ALBERT

Description: Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are abnormally sensitive to complement. Two membrane proteins, the C8 binding protein (C8bp) and the decay accelerating factor (DAF), which are expressed on normal cells, function to restrict lysis by homologous complement, and both of these proteins are absent from PNH erythrocytes. DAF is anchored to the plasma membrane on normal cells by a phosphatidylinositol linkage. The investigators found that a purified phosphatidylinositol-specific phospholipase C cleaved C8bp from the surface of normal lymphocytes and monocytes. This finding indicates that the abnormal complement sensitivity of PNH erythrocytes arises from a common defect, the inability to attach the phosphatidylinositol- containing anchor that is necessary for the membrane expression of both membrane complement regulatory proteins, the C8bp, and DAF.

Description: The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.

Description: Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are abnormally sensitive to complement. Two membrane proteins, the C8 binding protein (C8bp) and the decay accelerating factor (DAF), which are expressed on normal cells, function to restrict lysis by homologous complement, and both of these proteins are absent from PNH erythrocytes. DAF is anchored to the plasma membrane on normal cells by a phosphatidylinositol linkage. The investigators found that a purified phosphatidylinositol-specific phospholipase C cleaved C8bp from the surface of normal lymphocytes and monocytes. This finding indicates that the abnormal complement sensitivity of PNH erythrocytes arises from a common defect, the inability to attach the phosphatidylinositol- containing anchor that is necessary for the membrane expression of both membrane complement regulatory proteins, the C8bp, and DAF.

Description: Aerosol particles are essential constituents of the Earth's atmosphere, impacting the earth radiation balance directly by scattering and absorbing solar radiation, and indirectly by acting as cloud condensation nuclei. In contrast to most greenhouse gases, aerosol particles have short atmospheric residence times, resulting in a highly heterogeneous distribution in space and time. There is a clear need to document this variability at regional scale through observations involving, in particular, the in situ near-surface segment of the atmospheric observation system. This paper will provide the widest effort so far to document variability of climate-relevant in situ aerosol properties (namely wavelength dependent particle light scattering and absorption coefficients, particle number concentration and particle number size distribution) from all sites connected to the Global Atmosphere Watch network. High-quality data from almost 90 stations worldwide have been collected and controlled for quality and are reported for a reference year in 2017, providing a very extended and robust view of the variability of these variables worldwide. The range of variability observed worldwide for light scattering and absorption coefficients, single-scattering albedo, and particle number concentration are presented together with preliminary information on their long-term trends and comparison with model simulation for the different stations. The scope of the present paper is also to provide the necessary suite of information, including data provision procedures, quality control and analysis, data policy, and usage of the ground-based aerosol measurement network. It delivers to users of the World Data Centre on Aerosol, the required confidence in data products in the form of a fully characterized value chain, including uncertainty estimation and requirements for contributing to the global climate monitoring system.

Description: In order to assess the evolution of aerosol parameters affecting climate change, a long-term trend analysis of aerosol optical properties was performed on time series from 52 stations situated across five continents. The time series of measured scattering, backscattering and absorption coefficients as well as the derived single scattering albedo, backscattering fraction, scattering and absorption Ångström exponents covered at least 10 years and up to 40 years for some stations. The non-parametric seasonal Mann–Kendall (MK) statistical test associated with several pre-whitening methods and with Sen's slope was used as the main trend analysis method. Comparisons with general least mean square associated with autoregressive bootstrap (GLS/ARB) and with standard least mean square analysis (LMS) enabled confirmation of the detected MK statistically significant trends and the assessment of advantages and limitations of each method. Currently, scattering and backscattering coefficient trends are mostly decreasing in Europe and North America and are not statistically significant in Asia, while polar stations exhibit a mix of increasing and decreasing trends. A few increasing trends are also found at some stations in North America and Australia. Absorption coefficient time series also exhibit primarily decreasing trends. For single scattering albedo, 52 % of the sites exhibit statistically significant positive trends, mostly in Asia, eastern/northern Europe and the Arctic, 22 % of sites exhibit statistically significant negative trends, mostly in central Europe and central North America, while the remaining 26 % of sites have trends which are not statistically significant. In addition to evaluating trends for the overall time series, the evolution of the trends in sequential 10-year segments was also analyzed. For scattering and backscattering, statistically significant increasing 10-year trends are primarily found for earlier periods (10-year trends ending in 2010–2015) for polar stations and Mauna Loa. For most of the stations, the present-day statistically significant decreasing 10-year trends of the single scattering albedo were preceded by not statistically significant and statistically significant increasing 10-year trends. The effect of air pollution abatement policies in continental North America is very obvious in the 10-year trends of the scattering coefficient – there is a shift to statistically significant negative trends in 2009–2012 for all stations in the eastern and central USA. This long-term trend analysis of aerosol radiative properties with a broad spatial coverage provides insight into potential aerosol effects on climate changes.

Description: A distinct feature of multiple myeloma (MM) is the tight interaction between malignant plasma cells and their bone microenvironment, creating a niche suitable for MM growth. In particular, MM cells inhibit osteoblast (OB) differentiation and stimulate osteoclast (OC) function, resulting in imbalanced bone remodeling and osteolytic bone disease. Here we studied a novel cytokine, activin A, identified from a broad range of cytokines, in the development of MM bone disease. We next asked whether activin A inhibition could restore bone balance and suppress tumor growth. Activin, a member of the TNF-α superfamily, is a pleiotropic cytokine involved in bone remodeling. Here, we observed, that MM patients with multiple osteolytic lesions had a 4-fold increase in activin A expression levels in bone marrow plasma compared to MM patients with one or less osteolytic lesions and non-MM patients (average 123.6 ± 136 vs 26.4 ± 21.4 vs 30.6 ± 25.1 pg/ml respectively, p

Description: Phosphatidylinositide 3-kinase (PI3K) is a key enzyme implicated in intracellular signaling of diverse cellular responses including receptor-mediated responses and neutrophil activation. Several PI3K subunits have been cloned and shown to be localized to plasma membrane receptors, the cytosol, or intracellular vesicles or caveolae. We report the localization of PI3K to a distinct intracellular site, cytoplasmic lipid bodies, in leukocytes. In U937 monocyte cells, PI3K p85 regulatory and p110β catalytic subunits were localized to lipid bodies by immunocytochemistry and/or immunoblotting and enzyme assays of subcellular fractions. In RAW murine macrophages, p55, p85, and p85β PI3K subunits were present at isolated lipid bodies. PI3K p85 was also shown to colocalize and, by co-immunoprecipitation, to be physically associated with phosphorylated Lyn kinase in lipid bodies induced to form in human polymorphonuclear leukocytes. These findings, therefore, indicate a novel site for PI3K compartmentalization and suggest that PI3K-mediated signaling is active within cytoplasmic lipid bodies in leukocytes.

Description: We have previously shown that sublytic heterologous complement induces large but transient increases in erythrocyte membrane permeability. We now report that when erythrocytes are bystanders in zymosan-activated autologous serum, they increase their Na+ permeability 10-fold, indicating that autologous complement can also induce transient membrane lesions. When we isolated the effect of the C5b-9 membrane attack complex of complement by using human C5b-9 assembled from purified components, we found there was minimal lysis but efficient Na+ uptake. Suspension of the sublytically damaged erythrocytes in K+ medium caused the cells to lyse, which is consistent with the cells recruiting a compensatory K+ efflux similar to that observed when human erythrocytes were exposed to heterologous complement. Sublytic C5b-9 exposure also became lytic when extracellular Ca2+ was limited and when the cells were exposed to charybdotoxin, an inhibitor of the Ca(2+)- activated K+ channel. This indicates that Ca2+ is required for the functional termination of the C5b-9 lesion. We also show that the membrane hyperpolarization resulting from activation of the Ca(2+)- dependent K+ efflux does not influence the termination of the C5b-9 lesion. Thus, the influx of Ca2+ through the complement lesion initiates at least two apparently independent adaptive responses: (1) a process that terminates the leak; and (2) a K+ efflux that has a volume regulatory function. Our data support the potential of the sublytic C5b- 9 lesion to act as a physiologic mediator for autologous erythrocytes.

Description: Abstract 990 Introduction: This study aimed to determine activity and safety of weekly bortezomib (Takeda Inc) and temsirolimus (Pfizer Inc) in patients with relapsed/refractory Multiple Myeloma (MM). Methods: Eligibility criteria included: 1) patients with relapsed or relapsed/refractory MM with any prior lines of therapy including bortezomib, 2) no chemotherapy within 3 weeks, or biological/novel therapy within 2 weeks. Primary endpoint was the percent of patients with at least a minimal response (MR). Results: Twenty patients were enrolled on the phase I study and 43 on the phase II study. The MTD was determined at 1.6 mg/m2 bortezomib Days 1, 8, 15, and 22 every 35 days in combination with 25 mg IV temsirolimus Days 1, 8, 15, 22, and 29 every 35 days. Twenty % were stage III by ISS staging system in the phase I, and 21% in the phase II. The overall response rate of MR or better in the Phase II study was 20/43 (47%, 95%CI: 33,60), with 5% CR, 9% VGPR, 19% PR and 14% MR. Progression without any response occurred in just 1 patient (3%). One patient had an unconfirmed PR, but was included in the stable disease category. An additional 3 (6%) patients were unevaluable in the phase II trial because they did not complete their first cycle of therapy and had no follow up laboratory results for response. If these patients are excluded, the ORR including MR improves to 50% (95% CI: 36,64). The overall response rate in the phase I study was 20% with responses occurring in all the stages of the dose escalation. If three patients who were unevaluable in the phase I trial are excluded, then the response rate of evaluable phase I patients is 24% (95% CI: 9,46). Response was also evaluated by whether patients were bortezomib-refractory or not. These were defined as progressing while on therapy or progressing within 60 days of completing bortezomib therapy. Fifty-one patients had received bortezomib as part of prior treatment. Of these patients, 32 were refractory to bortezomib therapy immediately prior to study entry, and an additional 2 pts were refractory at prior time points. Responses observed among the 32 patients refractory to their most recent bortezomib therapy include 3 PR and 3 MR (ORR: 19%, 95% CI: 9,34). Another 21 patients had SD, 2 PD and 3 patients were unevaluable. Of the evaluable patients, the ORR was 6/29 (21%). Responses observed among the 19 patients who were not refractory to their last bortezomib treatment include 2 VGPR, 5 PR and 3 MR with 6 patients with SD, 0 PD and 3 unevaluable. The ORR among the evaluable patients who received bortezomib but were not refractory was 62%. Median time to response of MR or better (min, max) among all patients was 1.7 months (0.5,14.2) and among phase II patients 1.3 months (0.5,8.0). Median duration of MR or better (min, max) among all patients is 5.2 months (0.5,15.8) and among phase II patients is 4.6 months (0.5,10.8). Median duration of PR or better response (min, max) among all patients is 6.0 months (1.8,15.8) and among phase II patients is 5.2 months (1.8,10.8). The median time to progression for all patients in the phase I and II studies was 7.3 months (95% CI: 5.7 –17.2) and the median progression free survival was 6.4 months (95% CI: 4.8–7.4). The median overall survival in all phase I and II patients was 11.4 months (95%CI: 8.6-undetermined). Three deaths occurred during therapy in the phase I and II studies, 1 of septic shock, 1 with H1N1 infection, and 1 with cardiac amyloid and ventricular arrhythmia. The most common G1-4 toxicities that occurred in 〉 25% of patients included cytopenias, hypertrigyceridemia and diarrhea. Grade 3 and 4 thrombocytopenia occurred in 48% of patients in the phase I and II studies, G3 and 4 neutropenia occurred in 36%, and anemia in 26% of phase I and II patients. G3 and 4 hypertriglyceridemia occurred in 5% and diarrhea in 9%. Peripheral neuropathy (PN) was rare with no G3 or 4 neuropathy reported. Overall, there was 34% grade 1 and 2 PN seen. Conclusions: The combination of weekly bortezomib and temsirolimus showed an encouraging response rate in heavily pretreated patients with relapsed or refractory MM, with an overall response rate in evaluable patients as part of the phase II portion of the trial of 50%, and a 21% ORR including MR or better in evaluable bortezomib refractory patients. Cytopenias were the most common toxicities, specifically thrombocytopenia, as well as GI toxicity, with side effects proving manageable. Significant PN was rare in this study. Disclosures: Ghobrial: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Vij:Novartis: Honoraria; Eisai: Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Jakubowiak:Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Centocor Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 3051 Background: Lenalidomide (Revlimid®, Len) plus dexamethasone (Dex) is approved for the treatment of relapsed or refractory multiple myeloma (MM) patients following ≥1 prior therapy. RAD001, an mTOR inhibitor has been studied as a single agent in MM, and although well tolerated, did not have single agent activity. Informed by our previous studies demonstrating synergistic anti-MM activity of mTOR inhibitors when combined with Len, we studied this combination to evaluate its safety and activity as a non-steroid containing oral regimen in advanced MM. Methods: Patients with relapsed and refractory MM were assigned to Len and RAD001 to be taken for 21 days of a 28 day cycle (NCT00729638) in a standard 3+3 design. Patients were allowed to continue therapy until disease progression or unacceptable toxicity and were required to receive concomitant anti-thrombotic (aspirin 81 or 325 mg/day) therapy. Peripheral blood and bone marrow samples were collected before and after treatment for pharmacokinetic and pharmacodynamic studies. Results: Twenty-eight patients were registered on the trial between December 2008-December 2009. Two patients were inevaluable because of either rapidly progressive disease or failure to meet eligibility criteria on day 1. Data on 26 patients are therefore available. Pts had received a median of 4 prior lines of treatment.14 pts had received Len previously of which 11 pts had relapsed disease and 3 pts had relapsed/refractory disease. Both cohort 1 (Len: 10mg/day and RAD001: 5mg/every other day ×21 days of 28 day cycle) and 2 (Len: 15mg/day and RAD001: 5mg daily ×21 days of 28 day cycle) required expansion because of grade 3 neutropenia and grade 4 thrombocytopenia. Dose limiting toxicities included grade 4 neutropenia and thrombocytopenia in 2/3 patients in cohort 3 (Len: 20mg and RAD001: 5 mg × 21 days). The maximum tolerated dose (MTD) was therefore declared at 15 mg of len and 5mg of RAD001 for 21 days with a 7 day rest period. Most common (≥10%) Grade 1/2 events included nausea, fatigue, dyspnea, diarrhea, constipation, neuropathy and muscle cramps, all of which proved manageable with supportive care. One patient developed RAD related non-infectious pneumonitis requiring therapy discontinuation. Grade 3/4 adverse events (≥ 5%) included thrombocytopenia (11%) and neutropenia (22%). Nineteen patients completed 2 cycles and were evaluable for response. Median follow up is currently 8.7 months and median PFS is 4.3 months, with 12 patients receiving treatment at MTD. Overall response rate was 68% (13/19) (90% CI: [30-76%]) including CR(1) PR(2), MR(8) and SD(2). Four patients continue on the combination at 13, 14, 15 and 17 months respectively. Pharmacokinetic and pharmacodynamic data including immunohistochemistry for phosphorylated AKT, cytokine profiles, T cell subsets and transcription profile on MM cell pre and post treatment as well as correlation of response with pharmacodynamic studies will be presented. Conclusions: The combination of Len plus RAD001 is a well tolerated regimen with predictable hematological toxicities. Promising responses were noted in this heavily pretreated patient population with advanced MM. This combination thus provides an oral steroid free combination treatment strategy which warrants future evaluation in phase II studies. Disclosures: Richardson: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Adams:Novartis: Employment. Makrides:Celgene: Employment. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Raje:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Astra Zeneca: Research Funding; Acetylon: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Lenalidomide and RAD 001 for treatment of relapsed and refractory multiple myeloma.