- Email: [email protected]
Genetic variants harbored in the forkhead box protein 3 locus increase hay fever risk
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FIG 2. Images of representative cPLA2a immunoblotting experiments in patients with severe asthma carrying shorter (CA)12-18(T)17-38 or longer (CA)19-24(T)39-46 alleles of analyzed PLA2G4A promoter microsatellites (A) and in patients with severe asthma and nonsevere asthma and healthy controls (B).
SLC11A1 gene might have an impact on intracellular bacteria infection and leishmaniasis.11 Additional observations strengthening our initial hypothesis of cPLA2a contribution to asthma pathogenesis are data on comparative analysis of cPLA2a mRNA and protein expression in patients and healthy subjects randomly selected regardless of genotypes. Although we found similar expression of cPLA2a in patients with severe and nonsevere asthma, it was still strongly overexpressed compared with healthy controls. Obviously, the overall high prevalence of shorter alleles of studied microsatellites in patients with severe asthma, which was shown in our previous study,5 might influence such results. On the other hand, the selection was performed at random according to drawing of lots from all patients with asthma treated in our unit. Although high expression of cPLA2a in patients with asthma did not increase generation of eicosanoids by PBMCs, it might act locally in the bronchi,12 delivering arachidonic acid for enzymes of eicosanoids pathway at the direct site of inflammation.13 It could also influence other proinflammatory gene expression.14 In summary, our data suggest that (CA)n and (T)n microsatellites in the PLA2G4A promoter determine cPLA2a in vivo expression in patients with severe asthma and the overexpression of cPLA2a in persistent asthma might play a role in its pathogenesis. Milena Sokolowska, MD, PhDa Joanna Stefanska, MSca Karolina Wodz-Naskiewicz, MSca Malgorzata Cieslak, MDb Rafal Pawliczak, MD, PhDa
4. Sapirstein A, Bonventre JV. Specific physiological roles of cytosolic phospholipase A(2) as defined by gene knockouts. Biochim Biophys Acta 2000;1488:139-48. 5. Sokolowska M, Borowiec M, Ptasinska A, Cieslak M, Shelhamer JH, Kowalski ML, et al. 85-kDa cytosolic phospholipase A2 group IValpha gene promoter polymorphisms in patients with severe asthma: a gene expression and case-control study. Clin Exp Immunol 2007;150:124-31. 6. Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma (GINA) 2008. Available from: http://www.ginasthma.org. Accessed May 2009. 7. Proceedings of the ATS workshop on refractory asthma:current understanding, recommendations, and unanswered questions. American Thoracic Society. Am J Respir Crit Care Med 2000;162:2341-51. 8. Wu T, Ikezono T, Angus CW, Shelhamer JH. Characterization of the promoter for the human 85 kDa cytosolic phospholipase A2 gene. Nucleic Acids Res 1994;22: 5093-8. 9. Dolan-O’Keefe M, Chow V, Monnier J, Visner GA, Nick HS. Transcriptional regulation and structural organization of the human cytosolic phospholipase A(2) gene. Am J Physiol Lung Cell Mol Physiol 2000;278:L649-57. 10. Uhlemann AC, Szlezak NA, Vonthein R, Tomiuk J, Emmer SA, Lell B, et al. DNA phasing by TA dinucleotide microsatellite length determines in vitro and in vivo expression of the gp91phox subunit of NADPH oxidase and mediates protection against severe malaria. J Infect Dis 2004;189:2227-34. 11. Bayele HK, Peyssonnaux C, Giatromanolaki A, Arrais-Silva WW, Mohamed HS, Collins H, et al. HIF-1 regulates heritable variation and allele expression phenotypes of the macrophage immune response gene SLC11A1 from a Z-DNA forming microsatellite. Blood 2007;110:3039-48. 12. Nakatani N, Uozumi N, Kume K, Murakami M, Kudo I, Shimizu T. Role of cytosolic phospholipase A2 in the production of lipid mediators and histamine release in mouse bone-marrow-derived mast cells. Biochem J 2000;352(pt 2):311-7. 13. Choi IW, Sun K, Kim YS, Ko HM, Im SY, Kim JH, et al. TNF-alpha induces the late-phase airway hyperresponsiveness and airways inflammation through cytosolic phospholipase A(2) activation. J Allergy Clin Immunol 2005;116:537-43. 14. Pawliczak R, Logun C, Madara P, Lawrence M, Woszczek G, Ptasinska A, et al. Cytosolic phospholipase A2 group IValpha but not secreted phospholipase A2 group IIA, V, or X induces interleukin-8 and cyclooxygenase-2 gene and protein expression through peroxisome proliferator-activated receptors gamma 1 and 2 in human lung cells. J Biol Chem 2004;279:48550-61.
From athe Department of Immunopathology, Chair of Allergology, Immunology and Dermatology, Faculty of Biomedical Sciences and Postgraduate Training; and bthe Department of Immunology, Rheumatology and Allergy, Faculty of Medicine, Medical University of Lodz, Poland. E-mail: [email protected]. Supported by Polish Government grants N N401 225034 and N401 191 32/4009. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.
Genetic variants harbored in the forkhead box protein 3 locus increase hay fever risk
REFERENCES 1. Wenzel S. Severe asthma in adults. Am J Respir Crit Care Med 2005;172:149-60. 2. Leslie CC. Regulation of the specific release of arachidonic acid by cytosolic phospholipase A2. Prostaglandins Leukot Essent Fatty Acids 2004;70:373-6. 3. Adler DH, Cogan JD, Phillips JA 3rd, Schnetz-Boutaud N, Milne GL, Iverson T, et al. Inherited human cPLA(2alpha) deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction. J Clin Invest 2008;118:2121-31.
To the Editor: Disturbed regulation of T cells may lead to an imbalance between TH1 and TH2 cells and toward an elevated TH2 response, typically associated with allergy. Regulatory T cells are thought to be responsible for keeping T-cell populations and T-cell effects in balance, whereas modifications of regulatory T-cell signaling may lead to deviated immune responses. Recently, we had shown
Available online April 15, 2010. doi:10.1016/j.jaci.2010.02.016
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J ALLERGY CLIN IMMUNOL JUNE 2010
FIG 1. Depiction of gene structures on chromosome X, identified polymorphisms and LD (r2).The HapMap database was used to identify validated FOXP3 SNPs. In addition, the screened region was extended around the FOXP3 gene, thereby detecting SNPs in and around the neighbor genes CACNA1F, CCDC22, and PPP1R3F. Color coding for the linkage disequilibrium plot is given by Haploview: white (r2 5 0), shades of gray (0 < r2 < 1), and black (r2 5 1). Genotyped tagging SNPs are underlined. UTR, Untranslated region.
that T-cell–associated transcription factors influence the risk for asthma and allergy.1 The transcription factor forkhead box protein 3 (FOXP3; forkhead/winged helix transcription factor) seems to specifically control regulatory T-cell function.2 Deletions or change of function in the human FOXP3 gene result in the development of severe immune dysregulation with polyendocrinopathic and enteropathic X-linked syndrome,3 often associated with high serum IgE levels.4 Despite the prominent role T cells and T-cell regulation have in current asthma and allergy disease models, only 1 study has recently been published on FOXP3 genetics in that field to our knowledge.5 We now investigated 40 kilo base pairs of genomic sequence harboring the FOXP3 gene (Fig 1). On the basis of HapMap data (http://www.hapmap.org),6 we identified 4 tagging polymorphisms (rs2294020, rs2294021, rs3761548, and rs3761549) with a minor allele frequency (MAF) >0.10 capturing all essential genetic information of the FOXP3 gene locus using Haploview software.7 We studied the influence of these single nucleotide polymorphisms (SNPs) on atopic diseases in a cross-sectional population sample of 3099 German children age 9 to 11 years from Dresden and Munich as part of the International Study of Asthma and Allergy in
Childhood phase II. In that study, the prevalence of a doctor’s diagnosis of asthma, hay fever, and atopic dermatitis was assessed by self-administered questionnaires and by objective measurements such as lung function tests, skin prick tests, and serum IgE measurements as previously reported.8,9 Further information on technical details concerning matrix-assisted laser desorption/ionization time-of-flight genotyping or population characteristics is available from the corresponding author on request. Because of the X-chromosomal location, all SNPs were analyzed by fitting an additive effects-only logit model that equates the risks of male hemizygotes with female homozygotes. Hardy-Weinberg equilibrium did not deviate and was tested in females only. To correct for multiple testing, we used a Bonferroni correction in every phenotype. All tests were 2-sided, and the differences were considered significant with P <.05. Calculations were carried out with SAS (version 9.1.3) and SAS/Genetics software (SAS Institute, Inc, Cary, NC). Tagging SNP rs2294020 downstream of FOXP3 slightly increased the risk for atopy assessed by skin prick test in the study population and showed significant associations with hay fever (Table I). Also, when phenotypes related to the primary outcome
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TABLE I. Associations between atopic phenotypes and tagging SNPs showing odds ratios, 95% CIs, and P values (given for Wald test) Tagging SNPs and MAFs
rs 2294020 (MAF 0.31)
rs 2294021 (MAF 0.45)
rs 3761548 (MAF 0.43)
rs 3761549 (MAF 0.12)
Atopic sensitization (SPT1)
1.106 0.998-1.226 P5.0546
1.041 0.943-1.149 P5.4236
0.927 0.839-1.024 P5.1369
1.057 0.908-1.230 P5.4774
Asthmaà
1.100 0.942-1.286 P5.2294 1.028 0.914-1.157 P5.6441
1.072 0.924-1.245 P5.3594 0.928 0.828-1.039 P5.1939
0.967 0.831-1.125 P5.6607 1.066 0.953-1.193 P5.2644
0.951 0.749-1.208 P5.6811 1.022 0.86-1.216 P5.8027
1.256* 1.081-1.460 P5.0030 1.225 1.033-1.453 P5.0196
1.131 0.975-1.313 P5.1048 1.101 0.930-1.303 P5.2635
0.983 0.845-1.143 P5.8234 1.006 0.848-1.193 P5.9469
0.793 0.609-1.032 P5.0842 0.847 0.634-1.131 P5.2605
1.210* 1.069-1.371 P5.0027
1.059 0.937-1.197 P5.3582
0.962 0.851-1.089 P5.542
0.981 0.809-1.191 P5.8476
Atopic dermatitis§
Hay fever§
Hay fever and atopic sensitization
Current hay fever symptoms
SPT, Skin prick test. *Significant after correction for multiple testing. Defined by skin prick test against 6 common aeroallergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Alternaria tenuis, cat dander, and mixed grass and tree pollen). àDefined by a parental report of a physician’s diagnosis of asthma (at least once) or of spastic or asthmatic bronchitis (at least twice) in a self-administered questionnaire. §Defined by a parental report of a physician’s diagnosis of the respective diseases. Significant associations are shown in boldface.
hay fever were analyzed, significant associations between this SNP and hay fever with concomitant sensitization to inhalative allergens and current hay fever symptoms were observed. Associations for hay fever and current symptoms of hay fever remained statistically significant after correction for multiple testing. These results coincide with data from Zhang et al,5 who recently observed associations between FOXP3 SNPs (rs3761547 and rs3761548) and allergic rhinitis in a Chinese population. Common polymorphisms and microsatellite markers had been described in the FOXP3 gene and in its putative promoter and upstream untranslated region before. These gave conflicting association results with a number of complex immune and autoimmune diseases. Mutation events severely affecting FOXP3 function are rare and cause serious immune deficiency syndromes.3 The SNPs that we studied and that are covered by the tagging SNPs genotyped here are located within and downstream of the FOXP3 gene. When linkage disequilibrium (LD) analyses were extended, _ 0.9) were idenseveral other SNPs tagged by SNP rs2294020 (r2 > tified in and around neighboring genes coiled-coil domain containing 22 (CCDC22) and calcium channel, voltage-dependent, a-1F subunit (CACNA1F; Fig 1). Whereas CCDC22 is an unexplored gene with so far unknown biological function, CACNA1F is a voltage-gated calcium channel. Two polymorphisms in the allergic rhinitis associated LD block (rs2294020 located in exon 7 of CCDC22, and rs2075866 located in exon 28 of CACNA1F) were investigated further because of their position in exonic regions, putatively acting as exonic splicing enhancers. Indeed, using ESEfinder software, release 3.0 (http://rulai.cshl.edu/cgi-bin/tools/ ESE3/esefinder.cgi)10,11 allele-specific changes in score matrices for human serine/arginine-rich (SR) proteins were observed, which belong to a protein family essential for splicing (Fig 2). Also, in silico transcription factor binding analyses for promoter and intronic SNPs in the same LD block were performed that
predicted numerous SNP-dependent alterations of transcription factor binding (Alibaba 2.1 software12). However, a comparative genomic analysis searching for conserved noncoding sequences revealed only poor phylogenetic conservation in intergenetic and intragenetic regions of the locus (VISTA Genome browser13). Thus, further functional analyses are needed to determine whether the associations of SNPs in this LD block observed with allergic rhinitis may be influenced by splicing alterations in genes close to FOXP3 and/or locus regulatory elements in the area. Although the causal SNP for this associated LD block is not identified yet, our data and those of Zhang et al5 imply that genetic variations in the region of and around FOXP3 may modify the risk for allergic rhinitis. The association with hay fever we observed is consistent in our population and a second report. Also, when objective measurements were included in the definition of the phenotype, the association was present and specific. Genetic association reports with hay fever are sparse despite the fact that the disease has a very well defined phenotype, is very frequent, and imposes a heavy burden on affected individuals and public health. Within the atopic phenotypes, it is the disease most clearly associated with IgE-specific sensitizations and may represent a good candidate disease for genetically driven variation in regulatory T-cell function. Kathrin Suttner, MSca Martin Depner, MScb Martin Wetzke, MDa Norman Klopp, PhDc Erika von Mutius, MDb Thomas Illig, PhDc Tim Sparwasser, MDd Michael Kabesch, MDa From athe Center for Pediatrics, Clinic for Pediatric Pneumology and Neonatology, Hannover Medical School; bUniversity Children’s Hospital, Ludwig Maximilian’s University, Munich; cthe Institute of Epidemiology, Helmholtz Center Munich,
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FIG 2. Allele-specific values for score matrices of human SR proteins (SF2/ASF, SC35, SRp40, or SRp55) for SNP rs2294020 and SNP rs2075866 using ESEfinder software (release 3.0).10,11Analyzed exonic sequence along the x-axis with respective SNP position highlighted. The height of the bars reflects the motif scores for the different SR proteins, and the width of the bars represents the length of the motif (6, 7, or 8 nucleotides). Only high score values above the selected threshold defined by the ESEfinder software are shown. BRCA1, Breast cancer 1 gene; PO, polymorphic allele; WT, wild-type. Neuherberg; and dInstitute of Infection Immunology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover; a joint venture between the Helmholtz Centre for Infection Research (HZI) Braunschweig and the Hannover Medical School. E-mail: [email protected]. Supported by the National Genome Research Network (NGFN) research grant NGFN 01GS 0810 and the German research foundation as part of the transregional collaborative research program TR22 ‘‘Allergic Immune Responses of the Lung,’’ grant A15/Z3. Genotyping was performed in the Genome Analysis Center of the Helmholtz Zentrum Munich. Funding sources had no influence in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Disclosure of potential conflict of interest: E. von Mutius is a consultant for GlaxoSmithKline, UCB, and ProtectImmun and has received research support from Airsonett AB. M. Kabesch has financial interests in Roxall, Glaxo Wellcome, Novartis, Sanofi Aventis, and Allergopharma and has received research support from the DFG, BMBF, and the European Union. The rest of the authors have declared that they have no conflict of interest. REFERENCES 1. Suttner K, Rosenstiel P, Depner M, Schedel M, Pinto LA, Ruether A, et al. TBX21 gene variants increase childhood asthma risk in combination with HLX1 variants. J Allergy Clin Immunol 2009;123:1062-8.
2. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003;229:1057-61. 3. Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL, Buist N, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet 2001;27:18-20. 4. Gambineri E, Perroni L, Passerini L, Bianchi L, Doglioni C, Meschi F, et al. Clinical and molecular profile of a new series of patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: inconsistent correlation between forkhead box protein 3 expression and disease severity. J Allergy Clin Immunol 2008;122:1105-12. 5. Zhang L, Zhang Y, Desrosiers M, Wang C, Zhao Y, Han D. Genetic association study of FOXP3 polymorphisms in allergic rhinitis in a Chinese population. Hum Immunol 2009;70:930-4 6. The International HapMap Project. Nature 2003;426:789–796. 7. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263-5. 8. Weidinger S, O’Sullivan M, Illig T, Baurecht H, Depner M, Rodriguez E, et al. Filaggrin mutations, atopic eczema, hay fever, and asthma in children. J Allergy Clin Immunol 2008;121:1203-9. 9. Weiland SK, von Mutius E, Hirsch T, Duhme H, Fritzsch C, Werner B, et al. Prevalence of respiratory and atopic disorders among children in the East and West of Germany five years after unification. Eur Respir J 1999;14:862-70.
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10. Smith PJ, Zhang C, Wang J, Chew SL, Zhang MQ, Krainer AR. An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. Hum Mol Genet 2006;15:2490-508. 11. Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR. ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acid Res 2003;31:3568-71. 12. Grabe N. AliBaba2: context specific identification of transcription factor binding sites. Silico Biol 2002;2(suppl):S1-S15. 13. Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I. VISTA: computational tools for comparative genomics. Nucleic Acids Res 2004;32(Web Server issue):W273-9. Available online April 16, 2010. doi:10.1016/j.jaci.2010.02.017
Heterogeneity among characteristics of hypereosinophilic syndromes To the Editor: We read with interest the recent Journal article by Ogbogu et al1 reporting the results from a multicenter analysis of patients with hypereosinophilic syndrome (HES). In retrospective and prospective studies, we have analyzed clinical and laboratory data from 88 patients who satisfied the stringent criteria by Chusid et al2 for HES. We prospectively followed 55 patients; data from the remaining 33 were analyzed after chart review. The patients were recruited from 2002 to 2009 from 3 institutions that treated patients with hematologic disorders; the coordinating center was located at Silesian Medical University in Katowice, Poland. Before patients were referred to the hematology units, potential secondary causes of hypereosinophilia were excluded. Informed consent was obtained from all patients according to the Declaration of Helsinki, and the study was approved by the institutional ethics committee. All patients included in the study exhibited symptoms, signs, and imaging or histologic evidence of eosinophilic organ involvement. We performed complete blood count and differential analyses, routine chemistry tests, electrocardiograms, echocardiograms, chest x-rays, ultrasound/computed tomography/magnetic resonance studies, and analyses of bone marrow aspirates and biopsy samples. The methods of analysis of patients with HES, which included cytokine and tryptase measurements, immunophenotyping, and cytogenetic and molecular studies, are presented elsewhere.3 All laboratory tests were performed on treatment-naive patients. Statistical methods were reported previously.3 Because this was a retrospective study, some results were not available for all cases. Clinical responses were assessed after 1 month of treatment. A complete hematologic response was defined as a decrease in the absolute eosinophil count (AEC) to the normal range (<0.7 3 109/L) with resolution of organ involvement. A partial response _50% and/or symptomatic was defined as decrease in the AEC > improvement. ‘‘No response’’ was defined as a stable or increasing AEC and/or stable or progressive organ dysfunction. Forty-seven male and 41 female patients (median age at diagnosis, 56 years; range, 6-84 years) were included in the study. The median AEC was 6.6 3 109/L (range, 1.5-136 3 109/L), and a median 31% of cells were eosinophils (range, 7% to 80%). Patients were divided into 5 subgroups according to Klion et al4; their characteristics are presented in Table I. Of the 88 patients whose blood samples were tested by nested PCR analysis, 16 were found to have the FIP1L1–PDGFRA (Fip1like 1–platelet-derived growth factor receptor a; F/P) fusion transcript (18%; 14 male and 2 female patients). Three patients with F/P transcripts had no organ involvement. The remaining 13 cases
had the following abnormalities: splenomegaly (n 5 11), hepatomegaly (n 5 7), cardiac failure (n 5 3), lymph node enlargement (n 5 2), pulmonary infiltrate (n 5 2), and central nervous system involvement (n 5 1). In 10 patients (12%), the diagnosis of chronic eosinophilic leukemia (CEL) was established on the basis of an increased number of blasts in peripheral blood and/or in bone marrow (n 5 8) or the presence of molecular/cytogenetic markers, including 2 patients with the point mutation JAK2V617F (n 5 2). Splenomegaly was the most common form of organ involvement in this population. Twenty-six patients were found to have CEL (30%). Eight patients had a myeloproliferative variant of HES (etiology unknown) on the basis of at least 4 of the criteria proposed by Klion et al.4 Forty-two patients were tested for T-cell receptor (TCR) rearrangement by using multiplex PCR with heteroduplex analysis; clonality was detected in 18 patients (43%). Three patients were found to have aberrant T-cell phenotypes (by flow cytometry) and TCR clonal rearrangement (by PCR analysis). On the basis of the definition, these 3 patients (7%) were the only ones diagnosed with lymphocytic variant hypereosinophilic syndrome (L-HES).5 Fifty-one patients (including 15 not yet classified cases with clonal TCR) were diagnosed with idiopathic HES. The results of this analysis have been presented in detail.3 Serum tryptase and vitamin B12 levels were reported for 47 and 69 patients, respectively. Serum IgE levels were measured in 74 patients and were found to be increased compared with normal levels in 38 cases. Serum levels of IL-4 and IL-5 were documented for 49 and 51 patients, respectively. We compared the results of laboratory studies between patients with F/P1 and F/P– CEL and those of patients with HES (including idiopathic HES, L-HES, and myeloproliferative variant HES). Patients with CEL had significantly increased white blood cell counts, AECs, and levels of serum vitamin B12 and tryptase compared with patients with HES. Patients with HES had increased platelet counts and serum levels of IL-5 compared with patients with CEL (see this article’s Table E1 in the Online Repository at www.jacionline.org). Results of cytogenetic analyses were available for 48 patients (54%); karyotypes were normal in 84%. The cytogenetic abnormalities were as follows: -Y (n 5 2), complex karyotypes (n 5 2), translocation t(6;11) (n 5 1), -7 (n 5 1), and -17 (n 5 1). In our series, 76 patients (86%) began corticosteroid monotherapy at doses varying from 20 to 60 mg daily; clinical responses were observed in 64% (49 patients). Forty-one patients continued to receive a low dose of corticosteroid maintenance therapy (5-20 mg daily). Discontinuation of corticosteroids led to eosinophilia recurrence in most patients. Ten patients remained in remission even after therapy ended, but the follow-up period was less than 3 months (see this article’s Table E2 in the Online Repository at www.jacionline.org). Serum levels of IL-5 were available for 31 responders and 19 nonresponders. Median levels of serum IL-5 were significantly increased among patients who responded to steroids compared with nonresponders (18.6 vs 11.0; P 5 .01). Sixteen patients who had the F/P fusion transcript had a rapid response to imatinib at starting doses from 100 to 400 mg daily (100%; range, 6-65 months); they remained in sustained hematologic and molecular remission after a median of 42 months of therapy. Sixteen of 26 patients with CEL were resistant to corticosteroid monotherapy. The maximum starting dose was 1 mg/kg daily. The 10 patients with F/P– CEL were treated with a median number of 3
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 6
FIG 2. Images of representative cPLA2a immunoblotting experiments in patients with severe asthma carrying shorter (CA)12-18(T)17-38 or longer (CA)19-24(T)39-46 alleles of analyzed PLA2G4A promoter microsatellites (A) and in patients with severe asthma and nonsevere asthma and healthy controls (B).
SLC11A1 gene might have an impact on intracellular bacteria infection and leishmaniasis.11 Additional observations strengthening our initial hypothesis of cPLA2a contribution to asthma pathogenesis are data on comparative analysis of cPLA2a mRNA and protein expression in patients and healthy subjects randomly selected regardless of genotypes. Although we found similar expression of cPLA2a in patients with severe and nonsevere asthma, it was still strongly overexpressed compared with healthy controls. Obviously, the overall high prevalence of shorter alleles of studied microsatellites in patients with severe asthma, which was shown in our previous study,5 might influence such results. On the other hand, the selection was performed at random according to drawing of lots from all patients with asthma treated in our unit. Although high expression of cPLA2a in patients with asthma did not increase generation of eicosanoids by PBMCs, it might act locally in the bronchi,12 delivering arachidonic acid for enzymes of eicosanoids pathway at the direct site of inflammation.13 It could also influence other proinflammatory gene expression.14 In summary, our data suggest that (CA)n and (T)n microsatellites in the PLA2G4A promoter determine cPLA2a in vivo expression in patients with severe asthma and the overexpression of cPLA2a in persistent asthma might play a role in its pathogenesis. Milena Sokolowska, MD, PhDa Joanna Stefanska, MSca Karolina Wodz-Naskiewicz, MSca Malgorzata Cieslak, MDb Rafal Pawliczak, MD, PhDa
4. Sapirstein A, Bonventre JV. Specific physiological roles of cytosolic phospholipase A(2) as defined by gene knockouts. Biochim Biophys Acta 2000;1488:139-48. 5. Sokolowska M, Borowiec M, Ptasinska A, Cieslak M, Shelhamer JH, Kowalski ML, et al. 85-kDa cytosolic phospholipase A2 group IValpha gene promoter polymorphisms in patients with severe asthma: a gene expression and case-control study. Clin Exp Immunol 2007;150:124-31. 6. Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma (GINA) 2008. Available from: http://www.ginasthma.org. Accessed May 2009. 7. Proceedings of the ATS workshop on refractory asthma:current understanding, recommendations, and unanswered questions. American Thoracic Society. Am J Respir Crit Care Med 2000;162:2341-51. 8. Wu T, Ikezono T, Angus CW, Shelhamer JH. Characterization of the promoter for the human 85 kDa cytosolic phospholipase A2 gene. Nucleic Acids Res 1994;22: 5093-8. 9. Dolan-O’Keefe M, Chow V, Monnier J, Visner GA, Nick HS. Transcriptional regulation and structural organization of the human cytosolic phospholipase A(2) gene. Am J Physiol Lung Cell Mol Physiol 2000;278:L649-57. 10. Uhlemann AC, Szlezak NA, Vonthein R, Tomiuk J, Emmer SA, Lell B, et al. DNA phasing by TA dinucleotide microsatellite length determines in vitro and in vivo expression of the gp91phox subunit of NADPH oxidase and mediates protection against severe malaria. J Infect Dis 2004;189:2227-34. 11. Bayele HK, Peyssonnaux C, Giatromanolaki A, Arrais-Silva WW, Mohamed HS, Collins H, et al. HIF-1 regulates heritable variation and allele expression phenotypes of the macrophage immune response gene SLC11A1 from a Z-DNA forming microsatellite. Blood 2007;110:3039-48. 12. Nakatani N, Uozumi N, Kume K, Murakami M, Kudo I, Shimizu T. Role of cytosolic phospholipase A2 in the production of lipid mediators and histamine release in mouse bone-marrow-derived mast cells. Biochem J 2000;352(pt 2):311-7. 13. Choi IW, Sun K, Kim YS, Ko HM, Im SY, Kim JH, et al. TNF-alpha induces the late-phase airway hyperresponsiveness and airways inflammation through cytosolic phospholipase A(2) activation. J Allergy Clin Immunol 2005;116:537-43. 14. Pawliczak R, Logun C, Madara P, Lawrence M, Woszczek G, Ptasinska A, et al. Cytosolic phospholipase A2 group IValpha but not secreted phospholipase A2 group IIA, V, or X induces interleukin-8 and cyclooxygenase-2 gene and protein expression through peroxisome proliferator-activated receptors gamma 1 and 2 in human lung cells. J Biol Chem 2004;279:48550-61.
From athe Department of Immunopathology, Chair of Allergology, Immunology and Dermatology, Faculty of Biomedical Sciences and Postgraduate Training; and bthe Department of Immunology, Rheumatology and Allergy, Faculty of Medicine, Medical University of Lodz, Poland. E-mail: [email protected]. Supported by Polish Government grants N N401 225034 and N401 191 32/4009. Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.
Genetic variants harbored in the forkhead box protein 3 locus increase hay fever risk
REFERENCES 1. Wenzel S. Severe asthma in adults. Am J Respir Crit Care Med 2005;172:149-60. 2. Leslie CC. Regulation of the specific release of arachidonic acid by cytosolic phospholipase A2. Prostaglandins Leukot Essent Fatty Acids 2004;70:373-6. 3. Adler DH, Cogan JD, Phillips JA 3rd, Schnetz-Boutaud N, Milne GL, Iverson T, et al. Inherited human cPLA(2alpha) deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction. J Clin Invest 2008;118:2121-31.
To the Editor: Disturbed regulation of T cells may lead to an imbalance between TH1 and TH2 cells and toward an elevated TH2 response, typically associated with allergy. Regulatory T cells are thought to be responsible for keeping T-cell populations and T-cell effects in balance, whereas modifications of regulatory T-cell signaling may lead to deviated immune responses. Recently, we had shown
Available online April 15, 2010. doi:10.1016/j.jaci.2010.02.016
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J ALLERGY CLIN IMMUNOL JUNE 2010
FIG 1. Depiction of gene structures on chromosome X, identified polymorphisms and LD (r2).The HapMap database was used to identify validated FOXP3 SNPs. In addition, the screened region was extended around the FOXP3 gene, thereby detecting SNPs in and around the neighbor genes CACNA1F, CCDC22, and PPP1R3F. Color coding for the linkage disequilibrium plot is given by Haploview: white (r2 5 0), shades of gray (0 < r2 < 1), and black (r2 5 1). Genotyped tagging SNPs are underlined. UTR, Untranslated region.
that T-cell–associated transcription factors influence the risk for asthma and allergy.1 The transcription factor forkhead box protein 3 (FOXP3; forkhead/winged helix transcription factor) seems to specifically control regulatory T-cell function.2 Deletions or change of function in the human FOXP3 gene result in the development of severe immune dysregulation with polyendocrinopathic and enteropathic X-linked syndrome,3 often associated with high serum IgE levels.4 Despite the prominent role T cells and T-cell regulation have in current asthma and allergy disease models, only 1 study has recently been published on FOXP3 genetics in that field to our knowledge.5 We now investigated 40 kilo base pairs of genomic sequence harboring the FOXP3 gene (Fig 1). On the basis of HapMap data (http://www.hapmap.org),6 we identified 4 tagging polymorphisms (rs2294020, rs2294021, rs3761548, and rs3761549) with a minor allele frequency (MAF) >0.10 capturing all essential genetic information of the FOXP3 gene locus using Haploview software.7 We studied the influence of these single nucleotide polymorphisms (SNPs) on atopic diseases in a cross-sectional population sample of 3099 German children age 9 to 11 years from Dresden and Munich as part of the International Study of Asthma and Allergy in
Childhood phase II. In that study, the prevalence of a doctor’s diagnosis of asthma, hay fever, and atopic dermatitis was assessed by self-administered questionnaires and by objective measurements such as lung function tests, skin prick tests, and serum IgE measurements as previously reported.8,9 Further information on technical details concerning matrix-assisted laser desorption/ionization time-of-flight genotyping or population characteristics is available from the corresponding author on request. Because of the X-chromosomal location, all SNPs were analyzed by fitting an additive effects-only logit model that equates the risks of male hemizygotes with female homozygotes. Hardy-Weinberg equilibrium did not deviate and was tested in females only. To correct for multiple testing, we used a Bonferroni correction in every phenotype. All tests were 2-sided, and the differences were considered significant with P <.05. Calculations were carried out with SAS (version 9.1.3) and SAS/Genetics software (SAS Institute, Inc, Cary, NC). Tagging SNP rs2294020 downstream of FOXP3 slightly increased the risk for atopy assessed by skin prick test in the study population and showed significant associations with hay fever (Table I). Also, when phenotypes related to the primary outcome
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TABLE I. Associations between atopic phenotypes and tagging SNPs showing odds ratios, 95% CIs, and P values (given for Wald test) Tagging SNPs and MAFs
rs 2294020 (MAF 0.31)
rs 2294021 (MAF 0.45)
rs 3761548 (MAF 0.43)
rs 3761549 (MAF 0.12)
Atopic sensitization (SPT1)
1.106 0.998-1.226 P5.0546
1.041 0.943-1.149 P5.4236
0.927 0.839-1.024 P5.1369
1.057 0.908-1.230 P5.4774
Asthmaà
1.100 0.942-1.286 P5.2294 1.028 0.914-1.157 P5.6441
1.072 0.924-1.245 P5.3594 0.928 0.828-1.039 P5.1939
0.967 0.831-1.125 P5.6607 1.066 0.953-1.193 P5.2644
0.951 0.749-1.208 P5.6811 1.022 0.86-1.216 P5.8027
1.256* 1.081-1.460 P5.0030 1.225 1.033-1.453 P5.0196
1.131 0.975-1.313 P5.1048 1.101 0.930-1.303 P5.2635
0.983 0.845-1.143 P5.8234 1.006 0.848-1.193 P5.9469
0.793 0.609-1.032 P5.0842 0.847 0.634-1.131 P5.2605
1.210* 1.069-1.371 P5.0027
1.059 0.937-1.197 P5.3582
0.962 0.851-1.089 P5.542
0.981 0.809-1.191 P5.8476
Atopic dermatitis§
Hay fever§
Hay fever and atopic sensitization
Current hay fever symptoms
SPT, Skin prick test. *Significant after correction for multiple testing. Defined by skin prick test against 6 common aeroallergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Alternaria tenuis, cat dander, and mixed grass and tree pollen). àDefined by a parental report of a physician’s diagnosis of asthma (at least once) or of spastic or asthmatic bronchitis (at least twice) in a self-administered questionnaire. §Defined by a parental report of a physician’s diagnosis of the respective diseases. Significant associations are shown in boldface.
hay fever were analyzed, significant associations between this SNP and hay fever with concomitant sensitization to inhalative allergens and current hay fever symptoms were observed. Associations for hay fever and current symptoms of hay fever remained statistically significant after correction for multiple testing. These results coincide with data from Zhang et al,5 who recently observed associations between FOXP3 SNPs (rs3761547 and rs3761548) and allergic rhinitis in a Chinese population. Common polymorphisms and microsatellite markers had been described in the FOXP3 gene and in its putative promoter and upstream untranslated region before. These gave conflicting association results with a number of complex immune and autoimmune diseases. Mutation events severely affecting FOXP3 function are rare and cause serious immune deficiency syndromes.3 The SNPs that we studied and that are covered by the tagging SNPs genotyped here are located within and downstream of the FOXP3 gene. When linkage disequilibrium (LD) analyses were extended, _ 0.9) were idenseveral other SNPs tagged by SNP rs2294020 (r2 > tified in and around neighboring genes coiled-coil domain containing 22 (CCDC22) and calcium channel, voltage-dependent, a-1F subunit (CACNA1F; Fig 1). Whereas CCDC22 is an unexplored gene with so far unknown biological function, CACNA1F is a voltage-gated calcium channel. Two polymorphisms in the allergic rhinitis associated LD block (rs2294020 located in exon 7 of CCDC22, and rs2075866 located in exon 28 of CACNA1F) were investigated further because of their position in exonic regions, putatively acting as exonic splicing enhancers. Indeed, using ESEfinder software, release 3.0 (http://rulai.cshl.edu/cgi-bin/tools/ ESE3/esefinder.cgi)10,11 allele-specific changes in score matrices for human serine/arginine-rich (SR) proteins were observed, which belong to a protein family essential for splicing (Fig 2). Also, in silico transcription factor binding analyses for promoter and intronic SNPs in the same LD block were performed that
predicted numerous SNP-dependent alterations of transcription factor binding (Alibaba 2.1 software12). However, a comparative genomic analysis searching for conserved noncoding sequences revealed only poor phylogenetic conservation in intergenetic and intragenetic regions of the locus (VISTA Genome browser13). Thus, further functional analyses are needed to determine whether the associations of SNPs in this LD block observed with allergic rhinitis may be influenced by splicing alterations in genes close to FOXP3 and/or locus regulatory elements in the area. Although the causal SNP for this associated LD block is not identified yet, our data and those of Zhang et al5 imply that genetic variations in the region of and around FOXP3 may modify the risk for allergic rhinitis. The association with hay fever we observed is consistent in our population and a second report. Also, when objective measurements were included in the definition of the phenotype, the association was present and specific. Genetic association reports with hay fever are sparse despite the fact that the disease has a very well defined phenotype, is very frequent, and imposes a heavy burden on affected individuals and public health. Within the atopic phenotypes, it is the disease most clearly associated with IgE-specific sensitizations and may represent a good candidate disease for genetically driven variation in regulatory T-cell function. Kathrin Suttner, MSca Martin Depner, MScb Martin Wetzke, MDa Norman Klopp, PhDc Erika von Mutius, MDb Thomas Illig, PhDc Tim Sparwasser, MDd Michael Kabesch, MDa From athe Center for Pediatrics, Clinic for Pediatric Pneumology and Neonatology, Hannover Medical School; bUniversity Children’s Hospital, Ludwig Maximilian’s University, Munich; cthe Institute of Epidemiology, Helmholtz Center Munich,
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FIG 2. Allele-specific values for score matrices of human SR proteins (SF2/ASF, SC35, SRp40, or SRp55) for SNP rs2294020 and SNP rs2075866 using ESEfinder software (release 3.0).10,11Analyzed exonic sequence along the x-axis with respective SNP position highlighted. The height of the bars reflects the motif scores for the different SR proteins, and the width of the bars represents the length of the motif (6, 7, or 8 nucleotides). Only high score values above the selected threshold defined by the ESEfinder software are shown. BRCA1, Breast cancer 1 gene; PO, polymorphic allele; WT, wild-type. Neuherberg; and dInstitute of Infection Immunology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover; a joint venture between the Helmholtz Centre for Infection Research (HZI) Braunschweig and the Hannover Medical School. E-mail: [email protected]. Supported by the National Genome Research Network (NGFN) research grant NGFN 01GS 0810 and the German research foundation as part of the transregional collaborative research program TR22 ‘‘Allergic Immune Responses of the Lung,’’ grant A15/Z3. Genotyping was performed in the Genome Analysis Center of the Helmholtz Zentrum Munich. Funding sources had no influence in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. Disclosure of potential conflict of interest: E. von Mutius is a consultant for GlaxoSmithKline, UCB, and ProtectImmun and has received research support from Airsonett AB. M. Kabesch has financial interests in Roxall, Glaxo Wellcome, Novartis, Sanofi Aventis, and Allergopharma and has received research support from the DFG, BMBF, and the European Union. The rest of the authors have declared that they have no conflict of interest. REFERENCES 1. Suttner K, Rosenstiel P, Depner M, Schedel M, Pinto LA, Ruether A, et al. TBX21 gene variants increase childhood asthma risk in combination with HLX1 variants. J Allergy Clin Immunol 2009;123:1062-8.
2. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003;229:1057-61. 3. Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL, Buist N, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet 2001;27:18-20. 4. Gambineri E, Perroni L, Passerini L, Bianchi L, Doglioni C, Meschi F, et al. Clinical and molecular profile of a new series of patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: inconsistent correlation between forkhead box protein 3 expression and disease severity. J Allergy Clin Immunol 2008;122:1105-12. 5. Zhang L, Zhang Y, Desrosiers M, Wang C, Zhao Y, Han D. Genetic association study of FOXP3 polymorphisms in allergic rhinitis in a Chinese population. Hum Immunol 2009;70:930-4 6. The International HapMap Project. Nature 2003;426:789–796. 7. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005;21:263-5. 8. Weidinger S, O’Sullivan M, Illig T, Baurecht H, Depner M, Rodriguez E, et al. Filaggrin mutations, atopic eczema, hay fever, and asthma in children. J Allergy Clin Immunol 2008;121:1203-9. 9. Weiland SK, von Mutius E, Hirsch T, Duhme H, Fritzsch C, Werner B, et al. Prevalence of respiratory and atopic disorders among children in the East and West of Germany five years after unification. Eur Respir J 1999;14:862-70.
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10. Smith PJ, Zhang C, Wang J, Chew SL, Zhang MQ, Krainer AR. An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. Hum Mol Genet 2006;15:2490-508. 11. Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR. ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acid Res 2003;31:3568-71. 12. Grabe N. AliBaba2: context specific identification of transcription factor binding sites. Silico Biol 2002;2(suppl):S1-S15. 13. Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I. VISTA: computational tools for comparative genomics. Nucleic Acids Res 2004;32(Web Server issue):W273-9. Available online April 16, 2010. doi:10.1016/j.jaci.2010.02.017
Heterogeneity among characteristics of hypereosinophilic syndromes To the Editor: We read with interest the recent Journal article by Ogbogu et al1 reporting the results from a multicenter analysis of patients with hypereosinophilic syndrome (HES). In retrospective and prospective studies, we have analyzed clinical and laboratory data from 88 patients who satisfied the stringent criteria by Chusid et al2 for HES. We prospectively followed 55 patients; data from the remaining 33 were analyzed after chart review. The patients were recruited from 2002 to 2009 from 3 institutions that treated patients with hematologic disorders; the coordinating center was located at Silesian Medical University in Katowice, Poland. Before patients were referred to the hematology units, potential secondary causes of hypereosinophilia were excluded. Informed consent was obtained from all patients according to the Declaration of Helsinki, and the study was approved by the institutional ethics committee. All patients included in the study exhibited symptoms, signs, and imaging or histologic evidence of eosinophilic organ involvement. We performed complete blood count and differential analyses, routine chemistry tests, electrocardiograms, echocardiograms, chest x-rays, ultrasound/computed tomography/magnetic resonance studies, and analyses of bone marrow aspirates and biopsy samples. The methods of analysis of patients with HES, which included cytokine and tryptase measurements, immunophenotyping, and cytogenetic and molecular studies, are presented elsewhere.3 All laboratory tests were performed on treatment-naive patients. Statistical methods were reported previously.3 Because this was a retrospective study, some results were not available for all cases. Clinical responses were assessed after 1 month of treatment. A complete hematologic response was defined as a decrease in the absolute eosinophil count (AEC) to the normal range (<0.7 3 109/L) with resolution of organ involvement. A partial response _50% and/or symptomatic was defined as decrease in the AEC > improvement. ‘‘No response’’ was defined as a stable or increasing AEC and/or stable or progressive organ dysfunction. Forty-seven male and 41 female patients (median age at diagnosis, 56 years; range, 6-84 years) were included in the study. The median AEC was 6.6 3 109/L (range, 1.5-136 3 109/L), and a median 31% of cells were eosinophils (range, 7% to 80%). Patients were divided into 5 subgroups according to Klion et al4; their characteristics are presented in Table I. Of the 88 patients whose blood samples were tested by nested PCR analysis, 16 were found to have the FIP1L1–PDGFRA (Fip1like 1–platelet-derived growth factor receptor a; F/P) fusion transcript (18%; 14 male and 2 female patients). Three patients with F/P transcripts had no organ involvement. The remaining 13 cases
had the following abnormalities: splenomegaly (n 5 11), hepatomegaly (n 5 7), cardiac failure (n 5 3), lymph node enlargement (n 5 2), pulmonary infiltrate (n 5 2), and central nervous system involvement (n 5 1). In 10 patients (12%), the diagnosis of chronic eosinophilic leukemia (CEL) was established on the basis of an increased number of blasts in peripheral blood and/or in bone marrow (n 5 8) or the presence of molecular/cytogenetic markers, including 2 patients with the point mutation JAK2V617F (n 5 2). Splenomegaly was the most common form of organ involvement in this population. Twenty-six patients were found to have CEL (30%). Eight patients had a myeloproliferative variant of HES (etiology unknown) on the basis of at least 4 of the criteria proposed by Klion et al.4 Forty-two patients were tested for T-cell receptor (TCR) rearrangement by using multiplex PCR with heteroduplex analysis; clonality was detected in 18 patients (43%). Three patients were found to have aberrant T-cell phenotypes (by flow cytometry) and TCR clonal rearrangement (by PCR analysis). On the basis of the definition, these 3 patients (7%) were the only ones diagnosed with lymphocytic variant hypereosinophilic syndrome (L-HES).5 Fifty-one patients (including 15 not yet classified cases with clonal TCR) were diagnosed with idiopathic HES. The results of this analysis have been presented in detail.3 Serum tryptase and vitamin B12 levels were reported for 47 and 69 patients, respectively. Serum IgE levels were measured in 74 patients and were found to be increased compared with normal levels in 38 cases. Serum levels of IL-4 and IL-5 were documented for 49 and 51 patients, respectively. We compared the results of laboratory studies between patients with F/P1 and F/P– CEL and those of patients with HES (including idiopathic HES, L-HES, and myeloproliferative variant HES). Patients with CEL had significantly increased white blood cell counts, AECs, and levels of serum vitamin B12 and tryptase compared with patients with HES. Patients with HES had increased platelet counts and serum levels of IL-5 compared with patients with CEL (see this article’s Table E1 in the Online Repository at www.jacionline.org). Results of cytogenetic analyses were available for 48 patients (54%); karyotypes were normal in 84%. The cytogenetic abnormalities were as follows: -Y (n 5 2), complex karyotypes (n 5 2), translocation t(6;11) (n 5 1), -7 (n 5 1), and -17 (n 5 1). In our series, 76 patients (86%) began corticosteroid monotherapy at doses varying from 20 to 60 mg daily; clinical responses were observed in 64% (49 patients). Forty-one patients continued to receive a low dose of corticosteroid maintenance therapy (5-20 mg daily). Discontinuation of corticosteroids led to eosinophilia recurrence in most patients. Ten patients remained in remission even after therapy ended, but the follow-up period was less than 3 months (see this article’s Table E2 in the Online Repository at www.jacionline.org). Serum levels of IL-5 were available for 31 responders and 19 nonresponders. Median levels of serum IL-5 were significantly increased among patients who responded to steroids compared with nonresponders (18.6 vs 11.0; P 5 .01). Sixteen patients who had the F/P fusion transcript had a rapid response to imatinib at starting doses from 100 to 400 mg daily (100%; range, 6-65 months); they remained in sustained hematologic and molecular remission after a median of 42 months of therapy. Sixteen of 26 patients with CEL were resistant to corticosteroid monotherapy. The maximum starting dose was 1 mg/kg daily. The 10 patients with F/P– CEL were treated with a median number of 3