Lung symptoms in pseudohypoaldosteronism type 1 are associated with deficiency of the alpha-subunit of the epithelial sodium channel.

OBJECTIVE: To study patients with autosomal recessive pseudohypoaldosteronism type 1 and to relate pulmonary disease to gene mutations of the epithelial sodium channel (ENaC). STUDY DESIGN: Clinical and laboratory data were collected from 4 Swedish patients with pseudohypoaldosteronism type 1. The genes for ENaC and cystic fibrosis transmembrane conductance regulator were analyzed for mutations with methods including DNA sequencing. RESULTS: Three novel mutations were found in the alpha-gene of ENaC, 2 frameshifts (1449delC and 729delA) and 1 missense mutation resulting in the substitution of leucine for serine 562 in the alpha-chain (S562L). The 1449delC mutation was found in all patients in either homozygous or heterozygous form and seems to be the predominant cause of pseudohypoaldosteronism in Sweden. The allele coding for S562L also contained a transition converting tryptophan 493 to arginine (W493R), which seems to be a rare polymorphism. All patients had pulmonary symptoms to various degrees. The bacterial findings resembled, to some extent, those in cystic fibrosis, but development of chronic lung disease and progressive decline in lung function were not observed. CONCLUSIONS: Genetic deficiencies of the alpha subunit of the ENaC are associated with prominent lung symptoms, which are, however, clearly different from those in cystic fibrosis.

Intravenous synthetic alphaGal saccharides delay hyperacute rejection following pig-to-baboon heart transplantation.

Several oligosaccharides containing the terminal structure Gal(alpha)1-3Gal (alphaGal) and different side chains were tested in vitro for their ability to block natural anti(alpha)Gal antibodies. A di-and a trisaccharide (di(alpha)Gal and tri(alpha)Gal) were selected. A blood group B baboon, having IgG and IgM natural antipig titers of 1:256 and 1:1024 and a hemolytic titer (to pig red blood cells, RBCs) of 1:8, was chosen to measure pharmacokinetic parameters of the saccharides and to assess the extent of in vivo neutralization of the antibodies. Three grams each of the di(alpha)Gal and the tri(alpha)Gal dissolved in saline were administered by bolus intravenous (i.v.) injection. Blood samples were collected at various times and urine was collected at 8 and 24 h. Plasma and urine concentrations of the alphaGal saccharides were estimated by an ELISA specially developed for this study. A fast distribution phase followed by equilibrium and excretion phases were observed, indicating a T1/2 in the order of 1 h. Fifty-eight per cent of the saccharides were recovered in the urine within 24 h. Determination of antipig antibody binding by FACS analysis and of serum cytotoxicity titers for pig endothelial cells demonstrated that a 70% reduction in binding and cytotoxicity could be achieved with plasma saccharide levels of 300-400 microg/ml. Six months later, a pig heart was transplanted heterotopically into the baboon. A 3-g bolus of the saccharide mixture (1.5 g of each saccharide) was given i.v. before allowing blood reperfusion of the transplanted heart, followed by an i.v. infusion of 1 g/hr for 1 hr and 0.5 g/hr for the 3 succeeding hours. Blood concentrations of the saccharides, CH50, hematology and cytotoxicity for PK15 cells were estimated in blood samples taken at various times. Heart function was observed to be satisfactory for 8 h, but was found to have ceased at 18 h. Myocardial biopsies taken at 3 and 5 h showed congestion only, suggestive of minimal vascular rejection, but by 18 h demonstrated severe vascular rejection. In conclusion, alphaGal saccharide therapy given for a period of 4 h delayed, but did not totally prevent, the development of vascular rejection in the pig-to-baboon heart transplant model. alphaGal saccharide therapy may be one of several useful approaches for the prevention of hyperacute rejection in pig-to-primate organ transplantation.

Effects of exogenous stress protein 70 on the functional properties of human promonocytes through binding to cell surface and internalization.

The presence of antibodies against the major stress protein, Hsp70, in patients with autoimmune diseases led us to hypothesize that Hsp70 may occur extracellularly, and could exert chaperoning and regulatory effects on various cells. We examined the action of pure Hsp/Hsc70 on the main physiological functions of human promonocytic U-937 cells. The protein was isolated from calf muscle and was shown to be a mixture of inducible Hsp70 (60%) and constitutive Hsc70 (40%) isoforms. It was observed that the addition of the protein up-regulated two major monocyte/macrophage differentiation markers, CD11c and CD23, by 20-35%, while it had no effect on CD14. The experiments performed to investigate the influence of Hsp/Hsc70 on the reaction of U-937 cells to differentiation stimuli demonstrated that the addition of the protein prior to PMA was able to inhibit binding of proper transcription factors to double-symmetry and cAMP-response elements of the c-fos early response gene promoter. Administration of exogenous Hsp/Hsc70 prior to treatment with the tumor necrosis factor-alpha significantly lowered the number of apoptotic and necrotic cells. In no case did the control protein, ovalbumin, taken in the same concentration give a comparable effect on U-937 cells. Since the Hsp/Hsc70 effects occurred within the first hour of co-incubation, and therefore they might be explained by its interaction with the cell surface, we assayed binding of the biotinylated protein to U-937 cells by immunoenzyme assay, flow cytometry and indirect immunofluorescence. Using these three techniques we were able to detect Hsp/Hsc70 bound to cells after a 20 min incubation. According to flow cytometry data, at this time 32% of cells were positively stained with streptavidin-FITC. Immunofluorescence studies demonstrated Hsp/Hsc70 bound to the cell surface after a 20 min incubation followed by induction of patch and cap-like structures. One hour later, the majority of the protein had been internalized by U-937 cells.

Effects of interferons and tumour necrosis factor-alpha on human lung cancer cell lines and the development of an interferon-resistant lung cancer cell line.

Thirteen human lung cancer cell lines, 7 representing small cell lung cancer (SCLC) and 6 different types of non-SCLC, were tested for sensitivity to tumour necrosis factor alpha (TNF-alpha) and interferon alpha and gamma (IFN-alpha and gamma) using an automated fluorometric microculture cytotoxicity assay (FMCA). One SCLC line (H-82) was found to be sensitive to IFN-alpha in short-term (72 h) culture, whereas after prolonged (5 days) culture two additional SCLC cell lines responded to IFN-gamma. TNF-alpha inhibited the growth of one large cell carcinoma cell line (H-157), whereas all SCLC lines were found to be insensitive. The combination of IFN-gamma and TNF-alpha produced no further response compared with the single agents used alone. By continuous cultivation of the IFN-alpha-sensitive cell line H-82 in the presence of increasing concentrations of IFN-alpha, an IFN-alpha-resistant subline (H-82) was established. This line displayed a high degree of resistance ( > 100 fold) to IFN-alpha and cross-resistance to IFN-gamma. There was no alteration in the number of IFN binding sites, in the growth rate, the expression of selected surface markers for SCLC or the expression of multidrug resistance markers in the H-82R subline compared with the parental H-82 cell line. The results demonstrate a heterogeneous response of SCLC cell lines to IFN-alpha and gamma and TNF-alpha with only a minority of the cell lines responding to these agents by growth inhibition. The IFN-alpha and gamma H-82R subline may serve as a valuable tool in future studies on the mechanisms of IFN antitumour activity.