[Epidemiological situation of Afghanistan].
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):903-13.[Epidemiological situation of Afghanistan]. [Article in Polish]1.1Wojskowy Instytut Medyczny, Zak?ad Medycyny Morskiej i Tropikalnej w Gdyni. kktropmed@wp.plAbstractAfghanistan is one of the poorest countries and its demographic indicators are the worst in the world. Epidemiologic profile is dominated by vector-, water-, food-borne diseases and respiratory tract diseases. Significant threats pose also environmental factors, as an effect of high and low temperature, wind, sand, dust and local fauna. The whole of threats complete mines and unexploded ordnance scattered hundreds of square kilometers of Afghan land. Their number is assesses at 7 to 10 millions what makes Afghanistan one of the most mined and dangerous for traveling countries in the world. Information concerning current epidemiological hazards which can find travelers visited Afghanistan is presented in the article.PMID:
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/etc/nginx/nginx.conf.Gene expression analysis predicts insect venom anaphylaxis in indolent systemic mastocytosis - Niedoszytko - 2010 - Allergy -
Wiley Online Library
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To cite this article: Niedoszytko M, Bruinenberg M, van Doormaal JJ, de Monchy JGR, Nedoszytko B, Koppelman GH, Nawijn MC, Wijmenga C, Jassem E, Oude Elberink JNG. Gene expression analysis predicts insect venom anaphylaxis in indolent systemic mastocytosis. Allergy 2011; 66: 648&657.AbstractBackground:& Anaphylaxis to insect venom (Hymenoptera) is most severe in patients with mastocytosis and may even lead to death. However, not all patients with mastocytosis suffer from anaphylaxis. The aim of the study was to analyze differences in gene expression between patients with indolent systemic mastocytosis (ISM) and a history of insect venom anaphylaxis (IVA) compared to those patients without a history of anaphylaxis, and to determine the predictive use of gene expression profiling.Methods:& Whole-genome gene expression analysis was performed in peripheral blood cells.Results:& Twenty-two adults with ISM were included: 12 with a history of IVA and 10 without a history of anaphylaxis of any kind. Significant differences in single gene expression corrected for multiple testing were found for 104 transcripts (P&&&0.05). Gene ontology analysis revealed that the differentially expressed genes were involved in pathways responsible for the development of cancer and focal and cell adhesion suggesting that the expression of genes related to the differentiation state of cells is higher in patients with a history of anaphylaxis. Based on the gene expression profiles, a na&ve Bayes prediction model was built identifying patients with IVA.Conclusions:& In ISM, gene expression profiles are different between patients with a history of IVA and those without. These findings might reflect a more pronounced mast cells dysfunction in patients without a history of anaphylaxis. Gene expression profiling might be a useful tool to predict the risk of anaphylaxis on insect venom in patients with ISM. Prospective studies are needed to substantiate any conclusions.AbbreviationsFcfold change differenceGOgene ontologyIgEimmunoglobulin EISMindolent systemic mastocytosisIVAinsect venom anaphylaxisKEGGKyoto encyclopedia of genes and genomesMAPKmitogen-activated protein kinaseSPTskin-prick testVITvenom immunotherapyWntwingless int pathwayMastocytosis is an uncommon disease resulting from a pathological increase in mast cells in different tissues including skin, bone marrow, liver, spleen, and lymph nodes (). The clinical presentation of mastocytosis is heterogeneous, varying from sole skin presentation found in urticaria pigmentosa and mastocytoma, to different forms of systemic disease including indolent systemic mastocytosis (ISM), smoldering systemic mastocytosis, aggressive systemic mastocytosis and mast cell leukemia (). Of the adult patients with systemic mastocytosis, the large majority (ca. 90%) has the indolent form of the disease.Symptoms of the disease result from skin involvement, mast cell mediator release and massive mast cell infiltration, as found in aggressive variants of the disease. Symptoms of mast cell degranulation may vary from pruritus and flushing to anaphylaxis with profound hypotension and with occasionally even fatal outcome (). The cumulative prevalence of anaphylaxis in patients with mastocytosis has been reported to be as high as 50% (). Anaphylaxis is thought to be the most important burden for the majority of patients with mastocytosis.The most important eliciting factor of anaphylaxis in patients with mastocytosis is an insect sting (). It is estimated that 30% of patients with mastocytosis suffer from insect venom anaphylaxis (IVA) (). Thus, patients are advised to carry epinephrine auto-injectors and often start with venom immunotherapy (VIT), probably lifelong (). Because fatal anaphylaxis also has been reported, even without a history of previous anaphylactic sting reactions (), some authors postulate prophylactic treatment for all patients with mastocytosis (). However, because less than half of the patients with mastocytosis will develop anaphylaxis on insect stings during their lifetime, such a strategy of prophylactic treatment would inflict a significant burden on a large group of patients that is in no need for such an intervention. Therefore, it is of great interest to identify those patients at risk for IVA, ideally using a minimally invasive and highly sensitive assay. Unfortunately, it has not been possible so far to predict which patients with mastocytosis are at risk for anaphylaxis to an insect sting ().The mechanism(s) responsible for development of anaphylaxis in some and the lack of such reactions in other patients with mastocytosis are mainly unknown (). It has been hypothesized that the predisposition to anaphylaxis is related to activation of mast cell kinase pathways or calcium ion channels (). Although severe anaphylaxis may occur in patients with all variants of mastocytosis (), they are most frequent in the indolent form of mastocytosis. The mast cells in mastocytosis display several phenotypic abnormalities, among which the activating somatic mutation of the KIT receptor that also affects histamine release (). However, the contribution of these intrinsic alterations to the sensitivity for developing (insect venom) anaphylaxis is unknown.Better understanding of the molecular differences between mastocytosis patients with an IgE mediated anaphylaxis to insect venom and mastocytosis patients without such reactions might be helpful to predict the risk for anaphylaxis in those patients with mastocytosis who have not been stung yet. Analysis of gene expression allows for the direct recognition of gene activation. Whole-genome expression analysis has the potential to reveal differences in activation of gene networks involved in anaphylaxis between patients with and without IVA.The aim of this study therefore was to analyze whether in ISM differences in gene expression profiles can be identified in peripheral blood cells between patients with a history of anaphylaxis to insect venom who never received VIT and those patients without anaphylaxis of any kind.Materials and methodsPatientsPatients with ISM from the Department of Allergology, University Medical Center Groningen (UMCG) were included. ISM has been diagnosed in all patients according to the WHO criteria () by serum tryptase measurement and bone marrow investigation including histopathological, cytological, CD2 and CD25 flow cytometry and genetic examinations. None of these patients had been treated with VIT in the past. Subjects suffering from other severe chronic or/and malignant diseases and pregnant women were excluded.Patients were divided into two groups:Group 1: patients with a history of anaphylaxis caused by Hymenoptera venom. The diagnosis of IVA was based on medical history (grade IV reaction according to Mueller () and a positive skin tests and/or sIgE.Group 2: patients who did not experience any allergic reactions after insect stings (they were stung at least once after ISM has been diagnosed) or by other known or unknown factors in the past during at least 10&years of follow-up after the diagnosis of ISM has been made.The study was approved by the Medical Ethical Committee of the UMCG (METc ).Collection of blood samplesFrom all patients, RNA was isolated from whole blood using the PAXgene Blood RNA Tubes (Qiagen, Valencia, CA, USA). All tubes were immediately frozen and stored in &20&C till RNA isolation (maximal period 2&months). RNA was isolated using PAXgene Blood RNA Kit CE (Qiagen, Venlo, the Netherlands). All RNA samples were stored in &80&C till labeling and hybridization.The quality and concentration of RNA was determined using the 2100 Bioanalyzer (Agilent, Amstelveen, the Netherlands) with the use of Agilent RNA 6000 Nano Kit. Samples with a RNA integrity number &7.5 were used for further analysis on expression arrays.Gene expressionFor amplification and labeling of RNA with Illumina, TotalPrep 96 RNA Amplification kit was used (Applied Biosystems, Nieuwerkerk ad IJssel, the Netherlands). For each sample, we used 200&ng of RNA. The Human HT-12_V3_expression arrays (Illumina, San Diego, CA, USA) were processed according to the manufacturer&s protocol. Slides were scanned immediately using an Illumina BeadStation iScan (Illumina).Image and data analysisFirst-line check, background correction, and quantile normalization of the data were performed with Genomestudio Gene Expression Analysis module v 1.0.6 Statistics. Entities containing at least 75% of samples with a signal intensity value above the 20th percentile in 100% of the samples in at least 2 groups were included for the further analysis.Data analysis was performed using genespring package version 10.0.0 (Agilent Technologies, Santa Clara, CA, USA). Genes for which expression was significantly different between both groups were chosen based on a log2 fold change &2 in gene expression, t-test P-value &0.05 and a Benjamini&Hochberg false discovery rates &0.05. The P-values presented in the text were corrected for multiple testing. A na&ve Bayes prediction model was used to build a prediction model assessing the risk of anaphylaxis in patients with ISM (). A na&ve Bayesian classifier is a mathematical process computing the probability of classifying the patient in a group of risk of anaphylaxis or without risk based on the results of gene expression (). The expression of each particular gene is regarded as an independent predictor classifying patient to the particular group.Functional annotation of genes was described by the Go Process Analysis and pathways of the Kyoto encyclopedia of genes and genomes (KEGG) () with Genecodis functional annotation web-based tool ().Clinical data of this study were analyzed with statistica 8.0 (StatSoft, Tulsa, OK, USA). The chi-square was used to compare the number of patients with IVA in the studied groups. The mean difference of serum tryptase and urinary excretions of methylhistamine and methylimidazole acetic acid was analyzed using the U-Mann&Whitney test. P-values &0.05 were considered statistically significant.ResultsA total of 22 patients with ISM were included. Twelve patients (two men) had a history of IVA (group 1) and 10 patients (five men) had no history of anaphylaxis caused by insect venom or other factors (group 2). In group 2, one patient was asymptomatically sensitized to wasp and bee venom. No significant differences in clinical characteristics were found between both groups, except for sensitization to insect venom (P&=&0.0001) (). Ages at diagnosis of ISM and levels of serum tryptase and urinary excretions of methylhistamine and methylimidazole acetic acid were in medians and ranges in group 1: 49 (25-70) years, 29.4 (5.13-112) &g/l, 335 (153-1024) &mol/mol creatinine, and 3.3 (1.4-7.7) mmol/ and in group 2: 50 (34-64) years, 48.4 (4.62-155) &g/l, 452 (77-1046) &mol/mol creatinine, and 4.5 (1.4-10.9) mmol/mol creatinine, respectively.Table&1.&
&Clinical characteristics of the patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis (group 1) and without such a history (group 2) Group 1M50neg28.91531.40.09negposnegnegposnormalposnegF43adult UP34.13804.80.29negposposposposnormalposnegF64adult UP21.71663.10.11posposposnegposnormalposnegF47neg48.36046.50.17posposposposposnormalposnegF25adult UP27.410243.4n.d.n.d.n.d.n.d.posposnormalposnegF61neg15.22662.30.30negposnegposposnormalposnegF51neg74.51942.70.10posposposposposnormalposposF38adult UP5.131901.70.13posposnegposposnormalposnegM37adult UP1124047.7n.d.n.d.n.d.posposposnormalposnegF36neg31.35426.50.17posposposposposslightly hyperplasticposnegF70neg19.72892.70.10posposposnegposnormalposnegF54adult UP29.85934.1n.d.n.d.n.d.posposposnormalposnegGroup 2F64adult UP155104610.90.49posposposposposnormalnegnegF50adult UP20.42933.00.06posposposposposnormalnegnegM34adult UP55.44334.90.45posposposposposnormalnegnegM41juvenile UP1464704.01.61negposposposposnormalnegnegF64adult UP4.621021.60.10posposposnegposnormalnegnegM54neg28.2771.40.09posposposnegposnormalposposM50adult UP52.35306.4n.d.n.d.n.d.n.d.posposnormalnegnegF43adult UP36.58515.90.34posposposposposnormalnegnegM38neg44.43103.4n.d.n.d.n.d.posposposnormalnegnegF63adult UP1098236.3n.d.n.d.n.d.n.d.posposnormalnegnegComparison of gene expression profilesWhole-genome gene expression analysis was performed on RNA samples isolated from peripheral blood cells. From all 48.804 probes present on the array, 48.676 transcripts passed the quality control and were included for further analysis.Of all analyzed transcripts, 1951 showed a log2&&&2-fold difference in gene expression: 967 (49%) genes were increased and 984 (51%) were decreased in patients with IVA.To assess whether the differentially expressed genes were enriched for a specific cellular function, we performed genome ontology (GO) analysis using the Genecodis website. The main processes to which the differentially expressed genes map are signal transduction, multicellular organism development, transcription, cell differentiation, metabolic process, and ion transport (). Using the KEGG database (), we found that the pathways that are most significantly enriched for in our list of log2&&&2-fold differentially expressed genes are pathways involved in cancer, focal adhesion, cell adhesion, ubiquitin mediated proteolysis, Wnt signaling, and calcium signaling ().Table&2.&
&Gene co-occurrence annotation found by Genecodis () (GOSlim Process Function) for the genes differentially expressed (log2fc&2) between patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis and without anaphylaxis of any kind 88 genes)88 (1022)8.37904e-092.5975e-07GO:0007165: signal transduction50 genes&874 (37435)50 (1022)9.52928e-071.47704e-05GO:0007275: multicellular organismal development73 genes)73 (1022)2.37117e-062.45021e-05GO:0006350: transcription8 genes43 (37435)8 (1022)1.8644e-050.GO:0007165: signal transductionGO:0030154: cell differentiation29 genes471 (37435)29 (1022)4.93153e-050.GO:0008152: metabolic process30 genes503 (37435)30 (1022)6.56447e-050.GO:0006811: ion transport18 genes232 (37435)18 (1022)7.74516e-050.000343GO:0006629: lipid metabolic processTable&3.&
&Gene co-occurrence annotation found by Genecodis (Kyoto encyclopedia of genes and genomes [KEGG] pathways) () for the genes differentially expressed (log2fc&2) between patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis and without anaphylaxis of any kind 25 genes320 (37435)25 (1022)3.05203e-060.(KEGG) 05200: Pathways in cancer17 genes194 (37435)17 (1022)2.65238e-050.(KEGG) 04510: Focal adhesion13 genes131 (37435)13 (1022)6.47261e-050.(KEGG) 04514: Cell adhesion molecules (CAMs)&5 genes&19 (37435)&5 (1022)0.0.(KEGG) 05200: Pathways in cancer (KEGG) 04120:Ubiquitin mediated proteolysis12 genes129 (37435)12 (1022)0.0.(KEGG) 04120: Ubiquitin mediated proteolysis13 genes150 (37435)13 (1022)0.0.(KEGG) 04310: Wnt signaling pathway14 genes176 (37435)14 (1022)0.0.(KEGG) 04020: Calcium signaling pathway&9 genes&82 (37435)&9 (1022)0.0.(KEGG) 04512: ECM-receptor interaction15 genes206 (37435)15 (1022)0.0.(KEGG) 04810: Regulation of actin cytoskeleton&9 genes&96 (37435)&9 (1022)0.0.0130712(KEGG) 04912: GnRH signaling pathway&9 genes&98 (37435)&9 (1022)0.0.0139566(KEGG) 04916: Melanogenesis&7 genes&62 (37435)&7 (1022)0.0.0130655(KEGG) 00980: Metabolism of xenobiotics by cytochrome P45016 genes262 (37435)16 (1022)0.00245770.0190472(KEGG) 04010: MAPK signaling pathwaySubsequently, we analyzed the 104 transcripts that were significantly different between the two patient groups in single gene expression corrected for multiple testing (P&&&0.05) revealing a log2&&&3-fold difference in expression. In patients with a history of IVA, 37 transcripts (36%) were increased, whereas 67 transcripts (64%) were decreased in expression (). A hierarchical clustering of the differentially expressed genes is presented in . The most significant differences (P corrected for multiple testing &0.002) were found in eight transcripts: HS 552770, HS 41192, RBMY1A3P, DVL1, G0S2, HS 540329, HS 546027, and LOC283487. Except for HS 552770, which was up-regulated in patients with a history of IVA, the remaining seven transcripts were up-regulated in patients without anaphylaxis. Of these genes, it is known that DVL1 and G0S2 are related to the development of cancer, the function of the remaining transcripts is still unknown. GO analysis using the KEGG database on this list of 104 transcripts revealed the enrichment of genes mapping to two processes: pathways in cancer and the mitogen-activated protein kinase (MAPK) signaling pathway (). The function of 46 (39%) genes is unknown yet.Table&4.&
&List of genes that were differentially expressed (log2 fc&&&3, P&&&0.05 corrected for multiple testing by Benjamini&Hochberg method P&&&0.05) between patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis and without anaphylaxis of any kind ABI3BPAbi gene family, member 3 (nesh) binding protein0.00180.00940.263.81ANKRD6Ankyrin repeat domain 60.00990.01580.462.15B3GAT1Beta-1,3-glucuronyltransferase 1 (glucuronosyltransferase p)0.01020.01580.422.41C14ORF115Chromosome 14 open reading frame 1150.00250.00940.254.05C14ORF118Chromosome 14 open reading frame 1180.00340.01043.080.33C14ORF165Chromosome 14 open reading frame 1650.01080.01590.342.96C18ORF56Chromosome 18 open reading frame 560.02120.02690.422.41C20ORF106Chromosome 20 open reading frame 1060.00350.01043.130.32C20ORF132Chromosome 20 open reading frame 1320.00730.01382.360.42CCDC134Hypothetical protein flj223490.00950.01530.422.36CDC42BPAcdc42-binding protein kinase alpha (dmpk-like)0.00440.01120.392.56CEP57Centrosomal protein 57&kda0.01470.02041.980.51COL9A1Collagen, type ix, alpha 10.00890.01480.234.29CYORF15AChromosome y open reading frame 15a0.01620.02170.195.31CYORF15BChromosome y open reading frame 15b0.00800.01410.224.59CYORF15B&0.04170.04770.119.07DHX9Deah (asp-glu-ala-his) box polypeptide 90.00680.01382.730.37DVL1Dishevelled, dsh homolog 1 (drosophila)0.00010.00200.224.46EIF1AYEukaryotic translation initiation factor 1a, y-linked0.02940.03470.412.42EWSR1Ewing sarcoma breakpoint region 10.02580.03170.362.79FAM110AChromosome 20 open reading frame 550.00880.01482.120.47FBLN1Fibulin 10.00810.01410.551.81FBXL21F-box and leucine-rich repeat protein 210.00350.01040.244.19Hypothetical protein dkfzp434p0550.00380.01040.273.64G0S2G0/g1switch 20.00020.00230.137.74GLYATGlycine-n-acyltransferase0.00140.00890.402.49HGDHomogentisate 1,2-dioxygenase (homogentisate oxidase)0.00120.00890.402.52HLA-DRB4Major histocompatibility complex, class ii, dr beta 10.01840.02380.462.20HOXA6Homeobox a60.01680.02212.650.38HRBHiv-1 rev-binding protein0.00370.01045.050.20HS,107801&0.00900.01480.362.80HS,124514&0.00600.01380.283.55HS,134088&0.00710.01380.333.04HS,189987&0.01070.01593.230.31HS,41192&0.00000.00090.204.90HS,436654&0.00240.00940.372.68HS,527174&0.00220.00942.610.38HS,537553&0.00070.00550.244.20HS,540329&0.00010.00230.214.87HS,540415&0.00720.01380.234.31HS,541520&0.00030.00364.500.22HS,544017&0.00600.01382.350.43HS,545032&0.00840.01432.220.45HS,545163&0.00610.01380.244.15HS,545866&0.00200.00943.430.29HS,546019&0.03530.04093.310.30HS,546027&0.00010.00230.273.68HS,549742&0.01060.01590.244.15HS,552354&0.00150.00890.392.54HS,552770&0.00000.00064.810.21HS,562039&0.02870.03462.800.36HS,562265&0.00040.00444.820.21HS,563189&0.00070.00552.480.40HS,563982&0.01140.01660.204.90HS,565704&0.00180.00940.432.30HS,566231&0.01330.01872.510.40HS,572999&0.00140.00890.462.17HS,576804&0.00380.01043.770.27HS,580555&0.00020.00260.254.03HS,581365&0.00410.01100.462.18HS,581671&0.00150.00893.510.28HS,581933&0.00270.00940.313.23HS,583304&0.00230.00940.362.76HS,583989&0.00760.01390.382.66HS3ST4Heparan sulfate (glucosamine) 3-o-sulfotransferase 40.01310.01860.263.91IIP45Invasion inhibitory protein 450.00640.01380.342.96INOC1Ino80 complex homolog 1 (s, cerevisiae)0.00730.01380.462.17JARID1DSmcy homolog, y-linked (mouse)0.02160.02710.118.92JUPJunction plakoglobin0.01060.01592.460.41KLRC1Killer cell lectin-like receptor subfamily c, member 10.00540.01290.442.30LBHHypothetical protein dkfzp566j0910.00040.00422.940.34LBPLipopolysaccharide-binding protein0.00770.01400.224.48LIPJLipase-like, ab-hydrolase domain containing 10.00270.00942.900.34Hypothetical protein loc2834870.00020.00230.323.13Hypothetical loc3878850.00200.00940.352.87Hypothetical gene supported by bc035379; bc0421290.00070.00553.570.28Similar to protein tyrosine phosphatase, receptor type, u isoform 2 precursor0.00350.01045.550.18Hypothetical protein loc6417420.00630.01380.293.48Similar to atp-binding cassette sub-family d member 1 (adrenoleukodystrophy protein) (aldp)0.00640.01383.670.27Similar to mucin 6, gastric0.02910.03473.790.26Similar to hypothetical protein flj364920.00330.01042.360.42Similar to n-acylsphingosine amidohydrolase 20.01010.01580.392.57&0.02530.03140.109.90LRTM1Leucine-rich repeats and transmembrane domains 10.00690.01382.970.34LTKLeukocyte tyrosine kinase0.01600.02172.540.39MAP2K3Mitogen-activated protein kinase kinase 30.00250.00940.224.54MAP7Microtubule-associated protein 70.04330.04922.460.41MEGF8Egf-like domain, multiple 40.00360.01044.980.20N4BP2L1Hypothetical gene cg0180.01560.02140.323.16PDGFAPlatelet-derived growth factor alpha polypeptide0.00240.00940.263.84PKIBProtein kinase (camp-dependent, catalytic) inhibitor beta0.01650.02180.333.03PRKYProtein kinase, y-linked0.02760.03360.118.87RBM3Rna-binding motif (rnp1, rrm) protein 30.01890.02430.412.47RBMY1A3PRNA-binding motif protein, Y-linked, family 1, member A3 pseudogene0.000010.00170.244.20RGMBrgm domain family, member b0.03040.03562.430.41RHDrh blood group, ccee antigens0.00510.01283.000.33RP11-45B20,2Similar to hypothetical protein mgc489150.00160.00920.273.71SPAG17Sperm associated antigen 170.00690.01380.185.45SPNSialophorin (gpl115, leukosialin, cd43)0.01160.01670.501.99SUDS3Suppressor of defective silencing 3 homolog (s, cerevisiae)0.00710.01380.342.96TBPL2Tata box-binding protein like 20.00520.01280.372.69TMSB4YThymosin, beta 4, y-linked0.00260.00940.195.30TNFRSF4Tumor necrosis factor receptor superfamily, member 40.00810.01414.050.25TRAF4Tnf receptor-associated factor 40.00430.01120.402.51Figure&1. &Hierarchical clustering dendrogram of differentially expressed genes that were differentially expressed (log2 fc&3, P&&&0.05 corrected for multiple testing by Benjamini&Hochberg method P&& 0.05) between patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis and without anaphylaxis of any kind. Each column represents a patient sample, each row an individual gene. For each gene green color represents underexpression, red color overexpression, and black signal missing data.Subsequently, we used a prediction model that uses a na&ve Bayes (NB) classifier, based on the 104 most significant differentially expressed genes. This model was able to differentiate ISM patients with IVA from those without anaphylaxis with a sensitivity and specificity of 100%.To relate the whole-genome expression results to specific cell expression profiles, we analyzed the expression profiles of leukocyte-specific genes expressed in dendritic cells, B cells, memory T cells, mast cells, and basophils as described by Liu et al. (). A statistically significant difference in expression comparing both patient groups was found for the mast cell&specific genes CTTNBP2 encoding cortactin-binding protein 2, a central regulator of cytoskeletal rearrangement in mitosis (), which was up-regulated in patients with IVA (P&=&0.03) and SIGLEC6 encoding sialic acid&binding Ig-like lectin 6, playing a role in cell&cell interactions (), which was down-regulated in patients with IVA (P&=&0.04).DiscussionThe results of our study demonstrate that we are able to identify in ISM genes that are differentially expressed between the peripheral blood cells from patients with IVA and those from patients without a history of anaphylaxis. GO analysis reveals that the differentially expressed genes are enriched for genes that function in several pathways that regulate the balance between proliferation versus terminal differentiation: Wnt signaling pathway, focal and cell adhesion, calcium signaling, extracellular matrix interactions, pathways in cancer and MAPK signaling (). So, our data indicate that in spite of a high number of mast cells in all patients, the sensitivity to develop anaphylaxis might be correlated to the differentiation state of the mast cells. The functional annotation of the four most significantly differentially expressed genes between the two patient groups confirms this observation (). Together, these data indicate that ISM patients with a more differentiated mast cell phenotype are at risk for developing of IVA.Table&5.&
&Function of the most differently expressed genes with P&&&0.01 DVL 1Wnt signaling pathway ()Up-regulation of DVL1 was found in inflammatory bowel disease mucosa and also in colon cancer cells ()DVL1 is a key molecule in Wnt/planar cell polarity signaling pathway controlling tissue polarity and cell movement () and is up-regulated in various types of human cancers like melanoma, gastric and lung cancer, and neuroblastoma ()PDGFAUp-regulation of both PDGFA and PDGFRA was found in patients with gastrointestinal stromal tumors forming autocrine-paracrine loop (), and in prostate cancer, where the over expression was found not only in cancer but also in stromal cells ()MAPK signaling pathway ()G0S2Expressed i.e. in lymphocytes and monocytes during the switch from G0 to G1 phase of the cell cycle ()Up-regulation was found also in patients with vasculitis, psoriasis, rheumatoid arthritis and systemic lupus erythematosus ()MAP2K3Product of this gene MAP kinase kinase 3 activates p38 MAP kinase that induces IL1&, IL1& IL12 production (), B-cell proliferation (), and probably also enhances murine mast cell survival ()Further, our data indicate that gene expression profiling on peripheral blood cells from patients with ISM enables to differentiate patients with IVA from ISM patients without anaphylaxis in their medical history. It indicates that this procedure might be adapted to identify mastocytosis patients at risk for anaphylaxis to benefit from prophylactic treatment.It is assumed that baseline serum tryptase concentrations reflect the mast cell mass of the body. In patients without mastocytosis, an increased tryptase concentration is associated with an increased risk for the occurrence of insect venom allergy (), and in addition, it is also associated with the more severe reactions (). In patients with mastocytosis, higher basal tryptase values were also associated with a greater risk of anaphylaxis (). In our ISM group, however, we found in patients with IVA a trend toward lower levels of tryptase in serum and lower urinary excretion of the histamine metabolites methylhistamine and methylimidazole acetic acid, which are also thought to reflect mast cell burden. This difference might be because of the fact that we only included patients with ISM and excluded of patients with solely cutaneous mastocytosis (i.e. mastocytosis in the skin without systemic involvement). This is in contrast to the findings of Brockow who analyzed patients with both cutaneous and systemic mastocytosis. Our data might indicate that in patients with mastocytosis, the total mast cell load does not contribute to the particular risk for IVA or anaphylaxis of any kind. In fact, we postulate that the absence of anaphylaxis might be because of a more pronounced mast cell dysfunction in the patients with nonanaphylactic mastocytosis . This assumption is supported by the observation that in our group of Dutch and Polish patients with a more advanced (i.e. smoldering, aggressive, or leukemic) form of mastocytosis (n&=&10&+&13), none of the subjects is suffering from IVA (Kluin-Nelemans H., de Monchy J.G.R., van Doormaal J.J., Oude Elberink J.N.G. and Niedoszytko M. unpublished observation). The differences in expression of the described genes were not found in patients with IVA without mastocytosis as described previously by our group (). Therefore, these effects seem to be specific for patients with mastocytosis.The observation that the risk of anaphylaxis is related to more differentiated phenotype of mast cells may also fit very well to the recently developed concept of a mast cell activation syndrome (MMAS) (). Patients with mast cell activation syndrome suffer from repeated and severe anaphylaxis and show some criteria of systemic mastocytosis, but fail to meet sufficient criteria to be classified as mastocytosis. It might be possible that the mast cell phenotype in these patients is yet more differentiated compared to patients with mastocytosis, and it would be interesting to evaluate whether the findings of our study might also be of relevance in this new patient group ().Using a prediction model that uses na&ve Bayes (NB) classifier based on the 104 most significantly and most differentially expressed genes, it was possible to differentiate ISM patients with IVA from those without anaphylaxis. The value of single gene expression was regarded as an independent predictor of IVA. This differentiation was not possible using well-known measurements, such as serum tryptase, urinary histamine metabolites, and KIT mutation analysis.It is known that the prevalence of Hymenoptera sensitization in the general population is higher than the prevalence of IVA. Sensitization to insect venom as evidenced by skin-prick tests and specific IgE was also found in one ISM patient without a history of IVA. In some patients with IVA, especially with co-existing mastocytosis, it is difficult to detect the presence of specific IgE (). Here, it has been speculated that other mechanisms, besides specific IgE, might be involved in insect venom-associated activation of mast cells. As we have found no differences in other factors that could be related to the level of specific IgE (i.e. time elapsed since the last sting, number of stings, atopic status), the observed differences in gene expression may be related to the regulation of the production, secretion, binding to cells, and/or clearance of IgE.Our data indicate that it might be possible to construct a simple method based on expression values of several genes in peripheral blood cells that can be used in clinical practice to assess the risk of IVA by a minimally invasive technique in a similar way to predicting the response to chemotherapy in oncology (). Our results need to be replicated in independent populations, especially in a prospective way evaluating the natural history of anaphylaxis in patients with ISM.So far, it is not recommended to perform prophylactic VIT in mastocytosis patients, although this has been recently suggested by Rueff et&al. () who reported a patient with ISM and urticaria pigmentosa who died of an anaphylaxis after a yellow jacket sting without a history of previous anaphylactic sting reactions. Our study indicates that it might be possible to identify patients with mastocytosis at risk for anaphylaxis and might enable to define criteria for the selection of mastocytosis patients eligible for &prophylactic& VIT as has been suggested elsewhere ().The analysis of peripheral whole-blood cells was chosen because sampling is less invasive for patients compared to bone marrow aspiration. Furthermore, the standardized method of RNA isolation and analysis we used reduces potentially deleterious effects of sample handling on the result and allows reliable clinical application. Because of this methodology, the observed differences in expression were detected in other cell lines of peripheral blood than mast cells. The analysis of whole-blood cells might be justified because recent data show that KIT mutation can be present not only in mast cells but also in myeloid and lymphoid cell lineages ().We can only speculate whether the higher expression of genes related to the neoplastic transformation of cells is clinically relevant. At the one side, it is known that patients with ISM may develop haematological malignancies, but at the other side this phenomenon has proven to be very rare in our patient population that is presenting mainly with urticaria pigmentosa, osteoporosis, and/or anaphylaxis. The presenting symptom in all our patients with mastocytosis who have a haematological malignancy was that malignancy.In conclusion, we demonstrate that in ISM (), differentially expressed genes in patients with IVA are annotated to act in pathways favouring cellular differentiation over proliferation, indicating that the differentiation state of the mast cell might be a critical determinant of the sensitivity to anaphylaxis, and () gene expression profiling might be a useful tool in identifying patients who are at risk for IVA by a minimally invasive technique. Further studies in larger groups of patients are required to validate our approach for the development of a predictive tool to be used in clinical practice.AcknowledgmentThe research was supported by the Foundation for Polish Science and a grant of the Polish Ministry of Science and Higher Education N6 and N.
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