ABSTRACT
We have devised a method for the purification of human basophils from umbilical cord blood by FCM. Umbilical cord blood was collected from six healthy, full-term deliveries. After separation of red blood cells, mononuclear cells were isolated by Ficoll-Hypaque. Six samples followed by positive selection using flowcytometry (FCM) for CD203c(+)CD45(int+) cells. Purity and recovery of cells were measured. Purity and recovery of basophils by FCM with CD203c(+)CD45(int+) markers were 95.02 +/- 2.94% and 61.42 +/- 5.95%. Cell sorting for CD203c(+)CD45(int+) cells by FCM is an improved method for obtaining pure umbilical cord blood-derived basophils. We established cord blood-derived basophil purification technique as a source from which active basophils can be isolated for immunochemical characterization.
Subject(s)
Basophils/cytology , Fetal Blood/cytology , Flow Cytometry , Basophils/physiology , Cell Separation , Ficoll/chemistry , Humans , Leukocyte Common Antigens/immunology , Leukocyte Common Antigens/metabolism , Leukocytes/cytology , Leukocytes/immunology , Phosphoric Diester Hydrolases/immunology , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/immunology , Pyrophosphatases/metabolismABSTRACT
OBJECTIVE: To identify the genetic phenotypes of asthma based on the examination of the umbilical cord blood from neonates of the mothers with asthma. METHODS: Umbilical cord blood was collected from 39 asthmatic lying-in women and 46 healthy lying-in women. Eosinophils and basophils absolute counts, total immunoglobulin E, and interleukin (IL)-4 were examined. Mononuclear cells (MNCs) from the umbilical cord blood were cultured. IL-4 in the supernatant was test by ELISA. White cells were collected from the umbilical cord blood. Basophils and T lymphocytes were purified, cultured, and stimulated by hyper osmotic mannitol. RT-PCR was used to test the mRNA expression of IL-4 in the supernatant. RESULTS: There were no significant differences in the counts of basophils and eosinophils, total IgE, and IL-4 in the cord blood, and IL-4 in the supernatant of cultured MNCs. After stimulation of hyper-osmotic mannitol, equine anti-human IgE, and dust mite the HBDT positive rates were 64.10%, 17.95%, and 0% respectively in the neonates of asthmatic women group, the former two rates being both significantly higher than those in the neonates of healthy women group (15.22% and 2.17% respectively, P < 0.05 and P < 0.025). After stimulation of hyper-osmotic mannitol, IL-4 mRNA was markedly expressed in the purified basophils of both groups and was almost not expressed in the purified eosinophils of both groups. CONCLUSION: There is qualitative abnormalities in the basophils from the cord blood of neonates of asthmatic mothers that strongly express IL-4 mRNA. Such characteristics may be an asthma-related genetic phenotype.
