CXCR4 is a G protein-coupled chemokine receptor that has been implicated in the pathogenesis of primary immunodeficiency disorders and cancer. Autosomal dominant gain-of-function truncations of CXCR4 are associated with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a primary immunodeficiency disorder characterized by neutropenia and recurrent infections. Recent progress has implicated CXCR4-SDF1 (stromal cell-derived factor 1) signaling in regulating neutrophil homeostasis, but the precise role of IntroductionStromal cell-derived factor 1 (SDF1, CXCL12)-mediated activation 1 of the chemokine receptor CXCR4 is important for both normal and pathologic processes, including primordial germ cell migration, HIV pathogenesis, invasive migration of cancer cells, and leukocyte trafficking. 2-5 Therefore, there is substantial interest in understanding how CXCR4-SDF1 signaling regulates cell motility and how these mechanisms can be targeted to treat human disease. CXCR4 signaling is attenuated by receptor internalization, which is regulated by phosphorylation events and binding of regulatory proteins to the cytoplasmic tail. 6 The functional importance of CXCR4 internalization is highlighted by the dominantly inherited primary immunodeficiency, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, in which truncations of CXCR4 lead to altered signaling and gain of function. [7][8][9] WHIM syndrome is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis, a severe chronic neutropenia. 10,11 Substantial evidence supports the importance of CXCR4 signaling in regulating neutrophil homeostasis and release from the bone marrow (BM). 5 It has been postulated that the neutropenia in patients with WHIM syndrome results from both neutrophil retention in the BM and enhanced neutrophil apoptosis of retained neutrophils. 11 Direct evidence to support this hypothesis has been provided by a mouse model of WHIM syndrome induced by the ectopic expression of WHIM truncation mutations of CXCR4 in hematopoietic stem cells that show impaired neutrophil release into the blood and increased rates of apoptosis in the BM. 12 Previous reports indicate that neutrophils from patients with WHIM show increased signaling 13 and chemotaxis 8,9 in response to SDF1. However, some reports have suggested that the C-terminus of CXCR4 can both positively and negatively regulate cell motility 8,9,14 and, alternatively, may be involved in modulating the precise targeting of cells in vivo. 15 Despite the importance of CXCR4-SDF1 signaling, few animal models of WHIM syndrome are amenable to imaging or screening for drugs that modulate CXCR4-SDF1 function in vivo. Modeling WHIM syndrome is particularly attractive because CXCR4 signaling is important to many disease processes and is a direct result of aberrant chemokine signaling. Therefore, developing a model of WHIM syndrome in a system that allows the direct visualization of motility and chemotactic events in vivo would be a beneficial ...

Purpose-To evaluate the safety, toxicity, in vivo immunologic activation, and maximum-tolerated dose (MTD) of EMD 273063 (hu14.18-IL-2) in patients with metastatic melanoma.Patients and Methods-Thirty-three patients were treated with EMD 273063, a humanized anti-GD2 monoclonal antibody (mAb) linked to interleukin-2 (IL-2). EMD 273063 was given as a 4-hour intravenous infusion on days 1, 2, and 3 of week 1. Patients with stabilization or regression of disease could receive a second course of treatment at week 5. Dose levels evaluated were 0.8, 1.6, 3.2, 4.8, 6.0, and 7.5 mg/m 2 /d.Results-Nineteen of 33 patients completed course 1 with stable disease and went on to receive course 2. Eight patients had stable disease on completion of course 2. Grade 3 adverse events included hypophosphatemia (11 patients), hyperglycemia (three patients), hypotension (two patients), thrombocytopenia (one patient), hypoxia (three patients), elevated hepatic transaminases (two patients), and hyperbilirubinemia (one patient). Opioids were required for treatment-associated arthralgias and/or myalgias during 17 of 52 treatment courses. No grade 4 adverse events were observed. Dose-limiting toxicities at the MTD included hypoxia, hypotension, and elevations in AST/ ALT. Grade 3 toxicities were anticipated based on prior studies of IL-2 or anti-GD2 mAbs, and all resolved. Immune activation was induced, as measured by lymphocytosis, increased peripheral-blood natural killer activity, and cell numbers, and increased serum levels of the soluble alpha chain of the IL-2 receptor complex. Authors' disclosures of potential conflicts of interest are found at the end of this article. Authors' Disclosures of Potential Conflicts of InterestThe following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Acted as a consultant within the last 2 years: Ralph Reisfeld, EMD. Served as an officer or member of the Board of a company: Stephen D. Gillies, EMD. Received more than $2,000 per year from a company for either of the last 2 years: Stephen D. Gillies, EMD.Supported by grant Nos. CA32685, CA14520, CA87025, CA81403, and RR03186 from the National Institutes of Health and a grant from the Midwest Athletes for Childhood Cancer Fund. Partial personnel support was provided by EMD for data management required by EMD for this study, which was beyond the clinical research and data monitoring required for this National Cancer Institute-supported study. NIH Public Access

No abstract

Chemotaxis assays are essential tools for the study of gradient sensing and directed cell migration, and have the potential to aid in the diagnosis and characterization of patients with immune disorders. Current methods are limited in their ability to meet the more demanding requirements for clinical applications. Because patient samples have a short lifespan and sometimes a limited volume (e.g. pediatrics), the operational requirements for an efficient chemotaxis assay are increased in the clinical setting. Here we describe a microscale assay platform for gradient generation that overcomes these limitations. Passive fluidic methods are leveraged to provide a reliable microfluidic gradient generation device, operable in only three pipetting steps. In addition, arrayed imaging and advanced cell tracking algorithms enabled a 50-fold increase in throughput over current methods. These methods were employed to aid in the diagnostic evaluation of an infant who presented with severe, recurrent bacterial infections. Analysis of the infant's neutrophils revealed impaired cell polarization and chemotaxis in a gradient of the chemoattractant fMLP. The patient was subsequently diagnosed with an inhibitory mutation in the Rho GTPase, Rac2. The approach also enabled a microenvironmental screen of human primary neutrophil chemotaxis on fibronectin, fibrinogen and laminin with results suggesting that fibronectin, although commonly used, may not be the most appropriate matrix protein for chemotaxis assays. Together, these findings demonstrate the use of arrayed micro-devices to aid in the diagnosis of a primary immunodeficiency disorder, and illustrate the capability for increased throughput microenvironmental studies and screening targeted to specific human diseases.