Childhood asthma is associated with mutations and gene expression differences of ORMDL genes that can interact.

BACKGROUND: Genomewide association studies identified ORMDL3 as a plausible asthma candidate gene. ORMDL proteins regulate sphingolipid metabolism and ceramide homeostasis and participate in lymphocyte activation and eosinophil recruitment. Strong sequence homology between the three ORMDL genes and ORMDL protein conservation among different species suggest that they may have shared functions. We hypothesized that if single nucleotide polymorphisms (SNPs) in ORMDL3 alter its gene expression and play a role in asthma, variants in ORMDL1 and ORMDL2 might also be associated with asthma. METHODS: Asthma associations of 44 genotyped SNPs were determined in at least 1303 subjects (651 asthmatics). ORMDL expression was evaluated in peripheral blood mononuclear cells (PBMC) from 55 subjects (eight asthmatics) before and after allergen stimulation, and in blood (n = 60, 5 asthmatics). Allele-specific cis-effects on ORMDL expression were assessed. Interactions between human ORMDL proteins were determined in living cells. RESULTS: Sixteen SNPs in all three ORMDLs were associated with asthma (14 in ORMDL3). Baseline expression of ORMDL1 (P = 1.7 × 10(-6) ) and ORMDL2 (P = 4.9 × 10(-5) ) was significantly higher in PBMC from asthmatics, while induction of ORMDLs upon stimulation was stronger in nonasthmatics. Disease-associated alleles (rs8079416, rs4795405, rs3902920) alter ORMDL3 expression. ORMDL proteins formed homo- and heterooligomers and displayed similar patterns of interaction with SERCA2 and SPT1. CONCLUSIONS: Polymorphisms in ORMDL genes are associated with asthma. Asthmatics exhibit increased ORMDL levels, suggesting that ORMDLs contribute to asthma. Formation of heterooligomers and similar interaction patterns with proteins involved in calcium homeostasis and sphingolipid metabolism could indicate shared biological roles of ORMDLs, influencing airway remodeling and hyperresponsiveness.

Hepatocyte transplantation using the domino concept in a child with tetrabiopterin nonresponsive phenylketonuria.

Phenylketonuria is a metabolic disease caused by phenylalanine hydroxylase deficiency. Treatment is based on a strict natural protein-restricted diet that is associated with the risk of malnutrition and severe psychosocial burden. Oral administration of tetrahydrobiopterin can increase residual enzyme activity, but most patients with severe clinical phenotypes are nonresponders. We performed liver cell transplantation in a 6-year-old boy with severe tetrahydrobiopterin nonresponsive phenylketonuria who failed to comply with diet prescriptions. The transplanted hepatocytes were obtained in part from an explanted glycogen storage type 1b liver. Following two infusions, blood phenylalanine levels returned within the therapeutic target while the phenylalanine half-life assessed by loading tests decreased from 43 to 19 h. However, 3 months later, blood phenylalanine concentrations increased and the phenylalanine intake had to be reduced. Cell-based therapy is a promising therapeutic option in phenylketonuria, and the domino concept may solve the issue of cell sources for hepatocyte transplantation.

Interaction of bronchoalveolar lavage fluid with polyplexes and lipoplexes: analysing the role of proteins and glycoproteins.

BACKGROUND: Plasmid DNA complexed with cationic lipids (lipoplexes) or cationic polymers (polyplexes) has been used for gene transfer into the lung. Topical gene administration of lipoplexes or polyplexes into the lung after intratracheal instillation or aerosolisation could cause interaction of the complexes with extracellular substances of the airway surface liquid (ASL). These extracellular interactions might be causal for the observed inefficient transfection rate in vivo after topical administration. Therefore, we studied the impact of bronchoalveolar lavage fluid (BALF) on reporter gene expression mediated by non-viral gene vectors. BALF was considered as a model system to mimic possible interactions of the gene vectors with the ASL. METHODS: BALF was taken from 15 patients who underwent diagnostic bronchoscopy. Lipoplexes and polyplexes were incubated with increasing concentrations of BALF and major components of the BALF such as albumin, mucin and alpha(1)-glycoprotein, as a representative of glycosylated proteins. As cationic polymers, we tested dendrimers (fractured PAMAM) and polyethylenimine 25 kDa (PEI) and, as cationic liposomes, we used Lipofect-AMINE. The effect of BALF on polyplexes and lipoplexes was analysed by transfection experiments, fluorescence-quenching assay, 2-D-gel electrophoresis, SDS-PAGE, DNAse protection assay, size and zeta-potential measurements. RESULTS: BALF inhibited polyplex- and lipoplex-mediated gene transfer. Analysing components of BALF, we found that dendrimer-mediated gene transfer was not inhibited by any specific component. PEI-mediated gene transfer was dose-dependently inhibited by alpha(1)-glycoprotein, slightly inhibited by mucin, but not inhibited in the presence of albumin. Lipoplex-mediated gene transfer was inhibited by mucin at higher concentrations and by albumin, but not by alpha(1)-glycoprotein. 2-D-gel electrophoresis revealed that proteins of the BALF were adsorbed more intensively to lipoplexes than to polyplexes. In addition, mucin and alpha(1)-glycoprotein also adsorbed more intensively to lipoplexes than to polyplexes. Adsorption of BALF components led to a decrease in the positive zeta-potential of lipoplexes and led to a negative zeta-potential of polyplexes. Complement cleavage fragment C3 beta, and in the case of lipoplexes also the C3 alpha fragment, were found among the proteins opsonised on gene vectors. CONCLUSIONS: Our study shows that BALF contains inhibitory components for non-viral gene transfer. We could not detect a specific inhibitory component, but inhibition was most likely due to the change in the surface charge of the gene vectors. Interestingly, there is evidence for complement activation when the route of pulmonary gene vector administration is chosen. Consequently, shielding of gene vectors to circumvent interaction with the ASL environment should be a focus for pulmonary administration in the future.