Project description:Human basophils were purified from whole blood, pre-treatment (=v3) and mid-treatment (=v6) on omalizumab. In addition, basophils were isolated from non-treated, non-CSU subjects for a comparison of CSU vs. non-CSU.
Project description:A considerable proportion of patients with H1-antihistamine-resistant chronic spontaneous urticaria (CSU) fails to respond to omalizumab. However, there is yet no available biomarker that accurately predicts such a response outcome. The utility of blood basophil and eosinophil levels and exosomal and basophil micro(mi)RNAs as biomarkers of response to omalizumab in CSU was explored. In this prospective cohort study of twenty patients with antihistamine-resistant CSU, subjects were treated with 3-monthly doses of omalizumab, and their response assessed using the weekly urticaria activity score (UAS7) for a period of 12-weeks. Basophil and eosinophil levels in the blood and differentially expressed exosomal and basophil miRNAs of complete responders compared to non-responders were identified at baseline. Canonical pathways, altered by differentially expressed miRNAs, were identified using Ingenuity Pathway Analysis (IPA). Complete responders had higher baseline basophil levels (≥21 cells/L) in the peripheral blood compared to non-responders (P = 0.005). Baseline levels of eosinophils did not differ between the study groups. Complete responders in comparison to non-responders had an expression profile characterized by a significantly higher expression of six exosomal miRNAs (miR-6499-5p, miR-7848, miR-4494-3p, miR-450a-2-3p, miR-6877-3p, and miR-3976) and lower expression of miR-141-3p. Complete responders also had higher expression of three basophil miRNAs (miR-1200, miR-1236-3p, and miR-4496). The exosomal miRNA expression profile was predicted by IPA to alter Tec Kinase Signaling pathway activity (P = 0.003).
Project description:To find genes governing basophil development or functional maturation, we analyzed the gene expression profiles of basophil progenitors by RNA-sequencing.
Project description:Human basophils were examined in vitro for changes in their mRNA expression profiles during stimulation under a variety of conditions. Basophils were obtained from two sources prior to purification, residual cell packs from leukapheresis procedures (which represent the 80% of the sample results) or by venipuncture. For cells obtained by leukapheress, purification included application of elutration, 2-step Percoll gradients and negative selection on Miltenyi columns using StemSep basophil isolation antibodies (see J. Immunol. Methods, 385:51, 2012). For cells obtained by venipuncture, purification included application of 2-step Percoll gradients following by negative selection on Miltenyi columns using StemSep basophil isolation antibodies. The time from subject donation to the start of an in vitro study (first lysis for mRNA) ranged from 6-7 hours for leukapheresis packs to 4-5 hours for venipuncture. Basophil purities averaged 99% for these studies. For most of the studies, purified basophils were incubated in RPMI-1640 media supplemented with human serum albumin (0.03%), 20 µg/ml gentamycin and glutamine (as glutamax) for several hours to 3 days depending on the stimulus. For most experiments, the day 0 lysis acted as the initial control in order to examine profiles for changes due to simple culture. The study included stimulation with interleukin-3, inteleukin-33, interleukin-5, interleukin-2, nerve growth factor, anti-IgE antibody (6061P, Hybridoma Labs, Maryland) or fmet-leu-phe (FMLP) and the sample title indicates the mode and length of stimulation.
Project description:Human basophils were examined in vitro for changes in their mRNA expression profiles during stimulation under a variety of conditions. Basophils were obtained from two sources prior to purification, residual cell packs from leukapheresis procedures (which represent the 80% of the sample results) or by venipuncture. For cells obtained by leukapheress, purification included application of elutration, 2-step Percoll gradients and negative selection on Miltenyi columns using StemSep basophil isolation antibodies (see J. Immunol. Methods, 385:51, 2012). For cells obtained by venipuncture, purification included application of 2-step Percoll gradients following by negative selection on Miltenyi columns using StemSep basophil isolation antibodies. The time from subject donation to the start of an in vitro study (first lysis for mRNA) ranged from 6-7 hours for leukapheresis packs to 4-5 hours for venipuncture. Basophil purities averaged 99% for these studies. For most of the studies, purified basophils were incubated in RPMI-1640 media supplemented with human serum albumin (0.03%), 20 µg/ml gentamycin and glutamine (as glutamax) for several hours to 3 days depending on the stimulus. For most experiments, the day 0 lysis acted as the initial control in order to examine profiles for changes due to simple culture. The study included stimulation with interleukin-3, inteleukin-33, interleukin-5, interleukin-2, nerve growth factor, anti-IgE antibody (6061P, Hybridoma Labs, Maryland) or fmet-leu-phe (FMLP) and the sample title indicates the mode and length of stimulation.
Project description:To find genes regulated by Nfil3 during development or functional maturation, we analyzed the gene expression profiles of basophil progenitors by RNA-sequencing.
Project description:To investigate blood transcriptional changes induced by omalizumab treatment and to determine variations between omalizumab responders and non-responders.
Project description:In this study, we explored gene expression profiles of CD34-CLEC12Ahi pre-basophils isolated from the bone marrow as well as CD34-CLEC12Alo mature basophils from the bone marrow and CD34+ basophil progenitors. We revealed that the gene expression of mature basophils isolated from the bone marrow and spleen resembled each other whereas pre-basophils and mature basophils showed distinct gene expression profiles.