Interspecific transfer of mammalian artificial chromosomes between farm animals

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Intravenous immunoglobulin replacement treatment does not alter polymorphonuclear leukocytes function and surface receptors expression in patients with common variable immunodeficiency

The study of the expression of CD16, CD11b and Siglec 9 receptors and the oxidative burst provides insights on polymorphonuclear leukocytes (PMN) functionality in common variable immunodeficiency (CVID) and on the possible effects of intravenous immunoglobulin (IVIg) infusion. We evaluated in vivo before and soon after IVIg administration the CD16, CD11b and Siglec 9 expression on unstimulated and Escherichia coli-stimulated PMN and the oxidative burst induced by Escherichia coli and PMA. The E. coli stimulation up-regulated CD16 and Siglec 9 expression and it induced a strong CD11b up-regulation at baseline and soon after IVIg. The oxidative burst overlapped that observed in healthy donors when induced by Escherichia coli while it increased when induced by PMA. Soon after IVIg infusion, the oxidative burst decreased only when induced by PMA. Our results showed that the IVIg infusion in vivo had a minimal effect on CVID's PM

The lack of BTK does not impair monocytes and polymorphonuclear cells functions in X-linked agammaglobulinemia under treatment with intravenous immunoglobulin replacement

<div><p>The lack of BTK in X-linked agammaglobulinemia (XLA) patients does not affect monocytes and polymorphonuclear cells (PMN) phenotype and functions. In this study, we show that XLA patients had an increased frequency of the intermediate monocytes subset and that BTK-deficient monocytes and PMN had a normal expression of receptors involved in the activation and cellular responses. We demonstrate that BTK is not required for migration, phagocytosis and the production of reactive oxygen species (ROS) following engagement of FC gamma receptors (FcγR). XLA monocytes and PMN showed an efficient calcium (Ca²⁺)-independent activation of oxidative burst, suggesting that oxidative burst is less dependent by Ca²⁺ mobilization. The phagocytosis was functional and it remained unaltered also after Ca²⁺ chelation, confirming the independence of phagocytosis on Ca²⁺ mobilization. Intravenous immunoglobulin (IVIg) infusion exerted an anti-inflammatory effect by reducing the frequency of pro-inflammatory monocytes. In monocytes, the IVIg reduce the oxidative burst and phagocytosis even if these functions remained efficient.</p></div

PMN oxidative burst—Ca²⁺ chelation.

<p>Whole blood samples were pre-treated with 100 μM of the calcium chelator BAPTA-AM for 30 min and incubated in a water bath for 20 min at 37°C with opsonized <i>E</i>. <i>coli</i> (1-2x10⁹/ml) and PMA (1.62 mM). The conversion of DHR 123 to R 123 was used to evaluate the intracellular ROS production. FSC and SSC characteristics were used to identify the PMN population. (A) BAPTA-AM treatment cause a stronger reduction of <i>E</i>. <i>coli</i>-induced oxidative burst in HD than XLA (▪▪▪p = 0.003). (A) The average reduction in HD was about 75% (▪p = 0.01) and it was about 50% in XLA patients (▪▪p = 0.02). (D) BAPTA-AM did not affect the oxidative burst induced by PMA both HD and XLA patients. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean values and bars standard deviation. Statistical significance, indicated as <i>p</i> value, was determined by the nonparametric Mann-Whitney test and Wilcoxon Signed Rank test. <b>(</b>B and C) It shown PMN <i>E</i>. <i>coli-</i>induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+). (E and F) It shown PMN PMA-induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+).</p

Monocytes oxidative burst—Ca²⁺ chelation.

<p>Whole blood samples were pre-treated with 100 μM of the calcium chelator BAPTA-AM for 30 min and incubated in a water bath for 20 min at 37°C with opsonized <i>E</i>. <i>coli</i> (1-2x10⁹/ml) and PMA (1.62 mM). The conversion of DHR 123 to R 123 was used to evaluate the intracellular ROS production. FSC and SSC characteristics were used to identify the monocytes population. (A) BAPTA-AM treatment cause a stronger reduction of <i>E</i>. <i>coli</i>-induced oxidative burst in HD than XLA <i>(</i>▪▪▪p = 0.003). (A) The average reduction in HD was about 75% (▪p = 0.01) and it was about 50% in XLA patients (▪▪p = 0.02). (D) BAPTA-AM did not affect the oxidative burst induced by PMA both HD and XLA patients. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean values and bars standard deviation. Statistical significance, indicated as p value, was determined by the nonparametric Mann-Whitney test and Wilcoxon Signed Rank test. (B and C) It shown monocytes <i>E</i>. <i>coli</i>-induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+). (E and F) It shown monocytes PMA-induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+).</p

CD181, CD11b, CD11c and Siglec 9 expression on monocytes subsets from HD and XLA patients before and after IVIg infusion.

<p>Whole blood samples were analyzed for the expression of CD181, CD11b, CD11c and Siglec 9 before and after IVIg infusion. The expression of all surface receptors was evaluated by performing a staining at 4°C for 30 min with specific fluorochrome-labeled antibody. Samples were washed, suspended in ice-cold PBS and analyzed by flow cytometry. XLA patients show a similar CD181, CD11b, CD11c and Siglec 9 expression on monocytes subsets compared to HD. After IVIg infusion the expression of all receptors on monocytes subsets remained unaltered. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean values and bars standard deviation.</p

Frequencies of non classical, intermediate and classical monocytes increased from XLA patients before and after IVIg infusion respect to HD.

<p>(A and B) Monocytes subpopulations were phenotypically classified according to their expression of CD14 and CD16 into classical (CD14⁺⁺CD16^-), intermediate (CD14⁺⁺CD16⁺) and non classical monocytes (CD14⁺CD16⁺⁺) in a representative XLA patient before and after IVIg infusion. Percentages denote mean values. (C) Histograms show that non classical and classical monocyte frequencies from XLA patients are similar to that observed on HD and that IVIg infusion did not change their frequency. Intermediate monocytes percentage is increased in XLA patients (▪p = 0.01), IVIg infusion induce their reduction (▪▪p = 0.04) even if remained at higher level respect to HD (▪▪▪p = 0.01). (D) Histograms denote the percentage of increase of the three monocytes subsets of XLA patients respect to HD. Histograms denote mean values and bars standard deviation. Statistical significance as determined by the nonparametric Mann Whitney and Wilcoxon Signed Rank test is indicated as <i>p</i> value.</p