Judit Kramer

Transinhibition of C1 inhibitor synthesis in type I hereditary angioneurotic edema.

To ascertain the mechanism for decreased synthesis of C1 inhibitor (C1 INH) in certain patients with the autosomal dominant disorder hereditary angioneurotic edema, we studied expression of C1 INH in fibroblasts in which the mutant and wild type mRNA and protein could be distinguished because of deletion of exon 7 (delta Ex7). In the HANE delta Ex7 cells, the amount of wild type mRNA (2.1 kb) was expressed at 52 +/- 2% (n = 5) of normal, whereas the mutant mRNA was 17 +/- 1% (n = 5) of normal. Rates of synthesis of both wild type and mutant proteins (11 +/- 3 and 3 +/- 1% of normal, respectively) were lower than predicted from the mRNA levels. There was no evidence of increased C1 INH protein catabolism. These data indicate that there are multiple levels of control of C1 INH synthesis in type I hereditary angioneurotic edema. Pretranslational regulation results in < 50% of the mutant truncated 1.9-kb mRNA. In addition, translational regulation results in decreased synthesis of both wild type and mutatn C1 INH proteins. These data suggest a transinhibition of wild type C1 INH translation by mutant mRNA and/or protein.

A marked drop in the incidence of the null allele of the B gene of the fourth component of complement (C4B*Q0) in elderly subjects: C4B*Q0 as a probable negative selection factor for survival

SummaryThe incidence of allotypes of the genes of the fourth component (C4) and factor B of the complement system was compared in 252 persons under 45 years of fage (“young” group) with 482 people between 61 and 90 years of age (“old” group). One hundred people older than 90 years of age (nonagenarians) were also investigated. A striking difference was found between the “young” and “old” groups in the incidence (16.1% and 5.4%, respectively) of a silent gene of the C4B allele (C4B✸Q0). This difference was even more marked among “young” and “old” men (17.6% vs 3.4%). The incidence of the C4B✸Q0 allele in women dropped to the level of the men only in the nonagenarian group. The most probable explanation for this finding is that people carrying the C4B✸QO allele die from as yet unidentified disease(s) in their middle-age. Therefore, male (and to a lesser extent female) carriers of this allele may have a considerably shorter life expectancy than individuals without a silent gene in the C4B locus.

C4B*Q0 allotype as risk factor for myocardial infarction

The prevalence of the deficient, silent allotype of the C4B gene (C4B*Q0) is lower in elderly than in young healthy people, particularly in men.1 This may reflect increased mortality from some disease in middle aged carriers of the C4B*Q0 gene. We determined the presence of the gene in patients with acute myocardial infarction because myocardial infarction is the leading cause of death among middle aged Hungarians. We studied 181 consecutive patients with confirmed Q wave myocardial infarction admitted to four hospital departments between June 1992 and January 1993 (125 men, 56 women, aged 42-78), 93 consecutive patients with symptoms of angina pectoris (65 men, 28 women; aged 43-62) who were examined by coronary angiography (coronarography), and 737 previously tested healthy controls (252 young people aged 22-45 and 485 elderly people aged 60-99).1 Myocardial infarction was diagnosed as typical chest pain lasting …

Frequencies of certain complement protein alleles and serum levels of anti-heat-shock protein antibodies in cerebrovascular diseases

Background and Purpose A strong correlation exists between the intensity of atherosclerotic alterations in different arteries. Marked differences exist, however, in the age and sex distribution and risk factors for coronary heart disease (CHD) and cerebrovascular disease (CVD). We therefore performed genetic and immunologic studies in patients with CVD. Methods We studied 292 patients with CVD (stroke or transient ischemic attack) and as control either 198 healthy blood donors and 485 healthy elderly (aged >60 years) people (genetic study) or 94 blood donors aged 45 to 60 years and 49 healthy elderly (aged >60 years) people (anti–heat-shock protein [hsp] measurements). Allele frequencies of 3 genes (C4A, C4B, and C3) encoding proteins of the complement system were determined by electrophoresis and immunofixation. Serum concentration of autoantibodies against 60-kDa heat-shock protein (anti-hsp60) was measured by the enzyme-linked immunosorbent assay method. Results Marked differences were observed between CVD patients and controls in the genetic studies. In the CVD patients aged >60 years, the frequency (11.3%) of the deficient allele of the C4B gene (C4B*Q0) was significantly (P =0.0003) higher than that of the healthy controls (5.4%). By contrast, in the group aged 45 to 60 years, the frequency of the C4B*Q0 allele was lower in patients than in controls. Serum concentration of anti-hsp60 in the CVD patients did not differ from control values. Conclusions In previous studies C4B*Q0 frequency was reported to be higher in CHD patients aged 45 to 60 years than in aged-matched controls. Moreover, high anti-hsp60 levels were found in CHD patients. These findings contrast with our present report of lower frequency of C4B*Q0 in CVD patients. Therefore, genetic and immunologic factors may at least partly explain the differences between the natural history and risk factors of CHD and CVD.

Endothelial cells cultured from human brain microvessels produce complement proteins factor H, factor B, C1 inhibitor, and C4

The inflammatory mediators, cytokines and complement proteins are believed to regulate the sequential events during the development of lesions secondary to ischaemia and reperfusion. The endothelial cell monolayer of the brain microvasculature is the critical interface between the blood-borne mediators and brain tissue. The involvement of these cells in complement production and regulation has not been well documented. In the present study, expression of complement proteins (C1 inhibitor, factor H, factor B, C4) by cultured endothelial cells obtained from human brain microvessels has been characterized. Interferon gamma upregulates the production of all the complement factors studied. Serine proteases, plasmin and miniplasmin induce the expression of C4, decrease the level of ELISA detectable C1 inhibitor, and do not affect the production of factors H and B. These data indicate that complement proteins are expressed locally by the brain microvessels, and may modulate the inflammatory responses of brain tissue.

Usefulness of densitometry in typing of human complement component C4

C4, one of the early components of the classical complement pathway, is encoded within the class III region of the major histocompatibility complex of man at two highly polymorphic loci, C4A and C4B. Widespread use of C4 typing is, however, hindered by the lack of objective standardized methods. Objective evaluation of the C4 typing pattern by densitometry was first proposed by Christiansen and coworkers (1983) and Zhang and co-workers (1988). The use of this method in finding homoduplications of the C4 genes was described in a recent abstract (Truedsson et al. 1987). To the best of our knowledge, however, no systematic comparative study of densitometric vs visual evaluation of C4 typing has been performed. In the present work we measured the relative intensities of different C4A and C4B bands by densitometric scanning of pattern of 191 plasma samples. Ethylenediaminetetraacetate (EDTA) plasma samples were collected from paternity studies and stored at 7 0 °C until used. C4 phenotyping was carried out by the method of Awdeh and Alper (1980) using carboxypeptidase/B treatment as described by Sire and Cross (1986). Intensity of the Coomassie Brillant Blue R-250 staining of C4 bands in the gel was measured by scanning the gels with a Cello3 densitometer (Shandon, Ltd., Astmoor, United Kingdom). The C4A and C4B regions were evaluated as a whole, and the results were given as the staining intensity of the C4A region in the percentage of staining of the whole gel. Calculations were carried out by an IBM compatible computer (PC-420XT) using STATGRAPHICS program (STSC Inc., Plus+Ware, Braegen Grp. Inc., Toronto, Canada). Means of the groups were compared by the Mann-Whitney-Wilcoxon test. The probability that a value was correctly assigned to a group was calculated by the tail area probability analysis. Neuraminidase-treated EDTA plasma samples of 94 persons were typed for C4. The patterns were evaluated

Genetic regulation of the impaired immune response to hepatitis-B vaccine associated with low TCR density in end stage renal disease patients: contribution of complement C4 and factor B alleles.

We have studied the relationship between T-cell receptor (TCR) density, genetic factors and the specific immune response in 153 end stage renal disease (ESRD) patients on haemodialysis immunised with HBsAg vaccine. One-hundred and nineteen patients raised a protective (> 10 U/ml) antibody response to hepatitis-B vaccination (responder, R), while 34 patients were found to be non-responders (NR). The density of the T-cell receptors was determined by flow cytometry. Proliferation of the T-cells induced by autologous monocytes presenting HBsAg was also measured and expressed as a stimulation index (SI). MHC class I, II and III alleles of the patients were also determined. The densities of TCR/CD3 receptors in NR patients were found to be significantly decreased as compared to the R patients (189 +/- 22 vs. 282 +/- 58 arbitrary units, P = 1.3 x 10(-7). TCR/CD3 receptor densities were found to be strongly associated (Spearman correlation coefficient: 0.84, P < 0.000001) with the SI values. Both parameters were found to be under dual genetic control: (a) very low density of the TCR/CD3 receptors and very low SI were found mainly in NR patients carrying HLA-A1, HLA-B8 and HLA-DR3 alleles; and (b) TCR/CD3 densities and function in R group were found to be significantly lower in carriers than in non-carriers of two MHC class III complement protein alleles: C4A*6, and Bf*F. Non-responsiveness to hepatitis-B vaccination was found to be associated with extremely increased neopterin levels. These findings indicate that both genetic and acquired factors contribute to the hepatitis-B vaccination failure in ESRD patients.

Quantitative evaluation of gel electrophoretic patterns by videodensitometry

Abstract Videodensitometry based on television technique has been shown to be suitable for recording gel electrophoretic patterns. Its speed of operation is about 60 times as fast as that of conventional densitometers, whereas the patterns recorded are practically the same.

Structurally distinct mammalian calmodulins

Rabbits immunized with dinitrophenylated calmodulin produced monospecific antibody against CaM. Using the purified antibody, enzyme-linked immunosorbent assays (ELISAs) were carried out for calmodulin (CaM). The immunological heterogeneity of human and bovine CaMs from erythrocytes was investigated by means of indirect and inhibition ELISAs. The cross-reactivity between the two CaMs was found to be 50% in the indirect ELISA. An eight times higher concentration of human CaM was necessary to produce 30% inhibition in an inhibition assay. The effect of anti-bovine CaM on the stimulation of the red cell membrane calcium pump by human and bovine CaM has also been studied. We have found that (a) the human and bovine CaMs showed indistinguishable activator activities; (b) the antibody partially inhibited the stimulating effects of CaMs; (c) the inhibition was much less effective in the case of stimulation by human CaM. These results suggest that there is a difference in the reactivity of the anti-bovine CaM antibody with bovine and human CaMs. This difference can be attributed to a slight deviation in the antibody binding structure of the two mammalian CaMs in or near the antigenic site of the CaMs.