Kelly D. Stone

Cold Urticaria, Immunodeficiency, and Autoimmunity Related to PLCG2 Deletions

BACKGROUND Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. METHODS We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. RESULTS Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. CONCLUSIONS Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Hematopoietic prostaglandin D synthase defines a proeosinophilic pathogenic effector human TH2 cell subpopulation with enhanced function

BACKGROUND IL-5(+) pathogenic effector T(H)2 (peT(H)2) cells are a T(H)2 cell subpopulation with enhanced proinflammatory function that has largely been characterized in murine models of allergic inflammation. OBJECTIVE We sought to identify phenotype markers for human peT(H)2 cells and characterize their function in patients with allergic eosinophilic inflammatory diseases. METHODS Patients with eosinophilic gastrointestinal disease (EGID), patients with atopic dermatitis (AD), and nonatopic healthy control (NA) subjects were enrolled. peT(H)2 and conventional T(H)2 (cT(H)2) cell phenotype, function, and cytokine production were analyzed by using flow cytometry. Confirmatory gene expression was measured by using quantitative RT-PCR. Prostaglandin D2 levels were measured with ELISA. Gut T(H)2 cells were obtained by means of esophagogastroduodenoscopy. RESULTS peT(H)2 cells were identified as chemoattractant receptor-homologous molecule expressed on T(H)2 cells-positive (CRTH2(+)), hematopoietic prostaglandin D synthase-positive CD161(hi) CD4 T cells. peT(H)2 cells expressed significantly greater IL-5 and IL-13 than did hematopoietic prostaglandin D synthase-negative and CD161(-) cT(H)2 cells. peT(H)2 cells were highly correlated with blood eosinophilia (r = 0.78-0.98) and were present in 30- to 40-fold greater numbers in subjects with EGID and those with AD versus NA subjects. Relative to cT(H)2 cells, peT(H)2 cells preferentially expressed receptors for thymic stromal lymphopoietin, IL-25, and IL-33 and demonstrated greater responsiveness to these innate pro-TH2 cytokines. peT(H)2 but not cT(H)2 cells produced prostaglandin D2. In patients with EGID and those with AD, peT(H)2 cells expressed gut- and skin-homing receptors, respectively. There were significantly greater numbers of peT(H)2 cells in gut tissue from patients with EGID versus NA subjects. CONCLUSION peT(H)2 cells are the primary functional proinflammatory human T(H)2 cell subpopulation underlying allergic eosinophilic inflammation. The unambiguous phenotypic identification of human peT(H)2 cells provides a powerful tool to track these cells in future pathogenesis studies and clinical trials.

Immunomodulatory therapy of eosinophil-associated gastrointestinal diseases.

Eosinophil‐associated gastrointestinal disorders (EGIDs), including eosinophilic esophagitis (EE) and eosinophilic gastroenteritis (EG), are a spectrum of increasingly recognized inflammatory diseases characterized by gastrointestinal symptoms and eosinophilic infiltration of the gastrointestinal tract. Significant morbidity is associated with the development of esophageal strictures in some patients. Immune‐mediated reactions to food allergens appear to drive the inflammation in a subset of patients, especially those with solitary EE, but dietary interventions remain difficult in EE and are less effective in EG. Despite the increasing incidence of these disorders and their increased recognition by physicians, there are currently no medications that either United States or European Union regulatory agencies have specifically approved for use in EGIDs. This lack of safe and effective therapies for EGIDs is a major obstacle in the care of these patients and underscores the need for new therapeutic approaches. This review briefly discusses the currently available ‘off label’ drug treatments for EGIDs, most notably topical and systemic corticosteroids. Pathogenesis studies of EGIDs suggest possible therapeutic targets, and conversely, clinical trials of mechanistically‐targeted therapeutics give insight into disease pathogenesis. Thus, EGID pathogenesis is discussed as an introduction to mechanistically‐targeted immunotherapeutics. The two biologic categories that have been used in EGIDs, anti‐IgE (omalizumab) and anti‐IL‐5 (SCH55700/reslizumab and mepolizumab), are discussed. Because there are similarities in the pathogenesis of EGIDs with asthma and atopic dermatitis, biologic therapeutics currently in early trials for asthma management are also briefly discussed as potential therapeutic agents for EGIDs. Given the deficiencies of current therapeutics and the rapidly advancing knowledge of the pathogenesis of these disorders, EGIDs are an ideal model for translating recent advances in understanding immunopathogenesis into mechanistically‐based therapeutics. Further understanding of the early events in pathogenesis is also needed to develop preventive and disease‐modifying treatments.

Inhibition of TLR Activation and Up-Regulation of IL-1R-Associated Kinase-M Expression by Exogenous Gangliosides

Gangliosides, sialic acid-containing glycosphingolipids present in the outer leaflet of plasma membranes, are produced at high levels by some tumors, are actively shed into the tumor microenvironment, and can be detected in high concentrations in the serum of cancer patients. These tumor-shed molecules are known to be immunosuppressive, although mechanisms remain to be fully elucidated. In this study, we show that membrane enrichment of human monocytes with purified exogenous gangliosides potently inhibits ligand-induced activation and proinflammatory cytokine production induced by a broad range of TLRs, including TLR2, TLR3, TLR6, and TLR7/8, in addition to a previously identified inhibitory effect on TLR4 and TLR5. Inhibition of TLR activation is reversible, with complete restoration of TLR signaling within 6–24 h of washout of exogenous gangliosides, and is selective for certain gangliosides (GM1, GD1a, and GD1b), whereas others (GM3) are inactive. To characterize the inhibition, we assessed the expression of the TLR signaling pathway inhibitor, IL-1 receptor associated kinase-M (IRAK-M). In response to ganglioside enrichment alone, we observed striking up-regulation of IRAK-M in monocytes, but without concomitant proinflammatory cytokine production. This contrasts with endotoxin tolerance, in which IRAK-M up-regulation follows proinflammatory cytokine expression caused by LPS exposure. We hypothesize that ganglioside treatment induces a state of tolerance to TLR signaling, leading to blunted activation of innate immune responses. In the tumor microenvironment, shed tumor ganglioside enrichment of APC membranes may likewise cause these cells to bypass the normal TLR signaling response and progress directly to the inhibitory state.

Elevated basal serum tryptase identifies a multisystem disorder associated with increased TPSAB1 copy number

Elevated basal serum tryptase levels are present in 4–6% of the general population, but the cause and relevance of such increases are unknown. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase–encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.

Atopic Dermatitis in Children: Clinical Features, Pathophysiology, and Treatment

Atopic dermatitis (AD) is a chronic, relapsing, highly pruritic skin condition resulting from disruption of the epithelial barrier and associated immune dysregulation in the skin of genetically predisposed hosts. AD generally develops in early childhood, has a characteristic age-dependent distribution and is commonly associated with elevated IgE, peripheral eosinophilia, and other allergic diseases. Medications such as antihistamines have demonstrated poor efficacy in controlling AD-associated itch. Education of patients regarding the primary underlying defects and provision of a comprehensive skin care plan is essential for disease maintenance and management of flares.

Transplantation of human skin microbiota in models of atopic dermatitis

Atopic dermatitis (AD) is characterized by reduced barrier function, reduced innate immune activation, and susceptibility to Staphylococcus aureus. Host susceptibility factors are suggested by monogenic disorders associated with AD-like phenotypes and can be medically modulated. S. aureus contributes to AD pathogenesis and can be mitigated by antibiotics and bleach baths. Recent work has revealed that the skin microbiome differs significantly between healthy controls and patients with AD, including decreased Gram-negative bacteria in AD. However, little is known about the potential therapeutic benefit of microbiome modulation. To evaluate whether parameters of AD pathogenesis are altered after exposure to different culturable Gram-negative bacteria (CGN) collected from human skin, CGN were collected from healthy controls and patients with AD. Then, effects on cellular and culture-based models of immune, epithelial, and bacterial function were evaluated. Representative strains were evaluated in the MC903 mouse model of AD. We found that CGN taken from healthy volunteers but not from patients with AD were associated with enhanced barrier function, innate immunity activation, and control of S. aureus. Treatment with CGN from healthy controls improved outcomes in a mouse model of AD. These findings suggest that a live-biotherapeutic approach may hold promise for treatment of patients with AD.

Asthma morbidity among inner-city adolescents receiving guidelines-based therapy: role of predictors in the setting of high adherence.

BACKGROUND With the expanding effort to provide guidelines-based therapy to adolescents with asthma, attention must be directed to evaluating which factors predict future asthma control when guidelines-based management is applied. OBJECTIVE We evaluated the role of fraction of exhaled nitric oxide in parts per billion, markers of allergic sensitization, airway inflammation, and measures of asthma severity in determining future risk of asthma symptoms and exacerbations in adolescents and young adults participating in the Asthma Control Evaluation study. METHODS Five hundred forty-six inner-city residents, ages 12 through 20 years, with persistent asthma were extensively evaluated at study entry for predictors of future symptoms and exacerbations over the subsequent 46 weeks, during which guidelines-based, optimal asthma management was offered. Baseline measurements included fraction of exhaled nitric oxide in parts per billion, total IgE, allergen-specific IgE, allergen skin test reactivity, asthma symptoms, lung function, peripheral blood eosinophils, and, for a subset, airway hyperresponsiveness and sputum eosinophils. RESULTS The baseline characteristics we examined accounted for only a small portion of the variance for future maximum symptom days and exacerbations--11.4% and 12.6%, respectively. Future exacerbations were somewhat predicted by asthma symptoms, albuterol use, previous exacerbations, and lung function, whereas maximum symptom days were predicted, also to a modest extent, by symptoms, albuterol use, and previous exacerbations, but not lung function. CONCLUSION Our findings demonstrate that the usual predictors of future disease activity have little predictive power when applied to a highly adherent population with persistent asthma that is receiving guidelines-based care. Thus, new predictors need to be identified that will be able to measure the continued fluctuation of disease that persists in highly adherent, well-treated populations such as the one studied.

Germline hypomorphic CARD11 mutations in severe atopic disease

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor–induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

Matched Related and Unrelated Donor Hematopoietic Stem Cell Transplantation for DOCK8 Deficiency

We performed allogeneic hematopoietic stem cell transplantation in 6 patients with mutations in the dedicator-of-cytokinesis-8 (DOCK8) gene using a myeloablative conditioning regimen consisting of busulfan 3.2 mg/kg/day i.v. for 4 days and fludarabine 40 mg/m(2)/day for 4 days. Three patients received allografts from matched related donors and 3 patients from matched unrelated donors. Two patients received peripheral blood stem cells and 4 patients bone marrow hematopoietic stem cells. Tacrolimus and short-course methotrexate on days 1, 3, 6, and 11 were used for graft-versus-host-disease (GVHD) prophylaxis. All 6 patients are alive at a median follow-up of 22.5 months (range, 14 to 35). All patients achieved rapid and high levels of donor engraftment and complete reversal of the clinical and immunologic phenotype. Adverse events consisted of acute skin GVHD in 2 patients and post-transplant pulmonary infiltrates in a patient with extensive bronchiectasis pretransplant. Thus, a uniform myeloablative conditioning regimen followed by allogeneic hematopoietic stem cell transplantation in DOCK8 deficiency results in reconstitution of immunologic function and reversal of the clinical phenotype with a low incidence of regimen-related toxicity.