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1.
Scand J Immunol ; 76(2): 158-66, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22540226

ABSTRACT

Implementing precise techniques in routine diagnosis of chronic granulomatous disease (CGD), which expedite the screening of molecular defects, may be critical for a quick assumption of patient prognosis. This study compared the efficacy of single-strand conformation polymorphism analysis (SSCP) and high-performance liquid chromatography under partially denaturing conditions (dHPLC) for screening mutations in CGD patients. We selected 10 male CGD patients with a clinical history of severe recurrent infections and abnormal respiratory burst function. gDNA, mRNA and cDNA samples were prepared by standard methods. CYBB exons were amplified by PCR and screened by SSCP or dHPLC. Abnormal DNA fragments were sequenced to reveal the nature of the mutations. The SSCP and dHPLC methods showed DNA abnormalities, respectively, in 55% and 100% of the cases. Sequencing of the abnormal DNA samples confirmed mutations in all cases. Four novel mutations in CYBB were identified which were picked up only by the dHPLC screening (c.904 insC, c.141+5 g>t, c.553 T>C, and c.665 A>T). This work highlights the relevance of dHPLC, a sensitive, fast, reliable and cost-effective method for screening mutations in CGD, which in combination with functional assays assessing the phagocyte respiratory burst will contribute to expedite the definitive diagnosis of X-linked CGD, direct treatment, genetic counselling and to have a clear assumption of the prognosis. This strategy is especially suitable for developing countries.


Subject(s)
Chromatography, High Pressure Liquid/methods , Granulomatous Disease, Chronic/genetics , Membrane Glycoproteins/genetics , Mutation, Missense , NADPH Oxidases/genetics , Amino Acid Sequence , Base Sequence , Child, Preschool , Chromatography, High Pressure Liquid/economics , Cost-Benefit Analysis , Humans , Infant , Infant, Newborn , Male , Membrane Glycoproteins/chemistry , Molecular Sequence Data , NADPH Oxidase 2 , NADPH Oxidases/chemistry , Time Factors
2.
Scand J Immunol ; 75(1): 96-101, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21923742

ABSTRACT

We investigated the effects of viable, extended freeze-drying (EFD) or heat-killed (HK) Mycobacterium bovis bacillus Calmette-Guérin (BCG) in respiratory burst activity, gene expression of CYBB and NCF1 encoding components of the human phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase, TLR2 expression, and in IL-10 and TNF-α cytokine production by human peripheral blood mononuclear cells (PBMCs). Viable BCG significantly inhibited TLR2 and CYBB gene expression, as well as superoxide release by human PBMC. All BCG stimuli augmented IL-10 release, but only HK BCG or viable BCG increased TNF-α release by PBMCs. Our studies show that viable BCG can impair the NADPH oxidase system activation and the TLR2 route in human PBMCs. As well, different BCG preparations can distinctly influence cytokine production by human PBMCs.


Subject(s)
Adjuvants, Immunologic/pharmacology , BCG Vaccine/pharmacology , Leukocytes, Mononuclear/immunology , NADPH Oxidases/immunology , Respiratory Burst/immunology , Toll-Like Receptor 2/immunology , Adult , Enzyme-Linked Immunosorbent Assay , Freeze Drying/methods , Humans , Interleukin-10 , Leukocytes, Mononuclear/enzymology , Membrane Glycoproteins/immunology , NADPH Oxidase 1 , NADPH Oxidase 2 , RNA/chemistry , RNA/genetics , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 2/genetics , Tumor Necrosis Factor-alpha/immunology
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