Immunophenotyping of canine T cell activation and proliferation by combined protein and RNA flow cytometry.

The limited availability of canine-reactive monoclonal antibodies restricts the analyses of immune cell subsets and their functions by flow cytometry. The PrimeFlow™ RNA Assay may serve as a potential solution to close this gap. Here we report a blood immunophenotyping method utilizing combined protein- and RNA-based flow cytometry to characterize canine T cell activation and proliferation within individual cells. In this assay, CD69 expression was detected by an RNA probe and CD25 and Ki67 were detected by antibodies. Canine peripheral blood mononuclear cells (PBMCs) were stimulated with three agents with different modes of action, anti-CD3/CD28 antibodies, phytohemagglutinin, or phorbol myristate acetate /ionomycin. Robust T cell activation (CD25+ and/or CD69+) and proliferation (Ki67+) were detected. Both CD69 and CD25 appear to be robust and sensitive T cell activation markers with early induction and low background expression. Upon stimulation, T cell proliferation occurred later than T cell activation and was associated with CD25 expression. This canine T cell activation and proliferation immunophenotyping method was evaluated in 5 independent experiments using PBMCs from 10 different beagle dogs with satisfactory assay performance. This method can greatly facilitate the evaluation of immune disease pathogenesis and immunotoxicity risk assessment in nonclinical drug development in canine.

Microphysiological systems in early stage drug development: Perspectives on current applications and future impact.

Microphysiological systems (MPS) are making advances to provide more standardized and predictive physiologically relevant responses to test articles in living tissues and organ systems. The excitement surrounding the potential of MPS to better predict human responses to medicines and improving clinical translation is overshadowed by their relatively slow adoption by the pharmaceutical industry and regulators. Collaboration between multiorganizational consortia and regulators is necessary to build an understanding of the strengths and limitations of MPS models and closing the current gaps. Here, we review some of the advances in MPS research, focusing on liver, intestine, vascular system, kidney and lung and present examples highlighting the context of use for these systems. For MPS to gain a foothold in drug development, they must have added value over existing approaches. Ideally, the application of MPS will augment in vivo studies and reduce the use of animals via tiered screening with less reliance on exploratory toxicology studies to screen compounds. Because MPS support multiple cell types (e.g. primary or stem-cell derived cells) and organ systems, identifying when MPS are more appropriate than simple 2D in vitro models for understanding physiological responses to test articles is necessary. Once identified, MPS models require qualification for that specific context of use and must be reproducible to allow future validation. Ultimately, the challenges of balancing complexity with reproducibility will inform the promise of advancing the MPS field and are critical for realization of the goal to reduce, refine and replace (3Rs) the use of animals in nonclinical research.

Receptor occupancy and blocking of STAT5 signaling by an anti-IL-7 receptor α antibody in cynomolgus monkeys.

BACKGROUND: Interleukin-7 receptor α (IL-7Rα) is associated with autoimmune disease. Blocking its activation by interleukin-7 (IL-7) with a therapeutic monoclonal antibody may reduce pathogenic T cells and effectively control the autoimmune response in these disorders. METHODS: Two flow cytometry-based assays were developed and implemented to evaluate the interaction between cell surface IL-7Rα and an anti-IL-7Rα monoclonal antibody (Ab1). The receptor occupancy assay utilized competing and noncompeting commercial detection antibodies for "free" and "total" IL-7Rα, respectively. STAT5 phosphorylation (pSTAT5) was measured as a proximal biomarker of IL-7Rα inhibition by Ab1. RESULTS: Monkeys administered Ab1 had no free IL-7Rα detectable on the CD3+ T cell surface at 0.25 hours postdose through day 4, in all treatment groups. Ab1 treatment resulted in a significant reduction in total IL-7Rα, dropping to 53%, 44%, and 55% on day 4 at 0.3, 3, and 30 mg/kg, respectively, compared to predose levels. There were treatment-related decreases in the ability of IL-7 to induce STAT5 phosphorylation in both CD4+ and CD8+ T cells in monkey blood samples from all treated animals from 0.25 hours through Day 4 postdose. CONCLUSIONS: The nonclinical receptor occupancy assay was developed and applied to detect free and total IL-7Rα on the surface of CD3+ T cells in cynomolgus monkeys treated with Ab1. The results showed good correlation with the phosphorylation of STAT5 and serum concentration of Ab1. The approach for IL-7Rα occupancy and pSTAT5 measurements established in monkeys can be utilized in clinical trials for pharmacokinetic/pharmacodynamic evaluation of Ab1 effect in humans.

Investigation of the binding pocket of human hematopoietic prostaglandin (PG) D2 synthase (hH-PGDS): a tale of two waters.

The inhibition of hH-PGDS has been proposed as a potential target for the development of anti-allergic and anti-inflammatory drugs. Herein we describe our investigation of the binding pocket of this important enzyme and our observation that two water molecules bind to our inhibitors and the enzyme. A series of compounds were prepared to the probe the importance of the water molecules in determining the binding affinity of the inhibitors to the enzyme. The study provides insight into the binding requirements for the design of potent hH-PGDS inhibitors.

Human monoclonal antibodies to the insulin-like growth factor 1 receptor inhibit receptor activation and tumor growth in preclinical studies.

INTRODUCTION: The insulin-like growth factor type 1 (IGF-1) receptor contributes importantly to transformation and survival of tumor cells both in vitro and in vivo, and selective antagonists of the IGF-1 receptor (IGF-1R) activity represent an attractive experimental approach for human cancer therapy. METHODS: Using a phage display library, we identified several high-affinity fully human monoclonal antibodies with inhibitory activity against both human and rodent IGF.1Rs. RESULTS: These candidate therapeutic antibodies recognized several distinct epitopes and effectively blocked ligand-mediated receptor signal transduction and cellular proliferation in vitro. They also induced IGF-1R downregulation and catabolism following antibody-mediated endocytosis. These antibodies exhibited activity against human, primate, and rodent IGF-1Rs, and dose-dependently inhibited the growth of established human tumors in nude mice. CONCLUSION: These fully human antibodies therefore have the potential to provide an effective anti-tumor biological therapy in the human clinical setting.

Discovery of an Oral Potent Selective Inhibitor of Hematopoietic Prostaglandin D Synthase (HPGDS).

Hematopoietic prostaglandin D synthase (HPGDS) is primarly expressed in mast cells, antigen-presenting cells, and Th-2 cells. HPGDS converts PGH2 into PGD2, a mediator thought to play a pivotal role in airway allergy and inflammatory processes. In this letter, we report the discovery of an orally potent and selective inhibitor of HPGDS that reduces the antigen-induced response in allergic sheep.

Missing data on the Center for Epidemiologic Studies Depression Scale: a comparison of 4 imputation techniques.

BACKGROUND: Missing data are widespread in the medical sciences. Given their prevalence, researchers must be prepared to address problems that arise when data are missing. OBJECTIVES: The objectives were to (1) provide an estimate of bias for each imputation technique with known values from data engineered to be missing completely at random; (2) determine whether different Center for Epidemiologic Studies Depression (CES-D) Scale scores were obtained from item-mean, person-mean, regression, and hot-deck imputation techniques and whether they differed from the CES-D score obtained from complete cases; and (3) determine whether the variables that predicted the CES-D scores were the same for the complete cases and each of the 4 imputation techniques. METHODS: Depressive symptoms were assessed in patients (N=2,317) in an international clinical trial comparing high blood pressure treatments between April 1, 1999, and October 31, 1999. Patients were mailed surveys after randomization. Depressive symptoms were measured using the CES-D Scale. Respondents who completed all 20 items were compared with those who did not complete all 20 items, using independent t tests and chi-square. Z scores were used to determine CES-D mean differences, and multiple regression models were used to predict the CES-D scores for the 4 imputation techniques and the complete case data. RESULTS: Imputed CES-D mean scores ranged from 14.58 to 14.68. The 4 imputed CES-D mean scores were consistently, but not significantly, higher than the complete case CES-D mean of 14.06. Imputed mean scores were similar to each other and the complete case mean score. Four regression models predicting the imputed CES-D scores yielded similar predictions. With the exception of sex, the same variables predicted the complete case CES-D and the imputed CES-D scores. CONCLUSIONS: All the imputed means were similar to the complete case mean, with the exception of the regression imputation. Imputing missing data did not significantly alter the conclusions regarding which factors were associated with variations in CES-D scores. Since imputation has the potential to increase statistical power, researchers dealing with missing CES-D scores should consider imputing missing data.

Characterization of dendritic cells generated in vivo by an E. coli derived chimeric dual receptor agonist.

BACKGROUND: Progenipoietin-4 (ProGP-4) is an E. coli derived chimeric growth factor that activates the human Flt3 and G-CSF receptors. ProGP-4 possesses cross-species activity and treatment of mice with ProGP-4 results in increases in the number of WBC and Class II+/CD11c+ cells in both spleen and peripheral blood. Herein, we report morphologic, phenotypic and functional evaluation of Class II+/CD11c+ cells generated by in vivo administration of ProGP-4. MATERIAL/METHODS: C57BL/6 mice were injected daily with ProGP for 7 to 18 days. Leukocytes from spleen and peripheral blood were analyzed by flow cytometry to enumerate and characterize changes in DC populations. Spleens from ProGP treated mice were evaluated by immunocytochemistry and enriched CD11c+ populations were functionally assessed in a mixed lymphocyte assay and in an antigen dependent CTL assay. RESULTS: Administration of this dual receptor agonist to mice resulted in dose-dependent increases in the numbers of total white blood cells and Class II+/CD11c+ cells in spleen and peripheral blood. CD11c+ cells from ProGP-4 treated mice co-expressed DEC205 and also expressed CD80, CD86 and CD40, albeit at lower levels as compared to Class II+/CD11c+ cells from untreated animals. Despite lower co-stimulatory molecule expression, ProGP-4-generated Class II+/CD11c+ cells stimulated proliferation of allogeneic T cells and an antigen-specific T cell hybridoma as efficiently as bone marrow derived dendritic cells from untreated mice. CONCLUSIONS: The data presented in this report highlight the ability of E. coli derived ProGP-4 to expand large numbers of functional DC in the peripheral blood and lymphoid organs in vivo using a rodent model of hematopoiesis. E. coli derived chimeric receptor agonists such as ProGP-4 may enable further investigations of immunotherapeutic approaches to the treatment of diseases such as cancer and autoimmunity.