Peroxisome Proliferator–activated Receptors α and γ Down-regulate Allergic Inflammation and Eosinophil Activation

Allergic asthma is characterized by airway hyperresponsiveness, eosinophilia, and mucus accumulation and is associated with increased IgE concentrations. We demonstrate here that peroxisome proliferator–activated receptors (PPARs), PPAR-α and PPAR-γ, which have been shown recently to be involved in the regulation of various cell types within the immune system, decrease antigen-induced airway hyperresponsiveness, lung inflammation, eosinophilia, cytokine production, and GATA-3 expression as well as serum levels of antigen-specific IgE in a murine model of human asthma. In addition, we demonstrate that PPAR-α and -γ are expressed in eosinophils and their activation inhibits in vitro chemotaxis and antibody-dependent cellular cytotoxicity. Thus, PPAR-α and -γ (co)agonists might be of therapeutic interest for the regulation of allergic or inflammatory reactions by targeting both regulatory and effector cells involved in the immune response

Multiple immune abnormalities in tumor necrosis factor and lymphotoxin-α double-deficient mice

To investigate the roles of tumor necrosis factor (TNF) and lymphotoxin (LT)-α in the development and function of the immune system, the Tnf and Ltα genes were simultaneously inactivated in mice by homologous recombination. These mutant mice are highly susceptible to Listeria monocytogenes infection and resistant to endotoxic shock induced by the combined administration of D-galactosamine (D-GaIN) and lipopolysaccharide (LPS). Their splenic microarchitecture is disorganized, characterized by the loss of the clearly defined marginal zone, ill defined T and B cell areas, and absence of MAdCAM-1 and reduced ICAM-1, VCAM-1 and Mac-1 expression. They are devoid of peripheral lymph nodes and Peyer's patches, and show a strong reduction of lgA+ plasma cells in the intestinal lamina propria. The alymphoplasia is accompanied by a marked B lymphocytosis and reduced basal Ig levels. Ig depositions in the renal glomerulus and a strong up-regulation of MHC class I antigen expression on endothelial cells of different tissues are observed. The primary humoral immune response towards sheep red blood cells reveals a defective IgG isotype switch, while that against vescicular stomatitis virus is normal. The cytotoxic T cell responses are attenuated, although still effective, against vaccinia, lymphocytic choriomeningitis virus (LCMV-ARM) and LCMV-WE. In conclusion, the combined inactivation of Tnf and Ltα confirms their essential role in the normal development and function of the immune syste

Covalent binding of cyclosporine inhibits irreversibly T-lymphocyte activation

A diazirine derivative of cyclosporine (PL-CS) was used to photolabel recombinant human cyclophilin (rhCyp), the cytosolic receptor for the immunosuppressant cyclosporine. The affinity of PL-CS for rhCyp and the immunosuppressive activity were 10-fold reduced as compared to cyclosporine A. Whereas cyclosporine immunosuppression was fully reversible, UV cross-linking of PL-CS resulted in permanent inhibition of lymphocyte activation as shown by proliferation of anti-CD3 stimulated human peripheral lymphocyte, interleukin (IL)-2 gene transcription and IL-2 synthesis in the human T-leukemia cell line Jurkat. In vivo photolabeling of viable Jurkat cells revealed that a 21-kDa complex was the major radiolabeled product which was identified as a cyclophilin-cyclosporine complex. In addition, cyclophilin B (25 kDa) and proteins of an unidentified nature at 40, 46 and 60 kDa were observed in Jurkat cells. The cyclosporine-resistant human fibroblast cell line MRC5 displayed a different labeling pattern: cyclophilin B (25 kDa) and a 65-kDa protein were the major labeled products, while the 46- and 60-kDa components were not detectable and cyclophilin was only faintly labeled. In summary, covalent cyclosporine binding caused irreversible lymphocyte inactivation and revealed in addition to cyclophilin other specifically labeled proteins in lymphoid cells. The role and identity of these proteins is presently unknown

Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vivo

The immunosuppressant cyclosporine A (CSA) has been shown to bind to the ubiquitous cellular protein, cyclophilin, and to inhibit its rotamase activity. In the present study, 3H-cyclosporine diazirine analogue was used to photolabel viable human cells of lymphoid and fibroblast origin in order to identify the intracellular targets for the drug. While cyclophilin was strongly labeled in situ, additional minor cyclosporine-protein complexes of 25, 40, 46 and 60 kDa were identified in the T cell leukemia cell line Jurkat. These proteins bound specifically, since only active CSA but not inactive CSH or FK506 competed for binding. Photolabeling of MRC5 cells, a CSA resistant human fibroblast cell line, revealed a 25 kDa complex as the major product, while the 46 and 60 kDa bands were not detectable and cyclophilin labeling was only faint, even though both MRC5 and Jurkat cells contain similar cyclophilin concentrations. Thus, our data suggest that the intracellular targets of CSA and/or the accessibility to cyclophilin varies considerably in drug sensitive and resistant cell types, which may contribute to explaining the lymphocyte selectivity of the drug

Implementation of highly sophisticated flow cytometry assays in multi- center clinical studies: considerations and guidance

the item has no formal formal abstract as summary of the items the table of contents is given here: Table of contents 1 Introduction: 1.1 Technical overview and applications of flow cytometry 1.2 Use of flow cytometry for biomarker analysis in human clinical studies 2 Development and Validation of flow cytometry assays for clinical use 2.1 Summary of key challenges 2.2 Considerations for the Validation of PhosFlow assays used for PK/PD assessment 3 Whole blood flow cytometry assays in multi-center clinical studies: Immunophenotyping versus PhosFlow 3.1 On-site processing of whole blood samples 3.2 Stabilization of samples: stabilizing tubes or freezing of whole blood samples 3.3 Clinical study implementation 4 Summary and Future Perspectives

CSF-1 antibody Lacnotuzumab in a Phase 1 Healthy Volunteer Study and Mechanistic Investigation of Safety Outcomes

The colony-stimulating factor-1 (CSF-1) receptor pathway has been implicated in a variety of diseases, and CSF-1–dependent mechanisms are involved in bloodborne protein clearance. Lacnotuzumab is a novel, high-affinity, humanized, anti–CSF-1 monoclonal antibody that prevents CSF-1 mediated receptor activation. The presentedThis phase 1, two-part, double-blind study in healthy volunteers assessed the safety and tolerability of lacnotuzumab and its pharmacokinetic (PK) and pharmacodynamic properties. Part A (n = 36) was a single, ascending-dose assessment of eight lacnotuzumab doses (0.01 to 20 mg/kg); in part B (n = 16), lacnotuzumab was administered at either 5 or 10 mg/kg. In each study cohort, individuals were randomized 3:1 to lacnotuzumab or placebo. Lacnotuzumab was generally well tolerated. At higher doses (10 and 20 mg/kg), creatine kinase (CK) elevations (>5x the upper limit of normal, but asymptomatic and reversible) and mild transient periorbital swelling were reported. At all doses, theThe majority of adverse events (AEs) were low grade, no unexpected or novel AEs were observed, and there were no discontinuations for AEs. Serum-fFree/unbound lacnotuzumab serum concentration-time profiles showed nonlinear PK s across doses from 0.01–20 mg/kg, with faster apparent elimination at lower doses or concentrations; this was consistent with apparent target-mediated drug disposition. Lacnotuzumab also showed dose-dependent, on-target effects on multiple downstream biomarkers. Preclinical investigations of the CK elevation and periorbital swelling observed after lacnotuzumab administration suggest that these are reversible, non-pathological events linked to inhibition of the CSF-1 pathway. These data support further evaluation of lacnotuzumab in clinical studies