Using a cohort of more than 350 subjects with a wide variety of eosinophilic disorders, ranging from benign eosinophilia to eosinophilic leukemia, we have continued to identify and characterize novel subgroups of patients with eosinophilia. Episodic Angioedema with Eosinophilia (EAE; Gleichs Syndrome) is a rare disorder characterized by episodes of angioedema and eosinophilia that occur at monthly intervals and resolve spontaneously without therapy. To characterize the involvement of lineages other than eosinophils in the etiology and pathogenesis of EAE, nine subjects with suspected EAE were evaluated at the NIH. Cycling of multiple lineages, including neutrophils, lymphocytes and eosinophils, was observed in 5 subjects with EAE with a periodicity of 25-35 days. An aberrant CD3-CD4+ T cell population was detected in all 5 subjects, and TCR rearrangement studies showed a clonal pattern in 3 subjects. A peak of type II cytokines was detected in the serum of EAE subjects prior to onset of symptoms and eosinophil cycling and corresponded to ex-vivo type II cytokines detected intracellularly in CD3+CD4+CD154+ T cells. Although the etiology of EAE is not yet known, multiple lineages, including lymphocytes, neutrophils and mast cells, are involved and may be related to disease pathogenesis. Whether these cells act directly or promote eosinophilia and eosinophil activation remains to be elucidated. Other subgroups of eosinophilic subjects currently under study include families with autosomal dominant eosinophilia, pediatric patients, and a cohort of subjects with eosinophilic hepatitis. Therapy for eosinophilic disorders remains a primary focus of our group. In the past year, we have initiated two new clinical trials for the treatment of HES. The first of these is a placebo-controlled, double-blind trial of benralizumab (MedImmune/Astra Zeneca), an afucosylated antibody to IL-5 receptor alpha that has shown clinical efficacy in patients with eosinophilic asthma. The current trial stems, in part, from our prior pre-clinical work examining IL-5 receptor levels in patients with eosinophilia and/or mastocytosis (Wilson et al. J Allergy Clin Immunol 2011). To date 6 of 20 planned subjects have been enrolled. The second trial is an open-label trial of dexpramipexole (Knopp Biosciences) in patients with steroid-resistant HES. Dexpramipexole was developed for the treatment of amyotrophic lateral sclerosis (ALS). Although it was well tolerated, dexpramipexole failed to show efficacy in a large phase 3 trial for this disease. It did, however, appear to selectively lower blood eosinophils, which prompted the current proof-of-concept trial in HES. To date, 4 of the 10 planned subjects have been enrolled. In addition to the clinical trials described above, we have analyzed data from an ongoing clinical trial of imatinib in subjects with FIP1L1/PDGFRA-positive myeloproliferative neoplasm (FP), PDGFRA-negative HES with myeloproliferative features (MP) and steroid-resistant without myeloproliferative features (SR) (Khoury et al. presented at the annual meeting of the International Eosinophil Society; manuscript submitted). Overall, imatinib response rates were 100% in the FP group (n= 16), 50% in the MP group (n=13) and 0% in the SR group (n=16). The presence of ≥ 4 myeloproliferative features was the sole predictor of response. After ≥ 18 months in complete remission, imatinib was tapered and discontinued in 7 FP and 1 MP subjects. Five subjects remain in remission off therapy for a median of 13 months (range 2-21 months). We have also continued to enroll subjects in a double-blind clinical trial designed to assess the utility of a glucocorticoid challenge in determination of glucocorticoid responsiveness in HES and in a clinical trial of mepolizumab (humanized anti-IL-5 antibody) for treatment-refractory HES. We have also begun to explore the therapeutic potential of several new antibodies with the potential to target eosinophils, including antibodies to the eosinophil specific surface receptor, EMR1 (Legrand J et al. Allergy Clin Immunol, 2014), and the inhibitory surface receptor, siglec-8. In a pre-clinical study, analysis of blood and bone marrow cells from healthy and eosinophilic donors and in vitro-differentiated CD34(+) cells confirmed restriction of human EMR1 surface and mRNA expression to mature eosinophils. Tissue eosinophils also expressed EMR1. Although EMR1 was highly expressed on eosinophils from all subjects, surface expression was negatively correlated with absolute eosinophil counts (r = -0.46, P < .001), and soluble plasma levels correlated positively with absolute eosinophil counts (r = 0.69, P < .001), suggesting modulation of EMR1 in vivo. Nevertheless, afucosylated anti-EMR1 mAb (Kalobios, Inc.) dramatically enhanced natural killer cell-mediated killing of eosinophils from healthy and eosinophilic donors and induced a rapid and sustained depletion of eosinophils in monkeys. Helminth infections can associated with dramatic eosinophilia and complications that are indistinguishable from HES. This is particularly true in loiasis where symptoms are associated with increased eosinophil granule protein levels (indicative of eosinophil activation and degranulation) as well as elevated levels of eosinophil-associated cytokines (Herrick et al. Clin Inf Dis, in press). In some infections, eosinophilia and eosinophil-related symptoms can be exacerbated by effective treatment. For example, treatment of the filarial infection loiasis with diethylcarbamazine (DEC) can be associated with severe adverse events, including death, that are accompanied by a dramatic rise in eosinophilia and are positively correlated with the number of circulating microfilariae in the blood. In the past year, we have continued our double-blind, placebo-controlled study of the anti-IL-5 antibody, reslizumab, to prevent post-treatment side effects of DEC in patients with Loa loa infection. Because similar side effects have been seen in Loa infected patients during mass administration of ivermectin for onchocerciasis control, we also conducted a comparative study of post-treatment reactions following single dose ivermectin or single dose DEC. Although clinical symptoms were similar between the two groups post-treatment, there was a significant difference in peak percentage eosinophil change (GM change + 1.865 in DEC group versus + 2.572 in IVM group, p <.05) and the pattern of post-treatment neutrophil count between the two groups. The microfilarial counts decreased dramatically in both groups following therapy, but mf were cleared more effectively in the DEC group. Cytokine and chemokine profiles also suggest differences in the immunologic responses following the two drugs. Finally, in the past year, we have continued to look for biomarkers of eosinophilic disease activity for use in monitoring responses to therapy and helping to dissect out the mechanisms of eosinophil pathogenesis. To this end, a multiplex suspension array system was developed to simultaneously measure the concentrations of 4 eosinophil granule proteins (MBP, ECP, EDN and EPO) in serum and other biological fluids and culture supernatants (Makiya et al. J Immunol Methods 2014).