Michelle Makiya

The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan

Neutrophils, eosinophils and ‘classical’ monocytes collectively account for about 70% of human blood leukocytes and are among the shortest-lived cells in the body. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses and minimize the deleterious consequences of prolonged inflammation. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here we identify a long non-coding RNA that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. To control the lifespan of these cells, Morrbid regulates the transcription of the neighbouring pro-apoptotic gene, Bcl2l11 (also known as Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in individuals with hypereosinophilic syndrome, this long non-coding RNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.

Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor

Significance This study demonstrated that cross-neutralizing anti-poliovirus antibodies bind the site on poliovirus capsid surface that significantly overlaps the binding site of the cellular receptor. A second antibody with similar specificity was isolated by sequential phage display panning, suggesting that cross-reactive anti-poliovirus antibodies may be more prevalent in primates than previously recognized. Binding to the receptor recognition site explains unusually broad specificity of the antibodies. The antibodies bind type 1 and type 2 polioviruses at a slightly different angle, indicating that molecular details of virus–antibody interaction are different and suggesting that further screening or engineering may produce an antibody neutralizing all three serotypes of poliovirus. These results may be used for developing new antiviral strategies for the polio eradication campaign. Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus.

Chimpanzee-Human Monoclonal Antibodies for Treatment of Chronic Poliovirus Excretors and Emergency Postexposure Prophylaxis

ABSTRACT Six poliovirus-neutralizing Fabs were recovered from a combinatorial Fab phage display library constructed from bone marrow-derived lymphocytes of immunized chimpanzees. The chimeric chimpanzee-human full-length IgGs (hereinafter called monoclonal antibodies [MAbs]) were generated by combining a chimpanzee IgG light chain and a variable domain of heavy chain with a human constant Fc region. The six MAbs neutralized vaccine strains and virulent strains of poliovirus. Five MAbs were serotype specific, while one MAb cross-neutralized serotypes 1 and 2. Epitope mapping performed by selecting and sequencing antibody-resistant viral variants indicated that the cross-neutralizing MAb bound between antigenic sites 1 and 2, thereby covering the canyon region containing the receptor-binding site. Another serotype 1-specific MAb recognized a region located between antigenic sites 2 and 3 that included parts of capsid proteins VP1 and VP3. Both serotype 2-specific antibodies recognized antigenic site 1. No escape mutants to serotype 3-specific MAbs could be generated. The administration of a serotype 1-specific MAb to transgenic mice susceptible to poliovirus at a dose of 5 μg/mouse completely protected them from paralysis after challenge with a lethal dose of wild-type poliovirus. Moreover, MAb injection 6 or 12 h after virus infection provided significant protection. The MAbs described here could be tested in clinical trials to determine whether they might be useful for treatment of immunocompromised chronic virus excretors and for emergency protection of contacts of a paralytic poliomyelitis case.

Karyopherin alpha 1 is a putative substrate of the RAG1 ubiquitin ligase

The RAG1 recombinase, which participates in DNA manipulation during rearrangement of antigen receptor genes in developing immune cells, possesses ubiquitin ligase activity. The nuclear transport protein karyopherin alpha 1 (KPNA1) binds to RAG1 upstream of its ubiquitin ligase domain, but this interaction is not required for nuclear localization of RAG1. We found that the isolated ubiquitin ligase domain of RAG1 (amino acids 218-389) promoted ubiquitylation of purified KPNA1. While RAG1 auto-ubiquitylation is dependent on the ubiquitin conjugating enzyme CDC34, ubiquitylation of KPNA1 was best supported by UbcH2/Rad6 and UbcH5a. Ubiquitylation of KPNA1 required the lysine/arginine-rich region spanning RAG1 amino acids 218-263 upstream of the RAG1 ubiquitin ligase domain, but RAG1 was still able to undergo auto-ubiquitylation in this region even in the presence of KPNA1. This is the first putative substrate identified for the RAG1 ubiquitin ligase, and to our knowledge it is the first reported case of ubiquitylation of KPNA1.

Eosinophil-Associated Processes Underlie Differences in Clinical Presentation of Loiasis Between Temporary Residents and Those Indigenous to Loa-Endemic Areas

BACKGROUND Loa loa has emerged as an important public health problem due to the occurrence of immune-mediated severe posttreatment reactions following ivermectin distribution. Also thought to be immune-mediated are the dramatic differences seen in clinical presentation between infected temporary residents (TR) and individuals native to endemic regions (END). METHODS All patients diagnosed with loiasis at the National Institutes of Health between 1976 and 2012 were included. Patients enrolled in the study underwent a baseline clinical and laboratory evaluation and had serum collected and stored. Stored pretreatment serum was used to measure filaria-specific antibody responses, eosinophil-related cytokines, and eosinophil granule proteins. RESULTS Loa loa infection in TR was characterized by the presence of Calabar swelling (in 82% of subjects), markedly elevated eosinophil counts, and increased filaria-specific immunoglobulin G (IgG) levels; these findings were thought to reflect an unmodulated immune response. In contrast, END showed strong evidence for immune tolerance to the parasite, with high levels of circulating microfilariae, few clinical symptoms, and diminished filaria-specific IgG. The striking elevation in eosinophil counts among the TR group was accompanied by increased eosinophil granule protein levels (associated with eosinophil activation and degranulation) as well as elevated levels of eosinophil-associated cytokines. CONCLUSIONS These data support the hypothesis that differing eosinophil-associated responses to the parasite may be responsible for the marked differences in clinical presentations between TR and END populations with loiasis.

Development of a suspension array assay in multiplex for the simultaneous measurement of serum levels of four eosinophil granule proteins

The concentrations of major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO) have been associated with eosinophilic disease severity. Whereas a variety of techniques have been used to measure individual eosinophil granule protein concentration, none of these methods efficiently measures MBP, ECP, EDN and EPO simultaneously. A multiplex suspension array system was developed to simultaneously measure the concentrations of MBP, ECP, EDN and EPO in serum. The assay showed excellent inter- and intra-assay reliability, and serum levels of MBP, ECP and EDN from eosinophilic subjects analyzed by ELISA and multiplex were highly correlated (r=0.8579; P<0.0001, r=0.6356; P=0.0006 and r=0.8600; P<0.0001, respectively, Spearman rank correlation). Moreover, the multiplex assay required 500-fold less serum than a single ELISA to achieve comparable sensitivity. Absolute eosinophil count and eosinophil surface expression of the activation marker, CD69, were significantly correlated with concentrations of MBP, EDN and EPO, but not ECP, in serum from eosinophilic subjects. Furthermore, subjects with eosinophilic gastrointestinal disorder and normal peripheral absolute eosinophil counts (<0.5×10(9)/l) had significantly increased concentrations of MBP (P<0.0001), ECP (P<0.0001), EDN (P=0.0001) and EPO (P<0.0001) compared to normal donors. In summary, the eosinophil granule protein multiplex assay provides a rapid and reliable way to measure eosinophil granule protein levels and should prove useful in assessing patterns of degranulation in patients with eosinophilic disorders.

Somatic STAT5b gain-of-function mutations in early onset nonclonal eosinophilia, urticaria, dermatitis, and diarrhea

To the editor: Somatic, gain-of-function mutations in STAT3 and STAT5 have been described in leukemias and lymphomas[1][1][⇓][2][⇓][3][⇓][4][⇓][5]-[6][6] and, more recently, in CD3-CD4+ T cells from a patient with lymphocytic variant hypereosinophilic syndrome[7][7]; whereas germ line gain-

Clinical and Biological Markers in Hypereosinophilic Syndromes

Hypereosinophilic syndromes (HES) are rare, heterogeneous syndromes characterized by markedly elevated eosinophil counts in the blood and/or tissue and evidence of eosinophil-associated pathology. Although parasitic infections, drug hypersensitivity, and other disorders of defined etiology can present as HES (associated HES), treatment is directed at the underlying cause rather than the eosinophilia itself. A number of additional subtypes of HES have been described, based on clinical and laboratory features. These include (1) myeloid HES—a primary disorder of the myeloid lineage, (2) lymphocytic variant HES—eosinophilia driven by aberrant or clonal lymphocytes secreting eosinophil-promoting cytokines, (3) overlap HES—eosinophilia restricted to a single organ or organ system, (4) familial eosinophilia—a rare inherited form of HES, and (5) idiopathic HES. Since clinical manifestations, response to therapy, and prognosis all differ between HES subtypes, this review will focus on clinical and biological markers that serve as markers of disease activity in HES (excluding associated HES), including those that are likely to be useful only in specific clinical subtypes.

Structural basis of anthrax edema factor neutralization by a neutralizing antibody.

Fine epitope mapping of EF13D, a highly potent neutralizing monoclonal antibody specific for the anthrax edema factor (EF), was accomplished through random mutagenesis and yeast surface display. A yeast-displayed library of single point mutants of an EF domain III (DIII), comprising amino acids 624-800, was constructed by random mutagenesis and screened for reduced binding to EF13D. With this method, residues Leu 667, Ser 668, Arg 671, and Arg 672 were identified as key residues important for EF13D binding. They form a contiguous patch on a solvent-exposed surface at one end of the four-helix bundle of DIII. Computational protein-protein docking experiments between anEF13D model and a crystal structure of EF indicate that the EF13D heavy chain complementarity-determining region 3 (HCDR3) is deeply buried within a hydrophobic cleft between two helices of DIII and interacts directly with residues Leu 667, Ser 668, Arg 671 and Arg 672, providing an explanation for the high binding affinity. In addition, they show that the HCDR3 binding site overlaps with the binding site of the N-terminal lobe of calmodulin (CaM), an EF enzymatic activator, consistent with a previous finding showing direct competition with CaM that results in neutralization of EF. Identifying the neutralization epitope of EF13D on EF improves our understanding of the neutralization mechanism and has implications for vaccine development.

Dexpramipexole as an oral steroid-sparing agent in hypereosinophilic syndromes

Hypereosinophilic syndromes (HESs) are a heterogeneous group of disorders characterized by peripheral eosinophilia and eosinophil-related end organ damage. Whereas most patients respond to glucocorticoid (GC) therapy, high doses are often necessary, and side effects are common. Dexpramipexole (KNS-760704), an orally bioavailable synthetic aminobenzothiazole, showed an excellent safety profile and was coincidentally noted to significantly decrease absolute eosinophil counts (AECs) in a phase 3 trial for amyotrophic lateral sclerosis. This proof-of-concept study was designed to evaluate dexpramipexole (150 mg orally twice daily) as a GC-sparing agent in HESs. Dual primary end points were (1) the proportion of subjects with ≥50% decrease in the minimum effective GC dose (MED) to maintain AEC <1000/μL and control clinical symptoms, and (2) the MED after 12 weeks of dexpramipexole (MEDD) as a percentage of the MED at week 0. Out of 10 subjects, 40% (95% confidence interval [CI], 12%, 74%) achieved a ≥50% reduction in MED, and the MEDD/MED ratio was significantly <100% (median, 66%; 95% CI, 6%, 98%; P = .03). All adverse events were self-limited, and none led to drug discontinuation. Affected tissue biopsy samples in 2 subjects showed normalization of pathology and depletion of eosinophils on dexpramipexole. Bone marrow biopsy samples after 12 weeks of dexpramipexole showed selective absence of mature eosinophils in responders. Dexpramipexole appears promising as a GC-sparing agent without apparent toxicity in a subset of subjects with GC-responsive HESs. Although the exact mechanism of action is unknown, preliminary data suggest that dexpramipexole may affect eosinophil maturation in the bone marrow. This study was registered at www.clinicaltrials.gov as #NCT02101138.