Induction of gp91-phox, a component of the phagocyte NADPH oxidase, in microglial cells during central nervous system inflammation.

Gp91-phox is an integral component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex that generates reactive oxygen species (ROS) in activated circulating phagocytes. The authors previously demonstrated that gp91-phox knockout (KO) mice show significant protection from neuronal injury after cerebral ischemia--reperfusion injury, suggesting a pivotal role for this enzyme. Moreover, results from chimeric mice suggested that elimination of gp91-phox from both circulating phagocytes and a putative central nervous system (CNS) source were required to confer neuroprotection. In the current study, the authors demonstrated gp91-phox-specific immunostaining of perivascular cells in the CNS of control rats. However, after transient cerebral ischemia, gp91-phox-positive phagocytes were observed within the core ischemic region and activated microglial cells were positive in the penumbra. Such activated microglial cells were also gp91-phox-positive in the CNS of a chimpanzee with mild meningitis. Finally, in humans, both normal adult CNS tissues and isolated fetal microglial cells expressed gp91-phox mRNA. These microglia also expressed mRNA for the five other known components that comprise the NADPH oxidase complex. These data strongly suggest that microglial cells may contain a functionally active NADPH oxidase capable of generating ROS during CNS inflammation.

Plasma factor VII levels are influenced by a polymorphism in the promoter region of the FVII gene.

Genetic factors play a role in determining the variability of plasma factor VII (FVII) levels in healthy individuals. There is also evidence that high serum lipids are associated with high FVII levels in plasma. In the promoter region of the human FVII a DNA polymorphism has been described, originating from a decanucleotide insert present in the less frequent allele. This biallelic system, reflecting the absence (AA) or presence (Aa) of the decanucleotide, can be detected by a DNA enzyme immunoassay of PCR products. We evaluated the association between the polymorphic alleles and the levels of FVII:Ag and FVII:C in 100 healthy individuals and in 19 hypertriglyceridemic individuals. Among healthy individuals, mean FVII:Ag and FVII:C levels of those with the homozygous genotype (A/A; mean FVII:Ag 112%, mean FVII:C 109%) were significantly higher (P < 0.001) than the mean levels of those with the heterozygous genotype (A/a, mean FVII:Ag 80%, mean FVII:C 90%; P < 0.001). Similar genotype-associated differences for FVII:Ag and FVII:C were found in individuals with triglycerides above 250 mg/dl (P < 0.05). FVII:C and FVII:Ag levels were positively related to triglycerides only in individuals without the insert (P < 0.01); there was no significant relationship in those carrying the allele with the insert (A/a; P = 0.43 and 0.08). Our findings of genotype-associated differences in FVII levels and interactions with triglycerides are similar to those obtained with the amino acid dimorphism at position 353 of the factor VII protein.

Normal pituitary size in two patients with growth hormone gene deletion.

Somatotrope cells account for almost 50% of the volume of the pituitary gland, which is usually hypoplastic in subjects with growth hormone (GH) deficiency. Magnetic resonance imaging (MRI) was used to describe the sellar area of two siblings affected by GH gene deletion and hence absent GH secretion. Pituitary size and shape were normal in both subjects and there were no other abnormal findings in the sellar area. In particular, pituitary height (5.6 and 5.1 mm) and volume (268 and 229 mm3) in the two patients were within the normal range for age and higher than the usually reported values in GH deficient subjects. Our results suggest that the amount of GH secreted from the pituitary may have little influence on the dimensions of the gland.

Molecular study of human growth hormone gene cluster in three families with isolated growth hormone deficiency and similar phenotype.

The growth hormone (GH) gene (hGH-N) cluster was analysed using polymerase chain reaction, Southern and polymorphism analysis in five patients (including two pairs of siblings) with extreme short stature and absence of GH secretion. Patients 1 and 2 (siblings) were homozygous for a large deletion removing four genes of the cluster: hGH-N, hCS-L, hCS-A and hGH-V. Both siblings produced high anti-GH antibody levels in response to exogenous GH therapy, followed by growth arrest a few months after starting replacement therapy. In patient 3 we detected a heterozygous deletion which involved three genes of the cluster (hCS-A, hGH-V, hCS-B) and left an intact hGH-N gene. Direct sequencing of hGH-N specific amplified fragments excluded the presence of any point mutations in exons and splicing regions. In patients 4 and 5 (sisters) our study did not demonstrate any gene deletions. Analysis of polymorphic restriction patterns in this family demonstrated that both sisters inherited the same alleles from the father but different alleles from the mother, suggesting that the defect was not linked to the hGH-N gene. These results confirm the difficulty of clinical identification of subjects with hGH-N deletion and underline the importance of DNA analysis in patients with absence of GH secretion and extreme growth retardation.

Factor VII gene polymorphisms contribute about one third of the factor VII level variation in plasma.

To assess the role of genetic variation in determining factor VII (FVII) activity and antigen levels we studied a polymorphism located in the 5' region of the gene (5'F7), an intronic mutation (IVS7), and the 353Arg-Gln polymorphism. All the polymorphisms, which showed strong allelic association, analyzed separately or in combination by the one-way analysis of variance, were associated with significantly different FVII levels. The 5'F7 and 353Arg-Gln polymorphic systems, which have very similar allele frequencies, contributed to a similar extent to the total phenotypic variance, whereas the contribution of the IVS7 polymorphism was lower. Genetic variation at the FVII locus, evaluated on combined genotypes, accounted for up to 40% of the phenotype FVII variance. As also shown by the two-way analysis of variance, the use of two out of three markers is advisable, and since the 5'F7 polymorphism can be screened by a simple immunoassay, it should be preferred for population-based studies. No substantial differences between FVII activity and FVII antigen levels were found, thus suggesting that the variation was due to biosynthesis- or stability-mediated mechanisms. The genetic control of FVII levels described in this study plays an important role in determining plasma FVII level variability, which may influence the hemostatic balance.