Table1_Circulating serum profile of small non-coding RNAs in patients with anaphylaxis beyond microRNAs.docx

IntroductionAnaphylaxis is the most severe manifestation of allergic disorders. Currently, an increasing number of cells, pathways and molecules involved in the etiopathogenesis of anaphylaxis are being discovered. However, there are no conclusive biomarkers to confirm its diagnosis. Small non-coding RNAs (sncRNAs) are 18-200 nucleotide molecules that can be divided into: microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), transference RNA derived fragments (tRFs) and YRNA derived fragments (YRFs). These molecules participate in cell-cell communication modulating various physiological processes and have been postulated as non-invasive biomarkers of several pathologies. Therefore, in this study we characterized the serum circulating profile of other sncRNA beyond miRNAs in two populations of 5 adults and 5 children with drug- and food-mediated anaphylaxis, respectively.MethodsSamples were obtained from each patient under two different conditions: during anaphylaxis and 14 days after the reaction (control). The sncRNA analysis was carried out by Next Generation Sequencing (NGS).ResultsA total of 671 sncRNAs (3 piRNAs, 74 snoRNAs, 54 snRNAs, 348 tRFs and 192 YRFs) were identified in adults with drug-induced anaphylaxis, while 612 sncRNAs (2 piRNAs, 73 snoRNAs, 52 snRNAs, 321 tRFs and 164 YRFs) were characterized in children with food-mediated anaphylaxis. However, only 33 (1 piRNA, 4 snoRNAs, 1 snRNAs, 7 tRFs and 20 YRFs) and 80 (4 snoRNAs, 6 snRNAs, 54 tRFs and 16 YRFs) of them were statistically different between both conditions, respectively. Among them, only three (Y_RNA.394, Y_RNA.781 and SCARNA2) were common to both adults and children analysis.DiscussionThis study provides a differential profile of circulating serum sncRNAs beyond miRNAs in patients with anaphylaxis, postulating them as candidate biomarkers for this pathological event and as novel mediators of the reaction.</p

Table2_Circulating serum profile of small non-coding RNAs in patients with anaphylaxis beyond microRNAs.docx

IntroductionAnaphylaxis is the most severe manifestation of allergic disorders. Currently, an increasing number of cells, pathways and molecules involved in the etiopathogenesis of anaphylaxis are being discovered. However, there are no conclusive biomarkers to confirm its diagnosis. Small non-coding RNAs (sncRNAs) are 18-200 nucleotide molecules that can be divided into: microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), transference RNA derived fragments (tRFs) and YRNA derived fragments (YRFs). These molecules participate in cell-cell communication modulating various physiological processes and have been postulated as non-invasive biomarkers of several pathologies. Therefore, in this study we characterized the serum circulating profile of other sncRNA beyond miRNAs in two populations of 5 adults and 5 children with drug- and food-mediated anaphylaxis, respectively.MethodsSamples were obtained from each patient under two different conditions: during anaphylaxis and 14 days after the reaction (control). The sncRNA analysis was carried out by Next Generation Sequencing (NGS).ResultsA total of 671 sncRNAs (3 piRNAs, 74 snoRNAs, 54 snRNAs, 348 tRFs and 192 YRFs) were identified in adults with drug-induced anaphylaxis, while 612 sncRNAs (2 piRNAs, 73 snoRNAs, 52 snRNAs, 321 tRFs and 164 YRFs) were characterized in children with food-mediated anaphylaxis. However, only 33 (1 piRNA, 4 snoRNAs, 1 snRNAs, 7 tRFs and 20 YRFs) and 80 (4 snoRNAs, 6 snRNAs, 54 tRFs and 16 YRFs) of them were statistically different between both conditions, respectively. Among them, only three (Y_RNA.394, Y_RNA.781 and SCARNA2) were common to both adults and children analysis.DiscussionThis study provides a differential profile of circulating serum sncRNAs beyond miRNAs in patients with anaphylaxis, postulating them as candidate biomarkers for this pathological event and as novel mediators of the reaction.</p

Clinical data of patients included in this study.

<p>Clinical data of patients included in this study.</p

Lipid transfer proteins (LTP) included in the homemade array were separated by SDS-PAGE and stained with Coomassie Blue.

<p>Replicas were electrotransferred and incubated with polyclonal antibodies produced against peach LTP (dilution 1∶500). The name of the proteins corresponds to Table I.</p

Average weight of allergens included in the co-sensitization graph.

<p>Average weight of allergens included in the co-sensitization graph.</p

Recognition frequencies of food and pollen LTPs by geographical area.

<p>The recognition frequencies, shown as percentage of positive response (%), were obtained incubating the LTP microarray with single sera from allergic patients. Only LTPs with a positive response of more than 20% (taking all patients into account) are represented. Percentage positive responses and significant differences (p<0.05) are indicated.</p

Co-sensitization graph of LTP allergens.

<p>Each node represents one allergen (LTP, white ovals non-LTP allergens, blue squares) and the links represent co-sensitization of one or more sera for the linked allergens. The weight of each link, between 0 and 1, measures the degree of co-sensitization. For the sake of clarity, only the 35 links of weights greater than 0.55 of the total 190 existing links are plotted.</p

Purified proteins included in the LTP microarray.

<p>r recombinant protein;</p>*<p>non-LTPs or control proteins included in the array are marked with an asterisk.</p

Pollen counts (grains/m³ of air) of the regions included in the study.

*<p>Average pollen counts (grains/m³). The period is indicated in brackets.</p><p>Data were obtained as the average of the previous years (period in parentheses), from the Comité de Aerobiología-SEAIC (<a href="http://www.polenes.com/concentraciones.html" target="_blank">http://www.polenes.com/concentraciones.html</a>) and the PIA-Punto de información de Aerobiología-UAB (<a href="http://lap.uab.cat/aerobiologia/" target="_blank">http://lap.uab.cat/aerobiologia/</a>).</p

Frequency of LTP sensitization.

<p><b>A</b>. Recognition frequencies of food and pollen LTPs comparing fruit allergic patients with and without pollen sensitization (Pollen Fruit Allergy and Fruit Allergy, respectively). The recognition frequencies, shown as percentage of positive response (%), were obtained incubating the LTP microarray with single sera from allergic patients. <b>B</b>. Analysis of LTP crossreactivities by inhibition assays using the LTP microarray as solid phase and Pru p 3, Art v 3 and Cas s 8 (5, 1, 0.1, 0.01 µg/mL) as inhibitors. The inhibition percentage of the IgE binding capacity is indicated. Means (n = 3) and SDs (bars) are represented. All tests were performed in triplicate.</p