ALBERT

Description: We have developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 40-kd proteins has been analyzed during the first five seconds of platelet response to thrombin from 0.1 to 5.0 U/mL and compared with the progress of aggregation and serotonin secretion. The onset time for aggregation and phosphorylation of both proteins was less than one second, although with lowest (less than 0.5 U/mL) thrombin levels, a lag of up to 0.6 seconds occurred before 40K phosphorylation increased. The thrombin sensitivity of aggregation and 20K phosphorylation was approximately twice that of 40K phosphorylation, with Ka values of 0.51 and 0.53 v 1.10 U/mL, respectively. External calcium was necessary for maximal 20K phosphorylation, since EDTA inhibited this by 30%. The 40K phosphorylation was not affected by EDTA. Platelet activation by thrombin thus induced biochemical changes well before one second. The quenched-flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet function.

Description: Abstract 4465 The death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receives great interest as it targets and kills cancerous cells, but not non-transformed cells. While it is in phase I/II clinical trials for a range of solid tumours, the generally low sensitivity of leukemia cells to TRAIL makes it a less attractive therapeutic for these cancers. We found that doxorubicin and cytarabine, agents that induce DNA damage and impair cell cycle progression, can sensitize CML cells to TRAIL with CI

Description: Abstract 4760 Background: Anaplastic large cell lymphoma (ALCL) is a rare disease, comprising 2–3% of all non-Hodgkin lymphomas. Case reports of seroma associated ALCL of the breast in association with silicone breast implants have appeared in the literature since 1997, but no data on the incidence of this complication has been reported. We use three case reports, including two previously published, in conjunction with data derived from three separate entities of Partners HealthCare (Brigham and Women's Hospital; Massachusetts General Hospital; Faulkner Hospital) to establish an incidence estimate for this rare entity. Methods: Individual cases were identified by pathologists, surgeons and medical oncologists. We compared a list of patients from the institutions’ Cancer Registries, with the results of a query we ran on an institution-internal query tool. For MGH patients only, we were also able to compare cancers revealed through a natural language processing search result of institutional pathology reports. Two of the cases were in the overlap of Cancer Registry data, and query results. One case was not contained within these results as it was omitted from the Cancer Registry. Case Presentations: Case 1 was surgically treated for breast cancer and reconstruction at New England Medical Center. At an unknown time relative to her breast cancer and tissue expander placement, she received a McGhan 210 cc textured silicone implant to her left breast. At time of rupture this implant was replaced with a 270 cc McGhan textured silicone implant filled to 295 cc at Newton Wellesley Hospital (NWH). Her surgical course was complicated by recurrent seroma, and she was eventually switched to Mentor smooth implant, with 275 cc implant on the right and 375 filled to 425 cc on the left. However, a biopsy of tissue at the time of this implant revealed ALK-negative ALCL in the left breast. Implants were removed at NWH and she was treated at Massachusetts General Hospital (MGH). After 3 cycles of chemotherapy (CHOP plus radiation) she remains in CR now at 18 months after treatment. Case 2 presented at Brigham and Women's Hospital (BWH) after a surgically treated right breast cancer with recurrence and reconstruction with a McGhan 270 cc textured saline implant. In 2000 the patient presented with erythema at surgical site of her cancer and a biopsy confirmed ALCL. Due to age the patient was treated with radiation alone and this induced a sustained remission of her ALCL. Case 3 originally had bilateral augmentation mammoplasty in 1974 with bilateral McGhan 270cc textured saline implants. She presented at Northwest Medical Center in 2007 with what appeared to be an abscess at her left implant site but was positive for ALCL when biopsied. She was treated with CHOP and radiation at that institution. She recurred in 2008 in the right breast and presented to BWH for treatment. She received ESHAP, then radiation, then gemcitabine, cisplatin, and dexamethasone; despite these treatments, her disease progressed and the patient died this year. Results: A query of the comprehensive electronic health database of the Partners hospitals (RPDR) revealed 9,941 patients at our institutions, who had undergone full or partial reconstruction of the breast, or removal of a breast implant or tissue expander from 1992–2009. Database queries revealed 5778 patients at MGH, 4,968 at BWH, and 4780 at Faulkner Hospital (FH) with non-Hodgkin lymphoma. Cancer Registry data revealed 18 ALCL patients (4 women) at MGH, 73 ALCL patients (24 women) at BWH and 2 ALCL patients at FH (1 woman). Of our three cases one was treated entirely within our core healthcare system, one was referred from another Partners Institution (NWH) and one was referred for tertiary care of her lymphoma. Incidence is established as 2 cases of implant-associated ALCL per 9941 patients or 0.02%. Implant-associated disease comprises 3.2% of all ALCL cases and 10% of ALCLs presenting among women. Conclusions: Incidence of breast implant-associated ALCL may be more common than the rare case reports suggest. Evaluation of late complications of breast implant such as chronic seroma or abscess with consideration of this disease may improve case recognition. The fatality as a result of systemic dissemination of this disease has not previously been reported. Disclosures: No relevant conflicts of interest to declare.

Description: DNA methylation is one of the major epigenetic mechanisms that controls cellular differentiation. The ten-eleven translocation (TET) family of methylcytosine dioxygenases mediates active DNA demethylation through the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and subsequent intermediates. Here we demonstrate that TET2 regulates CD8+ T cell differentiation in vivo following acute and chronic viral infection. At steady-state, mice with a T-cell specific deletion of TET2 have intact thymic and peripheral T cell populations. Following acute viral infection with LCMV-Armstrong, TET2 loss enhances LCMV-specific CD8+ T cell memory differentiation in a cell-intrinsic manner without disrupting antigen-specific cell expansion or cytokine production. However, TET2-deficient memory CD8+ T cells exhibit altered recall responses with blunted re-expansion, retained expression of phenotypic memory markers and restricted re-expression of activation markers. During chronic viral infection with LCMV-clone 13, TET2 controls CD8+ T cell expansion and alters differentiation. Importantly, though mice with T-cell specific loss of TET2 developed similar levels of CD8+ T cell exhaustion as wild-type mice, TET2 loss specifically reduced PD-1 expression suggesting that TET2 may direct DNA demethylation of the PD-1 locus. Together, our data indicate that TET2 is an important regulator of CD8+ T cells following both acute and chronic viral infections and suggest targeting epigenetic regulators have potential for enhancing antiviral immunity. Disclosures No relevant conflicts of interest to declare.

Description: We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched- flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.

Description: We have developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 40-kd proteins has been analyzed during the first five seconds of platelet response to thrombin from 0.1 to 5.0 U/mL and compared with the progress of aggregation and serotonin secretion. The onset time for aggregation and phosphorylation of both proteins was less than one second, although with lowest (less than 0.5 U/mL) thrombin levels, a lag of up to 0.6 seconds occurred before 40K phosphorylation increased. The thrombin sensitivity of aggregation and 20K phosphorylation was approximately twice that of 40K phosphorylation, with Ka values of 0.51 and 0.53 v 1.10 U/mL, respectively. External calcium was necessary for maximal 20K phosphorylation, since EDTA inhibited this by 30%. The 40K phosphorylation was not affected by EDTA. Platelet activation by thrombin thus induced biochemical changes well before one second. The quenched-flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet function.

Description: We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched- flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.

Description: Regulation of DNA methylation is critical for proper T cell differentiation and function. Antigen-specific CD8+ T cells undergo global remodeling of DNA methylation following viral infection, suggesting that DNA methylation may direct antigen-specific T cell responses. TET2 is a member of the Ten-Eleven-Translocation (TET) family, which converts 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC) and subsequent intermediates ultimately leading to DNA demethylation. How TET2 regulates T cell differentiation and function is unknown. Here we demonstrate that TET2 expression is regulated by TCR signaling in primary murine T cells. Furthermore, using a novel flow cytometric assay to measure 5hmC levels on a single cell basis, we find that TCR signaling also regulates TET activity as evidenced by a rapid increase in global 5hmC levels after TCR stimulation that is blunted in TET2-deficient T cells. To determine the role of TET2 in T cell responses, we generated mice deficient in TET2 in the T cell compartment (TET2fl/flCD4Cre+) mice. These mice develop grossly normal thymic and peripheral T cell subsets. Given the regulation of TET2’s expression and activity by TCR stimulation, we used a murine model of acute viral infection, specifically LCMV-Armstrong, to test if TET2 regulates antigen-specific T cell responses in vivo. Following viral challenge, TET2fl/flCD4Cre+ mice have similar antigen-specific CD8+ T cell expansion and effector responses but exhibit significantly enhanced memory CD8+ T cell differentiation compared to control mice. These data demonstrate that TET2 plays a critical role in directing CD8+ T cell differentiation and function. Studies are ongoing to identify specific TET2 regulated genes important in the development of CD8+ T cell memory. Disclosures No relevant conflicts of interest to declare.

Description: Background African Americans experience higher rates of cardiovascular disease (CVD) and mortality from stroke and coronary heart disease. It is possible that genetic modifiers associated with African ancestry may confer higher risk of CVD. The sickle cell mutation results from a functional single nucleotide polymorphism (SNP) involving the substitution of GTG (valine) for a GAG (glutamic acid) in the gene encoding beta globin. Heterozygosity for the mutation (rs334) results in sickle cell trait (SCT) which is prevalent among 8% of African Americans, while homozygosity produces sickle cell anemia. Sickle cell trait has been deemed clinically benign yet recent evidence shows that it is associated with increased risk of chronic kidney disease, a 2 times increased risk for venous thromboembolism and a 4 times increased risk for pulmonary embolism. Further, there is evidence that individuals with SCT have higher serum levels of C-reactive protein, Fibrinogen 2.1 fragments and D-dimers. We hypothesized that African Americans with SCT have a higher risk for cardiovascular outcomes than those without SCT (homozygous wild-type hemoglobin). Methods Data were obtained from the Multi-Ethnic Study of Atherosclerosis (MESA), Jackson Heart Study (JHS) and Women’s Health Initiative (WHI). Incident myocardial infarction (MI) was defined as adjudicated non-fatal or fatal MI, coronary heart disease (CHD) was defined as a composite endpoint including adjudicated non-fatal MI, fatal CHD, or documented coronary revascularization procedure. Sickle cell trait was identified either by direct genotyping or imputation for rs334. Individuals who were homozygous for the sickle mutation or had a prior history of CHD events were excluded. Cox proportional hazards models were used to estimate a hazard ratio (HR) of incident MI or CHD comparing sickle cell trait carriers to non-carriers. Models were adjusted for age, sex and principal component of global ancestry. Analysis was performed separately in each cohort and the results were subsequently meta-analyzed using a fixed effect inverse variance weighted method. Results A total of 11,128 African American men and women were included in the pooled data base. The average age at baseline were 62.2 ± 10.1, 49.8 ± 11.8 and 61.4 ± 7.0 years for MESA, JHS and WHI respectively, with JHS participants significantly younger than the other two cohorts (p

Description: Cytokines regulate T cell development and function. Interleukin (IL)-4 is a cytokine classically associated with CD4+ T helper (TH) differentiation, specifically TH2 differentiation. However, IL-4 has recently been shown to drive the development of CD8+ innate-like lymphocytes (ILLs). CD8+ ILLs are non-conventional thymocytes that develop with characteristics typically associated with innate and/or memory immune cells, including surface expression of the activation/memory markers CD44 and CD122, expression of the T-box transcription factor Eomesodermin (Eomes) and the rapid production of interferon (IFN)-γ after ex vivo stimulation. Here we show that IL-4 is sufficient to promote Eomes expression in CD8 single-positive (SP) thymocytes in short-term in vitro culture and direct CD8+ ILL development in fetal thymic organ culture. Using genetic deficiency and pharmacologic inhibitors, we demonstrate that IL-4 up-regulation of Eomes in CD8SP thymocytes requires STAT6 and Akt signaling pathways. Next, we investigated the possibility that in addition to directing the fate of developing thymocytes, IL-4 might impact the function of mature CD8+ cells undergoing activation. We found that that in naïve peripheral CD8+ T the combination of IL-4 and low dose T cell receptor (TCR) stimulation is a potent inducer of Eomes. Futhermore, when combined, these stimuli promote the persistence of CD8+ T cells in an adoptive transfer model. Understanding how IL-4 directs CD8+ T cell differentiation may provide a novel way to enhance CD8+ T cell function in adoptive T cell therapies. Disclosures: No relevant conflicts of interest to declare.