Positive Subset Research Articles

Cancer stem cell-like characteristics of a CD133+ subpopulation in the J82 human bladder cancer cell line

Cancer stem cells (CSCs) are thought to be crucial for understanding the biological roots of cancer, and are of increasing importance as a target for new anticancer agents. According to an expression analysis of the cell surface antigens of various types of cancer, CD133 is considered to be a potential marker of cancer stemness. In this study, a human urinary bladder cancer cell line (J82) was used to analyze the cancer stem cell-like characteristics of CD133+ bladder cancer cells in vitro and in vivo. The CD133 expression in the J82 cells was examined and the cells were immunomagnetically categorized into positive and negative subsets. The CD133- and CD133+ subsets were phenotypically divergent with regard to the cell growth pattern, while CD133+ cells tended to colonize during their growth. In CD133+ cells, the pluripotent stem cell factors Oct-4 and Sox-2 were upregulated, and a statistically significant proliferation increase was observed when compared to CD133- cells. The CD133+ subpopulation was more tolerant to the chemotherapeutic agent cisplatin, and Bacillus Calmette-Guérin (BCG), an agent instilled intravesically to treat bladder cancer. In addition, CD133+ J82 cells were more resistant to radiation treatment when compared to CD133- cells. The in vivo tumorigenesis of the CD133- and CD133+ subsets of J82 cancer cells was also examined by subcutaneously injecting them into nude mice. The tumor growth was more aggressive in the CD133+ subpopulation, showing a significant difference in the tumorigenic potential in these subsets. In conclusion, J82 human bladder cancer cells include CD133- and CD133+ subpopulations, while the CD133 molecule is a potential marker of the potential malignancy of human bladder cancer. In the present study, the CD133+ subpopulation was herein demonstrated to have certain characteristics consistent with those of cancer stem cells.

The neural crest‐ and placodes‐derived afferent innervation of the mouse esophagus

The mouse is an invaluable model for mechanistic studies of esophageal nerves, but the afferent innervation of the mouse esophagus is incompletely understood. Vagal afferent neurons are derived from two embryonic sources: neural crest and epibranchial placodes. We hypothesized that both neural crest and placodes contribute to the TRPV1-positive (potentially nociceptive) vagal innervation of the mouse esophagus. Vagal jugular/nodose ganglion (JNG) and spinal dorsal root ganglia (DRG) neurons were retrogradely labeled from the cervical esophagus. Single cell RT-PCR was performed on the labeled neurons. In the Wnt1Cre/R26R mice expressing a reporter in the neural crest-derived cells we found that both the neural crest- and the placodes-derived vagal JNG neurons innervate the mouse esophagus. In the wild-type mouse the esophageal vagal JNG TRPV1-positive neurons segregated into two subsets: putative neural crest-derived purinergic receptor P2X(2) -negative/preprotachykinin-A (PPT-A)-positive subset and putative placodes-derived P2X(2) -positive/PPTA-negative subset. These subsets also segregated by the expression of TrkA and GFRα(3) in the putative neural crest-derived subset, and TrkB in the putative placodes-derived subset. The TRPV1-positive esophageal DRG neurons had the phenotype similar to the vagal putative neural crest-derived subset. The TRPV1-positive (potentially nociceptive) vagal afferent neurons innervating the mouse esophagus originate from both neural crest and placodes. The expression profile of the receptors for neurotrophic factors is similar between the neural crest-derived vagal and spinal nociceptors, but distinct from the vagal placodes-derived nociceptors.

Updates on B-cell immunotherapies for systemic lupus erythematosus and Sjogren's syndrome

Last year was marked by important clinical and mechanistic studies that improved our understanding of B-cell immunotherapy for systemic lupus erythematosus (SLE) and Sjogren's syndrome. Here, we will highlight the most relevant studies published in the last 18 months. The highlight of the year was the approval of belimumab on the basis of two major trials. On the flip side, the disappointing results of rituximab in lupus nephritis provided a clinical and mechanistic counterpoint in SLE. Still, major limitations in the LUpus Nephritis Assessment with Rituximab (LUNAR) trial, positive subset analysis and new open studies and registries continue to provide hope for and major insights into the use of B-cell depletion. In Sjogren's syndrome, the role of B-cell depletion has been further investigated, both for glandular and extraglandular manifestations of the disease with mixed results in a disease in which outcomes are notoriously hard to measure. The approval of anti-B cell activating factor therapy and an increasing body of open studies with rituximab as well as subset studies and secondary analysis of the Efficacy and Safety of Rituximab in Moderately-to-Severely Active Systemic Lupus Erythematosus (EXPLORER) and LUNAR trials provide hope for B-cell immunotherapy and significant insight into its mechanisms of action and utilization in a selected subset of patients. Ongoing clinical trials of other B-cell targeting agents are eagerly anticipated.

Regionally proximal relation of order [formula omitted] is an equivalence one for minimal systems and a combinatorial consequence

Inverse limits of nilsystems in topologically dynamical systems were characterized by Host, Kra and Maass recently. Namely, for each d∈N a certain generalization of the regionally proximal relation was introduced, and for a distal minimal system it was shown that such a relation is an equivalence one, which determines the maximal d-step nilfactor. One of the main results in this article is to show that the above results hold for a general minimal system.A combinatorial consequence is also deduced, which is the topological correspondence of the result obtained by Host and Kra for positive upper Banach density subsets using ergodic methods.

An observability estimate for the heat equation from a product of two measurable sets

This paper presents an observability estimate for the heat equation in Ω×(0,T), where Ω has a real analytic boundary ∂Ω. The observation region is ω×E, where ω and E are positive measurable subsets of Ω and (0,T) respectively. The building of this estimate is based on a slightly modified version of the new estimate established in Apraiz and Escauriaza (2012) [15], the technique developed in Phung and Wang (2012) [11] and the shortcut of the Lebeau–Robbiano strategy provided in Miller (2010) [3].

CD45/CD11b positive subsets of adult lung anchorage-independent cells harness epithelial stem cells in culture

Compensatory growth is mediated by multiple cell types that interact during organ repair. To elucidate the relationship between stem/progenitor cells that proliferate or differentiate and somatic cells of the lung, we used a novel organotypic ex vivo pneumoexplant system. Applying this technique, we identified a sustained culture of repopulating adult progenitors in the form of free-floating anchorage-independent cells (AICs). AICs did not express integrin proteins α5, β3 and β7, and constituted 37% of the total culture at day 14, yielding a mixed yet conservative population that recapitulated RNA expression patterns of the healthy lung. AICs exhibited rapid proliferation manifested by a marked 60-fold increase in cell numbers by day 21. More than 50% of the AIC population was c-KIT(+) or double-positive for CD45(+) and CD11b(+) antigenic determinants, consistent with cells of hematopoietic origin. The latter subset was found to be enriched with prosurfactant protein-C and SCGB1A1 expressing putative stem cells and with aquaporin-5 producing cells, characteristic of terminally differentiated alveolar epithelial type-1 pneumocytes. At the air/gel interface, AICs undergo remodeling to form a cellular lining, whereas TGF(β)1 treatment modifies protein expression properties to further imply a robust effect of the microenvironment on AIC phenotypic changes. These data confirm the active participation of clonogenic hematopoietic stem cells in a mammalian model of lung repair and validate mixed stem/somatic cell cultures, which license sustained cell viability, proliferation and differentiation, for use in studies of compensatory pulmonary growth.

Characterization of human thymocyte behavior in situ (111.46)

Abstract Thymocyte development occurs through the coordinated movement of developmental intermediates to defined anatomical locations within the thymus during which thymocytes sample surrounding cells and the microenvironment for signals to promote differentiation. The development of an experimental model in which purified, labeled cell populations migrate into a 3-dimensional living thymic slice gives us the first opportunity to visualize and monitor the behavior of human thymic subsets in situ. Double negative (DN) progenitors and CD4+CD8+ double positive (DP) human thymic subsets primarily localize to the cortex, whereas CD4+ and CD8+ single positive (SP) subsets localize appropriately to the medulla. In many respects, the migration of human thymic subsets mirror that of their murine counterparts: immature DP and DN cells move randomly with slow motility rates, DP cells migrate slower than DN, and both SP populations are fast and exhibit some directional movement presumably toward the medulla. These similarities between human and mouse thymocyte motility occur despite significant differences in the development and microenvironment of the thymus between the species. We are also examining cross-talk between mouse and human factors as well as the sensitivity of human thymic subsets to chemokine signals. The suitability of the thymic slice model to study human thymocyte development is a powerful system to study and manipulate processes that are otherwise inaccessible to researchers.

Optimized buffer system for detection of transcription factors and surface proteins in T-cells validated in high throughput screening of activated T-regulatory cells. (124.10)

Abstract Intracellular detection of antigens at the single cell level by flow cytometry requires optimized buffers and methods for detection of a given specificity and the compartment probed. Using a new buffer system tightly controlled for low non-specific staining, increased resolution, and significantly lower cell loss, we have developed an optimized system for detection of intracellular proteins by flow cytometry. This system works across multiple tissues from human and mouse models offering a one-method system for reliable antibody cross-reactivity tests. The optimized system detects proteins within the nucleus, endoplasmic reticulum, outer mitochondrial membrane, and the Golgi complex, with focus on optimal resolution of DNA-binding transcription factors. Intracellular targets detected included; FoxP3, T-bet, RORγT, GATA-3, Satb1, Bcl6, Bcl2, pH2AX, Ki-67, Themis, Nanog, Sox1, Sox2, Oct 3/4, Pax-6, Nestin, and various cytokines. In addition, fluorescent antibodies to cell surface antigens for multi-color flow were also found to be compatible with the optimized buffer system. Further verification of the buffer was done by high throughput screening of human cell surface markers with the BD LyoplateTM on activated human T-regulatory cells (Treg). GARP positive and negative Treg subsets were screened for various markers and the results are discussed in the poster.

Abstract 705: HER2 assessment to define outcomes for breast cancer patients treated with neoadjuvant therapy

Abstract Background: HER2 expression or amplification is now an established companion diagnostic test. Now, as HER2-targeting neoadjuvant therapy for breast cancer increases, the accuracy of the companion diagnostic tests are revisited. Specifically, we seek to determine with the level of expression of the target (HER2 protein) is associated with pathologic response. In this study, we quantitatively assess HER2 expression both before and after the neoadjuvant therapies to breast cancer in the BrUOG BR-211B (NCT00617942) trial. Methods: We evaluated core biopsy samples from breast cancer patients enrolled in a preoperative clinical trial using trastuzumab, nab-paclitaxel and carboplatin combination therapy. Tumor core biopsies were taken before and after 10 days of either trastuzumab or nab-paclitaxel treatment. Combination chemotherapy was given for 6 cycles. Automated quantitative analysis (AQUA), a fluorescent-based method for analysis of in situ protein expression, was used to assess HER2 and phospho-HER2 expression in the BruOG Cohort, a study where patients who serially received Herceptin and Abraxane were biopsied both before and after treatment. This study assesses the HER2 positive patient subset (n=27) Results: Of 18 evaluable patients, the 8 with a PathCR showed a mean HER2 levels of 10251 compared with 4766 in the patients without PathCR (p=0.0047). This is notable in that a HER2 score of 2000 is equivalent to HER2 2+ in previous studies. Measurement of Phospho-HER2 showed no difference in PathCR vs Non-PathCR groups. A small subset (9) cases were measured for HER2 immediately after treatment, but the study has accrued an insufficient number of cases to statistically determine if there is a reduction in HER2 immediately following treatment. Conclusions: High levels of HER2 are associated with PathCR in the neoadjuvant setting. Levels of Phospho-HER2 are not associated with response. Further studies are underway to determine changes in levels of HER2 immediately after treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 705. doi:1538-7445.AM2012-705

Abstract 1569: Multivalent melanoma vaccine using primary human melanoma cells and a vaccinia virus based antigen retrieval system

Abstract Melanoma is the fifth most diagnosed cancer in men and seventh most diagnosed cancer in women and it is estimated that there will be over 70,000 new cases and 8,000 deaths in 2011. Prevention of both primary and secondary melanomas requires the development of a melanoma specific multi-valent vaccine. We have been involved in developing a vaccinia virus based vaccine consisting of melanoma cell membrane antigens and integrated vaccinia viral proteins, which serve as a potent adjuvant. This melanoma vaccine, referred to as viral membrane oncolysate (VMO), is composed of five primary cell lines derived from patient tumors each independently infected with vaccinia virus and has been shown to induce anti-cancer immune response in clinical trials. Notable results were obtained in node positive subsets of patients that showed a positive response in survival distribution function suggesting that this preparation is an active therapeutic immunogen but the mechanism of immune activation needs to be elucidated. In order for us to define the immunogens that impart the clinical efficacy of the this melanoma VMO vaccine, it is essential to characterize the primary cell lines with respect to their phenotypic characteristics as well as to define the melanoma antigen repertoire so that effective vaccine strategies can be designed that do not rely exclusively on tumor derived primary cells. Our study reveals that all the five primary cell lines expressed antigens, such as gp100, CD146, and tyrosinase, similar to several commercially available melanoma cell lines although at varying concentrations. In addition, the five primary cell lines demonstrated varying migratory/invasive properties using a Boyden Chamber assay and their tumorigenic potential is being tested in nude mice. The cells were also characterized with respect to BRAF mutations. We observed several similarities between the antigenic profile of the primary and established cell lines as well as some unique characteristics. We conclude that using a combination of defined antigens from primary tumor cells is useful in designing a pan-antigen immunotherapeutic vaccine. This melanoma pan antigen vaccine can be an effective tool in primary prevention of melanoma presumably having the highest clinical efficacy when combined with unique antigens from primary cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1569. doi:1538-7445.AM2012-1569

Linear L–Positive Sets and Their Polar Subspaces

In this paper, we define a Banach SNL space to be a Banach space with a certain kind of linear map from it into its dual, and we develop the theory of linear $L$-positive subsets of Banach SNL spaces with Banach SNL dual spaces. We use this theory to give simplified proofs of some recent results of Bauschke, Borwein, Wang and Yao, and also of the classical Brezis-Browder theorem.

Validation of a multiplex chip-based assay for the detection of autoantibodies against citrullinated peptides

IntroductionAutoantibodies directed against citrullinated proteins/peptides (ACPAs) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognoses and may require different treatments, are characterized by different autoantibody profiles. The objective of this study was to develop a microarray for the detection of multiple RA-associated autoantibodies, initially focusing on responses against citrullinated epitopes on candidate autoantigens in RA.MethodsThe microarray is based on Phadia's ImmunoCAP ISAC system, with which reactivity to more than 100 antigens can be analyzed simultaneously, by using minute serum volumes (< 10 μl). Twelve citrullinated peptides, and the corresponding native arginine-containing control peptides, were immobilized in an arrayed fashion onto a chemically modified glass slide, allowing a three-dimensional layer with high binding capacity. The assay was optimized concerning serum dilution and glass surface, whereas each individual antigen was optimized concerning coupling chemistry, antigen concentration, and selection of spotting buffer. The performance of each peptide in the ImmunoCAP ISAC system was compared with the performance in enzyme-linked immunosorbent assays (ELISAs). Serum from 927 RA patients and 461 healthy controls from a matched case-control study were applied onto reaction sites on glass slides, followed by fluorescent-labeled anti-human immunoglobulin G (IgG) antibody. Fluorescence intensities were detected with a laser scanner, and the results analyzed by using image-analysis software.ResultsStrong correlations between the ImmunoCAP ISAC system and ELISA results were found for individual citrullinated peptides (Spearman ρ typically between 0.75 and 0.90). Reactivity of RA sera with the peptides was seen mainly in the anticyclic citrullinated peptide 2 (CCP2)-positive subset, but some additional reactivity with single citrullinated peptides was seen in the anti-CCP2-negative subset. Adjusting for reactivity against arginine-containing control peptides did not uniformly change the diagnostic performance for antibodies against the individual citrullinated peptides.ConclusionsThe multiplexed array, for detection of autoantibodies against multiple citrullinated epitopes on candidate RA autoantigens, will be of benefit in studies of RA pathogenesis, diagnosis, and potentially as a guide to individualized treatment.

Natural Killer Cells Recovery After Consolidation Chemotherapy in Elderly Patients with Acute Myeloid Leukemia (AML)

Abstract 2189 Background:Human Natural Killer (NK) cells are able to kill abnormal cells while preserving normal cells. Accumulating clinical and experimental data point toward a key role of these cells in the control and clearance of most if not all hematologic malignancies. Recent insights into NK have stimulated studies of innate immunity in haematological malignancies, as the role of NK cells in allogeneic transplantation. Better knowledge of the deficiencies of these effector cells can allow elaborating new protocols of immunotherapy in order to directly enhance their capacity to eliminate tumor cells.Hence, the mechanisms of recognition and killing of leukemic cells and their role in vivo have only been investigated very recently. Even though lysis of leukemic cells or leukemic cell lines by NK cells has been described in vitro, mechanisms underlying the interaction and destruction of these cells are not clearly defined. Some of these receptors are altered in AML patients at diagnosis and might be involved in the immune escape of AML blasts. However, the recovery of NK cells during consolidation chemotherapy treatment has not been studied. The present study monitored status of NK cells following patient's remission after chemotherapy in order to provide new targets for immunotherapy. Methods:We enrolled 29 elderly patients (mean: 70-years old) with non promyelocytic AML in first CR following induction and pre-consolidation chemotherapy. Patient peripheral NK cells were analyzed at diagnosis, before consolidation chemotherapy and every two weeks after treatment for 8 weeks. 6-colors flow cytometry was performed to investigate the expression of MHC receptors (CD158a, b, e, i, CD85j and NKG2A), activating receptors (NKp30, NKp46, NKG2D, CD16, DNAM-1, 2B4) as well as their differentiation status (perforin and granzyme expression). Their function, as determined by cytotoxicity (51Cr release and CD107 expression) and cytokine production (intracellular staining of IFN-g), was analyzed using purified NK cells stimulated by K562, or in redirected assays using NKp30, NKp46 and CD16 mAbs. The recovery of these receptors was then correlated with PFS and OS. Results:NK cell counts were depressed after induction and pre-consolidation chemotherapy as compared to NK cell counts of age-matched controls; they were further depressed during the first 2 weeks post-consolidation chemotherapy, but were back to pre-consolidation chemotherapy level at 4 weeks. NKp30 and NKp46 expression was lower at diagnosis as compared to controls but their levels restored progressively after induction and consolidation chemotherapy.NKG2D expression were depressed at pre-consolidation but increased after consolidation chemotherapy. For inhibitory receptors, CD158a or CD158b expressions were depressed at diagnosis, at post-induction and consolidation chemotherapy. In contrast, the NKG2A positive NK cell subsets increased progressively after consolidation chemotherapy. Moreover, sizes of perforin or granzyme positive NK cell subsets were increased in treated AML patients. K562 cytotoxicity was depressed after induction chemotherapy but increased after consolidation chemotherapy. In contrast, IFN-g secretion was decreased, at all time points.Finally, we try to correlate the recovery of these different receptors with OS and PFS. NKp30 and NKG2D recovery seems to be correlated with better PFS and OS. Conclusions:This study confirms that NK cells from AML patients displayed different phenotype and functional abnormalities at diagnosis. Chemotherapy seems to have different impact on the recovery of inhibitory or activatory NK receptors. The predominant data is that NK cells recovered rapidly after consolidation chemotherapy and seems to be more operational at that time. Immunotherapy of NK cells must be probably developed post consolidation chemotherapy when NK cells are ready and residual disease low. Antibodies to stimulate NK cells are actually evaluated in this setting. Disclosures:Anfossi:Innate Pharma: Employment. Andre:Innate Pharma: Employment. Breso:Innate Pharma: Employment. Perri:Innate Pharma: Employment. Romagne:Innate Pharma: Employment.

Outcomes for Adult Lymphoblastic Leukemia (ALL) Are Mainly Influenced by Age and Status of Minimal Residual Disease (MRD) by Multiparameter Flow Cytometry (MFC) After Therapy with the Modified Hyper-CVAD (with or without Rituximab) Regimen

Abstract 1524The hyper-CVAD regimen is an effective frontline program for de novo adult ALL [Kantarjian, JCO 18: 547, 2000; Kantarjian, Cancer 101: 2788, 2004]. Intensive cycles of hyper-CVAD (fractionated cyclophosphamide, vincristine [VCR], doxorubicin, dexamethasone) alternate with high dose methotrexate (MTX) and cytarabine every 21 days for 8 courses, followed by maintenance therapy with POMP (6-mercaptopurine, MTX, VCR, prednisone) interrupted by early and late intensifications. The regimen was modified in 1999 to include use of laminar air flow rooms during the induction phase for pts aged 60 years or older. Rituximab 375 mg/m2 (Days 1 & 11 of hyper-CVAD, Days 1 & 8 of MTX-cytarabine for 8 total doses) was administered for CD20 expression > 20% to counteract increased propensity for relapse [Thomas D, Blood 113: 6330, 2009]. Early anthracycline intensification with liposomal daunorubicin and cytarabine was incorporated from 1999–2000 then omitted from the regimen [Thomas D, Cancer 116: 4580, 2010]. CNS prophylaxis alternated intrathecal MTX day 2 with cytarabine day 7 of the first 3 courses for low CNS risk and first 4 courses for high CNS risk (in the absence of CNS disease). Maintenance therapy was extended from 24 to 30 months inclusive of early and late intensifications (hyper-CVAD followed by weekly MTX and L-asparaginase mos 6 & 7 then mos 18 & 19) to reduce incidence of late relapses. Results in the CD20 positive B-lymphoblastic subset treated with hyper-CVAD and rituximab were encouraging; 3-yr rates of CR duration (CRD) and survival (OS) were 60% and 50% overall, respectively. In younger (age < 60 years) CD20-positive subset, rates of CRD and OS were superior compared with standard hyper-CVAD (70% v 38%; P <.001% and 75% v 47%, P =.003) [Thomas D, JCO 28: 3830, 2010]. Historical experience in the adolescent and young adult (AYA) subset aged 15 – 30 yrs with CD20 positive B-lymphoblastic leukemia (now treated with pediatric augmented BFM regimen) showed that the addition of rituximab improved the 3-yr CRD rates from 26% to 65% (P =.001) and 3-yr OS rates from 47% to 75% (P =.05) compared with standard hyper-CVAD. There were no significant differences in outcome for the AYA subset by regimen (standard or modified) for the CD20 negative groups (all 3-yr rates > 70%). In contrast to the Burkitt leukemia experience, elderly pts with CD20 positive B-lymphoblastic leukemia treated with hyper-CVAD and rituximab did not benefit (rates of CRD 45% v 50%, P = NS and OS 28% v 32%, P = NS, respectively), related in part to deaths in CR. For the modified hyper-CVAD regimen (no anthracycline intensification), the rate of MRD negativity by 4- or 6-color MFC (sensitivity 0.01%, assay now 8-color MFC) at the time of CR in 95 evaluable pts was 72%. Overall, MRD positivity by MFC at the time of CR was associated with a higher relapse rate (52% v 21%, P =.01) and lower 3-yr CR duration rates (45% v 78%, P =.01). CD20 positive pts treated with rituximab had a higher rate of MRD negativity by MFC at CR than their CD20 negative counterparts (81% v 58%, P =.02). MRD positivity by MFC after hyper-CVAD and rituximab was associated with a significantly lower 3-yr CR duration rate (24% v 82%, P =.002), but survival rates were not statistically different (27% v 70%) likely due in part to deaths in CR in the older subset of the MRD-negative group. In contrast, for the CD20 negative subset, presence of detectable MRD by MFC at the time of CR did not influence 3-yr CR duration rate (58% v 63%). Additional doses of rituximab have been added to the consolidation cycles (Day 1 of cycles 5 – 8) and during the maintenance phase. Upfront dose reductions have been implemented for the induction-consolidation phase according to age and performance status in order to reduce deaths in CR. MTX-cytarabine cycles now include vincristine. All pts receive 8 IT treatments in the absence of CNS disease. Younger pts age 40 – 50 yrs receive pegylated asparginase (2000 Units/m2 with capping) during the induction-consolidation phase and early/late intensifications (augmented hyper-CVAD). Elderly pts or younger pts with B-lymphoblastic leukemia with contraindications to asparaginase are now treated with the hyper-CVAD and ofatumumab regimen regardless of CD20 expression. The cumulative experience of the serial modifications to the hyper-CVAD regimen stratified by age/MRD status and the preliminary experience with the frontline version of the augmented hyper-CVAD regimen will be presented. Disclosures:Off Label Use: Rituximab in Lymphoblastic Leukemia.

Interleukin-19: a constituent of the regulome that controls antigen presenting cells in the lungs and airway responses to microbial products.

BackgroundInterleukin (IL)-19 has been reported to enhance chronic inflammatory diseases such as asthma but the in vivo mechanism is incompletely understood. Because IL-19 is produced by and regulates cells of the monocyte lineage, our studies focused on in vivo responses of CD11c positive (CD11c+) alveolar macrophages and lung dendritic cells.Methodology/Principal FindingsIL-19-deficient (IL-19-/-) mice were studied at baseline (naïve) and following intranasal challenge with microbial products, or recombinant cytokines. Naïve IL-19-/- mixed background mice had a decreased percentage of CD11c+ cells in the bronchoalveolar-lavage (BAL) due to the deficiency in IL-19 and a trait inherited from the 129-mouse strain. BAL CD11c+ cells from fully backcrossed IL-19-/- BALB/c or C57BL/6 mice expressed significantly less Major Histocompatibility Complex class II (MHCII) in response to intranasal administration of lipopolysaccharide, Aspergillus antigen, or IL-13, a pro-allergic cytokine. Neurogenic-locus-notch-homolog-protein-2 (Notch2) expression by lung monocytes, the precursors of BAL CD11c+ cells, was dysregulated: extracellular Notch2 was significantly decreased, transmembrane/intracellular Notch2 was significantly increased in IL-19-/- mice relative to wild type. Instillation of recombinant IL-19 increased extracellular Notch2 expression and dendritic cells cultured from bone marrow cells in the presence of IL-19 showed upregulated extracellular Notch2. The CD205 positive subset among the CD11c+ cells was 3-5-fold decreased in the airways and lungs of naïve IL-19-/- mice relative to wild type. Airway inflammation and histological changes in the lungs were ameliorated in IL-19-/- mice challenged with Aspergillus antigen that induces T lymphocyte-dependent allergic inflammation but not in IL-19-/- mice challenged with lipopolysaccharide or IL-13.Conclusions/SignificanceBecause MHCII is the molecular platform that displays peptides to T lymphocytes and Notch2 determines cell fate decisions, our studies suggest that endogenous IL-19 is a constituent of the regulome that controls both processes in vivo.

P32 Neuropsychiatric lupus: a comparison of antiphospholipid antibody positive and negative subsets

P32 Neuropsychiatric lupus: a comparison of antiphospholipid antibody positive and negative subsets

HIV infection of thymocytes inhibits IL-7 activity without altering CD127 expression

BackgroundThymic function is altered in HIV infection and characterized by dysregulation of the thymic epithelial network, reduced thymic output and ultimately an impaired naïve T-cell pool. The IL-7/IL-7 receptor (IL-7R) signalling pathway is critical for the maturation and differentiation of thymocytes. HIV infection is associated with a decrease in IL-7Rα (CD127) expression and impaired CD127 signalling in circulating CD8+ T-cells; however, little is known about the effect of HIV on CD127 expression and IL-7 activity in the thymus. Therefore, the effect of in vitro HIV infection on CD127 expression and IL-7-mediated function in thymocytes was investigated.FindingsIn vitro HIV infection of thymocytes did not affect CD127 expression on either total thymocytes or on single positive CD4 or single positive CD8 subsets. However, HIV infection resulted in a decrease in the level of IL-7-induced STAT-5 phosphorylation and Bcl-2 expression in unfractionated thymocytes.ConclusionThese findings indicate that HIV infection alters IL-7 responsiveness of thymocytes by a mechanism other than CD127 downregulation and potentially explain the disruption in thymopoiesis observed in HIV infection.

Abstract 500: Activation of 5’-flanking sequences of the NANOG pseudogene (NANOGP8) in prostate cancer cell lines increases NANOG RNA expression, sphere formation, and clonogenicity

Abstract In cancer cells, in contrast to normal embryonic stem cells, NANOG is transcribed from the NANOG pseudogene, NANOGP8. The over-expression of NANOGP8 has been documented to promote the proliferation of NIH3T3 cells, the invasion of prostate PC3 cells, and the development of prostate tumors. In this study, we hypothesized that NANOGP8 transcription could be controlled by a promoter-like region in its 5’-flanking sequences and cells with activation of these sequences should exhibit NANOGP8 function, namely sphere formation and increased cell growth. To test this hypothesis, we first verified if the expressed NANOG in prostate cancer comes from NANOGP8. RT-PCR was conducted from RNA collected from the prostate cell lines PC3 and DU145 and primary culture of normal and cancerous prostate cells with primers spanning across both the whole coding region and the 3’UTR. The 1.6kb PCR products were sequenced verifying that the NANOG RNA was transcribed from NANOGP8 and not from NANOG in cancer cells and normal prostate cells. To examine if the 5’-flanking sequences of NANOGP8 have promoter-like function, we cloned a 5kb fragment of the 5’-flanking region of NANOGP8 from a PAC clone containing the whole NANOGP8 gene and inserted the fragment into the pEGFP vector to replace the CMV promoter. The EGFP expression plasmid driven by the 5’-flanking sequence was then transfected into PC3 and DU145 cells. After selection with G418, the EGFP positive cells were separated from EGFP negative cells by flow cytometry and expression of NANOG was analyzed with real-time RT-PCR in the two populations. The RNA expression of NANOG in EGFP positive DU145 and PC3 cells was 3.5 fold and 1.5 fold higher than in EGFP negative cells, respectively. In a DU145 stably transfected cell line the colony formation in the EGFP positive subset was 24% compared to 13.4% in the EGFP negative subset. Similarly, sphere formation in the EGFP positive DU145 cell subset was 40% more than that in the EGFP negative subset. Interestingly, in the spheres formed from the EGFP negative subset about 50% became EGFP positive and of the total spheres formed from both subsets, 82% were EGFP positive and only 18% of spheres were EGFP negative, suggesting that culture in serum-free medium without cell attachment promoted activation of the 5’-flanking region of NANOGP8. In conclusion, NANOG P8 is transcribed in both normal and cancerous prostate cells. The 5’-flanking sequence of NANOGP8 could play a role in expression of the NANOGP8 gene in prostate cancer cells and in regulation of stem-like properties of cancer stem cells. The mechanisms of the activation of the promoter-like 5’-flanking sequence of NANOGP8 is under investigation but could provide potential therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 500. doi:10.1158/1538-7445.AM2011-500

Antibodies to citrullinated α-enolase peptide 1 and clinical and radiological outcomes in rheumatoid arthritis

IntroductionThe anticyclic citrullinated peptide 2 (anti-CCP2) assay is a generic test for antibodies to citrullinated proteins, among which there is a subset of about 50% with antibodies to citrullinated enolase...

Effect of Elotuzumab on Circulating Lymphocytes, Chemokines, and Cytokines In Multiple Myeloma Patients

AbstractAbstract 4070Background: Elotuzumab is a humanized monoclonal IgG1 antibody directed against CS1, a cell surface glycoprotein which is highly and uniformly expressed on malignant plasma cells in multiple myeloma (MM). CS1 is also expressed at a lower level on the cell surface of natural killer (NK) cells, natural killer T-cells (NKT), and on a subset of CD8 positive T-cells, but not resting B cells, monocytes, or CD4 positive T-cells. Preclinical studies have previously demonstrated that elotuzumab kills myeloma cells via NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In addition, in whole blood assays elotuzumab treatment resulted in the elevation of chemokines and cytokines in culture supernatants. Elotuzumab is being studied in three phase 1 clinical trials in relapsed and/or refractory MM: 1) a monotherapy dose-escalation study, 2) a combination study with bortezomib, and 3) a combination study with lenalidomide and low-dose dexamethasone. Given that NK cells express CS1, there was a concern that elotuzumab treatment could potentially lead to NK cell depletion. Therefore, the pharmacodynamic goals of these clinical studies were to examine the effects of elotuzumab treatment on lymphocyte counts (in particular NK cells), chemokine levels, and cytokine levels. Methods: Absolute lymphocyte counts were determined in peripheral blood samples using the TruCOUNT™ flow cytometry assay. Serum levels of chemokines and cytokines were measured using a multiplex, bead-based assay (Luminex®). Results: In all 3 studies, we observed no depletion of total lymphocytes or lymphocyte subsets, including CS1 positive NK cells, with elotuzumab dosing either alone or in combination with bortezomib or lenalidomide/dexamethasone. A transient decrease in the absolute number of circulating total lymphocytes (approximately 75%–90% reduction from baseline) upon first elotuzumab dose was observed, followed by a recovery of these lymphocyte counts to baseline or near baseline levels as dosing cycles continued. The transient decrease in lymphocytes included both CS1 positive and CS1 negative cell subsets. This transient decrease in lymphocyte counts was associated with increased levels of circulating chemokines and cytokines following dosing. Post-dose serum samples from study subjects had a median 16-fold (range 1.3–270-fold) elevated levels of interferon inducible protein 10 (IP-10), a chemokine well known to induce lymphocyte trafficking. Other serum analytes were elevated following the initial elotuzumab dosing, including MCP-1, IL-6, and TNF-α, also known for their role in chemotaxis and inflammatory processes. The elevated levels of chemokines and cytokines were generally not seen at subsequent doses. Conclusions: A transient decrease in both CS1 positive and CS1 negative lymphocyte counts was observed following the first dose of elotuzumab in all phase 1 studies in patients with relapsed and/or refractory MM, which resolved during subsequent dosing. This transient decrease appears to be due to lymphocyte trafficking resulting from release of chemokines and cytokines. No evidence of elotuzumab-mediated depletion of CS1 positive lymphocytes was observed. Elotuzumab treatment thus does not appear to be associated with NK cell depletion. Disclosures:Neyer:Facet Biotech: Employment. Ding:Facet Biotech: Employment. Chen:Facet Biotech: Employment. Sheridan:Facet Biotech: Employment. Rice:Facet Biotech: Employment. Balasa:Facet Biotech: Employment. Keller:Facet Biotech: Employment. Fang:Facet Biotech: Employment. Albano:Facet Biotech: Employment. Tran:Facet Biotech: Employment. Zhao:Facet Biotech: Employment. Afar:Facet Biotech: Employment.