Pulse-shape discrimination against low-energy Ar-39 beta decays in liquid argon with 4.5 tonne-years of DEAP-3600 data.

The DEAP-3600 detector searches for the scintillation signal from dark matter particles scattering on a 3.3 tonne liquid argon target. The largest background comes from 39 Ar beta decays and is suppressed using pulse-shape discrimination (PSD). We use two types of PSD estimator: the prompt-fraction, which considers the fraction of the scintillation signal in a narrow and a wide time window around the event peak, and the log-likelihood-ratio, which compares the observed photon arrival times to a signal and a background model. We furthermore use two algorithms to determine the number of photons detected at a given time: (1) simply dividing the charge of each PMT pulse by the mean single-photoelectron charge, and (2) a likelihood analysis that considers the probability to detect a certain number of photons at a given time, based on a model for the scintillation pulse shape and for afterpulsing in the light detectors. The prompt-fraction performs approximately as well as the log-likelihood-ratio PSD algorithm if the photon detection times are not biased by detector effects. We explain this result using a model for the information carried by scintillation photons as a function of the time when they are detected.

First Results from the DEAP-3600 Dark Matter Search with Argon at SNOLAB.

This Letter reports the first results of a direct dark matter search with the DEAP-3600 single-phase liquid argon (LAr) detector. The experiment was performed 2 km underground at SNOLAB (Sudbury, Canada) utilizing a large target mass, with the LAr target contained in a spherical acrylic vessel of 3600 kg capacity. The LAr is viewed by an array of PMTs, which would register scintillation light produced by rare nuclear recoil signals induced by dark matter particle scattering. An analysis of 4.44 live days (fiducial exposure of 9.87 ton day) of data taken during the initial filling phase demonstrates the best electronic recoil rejection using pulse-shape discrimination in argon, with leakage <1.2×10^{-7} (90% C.L.) between 15 and 31 keV_{ee}. No candidate signal events are observed, which results in the leading limit on weakly interacting massive particle (WIMP)-nucleon spin-independent cross section on argon, <1.2×10^{-44} cm^{2} for a 100 GeV/c^{2} WIMP mass (90% C.L.).

Activation of the p53 pathway by the MDM2 inhibitor nutlin-3a overcomes BCL2 overexpression in a preclinical model of diffuse large B-cell lymphoma associated with t(14;18)(q32;q21).

p53 is frequently wild type (wt) in diffuse large B-cell lymphoma (DLBCL) associated with t(14;18)(q32;q21) that overexpresses BCL2. Nutlin-3a is a small molecule that activates the p53 pathway by disrupting p53-MDM2 interaction. We show that nutlin-3a activates p53 in DLBCL cells associated with t(14;18)(q32;q21), BCL2 overexpression and wt p53, resulting in cell cycle arrest and apoptosis. Nutlin-3a treatment had similar effects on DLBCL cells of activated B-cell phenotype with wt p53. Cell cycle arrest was associated with upregulation of p21. Nutlin-3a-induced apoptosis was accompanied by BAX and PUMA upregulation, BCL-XL downregulation, serine-70 dephosphorylation of BCL2, direct binding of BCL2 by p53, caspase-9 upregulation and caspase-3 cleavage. Cell death was reduced when p53-dependent transactivation activity was inhibited by pifithrin-α (PFT-α), or PFT-µ inhibited direct p53 targeting of mitochondria. Nutlin-3a sensitized activation of the intrinsic apoptotic pathway by BCL2 inhibitors in t(14;18)-positive DLBCL cells with wt p53, and enhanced doxorubicin cytotoxicity against t(14;18)-positive DLBCL cells with wt or mutant p53, the latter in part via p73 upregulation. Nutlin-3a treatment in a xenograft animal lymphoma model inhibited growth of t(14;18)-positive DLBCL tumors, associated with increased apoptosis and decreased proliferation. These data suggest that disruption of the p53-MDM2 interaction by nutlin-3a offers a novel therapeutic approach for DLBCL associated with t(14;18)(q32;q21).

Independent measurement of the total active 8B solar neutrino flux using an array of 3He proportional counters at the Sudbury Neutrino Observatory.

The Sudbury Neutrino Observatory (SNO) used an array of 3He proportional counters to measure the rate of neutral-current interactions in heavy water and precisely determined the total active (nu_x) 8B solar neutrino flux. This technique is independent of previous methods employed by SNO. The total flux is found to be 5.54_-0.31;+0.33(stat)-0.34+0.36(syst)x10(6) cm(-2) s(-1), in agreement with previous measurements and standard solar models. A global analysis of solar and reactor neutrino results yields Deltam2=7.59_-0.21;+0.19x10(-5) eV2 and theta=34.4_-1.2;+1.3 degrees. The uncertainty on the mixing angle has been reduced from SNO's previous results.

Effects of buthionine sulfoximine treatment on diaphragm contractility and SR Ca2+ pump function in rats.

The purpose of this study was to examine the effects of glutathione (GSH) depletion and cellular oxidation on rat diaphragm contractility and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in vitro under basal conditions and following fatiguing stimulation. Buthionine sulfoximine (BSO) treatment (n = 10) for 10 days (20 mM in drinking water) reduced (P < 0.05) diaphragm GSH content (nmol/mg protein) and the ratio of GSH to glutathione disulfide (GSH/GSSG) by 91% and 71%, respectively, compared with controls (CTL) (n = 10). Western blotting showed that Hsp70 expression in diaphragm was not increased (P > 0.05) with BSO treatment. As hypothesized, basal peak twitch force (g/mm(2)) was increased (P < 0.05), and fatigability in response to repetitive stimulation (350-ms trains at 100 Hz once every 1 s for 5 min) was also increased (P < 0.05) in BSO compared with CTL. Both Ca(2+) uptake and maximal SERCA activity (mumol.g protein(-1).min(-1)) measured in diaphragm homogenates that were prepared at rest were increased (P < 0.05) with BSO treatment, an effect that could be partly explained by a twofold increase (P < 0.05) in SERCA2a expression with BSO. In response to the 5-min stimulation protocol, both Ca(2+) uptake and maximal SERCA activity were increased (P < 0.05) in CTL but not (P > 0.05) in BSO diaphragm. We conclude that 1) cellular redox state is more optimal for contractile function and fatigability is increased in rat diaphragm following BSO treatment, 2) SERCA2a expression is modulated by redox signaling, and 3) regulation of SERCA function in working diaphragm is altered following BSO treatment.

Direct evidence for neutrino flavor transformation from neutral-current interactions in the Sudbury Neutrino Observatory.

Observations of neutral-current nu interactions on deuterium in the Sudbury Neutrino Observatory are reported. Using the neutral current (NC), elastic scattering, and charged current reactions and assuming the standard 8B shape, the nu(e) component of the 8B solar flux is phis(e) = 1.76(+0.05)(-0.05)(stat)(+0.09)(-0.09)(syst) x 10(6) cm(-2) s(-1) for a kinetic energy threshold of 5 MeV. The non-nu(e) component is phi(mu)(tau) = 3.41(+0.45)(-0.45)(stat)(+0.48)(-0.45)(syst) x 10(6) cm(-2) s(-1), 5.3sigma greater than zero, providing strong evidence for solar nu(e) flavor transformation. The total flux measured with the NC reaction is phi(NC) = 5.09(+0.44)(-0.43)(stat)(+0.46)(-0.43)(syst) x 10(6) cm(-2) s(-1), consistent with solar models.

Measurement of day and night neutrino energy spectra at SNO and constraints on neutrino mixing parameters.

The Sudbury Neutrino Observatory (SNO) has measured day and night solar neutrino energy spectra and rates. For charged current events, assuming an undistorted 8B spectrum, the night minus day rate is 14.0%+/-6.3%(+1.5%)(-1.4%) of the average rate. If the total flux of active neutrinos is additionally constrained to have no asymmetry, the nu(e) asymmetry is found to be 7.0%+/-4.9%(+1.3%)(-1.2%). A global solar neutrino analysis in terms of matter-enhanced oscillations of two active flavors strongly favors the large mixing angle solution.

Establishment and characterization of a new mantle cell lymphoma cell line, Mino.

Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin's lymphoma characterized by cyclin D1 overexpression and the cytogenetic abnormality, the t(11;14)(q13;q32). MCL cell lines have been difficult to establish and in vitro studies of these neoplasms are scarce. We describe the establishment and characteristics of a new MCL cell line, Mino. The cells are large, growing singly and in small clumps in vitro. By flow cytometry, the immunophenotype was compatible with MCL (i.e. CD5+CD20+CD23-FMC7+). Conventional cytogenetics showed hyperdiploidy with multiple complex karyotypic abnormalities, but no evidence of the t(11;14), proven to be present only by fluorescence in situ hybridization and polymerase chain reaction (PCR) methods. Western blots showed expression of cyclin D1 but no detectable cyclin D2 and cyclin D3; the retinoblastoma protein was predominantly phosphorylated. There was expression of tumor suppressor gene products including p53, p16(INK4a), and p21(WAF1). Sequencing of the TP53 gene revealed a mutation (codon 147(valine-->glycine)) in exon 5. Epstein Barr virus was absent. In summary, Mino is a new MCL cell line that may be useful to study the pathogenesis of MCL.

Differential expression of BCL-2 family proteins in ALK-positive and ALK-negative anaplastic large cell lymphoma of T/null-cell lineage.

Anaplastic large-cell lymphoma (ALCL) of T- or null-cell lineage, as defined in the revised European-American lymphoma classification, includes a subset of tumors that carry the t(2;5)(p23;q35) resulting in overexpression of anaplastic lymphoma kinase (ALK). Patients with ALK+ ALCL are reported to have a better prognosis than patients with ALK- ALCL. Because the mechanisms for this survival difference are unknown, we investigated the hypothesis that apoptotic pathways may be involved. We therefore assessed expression levels of the anti-apoptotic proteins BCL-2 and BCL-XL and the pro-apoptotic proteins BAX and BCL-XS in T/null-cell ALCL using immunohistochemical methods and correlated the findings with ALK expression and apoptotic rate (AR), the latter assessed by a modified Tdt-mediated dUTP nick-end labeling assay. ALK was detected in 21 of 66 (31.8%) ALCLs. BCL-2 was not detected in 21 ALK+ ALCLs but was present in 26 of 45 (57.8%) ALK- ALCLs (P < 0.0001). ALK+ and ALK- ALCLs also showed significant differences in expression of BCL-XL, BAX, and BCL-XS. ALK+ tumors less commonly had a high level of BCL-XL (1 of 17 versus 14 of 35, P = 0.01), and more commonly had high levels of BAX (13 of 18 versus 15 of 36, P = 0.05), and BCL-XS (11 of 16 versus 12 of 31, P = 0.05) compared with ALK- tumors. ALK+ tumors also had a higher mean AR than ALK- tumors (3.4% versus 1.1%, P = 0.0002). Differential expression of BCL-2 family proteins may be responsible for the higher AR observed in ALK+ ALCL and provides a possible biological explanation for the better prognosis reported for patients with ALK+ ALCL.

B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma involving bone marrow with an interfollicular pattern.

B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) may involve the bone marrow in nodular, interstitial, diffuse, or mixed patterns. However, B-cell CLL/SLL associated with large reactive germinal centers (the so-called interfollicular pattern) involving the bone marrow is not reported. We describe 2 examples of B-cell CLL/SLL that subtotally replaced the bone marrow with an interfollicular pattern. In both cases, the neoplasms were composed of small round lymphoid cells; proliferation centers also were present. The neoplasms surrounded large reactive germinal centers that were devoid of peripheral mantle zones. The germinal centers were paratrabecular and nonparatrabecular in case 1 and nonparatrabecular in case 2. Flow cytometry immunophenotypic studies done on bone marrow aspiration samples of both cases showed a uniform population of neoplastic cells positive for pan-B-cell antigens and the CD5 and CD23 antigens. Immunohistochemical studies done on bone marrow biopsy sections supported the flow cytometry results and demonstrated that the germinal centers were negative for BCL-2. B-cell CLL/SLL may rarely involve the bone marrow with an interfollicular pattern. Knowledge of this pattern will prevent confusion with follicle center lymphoma and large cell transformation, both of which initially were considered in the differential diagnosis of these cases.

Development of intermediate-grade (mantle cell) and low-grade (small lymphocytic and marginal zone) human non-Hodgkin's lymphomas xenotransplanted in severe combined immunodeficiency mouse models.

We have used severe combined immunodeficiency (SCID) (c.b.-17, ICR/SCID) mice to develop xenotransplantation (XT) models for human intermediate-and-low-grade non-Hodgkin's lymphomas (NHL). In the past, SCID mice have provided a variety of useful XT models for human hematopoietic neoplasms that primarily involve the acute leukemias and some nonhematopoietic tumors, but only rare reports exist on use of the SCID mouse model in the study of primary tumor cells from NHL. Intermediate-grade and low-grade NHL are the most common lymphomas seen in adults. There is no effective therapy for those types of NHL, and they have not been established in an animal model to date. The lack of an animal model has hampered studies that can evaluate the disease process in vivo as well as the definition of therapeutic parameters involved in treatment. We report in this study that primary patient samples of NHL ( intermediate grade and low grade) have been successfully established in SCID mice after XT. NHL include intermediate-grade (mantle cell lymphoma) and low-grade (eg, small lymphocytic lymphoma/chronic lymphocytic lymphoma and marginal zone lymphoma) forms. Studies have been directed toward creating appropriate conditions for the optimal grafting of these NHL in SCID mice so that the disease process in humans could be accurately simulated. These studies indicate that development of XT-human lymphoma cells in SCID mice appear to be linked to their biologic and/or clinical behavior, transplanted lymphoma cell number, and age, as well as to the natural killer cell status of the SCID mouse recipients. Evidence has also shown that NHL cells can exhibit homing or trafficking patterns in SCID recipients that resemble those observed in patients with gastrointestinal lymphomatous involvement (particularly that of mantle cell lymphoma). Our studies also indicate that artefactual influences, such as the outgrowth of Epstein-Barr virus-associated lymphoblastoid lesions, are rare occurrences in the human NHL/SCID models that we have established.

Increased tumor cell proliferation in murine tumors with decreasing dosage of wild-type p53.

A number of transgenic animal model systems have addressed the mechanistic role of p53 loss in tumor progression. However, many of these tumor models have analyzed p53 function in the context of other transgenes expressing activated oncogenes or defective tumor suppressor genes generated by gene targeting. To examine the role of p53 loss independent of other exogenous oncogenic influences, we analyzed some of the biological aspects of tumor formation and progression in p53-knockout mice containing a null germline p53 allele. We analyzed tumors from p53-/-, p53+/-, and p53+/+ littermates. Some of the p53+/- tumors had lost the remaining p53 allele (p53+/- loss of heterozygosity), whereas others retained the allele (p53+/-). In this report, we show that loss or absence of p53 conferred a tumor growth advantage by increasing the rate of cellular proliferation in a p53 dosage-dependent manner. The apoptotic levels in tumor tissue were found to be modest and not significantly dependent on p53 status. These results contrast with those from some other p53-deficient tumor models, in which p53 loss was associated with more rapid tumor progression through abrogated apoptosis. Finally, as p53 has been shown to regulate certain angiogenic factors, we examined the levels of angiogenesis in p53-containing and p53-deficient tumors. We found no p53-dependent differences in the levels of tumor angiogenesis measured by intratumoral microvessel density.

Retinoid-mediated inhibition of cell growth with stimulation of apoptosis in aggressive B-cell lymphomas.

Retinoids have been shown to modulate cell growth and differentiation in a variety of human tumor cell types, but their effects on B-cell non-Hodgkin's lymphomas (NHL-B) have not been explored. In this study, all-trans retinoic acid (ATRA) in the free form and liposome-encapsulated form (L-ATRA) were used to determine effects on fresh NHL-B patient cells as well as cell lines recently established from both HIV-negative and -positive NHL-B patient biopsies. Both ATRA and L-ATRA were found to inhibit cell proliferation in NHL-B cells. However, L-ATRA was found to be superior to free ATRA in inhibiting cell proliferation of NHL-B cells and resulted in greater than 90% cell growth inhibition in a dose-dependent manner. In addition, L-ATRA also induced high levels of apoptosis in NHL-B cells in vitro. To delineate the apoptotic pathways involved, the expression of the apoptosis suppressor oncogene bcl-2 was evaluated in different NHL-B cells with and without the t(14;18) chromosomal translocation. After L-ATRA exposure, more than a 50% reduction in the expression of bcl-2 protein was observed. bcl-2 message levels were also down-regulated in the L-ATRA-sensitive NHL-B cells. Bax protein levels were analyzed and found to be up-regulated in L-ATRA-sensitive NHL-B cells. Similar results were observed in sensitive AIDS/lymphoma cell lines. Experiments using an RAR-alpha antagonist (RO 41-5253) showed that both the proliferation inhibition and apoptosis induced by L-ATRA could be blocked in NHL-B cells. The findings of the present study indicate that L-ATRA may possess therapeutic potential in blocking cell proliferation, inducing apoptosis, and

Effects of genetic background on tumorigenesis in p53-deficient mice.

Mice with disrupted germline p53 alleles have been engineered by us and others and have been shown to have enhanced susceptibility to spontaneous tumors of various types. We monitored a large number of p53-deficient mice (p53+/- and p53-/-) and their wild-type littermates (p53+/+) of two different genetic backgrounds (129/Sv and mixed C57BL/6 x 129/Sv) up to 2 yr of age. p53+/- and p53-/- 129/Sv mice show accelerated tumorigenesis rates compared with their p53-deficient counterparts of mixed C57BL/6 x 129/Sv genetic background. The tumor spectra of the two strains of mice are similar except that almost half of 129/Sv p53-/- males develop malignant teratomas, whereas these tumors are rarely observed in C57BL/6 x 129/Sv mice and never in 129/Sv p53+/- males. In the study reported here, we further characterized the lymphomas that arose in the p53-nullizygous mice and found that over three-quarters of the lymphomas were of thymic origin and contained primarily immature (CD4+/CD8+) T-cells, whereas the remainder originated in the spleen and peripheral lymph nodes and were of B-cell type. The high incidence of early-onset lymphomas in the nullizygous mice makes these animals a good lymphoma model, whereas the heterozygous mice may be a useful model for Li-Fraumeni syndrome, a human inherited cancer predisposition.

Fas/APO-1 expression and function on malignant cells of hematologic and nonhematologic origin.

Fas/APO-1 is a cell-surface protein capable of inducing apoptosis in a variety of cell types upon specific antibody engagement. Antibodies against Fas/APO-1 have been used successfully for the treatment of several lymphoid malignancies in mice. Before apoptosis triggered by anti-Fas can be fully exploited as a clinical therapy, Fas/APO-1 distribution, function, and regulation must be further studied. In this study, we analyzed freshly isolated B-cell and T-cell lymphomas as well as nonhematological tumor cell lines for Fas/APO-1 expression and sensitivity to the growth-inhibitory effects of anti-Fas. Constitutive Fas/APO-1 was expressed at very low levels on only one of eight B-cell lymphomas analyzed. Expression was markedly up-regulated, however, by culture with high-molecular-weight B-cell growth factor (HMW-BCGF). Fas/APO-1 was constitutively expressed on one of two T-cell lymphomas examined at levels comparable to those of activated normal lymphocytes. However, neither the B-cell nor T-cell lymphomas positive for Fas/APO-1 expression were growth inhibited by anti-Fas. Furthermore, in the case of one HMW-BCGF-activated B-cell lymphoma, a significant growth enhancement was observed upon anti-Fas treatment. Nonhematologic tumor cell lines showed a similar spectrum of biologic responses to anti-Fas, being growth inhibited, growth stimulated or unaffected by antibody treatment. In summary, these studies suggest that engagement of Fas/APO-1 may trigger a diverse spectrum of biologic effects not unlike other members of the nerve growth factor receptor/tumor necrosis factor receptor superfamily.

Identification of a cDNA for a human high-molecular-weight B-cell growth factor.

Proliferation is necessary for many of the phenotypic changes that occur during B-cell maturation. Further differentiation of mature B cells into plasma cells or memory B cells requires additional rounds of proliferation. In this manuscript, we describe a cDNA for a human B-cell growth factor we call high-molecular-weight B-cell growth factor (HMW-BCGF). Purified HMW-BCGF has been shown to induce B-cell proliferation, inhibit immunoglobulin secretion, and selectively expand certain B-cell subpopulations. Studies using antibodies to HMW-BCGF and its receptor have suggested that HMW-BCGF, while produced by T cells and some malignant B cells, acts predominantly on normal and malignant B cells. The HMW-BCGF cDNA was identified by expression cloning using a monoclonal antibody and polyclonal antisera to HMW-BCGF. Protein produced from the cDNA induced B-cell proliferation, inhibited immunoglobulin secretion, and was recognized in immunoblots by anti-HMW-BCGF antibodies. The amino acid sequence of HMW-BCGF deduced from the cDNA predicts a secreted protein of 53 kDa with three potential N-linked glycosylation sites. The identification of this cDNA will allow further studies examining physiologic roles of this cytokine. We propose to call it interleukin 14.

Proliferating cell nuclear antigen (PCNA) expression in chronic lymphocytic leukemia (CLL).

Chronic Lymphocytic Leukemia (CLL) is usually an indolent disorder which in some patients assumes an aggressive clinical course. In order to assess at presentation the prognosis of a given patient, several staging systems and prognostic variables have been proposed including the expression of the Proliferating Cell Nuclear Antigen (PCNA). PCNA is a 36 kd nuclear protein, the regulation of which is cell cycle-dependent. In CLL, PCNA levels correlate with cell proliferation, clinical stage and the lymphocyte doubling time (LDT). Furthermore, preliminary data suggests that PCNA expression may also predict response to Fludarabine-based chemotherapy. Since PCNA is a cofactor for Delta DNA polymerase, PCNA overexpression in CLL may also reflect the intrinsic DNA repair activity of the leukemic cells and thus their resistance to chemotherapy. Further studies aiming at modulation of PCNA expression in CLL cells may clarify this issue and may offer a future new therapeutic strategy with which to treat this disorder.

Human B cell lymphomas: in vitro and in vivo studies on growth factors and cell growth.

The B cell non-Hodgkin's lymphomas (NHL-B) are a common, but heterogeneous group of human lymphoid neoplasms, consisting of monoclonal populations of neoplastic B lymphocytes demonstrating non-random chromosomal abnormalities, often associated with proto-oncogene translocations. Clinically and pathologically, these lymphomas are classified as low, intermediate, or high grade, according to the clinical aggressiveness of the NHL-B subtype. The clinical behavior can also be correlated with biological function regarding proliferative capabilities of the tumor cells. Our studies have shown that the low grade B cell lymphomas have low constitutive proliferative capacity in vitro and do not respond to cytokine growth factors (CGF), while the high grade NHL-B respond to the B cell growth factor (BCGF) family of CGFs. The high grade NHL-B also secrete BCGFs both in vitro and in vivo, as autocrine growth factors that may provide a target for new therapeutic approaches to therapy.

Studies on a type D retrovirus isolated from an AIDS patient lymphoma.

The development of AIDS-related lymphomas (ARL) has been on the rise in recent years. During an analysis of ARL from AIDS patients, one individual developed atypical syncytial variants of high-grade Burkitt's-type B-cell lymphomas, which prompted further study. However, the search for a HIV-1 retrovirus, which we hypothesized was infecting these cells, led to the subsequent discovery of a type D retrovirus in two early-passage lymphoma cell lines derived from this patient. Nucleotide and amino acid sequence analysis, as well as immunologic reactivity, indicated that the virus was closely related to Mason-Pfizer monkey virus (MPMV) or simian retrovirus type 1 (SRV-1). MPMV and SRV-1 are immunosuppressive type D retroviruses that cause an AIDS-like syndrome in rhesus macaques. Amplification of DNA from the patient's diagnostic bone marrow biopsy specimen by the polymerase chain reaction generated MPMV-specific fragments indicative of infection by a retrovirus similar to MPMV. Additionally, the patient's serum contained antibodies that recognized type D retroviral env proteins (gp20 and gp70) and gag proteins (p27 and p14) as assayed by immunoblot and radioimmunoprecipitation techniques. Although there have been reports of human cell lines infected with type D retroviruses and of type D reactive human sera, this is the first report of a type D retrovirus infection in a human confirmed by virus isolation, serum immunoreactivity, and viral DNA identification in tumor tissue.