 additional attractive styles
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Latest news for kenalog
Average Rating: 4.4 out of 5 based on 198 user reviews.
A fast and easy way to test your SRST configuration without taking down the WAN interface, is to add a static [kenalog] route to null 0 for any ip packets coming from the network the Callmanagers are on. Kenalog
this will cause phones to lose contact with all callmanagers and kenalog go into fallback mode kenalog, while still preserving data connectivity. Example: CCM Sub 10. 10. 0. 11/24 Add the following route: ip route 10. 10. 0. 0 255. 255. 255. 0 null0 Once you have completed your testing, remove the route, and phones will register back with Callmanager.


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To win "A special meet and greet with Paper Bird—private viewing of their sound check followed by Q&A" just leave a comment below, DM us on twitter, or leave a comment on our Facebook Page.
"Why do I love Paper Bird? Is it because they are the most adorable band ever? Yes. Is it because they are a big group of guys and gals living out their dreams? Yes. Is it because they play fun, sweet, folky music that's ahead of the curve? Yes.
I bought their CD off iTunes even though I could have burned a copy from Waylon. I bought tickets to the Boulder Theater show for myself and boyfriend even though I could have free tix through elephant's sponsorship. They're the kind of big hot lovely band you want to support. I first saw them at the Walk the Talk Show with Waylon Lewis in May. As if their music isn't good enough on its own, seeing them perform live is seriously magical (think modern version of the roaring '20s).
They are good people that don't care about material things or fame, they are doing what they love and doing a damn good job of it." ~Lindsey Block
Fri, Oct 2: Radio 1190, Boulder Weekly & elephantjournal.com present
w/ Nathaniel Rateliff + The Wheel & Drive River Yacht Club
GA / All Ages / $15.00
They're playing Belly Up in Aspen on October 28th. Halloween shows have just been announced. But if you haven't got your Paper Bird fix in since Walk the Talk Show (click here for video of their performance on our talk show), this Friday is your best best.
More info:
Paper Bird to release
Live Recorded EP on Junketboy
For the first time Colorado-based septet Paper Bird will be releasing their only live-recorded EP on October 6th, 2009 to national retail. The EP, titled Live at Twist & Shout, was recorded this summer at Denver's independent record store Twist & Shout. Junketboy will be releasing the EP to national independent stores. Paper Bird's decision to work with Junketboy reflects their interest in supporting the independent music scene. The band self-released their full-length album Anything Nameless and Joymaking (2007) and recent EP A Sky Underground (2009), both have been top selling records in local indie retail over the past eighteen months. Junketboy is an independent distribution company with a unique catalogue ranging from exclusive releases to live shows to limited-edition releases. The commitment of Junketboy is to supply independent retail with products and marketing tools needed to make cool record stores even cooler. www.thinkindie.com
The members of the band- Sarah Anderson, vocals and trumpet; sisters Esmé and Genny Patterson, vocals; Tyler Archuletta, trombone; Paul DeHaven, guitar; Caleb Summeril, banjo; Macon Terry, upright bass – bring a Folk, Americana sound that pays homage to the American Bandstand of the 1950's, with a hint of Roaring 20's flare. But don't be thrown off by their old-timey sound, Paper Bird maintains the Indie image and resonance of the current age with a progressive and complex style that keeps them on the forefront of the music scene. The band will be headlining the Boulder Theater on October 2nd. Tickets are available through www.bouldertheater.com.
For more information: Shoshanna Howard [email protected] or 303.998.0001
Online: www.paperbirdband.com or www.myspace.com/paperthebird
Bonus, our vid:
Walk the Talk Show: Paper Bird from Alex King & Mito Media on Vimeo.
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The story behind the Elephant-headed God. 456 shares September's Black Moon: The Rare, Powerful, Feminine Goddess Lilith. 73,836 shares Will & Grace are Back after 10 years & it's the Funniest 10 minutes of Election Commentary Ever. 17,934 shares October Energy Forecast: Prepare for Limitless, Unconditional Love. 7,895 shares The Fourth Kind of Love. 2,774 shares How Open-Hearted Men can Show Up for Strong, Independent Women. 3,686 shares What Teens need from their Parents. (Hint: It's not Grounding & Punishment.) 2,069 shares I am Who I am Thanks to You—The 5 Most Important People we Meet in Life. 2,128 shares 10 Good Reasons Not to Contact your Ex. 946 shares Nine Reasons to Love a Libra & One Secret they Keep. 1,478 share


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It's something that you hear time and time again, but when it fits it fits--there is nothing like a fat guy celebration! The Seattle defense is doing its job against Christian Ponder and the Minnesota Vikings with three sacks already in the first quarter.
Because he can (you know you love it)!


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Kentucky Derby winner Orb could face five of his beaten rivals in the May 18 Preakness Stakes in Baltimore.
The numbers for the 1 3/16-mile middle leg of the Triple Crown is notorious for fluctuating greatly. As it stands, the Derby horses besides Orb considered definite for the Preakness are: the D. Wayne Lukas-trained tandem of sixth-place Oxbow and eighth-place Will Take Charge and 17th place Goldencents. Fifth-place Mylute
a strong possibility, and 15th place Itsmyluckyday termed "60-40" to run by trainer Eddie Plesa Jr.
Preakness candidates who did not run in the Preakness are headed by Departing, the impressive Illinois Derby winner. Others under consideration are Sunland Derby winner Govenor Charlie, who skipped the Derby after missing some training with a foot bruise; Fear the Kitten, unable to run in the Derby as the 21st horse in preference; and Bellarmine, winner of a 1 1/16-mile allowance race on the Derby undercard.
Departing is co-owned by Claiborne Farm, the same Paris, Ky., farm where Orb was born and raised for Claiborne's long-time clients, the Phipps family and Stuart Janney III. When Orb is retired to a stallion career, it almost assuredly will be at Claiborne.
Hence, the Preakness plotlines will include Claiborne possibly ending the Triple Crown run of its clients and future stallion. In fact, Orb and Departing actually were raised in the same paddock at Claiborne.
Trainer Al Stall Jr. said Claiborne boss Seth Hancock called him Sunday morning and said "let's go forward."
"We were kind of pointing toward the Preakness no matter who was going to be there, really," Stall said. "I was unbelievably impressed with the way Orb trained and the way he ran. He's going to be a monster horse to try to beat. We just have our horse on a good schedule. He's fresh. I wanted to get a work into him before the Derby, which he did Thursday. We're very excited how that went and how he came out of it too. All that went into our decision. That's what made us firm it up today."
Over the years, Claiborne and Phipps horses have competed in racing's biggest stages. Forty Nine, Claiborne's 1988 Derby runner-up, defeated the Phippses' Seeking the Gold by a nose in both the Grade I Haskell and Grade I Travers.
"I'm sure they'd want it that way, Stuart and Mr. Phipps," Stall said of Departing going in the Preakness. "I mean, they're sporting guys. That's what they're here for. I doubt they'd say, 'Hey, get out of our way. We want the easy road.'… Forty Niner, Seeking the Gold – this is nothing new for them. It's a non-issue. We're just trying to keep our horse happy and he'll keep us happy."
Meanwhile, Orb came out of the Kentucky Derby, which he won by 2 ½-lengths over longshot Golden Soul, in good order. He was bright-eyed and attentive as a parade of fans, track officials and their families and media crowded out of the rain and into trainer Shug McGaughey's barn, many feeding Orb treats provided by McGaughey's wife, Alison, and having their picture taken with the 139th Kentucky Derby winner.
Orb left Churchill at 9:30 Sunday morning, headed to McGaughey's Belmont Park stable in New York. McGaughey said he expects to ship to Baltimore the Monday or Tuesday before the Preakness.
"I don't know where we're going from here," McGaughey said, speaking of what might be the outer limits of Orb's ability. "He continually impresses me with what he does and how he races. I think there's maybe more there than what we're getting. The maturity he showed yesterday and everything he did from his Derby Day, his experience in the paddock, which was tremendous, going to the post and the way he raced just kind of amazes me."
"I don't really know what I got. I know what I got now: I've got the Derby winner.
The Orioles are in town a couple of days during Orb's projected stay. The Maryland Jockey Club is a master at getting the Derby-winning trainer to throw out a ceremonial first pitch. Is McGaughey going to practice?
"I'll probably warm up a little bit this week and stuff," he said cheerfully while at the barn before Orb's departure. "I'm excited about this. I was excited all last week. I wasn't nervous (Saturday). The only time I was nervous was when I was home getting dressed and wasn't here. I'm going to be excited going on.
"I don't really know what I got. I mean, I know what I've got now: I've got the Derby winner. I'm looking forward to running him in two weeks from now, everything being good."
Lukas said Orb's rivals will have to get better to beat him in Baltimore. Orb came from about 20 lengths back to make a monster move around the far turn and through the stretch.
"I think the Preakness will be the biggest hurdle for him for the Triple Crown," he said. "If he gets by that, he gets to go back home to Belmont and run right out of his stall… He's got the pedigree and he's got a style that really equates to winning these things. The way he finishes his races, starting back to the Florida Derby and now this one, is going to make him awful tough."
Lukas thought the sloppy track conditions hampered his horses, but also said, "I think the best horse won, dry or not. Any time a horse comes home the last quarter like he did, you're going to win probably a Derby.
"It was a wonderful victory for them. I said all week that there's a certain amount of karma in this race that rewards people who have been stalwarts in the industry. It seems like the racing gods look down and say, 'This year we're going to give it to Stuart Janney and Dinny Phipps.'
"In spite of the weather, it was a great show. I was not disappointed. You have to get realistic on these things. At the top of the stretch I thought Oxbow might kick on and get it, but I could see they were coming on the outside. Will Take Charge didn't have a clean trip, but he ran pretty darn good."
Trainer Doug O'Neill, who won last year's Derby with I'll Have Another, said he was proud of Goldencents, who started out near the wicked pace set by the newly blinkered Palace Malice but then was taken back a bit by jockey Kevin Krigger.
"We're still scratching our head on how the race unfolded," he said. "I think he just didn't enjoy the kickback, the slop and where he was. Kevin did a great job. When Palace Malice flew out of there, there was no sense in chasing him. It just wasn't his day. I think the (Preakness') shorter distance, and we'll just take the approach of 'come and get us' there. A mile and a quarter, the long stretch, it's a tough journey here at Churchill."
Of Orb, O'Neill said, "He's an unbelievable horse. He's got Triple Crown talent written all over him. I think if you're going to beat him, Baltimore would be the place. I think having Belmont being his home and his style, it sure looks like he'll be ultra tough there. He's a super horse."
Plesa blamed Itsmyluckyday's disappointing performance on the wet track. "You could see where he was struggling with the racetrack all the way around," he said.
Golden Soul will train up to the Belmont, said trainer Dallas Stewart. "He ran great, but nobody was going to beat the winner," he said. "If we could get one of the three, we'd be real happy. I'm sure he'll get a mile and a half."
Third-place Normandy Invasion will skip the Preakness and point toward Saratoga's Travers Stakes. "I don't know what I'll do between now and then," said trainer Chad Brown.


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Francisco Reyes
Height: 5'8
Weight: 150 lbs
Position: Bottom
Francisco's smooth lips, hot body, and spunky attitude make for an amazing Lucas Entertainment debut in Barcelona Nights. A Barcelona native, he works in PR and in his spare time loves swimming, sports, travel... and getting fucked! He hopes his fans will be easy on him for his first time on camera!


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While oil prices continue their seemingly unrelenting march upwards, and with winter soon upon us. why are there no fuel-surcharges in the US?
BA has just raised their surcharge on long-haul tickets sold here in the UK from £6.00 ($10.75) to £10.00 one-way ($18.00). In addition, for short-haul, the increase has gone from £2.50 to £4.00 one-way. Seems all other EU carriers, except for LCC's, are doing the same.
BA says their fuel expenses will increase by £225M (Although they say they have hedged 72% of fuel thru March, 2005). But what about US carriers? I would assume they all buy from the same market.
Some reports suggest oil will shortly reach $60/Barrel. If. more likely when, that happens, what will be the effect on carriers like UA & US, who cannot hedge, and DL, which sold their hedged oil contracts to raise cash?
While a recent increase in low-end domestic fares are fuel related, I was wondering why they don't levy a seperate surcharge across the board.


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Popular Diets
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Joy Wilson, of the widely-recognized food blog Joy the Baker and author of "Joy the Baker Cookbook: 100 Simple and Comforting Recipes," is one of my absolute favorite bloggers. Her delicious recipes, candid narrative and quirky personality are enough to make me swoon over everything she shares.
One of Joy's latest projects is the "Joy the Baker Podcast" on which she discusses recent happenings in the kitchen, in friendships and in life.
On her latest episode titled 'The Friend Diet,' Joy discussed a new diet she's trying out to become more aware of what she's eating on a day-to-day basis. It's called the 'friend diet,' and it's a system she and her blogger friend, Tracy of Shutterbean, dreamed up one day.
The pair essentially takes photographs of everything they eat and text it to each other for accountability. As for whether or not it actually works, we talked with Joy to find out.
What sparked the idea of the friend diet?
Let's see, I text message with my best friend Tracy all day long. It's nonsense really. We noticed that a hearty majority of our texts revolved around the food that we were feeling guilty about eating. One day we just decided to flip the script. Now we tell each other, with pictures and with words, exactly what we're eating all day long. From a huge cheeseburger lunch to a handful of granola, we lay it all out there. We needed a way to be accountable to ourselves and bringing someone else in the loop seemed like the best friend way to do it. (more…)
Two new weight-loss products that use text messages launched this summer: Self's Diet Tapper and textWeight. Both send you regular messages that are intended to keep you on track and motivated to reach your weight loss goals. I signed up for three weeks of both services to see how they compare.
Beyond the fact that both services utilize text messages and provide weight-loss tips, the similarities don't go much further.
TextWeight asks you to send in your weight, and with the data you reply, creates a graph of your weight loss progress. Each time you send in your weight, you will receive a tip, which will be on a subject that you have identified as an area you need to improve to lose weight. You can log into textWeight.com to see your progress or modify your settings, such as how often you wish to be texted.
Diet Tapper will send you five texts (or "taps") per day. You will receive three meal suggestions, a drink suggestion and a workout suggestion. Many of these messages have links to a specific page on Self's website, directing you to a more detailed workout or a full recipe.


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$0.99The bestselling author ofThe O'Reilly Factor, The No Spin Zone, and Who's Looking Out for You? talks straight to kids this time. He is as demanding, direct, and wry asever—but he's also more revealing too, sharing candid snapshots of his own childhood throughout.Bill O'Reilly, a former schoolteacher, now an award-winning broadcast news journalist, husband, and father of two, joins forces with an
$8.49Four-time #1 bestselling author and veteran television news journalist Bill O'Reilly has more than 5 million copies of his books in print to date! His first book for young fans, The O'Reilly Factor for Kids, held the honorable distinction of being the #1 bestselling nonfiction title for kids in 2005 according to Nielsen's The Book Standard.Back again with a dialogue on rights that will have
The Ten Most Amazing Ideas of Modern ScienceWorld-altering discoveries that reveal a universe of uncertainty and constant changeWhether probing the farthest reaches of the vast universe or exploring the microscopic world of genetics and the subatomic world of quantum mechanics, Instability Rules is a remarkably informative and engaging look at ten milestone discoveries and their discoverers-a wide range of very human personalities whose
Our True Story of a Polygamous MarriageFrom a familythat inspired Big Love's story of Bill Henricksonand his three wives, this first-ever memoir of a polygamous family captures theextraordinary workings of a unique family dynamic, and argues forthe acceptance of plural marriage as an alternative lifestyle. Readers ofCarolyn Jessop's Escape, Elissa Wall's StolenInnocence, and James McGreevey's Confession,as well as fans of shows like
Parallel Universes and the Deep Laws of the CosmosFrom the best-selling author of The Elegant Universe and The Fabric of the Cosmos comes his most expansive and accessible book to date—a book that takes on the grandest question: Is ours the only universe?There was a time when "universe" meant all there is. Everything. Yet, in recent years discoveries in physics and cosmology have led a number of scientists to conclude that our universe may be one
Reflections on Family, Faith, and FlagIn the fall of 2009, with the publication of her #1 national bestselling memoir, Sarah Palin had the privilege of meeting thousands of everyday Americans on her extraordinary 35-city book tour. Inspired by these encounters, her new book, America By Heart: Reflections on Family, Faith, and Flag, celebrates the enduring strengths and virtues that have made this country great.Framed by her strong
How Life and Consciousness are the Keys to Understanding the True Nature of the UniverseRobert Lanza is one of the most respected scientists in the world-a US News & World Report cover story called him a genius" and a renegade thinker," even likening him to Einstein. Lanza has teamed with Bob Berman, the most widely read astronomer in the world, to produce Biocentrism, a revolutionary new view of the universe.Every now and then a simple yet radical idea shakes the very foundations of
The Hidden World of Mormon PolygamyWhat do we really know about modern practicing polygamists-not fictional ones like the Henrickson family on HBO's Big Love? We've seen the Fundamentalist Church of Jesus Christ of Latter Day Saints in the news, the underage brides in pioneer dresses on a Texas ranch. But the FLDS is just one of many groups that have broken with mainstream Mormonism to follow those parts of Joseph Smith's doctrine
150 Deep, Beautiful, and Elegant Theories of How the World WorksIn This Explains Everything, John Brockman, founder and publisher of Edge.org, asked experts in numerous fields and disciplines to come up with their favorite explanations for everyday occurrences. Why do we recognize patterns? Is there such a thing as positive stress? Are we genetically programmed to be in conflict with each other? Those are just some of the 150 questions that the world's best


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By Catholic Online (NEWS CONSORTIUM)
1/29/2013 (3 years ago)
Catholic Online (www.catholic.org)
Americans love their cats, as anyone who has spent time on a social network can attest. Saturdays have become "caturdays" and meme artwork sporting cats with amusing quotations have reared up on many social forums. Widely regarded as cuddly, lazy, and sometimes, silly, most Americans would be surprised to know the truth about what cats prefer to do with their time.
By Catholic Online (NEWS CONSORTIUM)
Catholic Online (www.catholic.org)
1/29/2013 (3 years ago)
Published in Green
Keywords: cats, murder, conservation, spay, neuter, native species
LOS ANGELES, CA (Catholic Online) - According to a report from the Smithsonian Conservation Biology Institute and the Fish and Wildlife Service, domestic cats prefer to sit around and think of murder all day.
And researchers say, when let out of the house, they typically do it.
Cats are predatory animals by nature, but their evolutionary habitat is the Middle East and parts of Northern Africa. They were domesticated in the Middle East and Egypt as sacred animals. However, in the past 10,000 years, they have spread around the world, brought along by humans as pets.
Despite their domestication, their wild side still flourishes and cats love to hunt.
Unfortunately, many native species in places such as North America, have not evolved next to cats and are unable to avoid them. The result is that cats are hunting and killing many native species of birds and small mammals in quantities that alarm wildlife conservationists.
New estimates suggest that both feral and domestic cats murder no less than 2.4 billion birds and 12.3 billion mammals per year. Unfortunately, other invasive vermin like Norway rats, for example, are not the creatures being hunted. So cats are killing off beneficial, native species, allowing the vermin to flourish in their absence.
Researchers who compiled the most recent data say there is some uncertainty with their study because it draws upon decades of research from many smaller, less comprehensive studies and stitches together estimates from across the country. However, even if the estimates provided by their scientific model are high, the conclusion is the same.
Cats are murderous animals and they're impacting the environment in a big way.
The report is likely to cause many agencies to reevaluate how cats, in particular feral populations, are treated. Currently, many communities tolerate feral populations of cats. Nearly every park and suburban community has some. However, if the cats are recognized as both an invasive species and harmful for the native fauna, then conservationists may call for their removal.
This will undoubtedly result in opposition from cat lovers who feed feral cats in their community.
Conservationists criticize these animals as "subsidized predators."
While such feedings may sustain the cats and prevent them from eating other animals, cats can't escape their instinct. Science says, when faced with potential prey, your loving feline friend is apt to pounce.
Ultimately, the best response is to take steps to ensure responsible adoption and ownership practices. Cats should be spayed and neutered, and they should never be abandoned. Cats that roam freely may have to be enclosed. The subsequent alternative for feral cat populations would be to euthanize them.
Regardless, for many native species, the status quo may not be sustainable. Clearly, both conservationists and cat lovers will need to come together to develop a practical solution.
Simple solutions might be to adorn cats with bells on their collars, to make sneaking up on prey more difficult. Meanwhile, feral populations should be trapped and fixed to prevent reproduction. Finally, animal control agencies should be endowed with the resources necessary to locate irresponsible owners who abandon their pets, and levy fines against them as well as ownership bans.
Such steps may reduce the need for more draconian measures against cat populations while helping to sustain native species in the process. Encouraging owners to do better by their pets would also be a significant benefit.
In the meantime, keep your eyes open around fluffy. It's hard to know what's behind that smile of his.
By Marshall Connolly (CALIFORNIA NETWORK)
Tropical storm Matthew has formed in the Caribbean and appears likely to brush the east coast of Florida. The storm is on track to strike most of the islands in the Caribbean. LOS ANGELES, CA (California Network) - Tropical storm Matthew has formed and is taking aim at ... continue reading
By Marshall Connolly (CALIFORNIA NETWORK)
An event that some say will usher in the apocalypse is about to take place on Friday. A Black Moon will rise on September 30 over North America, and some think it signals the second coming of Jesus Christ. LOS ANGELES, CA (California Network) - A rare Black Moon will ... continue reading
By Marshall Connolly (CALIFORNIA NETWORK)
Global warming is real and getting worse. There is no more credible scientific debate on this issue. However, there is plenty of debate over how we should approach the problem. One professor has offered a provocative solution, stop having children. LOS ANGELES, CA ... continue reading
By Marshall Connolly (CALIFORNIA NETWORK)
Every town is Flint. That's the fear following a report on America's water quality. Many cities and towns have such poor, neglected water infrastructure that millions of Americans are already drinking poisonous water. LOS ANGELES, CA (California Network) - Millions of ... continue reading
By Marshall Connolly (CALIFORNIA NETWORK)
Sixty-five million years ago, the sky tore open in a blinding flash of light. A second later, a tremendous asteroid collided with the Earth somewhere off the prehistoric Yucatan peninsula. The impact threw so much dust and soot into the air it blotted out the sun for ... continue reading
By Marshall Connolly (CALIFORNIA NETWORK)
The webcomic "xkcd" has produced a timeline of global warming that will startle most people. The comic follows the timeline of Earth's temperature from 20,000 B.C. to the present day and it shows why global warming is such a major issue. LOS ANGELES, CA (California ... continue reading
By Kenya Sinclair (CALIFORNIA NETWORK)
Miraculous events and sightings have been recorded throughout history and Peru may have just experienced one of them. LOS ANGELES, CA (Catholic Online) - The Mexican station Misterio Canal (Mysterious Channel) uploaded a video to their YouTube Channel revealing what ... continue reading
By Marshall Connolly (CALIFORNIA NETWORK)
On February 15, 2013, a previously undetected near-Earth asteroid slammed into the Earth. The object, weighing about 14,000 tons, ripped through the air at a shallow angle, saving the planet from catastrophe. The meteor then exploded over a populated area, sending ... continue reading
By Kenya Sinclair (CALIFORNIA NETWORK)
US President Barack Obama has just quadrupled the size of Papahānaumokuākea, Hawaii territory - but why? LOS ANGELES, CA (Catholic Online) - The island of Papahānaumokuākea, pronounced "Pa-pa-hah-nou-mo-koo-ah-keh-ah," is a Marine National Monument ... continue reading
By Kenya Sinclair (CALIFORNIA NETWORK)
One Giant Panda mother surprised zookeepers by giving birth to twins! LOS ANGELES, CA (Catholic Online) - When zookeepers at Austria's Schönbrunn Zoo, also called "Vienna Zoo," discovered Giant Panda Yang Yang was pregnant, they were ecstatic.<a ... continue reading


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Located in Bur Dubai within a short walk of BurJuman Mall, Pearl Residence Hotel Apartments is within a mile (2 km) of other popular sights such as Dubai Museum. This 84-room aparthotel welcomes guests with a restaurant and conveniences like an outdoor pool and free self parking. Located in central Dubai, the aparthotel is also near Grand Mosque and The Dubai Heritage Village.
This aparthotel is home to a restaurant and a bar/lounge. Savor a meal from the privacy of your room with 24-hour room service, or get a pick-me-up at the coffee shop/café. For your convenience, a buffet breakfast is available for a fee.
The 84 air-conditioned apartments feature kitchenettes. Guests can expect WiFi and TVs with satellite channels. Other standard amenities include safes and desks.
Property features
At Pearl Residence Hotel Apartments, guests enjoy an outdoor pool, a fitness center, and a sauna. You can take advantage of free parking, along with an airport shuttle (available 24 hours). The 24-hour front desk has staff standing by to help with tours/tickets and securing valuables. Additional amenities include a rooftop terrace, WiFi in public areas, and conference space.
- Walking distance to BurJuman Mall
- Free parking
- Onsite dining
- Outdoor pool
Pearl Residence Hotel Apartments18 Al Mankhool Street Dubai 47385
Hotel Amenities
Hotel Amenities
In addition to an outdoor pool, Pearl Residence Hotel Apartments provides a sauna and a fitness center. The aparthotel offers a restaurant and a coffee shop/café. A bar/lounge is on site where guests can unwind with a drink. High-speed wireless Internet access is available for a surcharge.
Business-related amenities consist of a business center and a meeting room. This business-friendly aparthotel also offers a rooftop terrace, tour/ticket assistance, and complimentary newspapers in the lobby. Complimentary self parking is available on site.
Pearl Residence Hotel Apartments is a smoke-free property.
- 24-hour front desk
- Total number of rooms - 84
- Number of floors - 8
- Bar/lounge
- Breakfast available (surcharge)
- Business center
- Coffee shop or café
- Free newspapers in lobby
- Conference space
- Elevator/lift
- Express check-in
- Express check-out
- Fitness facilities
- Airport transportation
- Tours/ticket assistance
- WiFi (surcharge)
- One meeting room
- Rooftop terrace
- Restaurant
- Safe-deposit box at front desk
- Year Built 2000
- Free self parking
- Sauna
- Smoke-free property
- Number of buildings/towers - 1
- Steam room
- Outdoor pool
Family Friendly Amenities
- Outdoor pool
- Kitchenette
- Refrigerator
Available in all rooms: Wireless internet access ( AED 45 per day )
Available in some public areas: Wireless internet access ( AED 45 per day )
Free self parking
Room Amenities
- In-room climate control (air conditioning)
- Air conditioning
- Kitchenette
- Refrigerator
- Hair dryer
- In-room safe
- Television
- Desk
- Satellite TV service
- WiFi (surcharge)
- Room service (24 hours)
Where to Eat
Buffet breakfasts are available for a surcharge.
Pearl - This restaurant specializes in international cuisine.
24-hour room service is available.
Nearby Things to Do
Recreational amenities at the aparthotel include an outdoor pool, a sauna, and a fitness center.
To make special requests for accessibility needs, please note them at check-out when you book your room.
- Accessible bathroom
Hotel Policies
Check-in time starts at 2 PM
Check-in time ends at noon
Special check-in instructions:
Check-out time is noon
Payment types
- No pets or service animals allowed
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
No rollaway/extra beds available
No cribs (infant beds) available
No pets or service animals allowed
- Tourism fee: AED 10 per accommodation, per night
Optional extras
- Fee for buffet breakfast: AED 29 per person (approximately)
- Fee for in-room wireless Internet: AED 45 per day (rates may vary)
- Fee for wireless Internet in public areas: AED 45 per day (rates may vary)
Hotel Name
- Pearl Residence
- Pearl Residence Dubai
- Pearl Residence Hotel Apartments
- Pearl Residence Hotel Apartments Dubai
We should mention
Only registered guests are allowed in the guestrooms. No pets and no service animals are allowed at this property.


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A new poll suggests U.S. troops are more likely to vote for Donald Trump or Gary Johnson than for Hillary Clinton.
Jake Novak says if the election were held today, Trump would beat Clinton. The question is: Can he hang on to the momentum? Or, will he blow it?
New polls released this week suggests Hillary Clinton has a growing problem with millennial voters, NBC News reports.
Gary Johnson and Jill Stein will not appear in the first presidential debate after failing to meet qualification criteria.
Here's a scary truth for Clinton and Trump: More than one-third of young voters plan to vote for a third-party candidate. And that could be lethal for Clinton, says Jake Novak.
The Hillary Clinton and Donald Trump race is looking a whole lot closer all of a sudden, Vox reports.
Solomon Trujillo, Trujillo Group Investments, doesn't think there is enough talk of growth on the campaign trail, and shares his thoughts on the changing demographics in the U.S.
Hillary Clinton leads Donald Trump by just 2 points. Libertarian Gary Johnson maintains 11 points and Green Party candidate Jill Stein maintains 4 points.
Gary Johnson's foreign policy stumble may help Hillary Clinton more than it aids her rival Donald Trump.
WASHINGTON, Sept 7- A sheriff's department in North Dakota is investigating possible charges against the U.S. Green Party presidential candidate, Jill Stein, for damaging equipment during protests on Tuesday over construction of an oil pipeline. Stein was part of a group protesting the Dakota Access pipeline and spray-painted construction equipment, the...
WASHINGTON, Sept 7- Authorities in North Dakota are investigating possible charges against U.S. Green Party presidential candidate for allegedly damaging equipment during protests on Tuesday over construction of an oil pipeline in the state. Jill Stein allegedly was part of a group protesting the building of the Dakota Access pipeline and spray painted...
A new CNN/ORC poll shows Donald Trump slightly ahead of Hillary Clinton among likely voters.
Sept 2- Republican presidential candidate Donald Trump has pulled into an effective tie with Democratic rival Hillary Clinton, erasing a substantial deficit as he consolidated support among his party's likely voters in recent weeks, according to the latest Reuters/Ipsos national tracking poll released Friday. The poll showed 40 percent of likely voters...
Hillary Clinton is the only candidate with a "coherent" tech platform that will create jobs and grow the economy, says CTA CEO Gary Shapiro.
GOP nominee Donald Trump may be in second place among voters, but among the nation's economists he's coming in third.
CNBC's John Harwood talks to Libertarian candidate Gary Johnson about this fight to gain a spot on the debate stage next month.
Libertarian Party presidential candidate Gary Johnson sat down with CNBC's John Harwood to discuss his old party, marijuana and making the debates.
Entitlement spending and pension funding are huge issues no one is addressing right now, Libertarian presidential candidate Gary Johnson tells CNBC.
Libertarian Party presidential nominee Gary Johnson, says he is optimistic he will get to the 15-percent needed to be part of the presidential debate. Also Johnson talks about his party's platform, including tax reform, entitlement reform, and balancing the federal budget, which would include reducing military spending. And Johnson weighs in on his stance to legalize marijuana.
Illusionist Penn Jillette criticized his former "Celebrity Apprentice" co-worker and Republican presidential nominee Donald Trump.


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A soaring tower and charming interior courtyard add style and elegance to this spacious home. The formal living room includes a cozy fireplace and combines with the dining room to make a spacious area for entertaining. Gourmet kitchen has large center island, walk-in pantry, sunny breakfast area and built-in desk. Spacious family room has access to one of the two covered patios. Luxurious master bedroom suite features dual walk-in closets, plus sumptuous bath with split vanities, large tub, and walk-in shower. The private guest casita includes a living room, bedroom, and bath.
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From Loop 101: Exit at Scottsdale Road and travel north. Proceed 5 miles and turn left onto Pinnacle Vista Drive. Take the first right onto 71st Place and proceed through the gates and the sales office and model will be on your left.
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Saugatuck Jobs
Looking for jobs in Saugatuck? Snagajob has great opportunities with the area's leading companies. And applying for Saugatuck jobs has never been easier. Look at the job listings below, click on a job title that interests you and hit the "apply now" button. Part-time jobs, teen jobs, student jobs and summer jobs are now just a click away. Start your job search now.
Saugatuck, MI Job Openings
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Cities within 20 miles of Saugatuck, MI
- Holland, MI (181 jobs)
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- Zeeland, MI (6 jobs)
- Grand Rapids, MI (6 jobs)
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Eric Manalastas and Jayson Vega!
Congratulations, Eric and Jayson! I will email you soon to ask for your mailing address.
The Sibling Reality: When Picture Books Stop Being Nice and Start Getting Real - I love it when a blog title makes me sound old. Now that my kids have reached the ripe ages of five and two, I'm finding myself more interested in picture ...
3 days ago


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Top Definition
the 2-3 ply you use to wipe up with after taking a deuce
I'm all out in here, could you pass me some more rim towel.
Shlay4 tarafından 25 Mayıs 2008, Pazar
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Jerry Cantrell
~ Person
Legal name: Jerry Fulton Cantrell
Jerry Fulton Cantrell Jr. (born March 18, 1966) is an American musician who is best known as the lead guitarist, co-lead vocalist and main songwriter for the rock band Alice in Chains. He also has a solo career and released the albums Boggy Depot in 1998 and Degradation Trip Volumes 1 & 2 in 2002.
Showing official release groups by this artist. Show all release groups instead, or show various artists release groups.