Ficoll Gradient Centrifugation Research Articles

Defining the effects of prematurity on the lymphocyte transcriptome

Methods Peripheral blood samples were collected from premature infants at the time of hospital discharge at multiple sites and shipped to a central laboratory. Freshly purified PBMCs were isolated by Ficoll gradient centrifugation, sorted into individual lymphocyte cell types, and processed for total RNA. RNA isolated from CD8 lymphocytes (n=79) was used for RNA-Seq analysis using the Illumina HiSeq2500. Sequences were aligned using the SHRiMP algorithm and expression values were summarized using HTSeq. Normalized gene expression data were analyzed for significant changes in expression using various statistical approaches. Ingenuity Pathway Analysis software was used for gene set interpretation.

A comparison of umbilical cord blood-derived endothelial progenitor and mononuclear cell transplantation for the treatment of acute hindlimb ischemia

Acute lower limb ischemia is a common peripheral artery disease whose treatment presents many difficulties. Stem cell transplantation is considered a novel and promising method of treating this disease. Umbili- cal cord blood (UCB) is rich in stem cells, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). However, historically, banked umbilical cord blood has been used mainly to treat blood-related diseases. Therefore, this study compared the efficacy of umbilical cord blood- derived mononuclear cells (UCB-MNCs) with EPC transplantation for the treatment of acute hindlimb ischemia (ALI) in mouse models. MNCs were isolated from UCB by Ficoll gradient centrifugation, after which the EPCs were sorted based on CD34+ and CD133+ markers and cultured according to a previously published protocol. To induce ALI, mice were immuno-suppressed using busulfan (BU) and cyclophosphamide (CY), after which the femoral arteries were burned. Induction of ALI in the immune suppressed mice was confirmed by the grade of tissue damage, pedal frequency in water, tissue edema, changes in histology, total white blood cell count, and white blood cell composition. Model mice were injected with a dose of MNCs or EPCs and un-treated control mice were injected with phosphate buffered saline. The efficiency of treatment was evaluated by comparing the grade of tissue damage between the three groups of mice. Mice aged 6-12 months were suitable for ALI, with 100% of mice exhibiting ischemia from grade I 10%, grade III 50%, grade IV 40%. For all ALI mice, a gradual in- crease in pedal frequency in water, increased tissue edema, necrosis of muscle tissue, and loss of hindlimb func- tion were observed after 20 days. Transplanted MNCs and EPCs significantly improved hindlimb ischemia com- pared with control treatment. Moreover, EPC transplantation significantly improved hindlimb ischemia compared with MNC transplantation. Following EPC and MNC transplantation, 44.44% and 33.33% of the mice recovered fully (grade 0), respectively. Specifically, all recovered mice exhibited hindlimb activities similar to those of nor- mal mice. Transplantation of UCB-derived MNCs and EPCs are promising therapies for hindlimb ischemia.

Philadelphia Chromosome (Ph)-Positive Leukemia Patients Who Fail ABL1 Tyrosine Kinase Inhibitors Without BCR-ABL1 Kinase Domain Point Mutations Demonstrate Sensitivity To PI3-K/AKT Inhibitors

Despite the impressive success of imatinib in chronic myeloid leukemia (CML), resistance to therapy remains an issue for approximately 15-20% of newly diagnosed chronic phase patients by 5 years and transient responses are the rule for patients with blast crisis CML or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The most frequent and well-characterized mechanism of resistance to imatinib is acquisition of point mutations in the BCR-ABL1 kinase domain which compromise drug binding. This form of resistance is largely well-handled by the newer, more potent ABL1 inhibitors nilotinib, dasatinib, and ponatinib. However, in the remainder of patients with resistance sustained inhibition of BCR-ABL1 kinase activity is necessary but no longer sufficient to inhibit cell growth, implicating activation of additional, thus far, poorly understood BCR-ABL1-independent mechanisms of growth and survival. In an effort to identify and validate novel pathways important in BCR-ABL-independent resistance to ABL1 kinase inhibitors, we screened a cohort of 30 patients with CML or Ph+ ALL exhibiting clinical resistance to at least one ABL1 kinase inhibitor without evidence of a drug-resistant BCR-ABL1 kinase domain mutation. Following informed consent, fresh primary mononuclear cells were isolated from each sample by Ficoll gradient centrifugation, plated ex vivo in the presence of a panel of clinical and pre-clinical small-molecule kinase inhibitors, and assessed for effects on viability after 3 days. Ex vivo resistance to ABL1 kinase inhibitors largely tracked with clinical resistance profiles, and considerable variation in sensitivity profiles for other inhibitors was observed. Among these data, we found a particularly interesting subset of patients whose cells demonstrated ex vivo sensitivity to one or more PI3-K/AKT inhibitors (n=6/30; 20%) including PI-103, BEZ235, CAL-101, and MK-2206. These findings are consistent with previous studies demonstrating PI3-K activation in select ABL1 kinase inhibitor-resistant CML cell lines (Quentmeier et al., J Hem Onc 2011) and in response to imatinib in pre-overt resistance (Burchert et al., Leukemia 2005). Among the effective PI3-K/AKT inhibitors, the most prominent were the dual class IA PI3-K/mTOR inhibitor PI-103 (IC50 range:38-110 nM) and BEZ235 (IC50 range: 20-210 nM). To further interrogate the mechanism(s) behind dependence on this pathway, samples of interest were analyzed by deep sequencing using a custom capture library encompassing ∼2000 kinases, phosphatases, and adaptor proteins. However, in contrast to previous reports of activating PIK3CA mutations in imatinib-resistant KCL-22 cells, we found PIK3CA to be wild-type in all samples. Despite a common sensitivity to PI3-K/AKT pathway inhibition, we found sequence variants were somewhat heterogeneous and direct kinase targets in this pathway were wild-type, suggesting other causative lesions which activate this pathway. Variants were prioritized based on known associations to this pathway, generated and evaluated in vitro for Ba/F3 cell transformation capacity, and validated for pathway activation and kinase inhibitor sensitivity. These results will be presented. Taken together, our findings suggest that a subset of patients with Ph+ leukemia who become refractory to ABL1 kinase inhibitors without a BCR-ABL1 kinase domain mutation demonstrate acquired dependence on the PI3-K/AKT axis, warranting further investigation of inhibitors of this pathway alone and in combination with ABL1 inhibitors as a molecularly targeted therapeutic strategy in patients. Disclosures:Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Deininger:BMS: Consultancy, Research Funding; ARIAD: advisory board, advisory board Other, Consultancy; Novartis: advisory board, advisory board Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead: Research Funding. Tyner:Incyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, ARIAD & Incyte: Clin trial funding. OHSU holds contracts; Druker receives no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict has been reviewed and managed by OHSU. Other.

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies. Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production. DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 °C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4-20 x 10(6) cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

Lineage Classification of Childhood Acute Lymphoblastic Leukemia according to the EGIL Recommendations: Results of the ALL-BFM 2000 Trial

Flow cytometry immunophenotyping (FCM) is an undispensable tool for the diagnosis and for the treatment stratification of childhood acute lymphoblastic leukemia. The correlation of the EGIL-classification with prognostically relevant parameters like age, prednisone response and risk group is analyzed. Between March 2000 and June 2009 12 patients less than 1 year of age, 1 836 patients with 1 to less than 6 years, 620 patients with 6 to less than 10 years, 615 patients with 10 to less than 15 years and 275 patients with 15 to less than 19 years were analyzed with a comprehensive 4-color antibody panel and classified according to the EGIL recommendations. Bone marrow or peripheral blood mononuclear cells were isolated by ficoll gradient centrifugation, washed and stained with fluorochrome-conjugated antigen-specific monoclonal antibodies. Cell preparations were acquired and analyzed on a flow cytometer. Centralized FCM was performed for 2 775 patients (82.6%) with B-cell precursor acute lymphoblastic leukemia, 493 patients (14.7%) with T-cell acute lymphoblastic leukemia and 90 patients (2,7%) with biphenotypic acute leukemia. There was a slight overall predominance of male (56.1%) over female (43.9%) patients. Patients with B-cell precursor ALL had a slightly more favourable outcome with a 10 y pEFS of 78 ± 1.0%, compared to patients with a T-ALL or BAL (biphenotypic acute leukemia) phenotype with a 10 y pEFS of 74 ± 1.8% (n.s.) or 69 ± 9.0% (p<0.009), respectively. FCM according to the EGIL recommendations not only provides diagnostic lineage determination and subclassification but also enables an initial prognostic orientation before MRD (minimal residual disease)-based risk stratification becomes available.

Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells

One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling.

Abstract 2852: Obinutuzumab (GA101) efficacy in chronic lymphocytic leukemia in vitro is not diminished in high risk patients.

Abstract Introduction : we and others have already demonstrated the superiority of obinutuzumab (GA101) over rituximab (RTX) in in vitro depletion assays against chronic lymphocytic leukemia (CLL) cells. It has been recently suggested that complex karyotype (3 or more chromosomal abnormalities) and recurrent somatic mutations of the TP53, NOTCH1 and SF3B1 genes, influenced treatment-free and overall survivals in CLL patients. We address the question whether these factors may also influence the efficacy of anti-CD20 directed immunotherapy. Aim: To assess in vitro pre-clinical activity of RTX and GA101 against CLL cells in freshly isolated PBMCs, and correlate depletion efficacy with modern and classical (FISH, IgVH mutational status, age, gender, Binet stage, b2-microglobulin, bulky adenopathies&amp;gt;5cm) prognostic parameters in 95 CLL patients. Moreover, CD20 antigen density was assessed in 24 patients. Methods: PBMCs were isolated from blood samples by Ficoll gradient centrifugation. Antibody-mediated B cell depletions were determined by enumerating trypan blue negative, flow cytometrically CD19-positive B lymphocytes after treatment with 10 μg/ml of anti-CD20 antibodies. The Quantibrite® kit was used to determine CD20 density, measured as quantity of antibodies bound per cell (CD20-ABC). The Mann-Whitney test was used to compare median depletion according to relevant clinical and biological data. Results: patients characteristics were as follows: 69% males, median age 66y (36% &amp;gt;70y), advanced Binet stage B/C in 39% (31.7% had NCI2008 criteria for treatment), 22.6% had lymph nodes&amp;gt;5cm (56% had b2-microglobulin&amp;gt;3.5mg/l). Molecular and cytogenetics studies were as follows: unmutated IgVH in 42.7%, del13q/tri12/del11q/del17p=25.6%/22%/20%/5.8% respectively, complex karyotype (CK) was found in 20.8% of cases, and somatic mutation of TP53/NOTCH1/SF3B1 in 11.1%/12.6/4.8% of patients. The median B-CLL depletion in freshly isolated PBMC fraction due to treatment with were 61% for GA101 vs 21.5% for RTX (n=95, p&amp;lt;0.001). GA101 had higher activity than RTX in 83% of patients, yielding B-cell depletion&amp;gt;50% in 64% of cases. As shown in Figure 1, in all categories of patients, GA101 was superior to RTX. This was particularly noticed in the patients with TP53 deletion and/or mutation (n=7). Median CD20-ABC was 8100 in 24 patients, who were categorized as high or low CD20 expression based on that median. Low CD20-ABC patients had significantly less antibody-induced B-cell depletion, 12.8% (vs 25%, p=0.37) for RTX, and 42.5% (vs 67.5%, p=0.048) for GA101. CD20-ABC was not found to be correlated with clinicobiological parameters described above. Conclusions: according to modern prognostic parameters, GA101 confirms its higher efficacy than RTX (three-fold), and deserves further combination with chemotherapy or non-chemotherapy entities in CLL management. Citation Format: Loic Ysebaert, Emilie Laprévotte, Christian Klein, Jean-Jacques Fournié, Anne Quillet-Mary. Obinutuzumab (GA101) efficacy in chronic lymphocytic leukemia in vitro is not diminished in high risk patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2013-2852

Abstract 4736: Generation of tumor-specific antibodies from sentinel lymph nodes of breast cancer patients using hybridoma technology.

Abstract The field of targeted therapy is providing an entirely new generation of highly active anticancer drugs. For example, addition of a targeted drug to a chemotherapy regimen has shown improved survival in the treatment of breast cancer (Herceptin) and colorectal cancer (Avastin). Main problems with this kind of therapy are that many patients are not candidates because their tumors do not overexpress the drug target and that patient develop resistance to the targeted drug. A method to rapidly develop different sets of therapeutic antibodies (Abs) would greatly contribute to the field of targeted anticancer therapy. Even more novel would be methods that can develop Abs derived from individual patients. The rationale behind this concept is solidly supported by a large number of SEREX studies demonstrating that the sera of cancer patients do contain autoantibodies against their own tumors. These autoantibodies may show greater cancer specificity as tolerance mechanisms usually inhibit Ab production against normal cells. We generated hybridomas from the sentinel nodes of 29 breast cancer patients. Surgically removed fresh breast tumors and sentinel nodes were used for this study. Sentinel lymph node cells were harvested by either scraping the cut surface or by removing a 2 mm slice of the nodes. Lymph nodes were mechanically disaggregated and sieved before Ficoll gradient centrifugation. The yield of lymphocytes varied from 0.5 to 50x106 cells. Following stimulation, mononuclear cells were fused with plasmacytoma P3X63.Ag8.653 under hypo-osmolar condition, using BTX Electro Cell Manipulator and microslide electrodes. Each immunoglobulin secreting clone was evaluated with ELISA for binding to a panel of breast cancer cell lines representing the 4 subclasses of breast cancer, and 2 non-cancer breast epithelial cell lines. Tumor cell lysate-binding was observed in 28% of the hybridoma clones. The lysate-binding clones were further evaluated with whole cell imaging, flow cytometry, and Western blotting. Approximately 10% of the tumor-binding clones were found to bind whole tumor cells with unique binding patterns. These clones were also found to bind to the patient's own tumor. Thus, we have identified several tumor-specific autoantibodies from breast cancer patients. Successful outcome of these experiments demonstrate the feasibility of rapidly generating human monoclonal Abs that have a great potential for tumor target discovery and their use in cancer therapy and diagnosis. The innovative aspect of this strategy is the exploitation of the cancer patient's own immune repertoire for generating therapeutic Abs. This is in contrast to most of the currently available therapeutic Abs for clinical use, which are derived from chimerization and humanization of rodent Abs. No currently available antibody is developed through the selective and regulatory processes of the human immune system. Citation Format: Girja S. Shukla, Stephanie C. Pero, Yu-Jing Sun, Chelsea Carman, David N. Krag. Generation of tumor-specific antibodies from sentinel lymph nodes of breast cancer patients using hybridoma technology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4736. doi:10.1158/1538-7445.AM2013-4736

Bone Marrow–Derived Cell Therapy After Myocardial Infarction

We would like to clarify several points in the editorial (1) accompanying our publication of the TIME report (2). First, the automated system for extracting bone marrow-derived mononuclear cells (MNCs) used for Cardiovascular Cell Therapy Research Network (CCTRN) clinical trials (2,3,4) employs Ficoll gradient centrifugation, similar to the manual method used for REPAIR AMI (5). Thus, the difference lies in improved reproducibility and standardization by the automated system, not some other fundamental characteristic. Second, our original publication erroneously listed the heparin concentration at 0.1 units/ml. In fact, no heparin was added to the automated system, and cells used in all CCTRN studies contained only residual heparin from bone marrow aspiration, estimated to be not more than 0.01 units per ml (Data Correction 6). This is five times below the lowest concentration evaluated (7) in a stromal cell-derived factor-1- (SDF-1)-stimulated invasion capacity assay, and 200-2000 times lower than heparin concentrations used in other functional assays reported by this group. Accordingly, the statement: “… the cell product used in TIME contained heparin at a dose that has been shown to impair the migratory capacity of MNCs” is no longer supported by the facts. We regret the confusion caused by our error. Third, the statement, “MNCs isolated by the automated system used in this study have not been tested in an animal model of MI, and cell bioactivity has not been verified in any human studies of heart disease” should be considered in view of the following: (a) MNCs from automated and manual preparations were found to have similar reperfusion activity in a murine hind-limb ischemia model (2); (b) A Cochrane review of stem cell studies (8) reported ten clinical trials cells were administered in a solution of “standard saline containing heparin.” Of these ten studies, we note that seven disclosed positive results (e.g., increased left ventricular ejection fraction, LVEF);(c) in the CCTRN FOCUS trial we found that LVEF increased 2.7% in patients receiving MNCs versus placebo, and that this increase was associated with higher percentages of CD34+ and CD133+ MNCs (3). Additionally, pre-clinical studies by CCTRN investigators demonstrated retention of SDF-1 migration and CFU-activity when MNCs isolated by the automated system were compared with MNCs from the manual method (unpublished observations). Thus, cell bioactivity was observed not only in one CCTRN trial (with miniscule residual heparin) but also in seven other trials in which cells were re-suspended in heparin. One explanation for this retention of bioactivity is that “…the effects of heparin can be ameliorated by excessive serum in vitro…” (7). Summarizing, the available evidence does not support the contention that bioactivity of cells administered in the TIME study was impaired by processing techniques or exposure to heparin. We believe that the TIME results are consistent with a limited effect of bone marrow cells on recovery of LV function, and support previous findings (9) demonstrating that MNCs have reduced cytokine and growth factor production compared to other bone marrow-derived stem and progenitor cells.

Effects of exogenous interleukin-2, interleukin-23 on differentiation of IRBP 1-20-specific T cells toward Th1 and Th17 and comparison of the pathogenicity of IRBP 1-20-specific T cells

To explore the effects of exogenous interleukin (IL)-2 and IL-23 on differentiation of IRBP 1-20-specific T cells originated from C57BL/6 mouse with experimental autoimmune uveitis (EAU) and to investigate the difference in pathogenicity. Experimental study. Thirty C57BL/6 mice were immunized subcutaneously with 200 µl emulsion containing 200 µg interphotoreceptor retinoid-binding protein (IRBP) 1-20 and 0.8 mg mycobacterium tuberculosis in complete Freund's adjuvant, distributed over six spots at the tail base and on the flank. On postimmunization Day 13, spleens and draining lymph nodes were removed from mice, and a part of spleens, as the control group, was reserved for examining the expression of IFN-γ mRNA and IL-17 mRNA by RT-PCR. T cells were isolated from the rest of spleens and lymph nodes by passage through a nylon wool column, and then T cells were stimulated for 48 hours with IRBP 1-20 in the presence of antigen-presenting cell and mouse recombinant cytokine IL-2 or IL-23. The IRBP 1-20-specific T cells were separated by Ficoll gradient centrifugation, the expressions of IFN-γ mRNA and IL-17 mRNA were assessed by RT-PCR, and IFN-γ and IL-17 in T cells supernatant were detected using a commercial ELISA kit. The T cells were cultured for another 48 hours, and then the proportions of IL-17(+)γδ(+)T cells were analyzed by flow cytometry. EAU models were built in 30 C57BL/6 mice, T cells from which were randomly divided into two groups according to the methods mentioned above: IL-2 group and IL-23 group. Transfer EAU models were built in other 6 mice and divided into two groups, IL-2 group and IL-23 group, by intraperitoneal injection of 3.5×10(6) IRBP-special-T cells from IL-2 group or IL-23 group respectively. Clinical changes were observed by indirect ophthalmoscopy on postimmunization day 3, 7, 14. For histopathological evaluation, whole eyes were collected on postimmunization day 21. Rank sum test, one-way ANOVA and paired t test were used to analyze the results. A comparison of pathogenicity was made between Th1 and Th17 through clinical observation and histopathological evaluation of B6 mouse. Rosette formation was found among T cells on poststimulation day 2. There was a statistical difference in the expression of IFN-γ mRNA between the IL-2 group and normal group or IL-23 group (0.26 ± 0.02 vs. 0.12 ± 0.05 or 0.10 ± 0.00) (F = 80.51, P = 0.003); however, the expression of IFN-γ in the IL-2 group [(13 124.67 ± 107.73) µg/L] was higher than that of the IL-23 group [(3953.67 ± 117.34) µg/L] (t = 169.61, P = 0.000); and the expression of IL-17 mRNA in the IL-2 group [(588.67 ± 77.43) µg/L] was lower than that of the IL-23 group [(5038.33 ± 88.00) µg/L] (t = -361.71, P = 0.000). Flow cytometer showed that the concentration of IL-17 in the IL-2 group [(3.23 ± 0.28)%] was significantly lower than that in the IL-23 group [(9.93 ± 0.55)%] (t = -33.18, P = 0.001). There was a significant difference in the proportion of IL-17(+)γδ(+)T cells between the IL-23 group and IL-2 group (9.93 ± 0.55 vs. 3.23 ± 0.28) (t = -33.18, P = 0.001). Inflammatory response could not be detected in either group three days after injection of IRBP 1-20-specific T cells. Both groups of mice had the most severe inflammatory response on the 14 th day, of which that of the IL-23 group was significantly more severe. IL-23 facilitates IRBP 1-20-specific T cells to differentiate into Th17, whereas IL-2 inhibits this process and induces these cells to differentiate towards Th1. Further studies showed that the pathogenicity of Th17 cells was significantly higher than that of Th1 cells.

Abstract B7: MDSC frequencies in peripheral blood of ipilimumab-treated melanoma patients correlate with clinical response and overall survival.

Abstract Background: Metastatic melanoma has a poor prognosis, with high resistance to chemotherapy and irradiation. Recently, immunotherapy has shown clinical efficacy. The anti-CTLA-4 antibody ipilimumab (Yervoy©) was the first agent to significantly prolong the overall survival of stage III/IV melanoma patients, and received FDA approval in March 2011. About 10-13% of patients develop a clinical response during the treatment. A major focus of patient monitoring is to identify biomarkers that predict clinical outcome. We studied Myeloid Derived Suppressor Cells (MDSC) in ipilimumab treated stage III/IV melanoma patients in order to analyze drug-induced changes in the myeloid cell compartment and possible correlations with clinical outcome. Methods: Before and after treatment with ipilimumab, PBMC were isolated by Ficoll gradient centrifugation from fresh blood samples of patients with stage III/IV melanoma. Freshly isolated cells were characterized by multicolor flow cytometry. Results: Lin- CD14+ HLA-DR- monocytic and lin- CD15+ HLA-DR- granulocytic MDSC were enriched in patients, as compared to healthy controls. During ipilimumab treatment, MDSC frequencies remained stable compared to baseline levels, with high patient-to-patient variability. After tumor resection, lin- CD14+ HLA-DR- cells did not change, whereas frequencies of lin- CD15+ HLA-DR- cells decreased. Our data show that frequencies of lin- CD14+ HLA-DR- cells were independent of serum LDH levels, but were significantly enriched in patients with severe metastatic disease (M1c) compared to patients with metastasis in distant skin or lymph nodes only (M1a). Interestingly, clinical responders to ipilimumab treatment showed significantly less lin- CD14+ HLA-DR- cells during and after treatment, compared to non-responders. This difference was found regardless of the metastatic stage. The relevance of low monocytic MDSC levels (less than 1.00%) was further confirmed by the finding that these patients had a significantly prolonged overall survival. Conclusion: Malignant melanoma is associated with increased frequencies of lin- CD14+ HLA-DR-monocytic and lin- CD15+ HLA-DR- granulocytic MDSC. The frequency of monocytic MDSC in ipilimumab treated patients during and after treatment may be used as predictive marker, since low frequencies identify patients that are more likely to benefit from the treatment in terms of tumor regression and prolonged overall survival. Citation Format: Christiane Meyer, Laurene Cagnon, Carla M. Costa Nunes, Petra Baumgaertner, Nicole Montandon, Loredana Leyvraz, Olivier Michielin, Emanuela Romano, Daniel E. Speiser. MDSC frequencies in peripheral blood of ipilimumab-treated melanoma patients correlate with clinical response and overall survival. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B7.

Mixed Results for Bone Marrow–Derived Cell Therapy for Ischemic Heart Disease

TWO EARLY-PHASE STUDIES REPORTED IN JAMA 1,2 ADdress the same general question: what is the effect of bone marrow–derived cell therapy on safety and surrogate end points in patients after myocardial infarction (MI)? Other than this thematic similarity, the Timing In Myocardial infarction Evaluation (TIME) trial and the POSEIDON trial have little in common. The TIME trial examined 120 patients with a recent (3-7 days) acute MI (AMI) and autologous mononuclear cells (MNCs) delivered via the intracoronary route with the intervention on day 3 (n=67) or on day 7 (n=53). This was a placebocontrolled, multicenter trial with a fixed dose of MNCs. The major motivation was to test the concept that timing of MNC therapy within the first week after MI influences outcome. The POSEIDON trial involved patients with ischemic cardiomyopathy after distant MIs (years earlier) and used autologous or allogeneic mesenchymal cells (MSCs) administered transendocardially via injection catheters. Two centers recruited 30 patients and randomly assigned them into 1 of 6 permutations of cell origin (self or donor) and dosage (n=5 patients each), with no controls. The goal of the trial was to compare the safety and efficacy of self-derived MSCs with donor MSCs in chronic ischemic cardiomyopathy. TIME is the latest in a series of studies from the Cardiovascular Cell Therapy Research Network (CCTRN), a consortium funded by the National Institutes of Health, which was mandated to conduct and complete trials of cell therapy within its initial 5-year funding period. Three trials (LateTIME, FOCUS-CCTRN, and now TIME) of autologous MNCs have been conducted. The goal of these trials has been to follow up on earlier positive phase 1 and 2 studies of MNCs; however, the CCTRN studies (including TIME) have all had negative results. The CCTRN used a convenient automated system for extracting MNCs from bone marrow aspirates. The older motivating studies, including the Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) multicenter trial of MNC therapy, relied instead on laborious manual Ficoll gradient centrifugation of bone marrow aspirates. TIME followed up on the subgroup analysis of REPAIR-AMI, hinting at superior functional outcomes in patients who had received MNCs more than 4 days after MI vs those who received therapy earlier. However, in TIME no significant incremental treatment effects on function, volumes, or infarct size were observed in patients treated with MNCs either 3 or 7 days after MI. The lack of benefit of MNC therapy in TIME and in the other CCTRN studies raises 2 possibilities: MNCs do not work, in which case the previous positive clinical studies were red herrings, or the CCTRN trials used an inactive cell product. The latter possibility cannot be discounted because MNCs isolated by the automated system used in this study have not been tested in an animal model of MI, and cell bioactivity has not been verified in any human studies of heart disease. In addition, the cell product used in TIME contained heparin at a dose that has been shown to impair the migratory capacity of MNCs. Such details of cell manufacturing are known to influence MNC potency substantially. Given these uncertainties, TIME (and the other 2 CCTRN studies) are well-intentioned, nicely designed, and impeccably executed, but difficult to interpret. POSEIDON, on the other hand, follows a more conventional development pathway, with ample preclinical validation and early proof-of-concept data in patients. The patients with chronic ischemic cardiomyopathy included in POSEIDON have a compelling clinical need. Their functional status is depressed and their ventricles have undergone extensive deleterious structural remodeling. The major finding of POSEIDON is that therapy with allogeneic (ie, donor-derived) MSCs appears to be safe and at least as active as therapy with autologous (ie, selfderived) MSCs. Allogeneic (but not autologous) MSCs reduced end-diastolic volume, whereas both allogeneic and autologous MSCs reduced early enhancement defect (a controversial measure of infarct size derived from computed tomography). The apparent changes in myocardial structure were not matched by improvements in global ventricular function; neither allogeneic nor autologous MSCs showed any significant increase in ejection fraction when analyzed individually or pooled together.

Dishevelled Proteins of Wnt Signaling Pathways Are Highly Expressed in Chronic Lymphocytic Leukemia

AbstractAbstract 4583Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world and is characterized by progressive accumulation of CD5+, CD19+, and CD23+ B cells in the blood, bone marrow, and secondary lymphoid organs. The progressive accumulation of malignant B cells is partly due to defective apoptosis. An important constitutively activated signaling pathway in CLL cells might be the Wnt signaling cascades that include the Wnt/b-catenin pathway and the non-canonical Wnt pathway. These pathways might be activated downstream of the constitutively phosphorylated ROR1. Dishevelled family proteins (Dvl1, Dvl2, and Dvl3) are important cytoplasmic mediators of Wnt signaling and have recently been shown to be expressed in many cancer types. The expression and precise roles of individual Dvl proteins are however not clear.The aim of the study was to characterize the expression of Dvl1, Dvl2 and Dvl3 proteins in progressive and non-progressive CLL and compare to normal white blood cells (PBMC) and to assess the expression of other important proteins of the canonical pathway as b-Catenin and GSK-3β as well as the non-canonical pathways as GSK-3α, PKC, ERK and AKT respectively.Leukemic cells from progressive (n=9) and non-progressive (n=9) CLL patients were isolated by Ficoll gradient centrifugation as well as PBMC from healthy donors (n=9). Six cell lines including four CLL cell lines EHEH, CII, I83, 232 B4, and the Jurkat and Lucas cell lines were also used. Dvl 1, Dvl 2, Dvl 3 proteins were analyzed by Western blot and densitometric calculations as well as PKC, GSK-3α, AKT and ERK using antibodies against the total protein and the phosphorylated protein respectively. Immunoprecipitation was performed to check the phosphorylation status of Dvl proteins.Overexpression of Dvl1, Dvl2, and Dvl3 was detected in all progressive and non-progressive CLL patients and the six cell lines. There was a significantly higher expression of Dvl1 and Dvl3 in progressive compared to non-progressive CLL patients (p<0.01 and p<0.01 respectively). In contrast to Dvl1 and Dvl3, Dvl2 was highly expressed in non-progressive as compared to progressive CLL patients. Dvl1 was phosphorylated at serine residues while Dvl2 was phosphorylated at tyrosine residues. b-Catenin followed the same expression pattern as Dvl2 being highly expressed in non-progressive CLL as compared to progressive CLL patients (p<0.001) whereas GSK-3β phosphorylation was significantly higher in progressive than non-progressive CLL patients (p<0.01). There was no significant difference in the degree phosphorylation of PKC and GSK-3α comparing non-progressive CLL, progressive CLL and healthy donors.In summary Dvl1, Dvl2 and Dvl3 are highly upregulated in CLL patients and CLL cell lines but the expression in normal PBMC is almost negligible. Dvl1, Dvl2 and Dvl3 may have a key role in the transduction of Wnt mediated signals in CLL. Disclosures:No relevant conflicts of interest to declare.

Inhibitory human antichimeric antibodies to rituximab in a patient with pemphigus

Inhibitory human antichimeric antibodies to rituximab in a patient with pemphigus

Abstract 3027: Dishevelled proteins are highly upregulated in chronic lymphocytic leukemia

Abstract Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia and is characterized by the progressive accumulation of CD5+ B cells. An important signaling pathway in CLL cells might be Wnt signaling pathways. Wnt signaling pathways include the Wnt/ β-catenin pathway and the non-canonical Wnt pathways. Dishevelled proteins (DVL1, DVL2, and DVL3) are important mediators in Wnt signaling and have been shown in to be expressed many cancer types but their precise role is not clear. The aim of the study was to characterize the expression of DVL1, DVL2 and DVL3 proteins in indolent and progressive CLL and compare to normal white blood cells (PBMC) as well as to assess the expression of other important proteins of the canonical pathway as β-catenin and the non-canonical pathways as GSK-3β, PKC, ERK and AKT respectively. Leukemic cells from indolent (n=9) and progressive (n=9) CLL patients were isolated by Ficoll gradient centrifugation as well as PBMC from healthy donors (n=9). Six cell lines including four CLL cell lines EHEH, CII, I83, 232 B4, and the Jurkat and Lucas cell lines were also used. DVL 1, DVL 2, DVL 3 were analyzed by Western blot and densitometric calculations as well as PKC, GSK-3β, AKT and ERK using antibodies against the total protein and the phosphorylated protein respectively. Overexpression of DVL1, DVL 2, and DVL3 was seen in all indolent and progressive CLL patients and the six cell lines. There was a significantly higher expression of DVL 1 and DVL 3 in indolent compared to progressive CLL patients (p&amp;lt;0.01 and p&amp;lt;0.01 respectively). In contrast to DVL 1 and DVL 3, DVL 2 was highly expressed in indolent as compared to progressive CLL patients. ≤ -catenin followed the same expression pattern as DVL2 being highly expressed in indolent CLL as compared to progressive CLL patients (p&amp;lt;0.001). There was no significant difference in the degree phosphorylation of PKC and GSK-3β between indolent CLL, progressive CLL and healthy donors.The degree of AKT and ERK phosphorylation was significantly higher in indolent than in progressive CLL patients (p&amp;lt;0.001 and p&amp;lt;0.01 respectively). In summary DVL1, DVL2 and DVL3 are highly expressed in CLL patients and CLL cell lines but their expression in normal PBMC is almost negligible but expression of other canonical and non-canonical pathways proteins are seen both in CLL and healthy donors. DVL1, DVL2 and DVL3 may have a key role in the transduction of Wnt mediated signals in CLL. 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 3027. doi:1538-7445.AM2012-3027

Dendritic Cell Depletion in Burn Patients Is Regulated by MafB Expression

Studies have shown that monocytes are hyporesponsive and that dendritic cells (DCs) are depleted in burn patients. We have recently shown in a mouse model that burn injury alters the transcriptional regulation in bone marrow progenitors and inhibits myeloid-derived DC (mDC) production. In the present study, using human burn patient peripheral blood mononuclear cells, we have shown an overexpression of MafB with a corresponding reduction in peripheral blood mononuclear cell-derived mDCs. We isolated mononuclear cells from burn patient (23–68% TBSA) and control volunteer peripheral blood samples by Ficoll gradient centrifugation and cultured mDCs by using a standard ex vivo culture system. Fluorescence-activated cell sorter analysis was used to select myeloid cells based on the cell surface expression of CD45+. The mDC fraction was identified by the expression of human leukocyte antigen (HLA)-DR+CD11c+, and we found a significant reduction in HLA-DR+ leukocytes for up to 4 weeks postburn. MafB expression was then examined in HLA-DR+CD14+ monocytes. Burn injury alters the phenotype of CD14+ monocytes augmenting MafB expression and reducing their differentiation into mDCs. MafB was then silenced in ex vivo culture prior to DC differentiation by using small interfering RNA technique. MafB gene silencing improved the differentiation potential of CD14+ cells into mDCs, increasing the percentage of mDCs by >75%. Furthermore, GATA-1+ and HLA-DR+ mDCs were increased following MafB silencing. Although burn injury augments the number of peripheral blood monocytes, the frequency of mDC is reduced. This impairment is likely secondary to the down-regulation of mDC differentiation by high MafB-expressing monocytes following burn injury.

Abstract 17094: “Trojan Horse” Monocytes: a New Approach for Gene Delivery to Angioplastied Arteries

Introduction: Monocyte-derived macrophages are implicated in sustained inflammation typical of atherosclerotic and restenotic arteries. Since the role of macrophage-mediated inflammatory reactions in the progression of atherosclerosis and post-angioplasty restenosis is firmly established, multiple approaches have been devised to decrease the number and/or functionality of mononuclear phagocytic cells residing in atherosclerotic and stented arteries. However, no prior research has exploited the capability of monocytes to target sites of vascular injury in order to introduce pro-healing “therapeutic” genes into diseased vasculature. Here we report preliminary results demonstrating feasibility of this concept. Methods and Results: Monocytes were isolated from heparinized rat blood by sequential Ficoll and Percoll gradient centrifugation. The cells labeled with CM-DiI dye (5x10 5 cells/ml) were suspended in complete RPMI1640 medium and administered via tail vein to rats that underwent balloon denudation injury of carotid arteries 4 days prior to cell therapy. The animals were euthanized on day 8 after balloon injury (day 4 after monocyte delivery). The CM-DiI-labeled cells were found in the nascent neointima and adventitia of balloon-injured arteries (373±90 and 77±37 per mm 2 of tissue, respectively). Comparison of CM-DiI labeling with immunohistochemical staining results (CD 68) has demonstrated that up to 30% of all macrophages in the neointimal tissue of rats treated with ex vivo modified monocytes derive from the delivered cells. To verify functional competence of delivered cells, monocytes isolated as above were transduced with adenovectors driving synthesis of firefly luciferase and administered to rats that underwent Fogarty balloon injury 4 days prior to the cell delivery. One day later, luciferase expression in balloon injured arteries was confirmed by bioluminescence imaging. Conclusions: These pilot studies demonstrate the feasibility of using ex vivo transfected autologous or syngeneic monocytes as a means to introduce prohealing factors to diseased vasculature.

Novel Double Labeled Mixed Lymphocyte in Vitro Assay to Predict the Dominant Graft in 2 Unit UCB Transplantation

Abstract 4699 Introduction:Umbilical cord blood cells (UCB) from allogeneic donors have been established as an alternative source for HSC transplantation in patients who lack suitably HLA matched bone marrow or peripheral blood stem cells from adult donors. Transplantation using 2 unit UCB has been shown to compensate the low engraftment and slow hematopoietic recovery resulting from 1 unit UCB transplantation in full stature adult patients. At present, there are no unit specific factors that reliably predicts for the “winning unit” in 2 unit UCB transplantation, e.g. cell viability, number of infused total nucleated cells, CD34+ or CD3+ cells, sex mismatch, ABO blood group, and degree of HLA mismatch. In vivo mouse models suggest that CD34 negative subsets play an important role. Among CD34 negative subsets, CD8 T subset accounts for approximately 34.0+/−23.3% of T lymphocytes from UCB. In bone marrow transplantation CD8 T cells have been found to facilitate donor hematopoietic cell engraftment. Moreover, it has been reported that 1 dominant unit coincides with a specific CD8 T cell response against the non-engrafted unit which was not observed from CD4 or NK cells. Methods:In this study, we used volunteer donated UCB research units (kindly provided by P. Rubinstein, MD, New York Blood Center). Mononuclear cells (MNC) were purified by Ficoll gradient centrifugation, and CD3 T cells were isolated with CD3 MicroBeads (Miltenyi Biotec; autoMACS). The purified CD3 (confirmed by FACS >95% purity) cells were labeled with CFSE and DDAO-SE. After labeling, the cells from two different donors were mixed in 96-well U-bottom plates for continued culture in 37 °C 5% CO2. The expansion from each labeled donor cells was evaluated using flow cytometry; the dead cells were gated out using propidium iodide, and the data was analyzed using FlowJo software. For proper T cells activation, we also compared different activation conditions using i.) anti-CD3/CD28 Beads, ii.) anti-CD3 antibody plus anti-CD28 antibody, and iii.) cytokine IL-2. The schematic illustration of methods is shown in Figure 1. [Display omitted] Results and discussion:We noted that T cells from UCB are primarily at naïve stage as determined by CD45RA (93.8 +/− 7.11%) and CCR7 (84.9 +/− 12.0%) expression. We also determined the optimal activation condition using a modified mixed lymphocyte reaction from 2 UCB units. Four days after incubation, the proliferation from 2 units labeled with CFSE and DDAO-SE could be reproducibly distinguished using FL1 channel for CFSE and FL4 channel for DDAO-SE (Figure 1). The optimal concentration for labeling using CFSE (1 mM) and DDAO (1 μM or 3 mM) was determined by titration. To avoid cell toxicity resulting from CFSE and DDAO-SE labeling, as well as self-crossing from each donor using two dyes, we examined additional mixed lymphocyte analyses in which each donor was labeled with CFSE or DDAO-SE respectively and vice versa. As shown in Figure 1, we found consistently that the predicated dominant unit accounted for the majority of culture (73.2% stained with DDAO; 63.5% stained with CFSE) after 4 days co-culture. The dominance was not correlated with cell proliferation indicated by the proliferation index (1.12 for dominant and 1.48 for another unit). After confirmation of this in vitro assay, further studies were conducted to evaluate the IFN-γ release of 2 UCB units in this optimized mixed lymphocyte assay in the condition using cytokine IL-2. Interestingly, we could only detect IFN-γ by intracellular staining in one unit when co-culture was set-up using CD3 T cells from each unit; the expression of IFN-γ was not detected when we used CD3 T cells from 1 unit. The correlation between dominance and the expression of IFN-γ is currently under investigation. Conclusion:UCB Transplantation is an important alternative for patients lacking bone marrow or peripheral blood stem cell donors. With the establishment of this novel modified mixed lymphocyte in vitro assay for prediction of the “winning” immune dominant unit, routine analyses can be performed to guide unit selection. Further interventions can be exploited to preferentially treat the expected dominant unit with glycosylation, cytokines, prostaglandins, or C3a compliments to further enhance hematopoietic stem cells trafficking and engraftment to the marrow. Disclosures:No relevant conflicts of interest to declare.

Complement component C5a Promotes Expression of IL-22 and IL-17 from Human T cells and its Implication in Age-related Macular Degeneration

BackgroundAge related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly populations worldwide. Inflammation, among many factors, has been suggested to play an important role in AMD pathogenesis. Recent studies have demonstrated a strong genetic association between AMD and complement factor H (CFH), the down-regulatory factor of complement activation. Elevated levels of complement activating molecules including complement component 5a (C5a) have been found in the serum of AMD patients. Our aim is to study whether C5a can impact human T cells and its implication in AMD.MethodsHuman peripheral blood mononuclear cells (PBMCs) were isolated from the blood of exudative form of AMD patients using a Ficoll gradient centrifugation protocol. Intracellular staining and enzyme-linked immunosorbent assays were used to measure protein expression. Apoptotic cells were detected by staining of cells with the annexin-V and TUNEL technology and analyzed by a FACS Caliber flow cytometer. SNP genotyping was analyzed by TaqMan genotyping assay using the Real-time PCR system 7500.ResultsWe show that C5a promotes interleukin (IL)-22 and IL-17 expression by human CD4+ T cells. This effect is dependent on B7, IL-1β and IL-6 expression from monocytes. We have also found that C5a could protect human CD4+ cells from undergoing apoptosis. Importantly, consistent with a role of C5a in promoting IL-22 and IL-17 expression, significant elevation in IL-22 and IL-17 levels was found in AMD patients as compared to non-AMD controls.ConclusionsOur results support the notion that C5a may be one of the factors contributing to the elevated serum IL-22 and IL-17 levels in AMD patients. The possible involvement of IL-22 and IL-17 in the inflammation that contributes to AMD may herald a new approach to treat AMD.

High-throughput Screening Uncovers a Compound That Activates Latent HIV-1 and Acts Cooperatively with a Histone Deacetylase (HDAC) Inhibitor

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.