Ann Georgelas

Mepolizumab as a corticosteroid-sparing agent in lymphocytic variant hypereosinophilic syndrome

BACKGROUND Mepolizumab, a monoclonal anti-IL-5 antibody, is an effective corticosteroid-sparing agent for patients with Fip1-like 1/platelet-derived growth factor receptor α fusion (F/P)-negative hypereosinophilic syndrome (HES). Lymphocytic variant hypereosinophilic syndrome (L-HES) is characterized by marked overproduction of IL-5 by dysregulated T cells. OBJECTIVE To determine whether patients with L-HES respond to mepolizumab in terms of corticosteroid tapering and eosinophil depletion to the same extent as corticosteroid-responsive F/P-negative patients with HES and a normal T-cell profile. METHODS Patients enrolled in the mepolizumab trial were evaluated for L-HES on the basis of T-cell phenotyping and T-cell receptor gene rearrangement patterns, and their serum thymus-and-activation-regulated chemokine (TARC) levels were measured. Response to treatment was compared in patient subgroups based on results of these analyses. RESULTS Lymphocytic variant HES was diagnosed in 13 of 63 patients with HES with complete T-cell assessments. The ability to taper corticosteroids on mepolizumab was similar in patients with L-HES and those with a normal T-cell profile, although a lower proportion of patients with L-HES maintained eosinophil levels below 600/μL. Increased serum TARC levels (>1000 pg/mL) had no significant impact on the ability to reduce corticosteroid doses, but a lower proportion of patients with elevated TARC achieved eosinophil control on mepolizumab. CONCLUSION Mepolizumab is an effective corticosteroid-sparing agent for patients with L-HES. In some cases however, eosinophil levels remain above 600/μL, suggesting incomplete neutralization of overproduced IL-5 or involvement of other eosinophilopoietic factors.

Exploring potential noninvasive biomarkers in eosinophilic esophagitis in children

Background and Aims: Eosinophilic esophagitis (EE) continues to present clinical challenges, including a need for noninvasive tools to manage the disease. To identify a marker able to assess disease status in lieu of repeated endoscopies, we examined 3 noninvasive biomarkers, serum interleukin (IL)-5, serum eosinophil-derived neurotoxin (EDN), and stool EDN, and examined possible correlations of these with disease phenotype and activity (symptoms and histology) in a longitudinal study of children with EE. Subjects and Methods: Children with EE were studied for up to 24 weeks (12 weeks on 1 of 2 corticosteroid therapies and 12 weeks off therapy). Twenty children with normal esophagogastroduodenoscopies with biopsies were enrolled as controls. Serum IL-5, serum EDN, and stool EDN were measured at weeks 0, 4, 12, 18, and 24 in children with EE, and at baseline alone for controls. Primary and secondary statistical analyses (excluding and including outlier values of the biomarkers, respectively) were performed. Results: Sixty subjects with EE (46 [75%] boys, mean age 7.5 ± 4.4 years) and 20 normal controls (10 [50%] boys, mean age 6.7 ± 4.1 years) were included. Significant changes in serum EDN (significant decrease from baseline to week 4, and then rebound from week 4 to week 12) occurred. Serum EDN levels were stable after week 12. Serum IL-5 and stool EDN levels in subjects with EE were not statistically different from those of the control subjects when each time point for the cases was compared with the controls’ 1-time measurement. Conclusions: Serum EDN levels were significantly higher in subjects with EE than in controls, and the results suggest a possible role, after additional future studies, for serum EDN in establishing EE diagnosis, assessing response to therapy, and/or monitoring for relapse or quiescence.

Modulation of hematopoietic stem/progenitor cell engraftment by transforming growth factor β

OBJECTIVE To investigate if cell cycle progression plays a role in modulating the engraftment potential of mouse hematopoietic stem and progenitor cells (HSPC). MATERIALS AND METHODS HSPC were isolated from adult mouse bone marrow, cultured in vitro under conditions promoting cell cycle arrest, and subsequently were evaluated for cell cycle status, clonogenic activity, and transplant potential. RESULTS In the presence of steel factor (STL) as a survival cytokine, transforming growth factor beta (TGF-beta) increased the G0/G1 fraction of cycling progenitor cells (Rh(high)) after a 20-hour culture. Clonogenic activity of quiescent long-term repopulating (Rh(low)) HSPC was unaffected by this culture, whereas clonogenic potential of Rh(high) cells decreased by about 30%. In competitive repopulation assays, Rh(low) cells cultured in STL + TGF-beta engrafted better than cells cultured in STL alone. However, culture in STL + TGF-beta did not overcome the failure of Rh(high) cells to engraft after transplant. We also utilized a two-stage culture system to first induce proliferation of Rh(low) HSPC by a 48-hour culture in STL + interleukin 6 + Flt-3 ligand, followed by shifting the culture to STL + TGF-beta for 24 hours to induce cycle arrest. A competitive repopulation assay demonstrated a relative decrease in repopulating potential in cultures that were cycle arrested compared to those that were not. CONCLUSION Cell cycle progression by itself cannot account for the decrease in repopulating potential that is observed after ex vivo expansion. Other determinants of engraftment must be identified to facilitate the transplantation of cultured HSPC.

Immunologic response to fungus is not universally associated with rhinosinusitis

OBJECTIVE: Immunologic response to fungal antigens has been cited as an etiologic factor in chronic rhinosinusitis (CRS). Previous work demonstrated a significant cytokine response in CRS patients that did not correlate with an immunoglobulin E (IgE) response. This study was performed in an effort to replicate these findings in a more geographically diverse population. DESIGN: Prospective in vitro study. SETTING: Two academic tertiary rhinologic practices in Texas and Utah. METHODS: Serum and peripheral blood monocytes (PBMC) were obtained from 10 CRS patients and seven controls. Total IgE and fungal-specific IgE levels were determined. Cytokine levels were measured after PBMC exposure to Alternaria, Aspergillus, Cladosporium, and Penicillium extracts. Correlations between cytokine responses and presence of CRS as well as IgE and IgG were determined. RESULTS: Interleukin-5 (IL-5) was produced after Alternaria extract exposure in both CRS patients and controls, but the production was heterogenous and did not correlate with the presence of CRS. IL-5 levels after Alternaria extract exposure correlated strongly with levels of Alternaria-specific IgE in both CRS patients and controls. IL-5 production did not correlate with IgG levels. IL-4, IL-13, and interferon-gamma production did not differ between CRS patients and controls. CONCLUSIONS: In contrast to previously reported data, IL-5 responses to Alternaria extract were not predictive of CRS presence. Our results in patients from Utah and Texas significantly differ from previously published findings in predominantly Midwestern patients. The immunologic response to fungal extracts appears to be heterogenous and may differ based on geography, allergy status, and/or other as-yet unknown factors.

Penaeus monodon tropomyosin induces CD4 T-cell proliferation in shrimp-allergic patients.

Shellfish allergy affects approximately 2% of the population and can cause immediate hypersensitivity reactions such as urticaria, swelling, difficulty breathing, and, in some cases, anaphylaxis. Tropomyosin is the major shrimp allergen and binds IgE in two-thirds of patients. A total of 38 shrimp-allergic patients and 20 negative control subjects were recruited and evaluated on the basis of history, skin prick testing, specific immunoglobulin E (IgE) levels, and peripheral blood mononuclear cell proliferation in response to shrimp tropomyosin or shrimp tropomyosin-derived peptides. Of the classically allergic patients by history, 59% tested positive for serum shrimp IgE antibodies. Of patients with shrimp-specific IgE in sera, 70% also had significant IgE levels specific for shrimp tropomyosin. Peripheral blood mononuclear cells from classically shrimp-allergic patients proliferated in a dose-dependent manner in response to to tropomyosin. In addition, a T-cell line derived from a shrimp-allergic patient proliferated specifically in response to tropomyosin-derived peptides. These studies suggest a strategy for immunotherapy using a tropomyosin-derived T-cell epitope vaccination.

High quality and quantity Genome-wide germline genotypes from FFPE normal tissue

BackgroundAlthough collections of formalin fixed paraffin embedded (FFPE) samples exist, sometimes representing decades of stored samples, they have not typically been utilized to their full potential. Normal tissue from such samples would be extremely valuable for generation of genotype data for individuals who cannot otherwise provide a DNA sample.FindingsWe extracted DNA from normal tissue identified in FFPE tissue blocks from prostate surgery and obtained complete genome wide genotype data for over 500,000 SNP markers for these samples, and for DNA extracted from whole blood for 2 of the cases, for comparison.Four of the five FFPE samples of varying age and amount of tissue had identifiable normal tissue. We obtained good quality genotype data for between 89 and 99% of all SNP markers for the 4 samples from FFPE. Concordance rates of over 99% were observed for the 2 samples with DNA from both FFPE and from whole blood.ConclusionsDNA extracted from normal FFPE tissue provides excellent quality and quantity genome-wide genotyping data representing germline DNA, sufficient for both linkage and association analyses. This allows genetic analysis of informative individuals who are no longer available for sampling in genetic studies.

Extracellular Eosinophil Granule Protein Deposition in Ringed Esophagus with Sparse Eosinophils

BackgroundEosinophilic esophagitis (EoE) remains difficult to classify because of varying presentations. Not uncommonly, patients present with symptoms of esophageal dysfunction and have esophageal changes on endoscopy resembling EoE but without >15 eosinophils/HPF. Patients with low numbers of eosinophils in esophageal biopsy specimens may have esophageal changes and symptomatic disease brought about by eosinophil granule protein deposition without recognizable intact cells.AimTo determine whether extracellular eosinophil granule protein deposition is present in the esophagi of patients with low eosinophil numbers who have clinical symptoms and characteristic endoscopic esophageal changes of EoE including ringed esophagus (RE).MethodsEsophageal biopsy specimens were studied from eight EoE patients with >15 eosinophils per high power field (HPF) and nine patients with RE (<15 eosinophils/HPF). The specimens were analyzed for eosinophil granule proteins, major basic protein 1 (eMBP1) and eosinophil-derived neurotoxin (EDN), by indirect immunofluorescence.ResultsBoth EoE and RE showed positive EDN and eMBP1 extracellular deposition; control esophagus showed minimal or none. Comparing EoE and RE, extracellular EDN and eMBP1 were similar except that EDN in EoE was greater in the distal esophagus.ConclusionsThis study highlights the importance of assessing eosinophil granule protein deposition in esophageal disease with potential eosinophil involvement. Persistent/progressive esophageal changes may be brought about by eosinophil granule proteins despite low numbers of intact cells. The meaning of “resolution” in EoE may need to be redefined based on numbers of esophageal eosinophils, extracellular eosinophil granule protein deposition, and subsequent clinical course of patients.

Characterization of myeloid and lymphoid subsets in dendritic cells derived from cord blood and adult blood

Abstract Dendritic cells (DC) are antigen presenting cells that play a critical role in T cell dependent immune responses. Two distinct DC subsets have been identified based on surface phenotype, cytokine requirements, and immunologic function. Myeloid DCs are GM-CSF dependent and express CD11c + and CD45RO + . Lymphoid DCs are IL-3 dependent and express CD45RA + and CD123 + . Myeloid DCs initiate Th1 immune responses, whereas lymphoid DCs regulate Th2 responses. We sought to characterize these subsets in freshly isolated CD4 + DCs and in monocyte-derived DCs from cord and adult blood. CD4 + DCs were isolated from blood mononuclear cells using antibody-linked magnetic microbeads, first to deplete B cells, T cells, monocytes and NK cells, then to select for CD4 + DCs. Monocytes were isolated from blood by positive selection using anti-CD14 linked magnetic microbeads. Monocytes were cultured in GM-CSF and IL-4 for 7 days. TNF-α was added on day 5. Surface phenotypes of CD4 + DCs and monocyte-derived DCs were evaluated by flow cytometry for lineage, MHC, adhesions, co-stimulatory, and cytokine receptor molecules. We found that CD4 + DCs included both subsets with a predominance of lymphoid DCs. In contrast, monocyte derived DCs exhibited a myeloid surface phenotype (CD11c + and CD45RO + ). No differences in DC subsets were observed between cord blood and adult blood. We conclude that distinct DC subsets can be generated from blood based upon the method of isolation. We speculate that DC-based strategies might be feasible for the prevention and treatment of infectious diseases in newborn infants.

S1858 Eosinophil Degranulation Differentiates Eosinophilic Esophagitis (EoE), Possible EoE and GERD Patients

for each feature). EE patients also had significantly higher epithelial scores (mean of 7.33 as compared to 0.52 in GERD, p<0.001) and higher LP maximum scores (mean 4.5 versus 1 in GERD, p<0.001). Epithelial histologic features correlated well with endoscopic features of furrows/thickening, plaques, and pallor (r=0.62-0.82, p<0.001 for each) while LP scores correlated best with the endoscopic feature of thickening/furrows (r=0.64, p=0.002). Symptom complaints of dysphagia + anorexia/early satiety correlated best with epithelial scores (r=0.32, p=0.01); dysphagia correlated best with LP scores (r=0.45, p=0.04). None of the other symptoms assessed correlated with histologic or endoscopic findings. Conclusion: EE patients have significantly more severe endoscopic and histologic changes as compared with GERD patients. The objective endoscopic and epithelial/LP histologic features correlate well with one another. However, of subjective complaints, only dysphagia and anorexia/early satiety correlate with histologic and endoscopic findings.