Serum C4 levels in patients with systemic lupus erythematosus in remission.

Abstract Twenty-six patients with systemic lupus erythematosus (SLE) showing systemic lupus activity measure (SLAM) and SLE disease activity index (SLEDAI) scores ≤2, as well as a lower C4 concentration than the mean C4 levels of healthy controls, were selected to evaluate the C4 levels of SLE patients in remission. Serum complement (CH50), complement components (C4, C3, and B), complement split products (C4d, iC3b, and Bb), phenotypic expression of C4 allotype, C4 production by peripheral blood monocytes, peripheral blood lymphocyte subpopulation, and interferon-gamma (IFN-γ) production were examined. In patients with SLE in remission, the C4 consumption (C4d/C4) was found to increase, and this was considered to be the most important factor for determining the serum concentration of C4. However, the relevance of the C4 allotypic expression was minimal. The IFN-γ-stimulated production of C4 by peripheral blood monocytes in SLE patients in remission was also less than that of the healthy controls. The IFN-γ-stimulated production of C4 in SLE patients in remission correlated with the peripheral blood CD4-positive cells. Less IFN-γ was produced by lymphocytes of SLE in remission than by those of healthy adults. We conclude that the serum C4 levels in SLE patients in remission reflect the degree of C4 consumption as well as the disease state, rather than genetic influences such as a C4A defect.

Geographical north-south decline in DNASE1*2 in Japanese populations.

Allele frequencies for human deoxyribonuclease I (DNase I) phenotypes were determined using blood samples from about 2000 Japanese subjects living in nine prefectures, and compared with one another. DNase I phenotyping was performed principally using isoelectric focusing electrophoresis and activity staining. The DNase I system was shown to have enhanced potential for anthropologic, genetic, and clinical studies of Japanese populations. DNase I phenotypes were analyzed to evaluate the degree of genetic variation at the DNASE1 locus. Our examination of DNase I types revealed a decreasing north-to-south gradient in the DNASE1 allele.

Distribution of ABO genotypes and allele frequencies in a Korean population.

The genotypes of the ABO blood group system were investigated in Korean living in Kangwon-Do area by PCR-RFLP analysis of the seven polymorphic nucleotide positions 261, 467, 526, 646, 703, 796 and 803 of the cDNA from A1 transferase. In 253 unrelated Korean individuals, 15 genotypes were found and the allele frequencies of A(Pro), A(Leu), B, O(T) and O(A) were 0.022, 0.209, 0.209, 0.360 and 0.200, respectively, with no deviation from Hardy-Weinberg expectations (chi 2 = 2.145, d.f. = 6, 0.90 < p < 0.95). As for the distribution of allele frequencies, a significant difference was noticed between the Korean and a Japanese (chi 2 = 30.87, d.f. = 4, p < 0.001) and a German (chi 2 = 127.76, d.f. = 4, p < 0.001) populations.

Immunohistochemical detection of truncated APC protein in sporadic human colorectal adenomas and adenocarcinomas.

Mutations of the APC gene frequently occur in sporadic forms of colorectal adenomas and adenocarcinomas. Phenotypically, the vast majority of these mutations result in the truncation of the APC protein. To demonstrate the defective APC gene product in human colorectal tumors, rabbit region-specific antisera raised against the APC protein of amino acid sequences between 371 and 390 (SPI) and between 1821 and 1840 (SP3) were used to exhibit the truncated APC protein. In all, 86 lesions from 67 cases of sporadic adenoma and adenocarcinoma were examined; abnormal staining patterns were distinguished in 43 lesions (50%); the incidence of abnormalities was not significantly different between adenomas and carcinomas. The majority, 75% exhibited epitopic change with the SPI-positive and SP3-negative phenotype (type P1), and 25% exhibited neither of these phenotypes (type P2). The staining pattern in all lesions was uniform, and studies of carcinomas arising in adenomas showed the same pattern of staining. These findings supported the view that the APC lesion is a very early event in colorectal carcinogenesis. Furthermore, this simple immunohistochemical approach demonstrated that different adenomas from the same patient showed different staining patterns.

Suballeles of the ABO blood group system in a Japanese population.

The nucleotides (nt) at positions 467 and 646 of the ABO blood group system were analyzed in a Japanese population by means of the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods. Two types at nt467, tentatively designated 'Pro' and 'Leu', were found in the common A (= A1) alleles, and two types at nt646 named 'T' and 'A' were found in O alleles. The types at nt467 of B and O alleles were Pro and those at nt646 of A and B alleles were T. Therefore, A alleles were divided into A(Leu) and A(Pro) suballeles and O alleles were divided into O(T) and O(A). The allele frequencies in the present survey were calculated as ABO*A(Pro) = 0.0712, ABO*A(Leu) = 0.2163, ABO*B = 0.1779, ABO*O(T) = 0.2731 and ABO*O(A) = 0.2615. No O2 (or O(3)) allele was observed in the population samples. At least five alleles with polymorphic frequencies and 15 genotypes are present in the Japanese sample.

Genotyping of the ABO blood group system: analysis of nucleotide position 802 by PCR-RFLP and the distribution of ABO genotypes in a German population.

Genotypes of the ABO blood group system were studied by PCR-RFLP analysis of the eight polymorphic nucleotide positions (nps) 261, 467, 526, 646, 703, 796, 802 and 803 of the cDNA from A transferase. In 169 unrelated German individuals, 17 genotypes were found and the calculated allele frequencies of A(Pro), A(Leu), B, O(T), O(A) and O2 were 0.2130, 0.0770, 0.0473, 0.4260, 0.2160 and 0.0207, respectively. These frequency data may provide useful additional information for disputed paternity and stain testing. A variant O allele, O2, was fout at a polymorphic frequency. As the nucleotide (np 261) of the O2 allele is the same as that of A and B alleles, the analysis of at least three nucleotide positions, i.e. nps 261, 526 and 802, is necessary to avoid mistyping of the ABO genotype.

Genotyping of ABO blood groups by PCR and RFLP analysis of 5 nucleotide positions.

The genotyping of ABO blood groups was performed using the polymerase chain reaction (PCR) method. The 4 DNA fragments containing the nucleotide position 261, 526, 703 and 796 of cDNA from A-transferase were amplified by PCR, and the amplified DNA subjected to restriction fragment length polymorphism (RFLP) analysis. The different nucleotide at position 803 was clearly distinguished by electrophoresis of the PCR products amplified with allele-specific primers. By analyzing the electrophoresis patterns, ABO genotyping was conclusively accomplished. The frequencies of ABO genotypes found in Japanese blood donors with A and B phenotypes were as follows: in the phenotype A group, AA = 19.8% and AO = 80.2%; and in the phenotype B group, BB = 12.8% and BO = 87.2%.

Isolation of mouse complement component C7.

Mouse complement component C7 was purified from serum by a sequential procedure of fractionation precipitation by ammonium sulfate, followed by DE-52 anion exchange chromatography. Protein G affinity column chromatography, Mono S cation exchange chromatography and Superdex 200 gel filtration. The final product contained a highly purified mouse C7 component showing a single band on SDS-PAGE at the apparent Mrs of 90 kDa and 100 kDa under non-reduced and reduced conditions respectively. The yield of C7, which was measured by the biological activity, was 7.0%

Activation of complement in normal serum by hydrogen peroxide and hydrogen peroxide-related oxygen radicals produced by activated neutrophils.

Neutrophils activated by soluble particulate stimuli generate superoxide anion and subsequently form hydrogen peroxide and other oxygen radicals. The effect of hydrogen peroxide on the complement system in normal serum was investigated. Treatment of normal serum with hydrogen peroxide resulted in a diminution of the haemolytic activity of the total and alternative complement pathways and the haemolytic titres of C3 and C5 but not of C2, in normal serum. These decreases in complement activity depended on the concentration of hydrogen peroxide added to the serum. Immunoelectrophoretic analysis of hydrogen peroxide-treated serum showed that C3 and C5 proteins were activated. Complement degradation products C3a and C5a were produced in normal serum treated with hydrogen peroxide, and 20 mM EDTA abolished C3a and C5a production in hydrogen peroxide-treated serum but 20 mM Mg-EGTA did not. Catalase completely abolished and dimethylsulphoxide and D-mannitol, hydroxyl radical scavengers, partially inhibited the hydrogen peroxide-mediated complement activation. Hypochlorite, incubated with normal serum, significantly inhibited serum haemolytic activity, and sodium thiosulphate, a reducing agent, abolished the effect of hypochlorite. Normal serum incubated with activated neutrophils showed neutrophil chemotactic activity and decreased serum haemolytic activity, and the addition of catalase or methionine (5 mM) completely abolished the effects of activated neutrophils. These results suggest that hydrogen peroxide activates complement via an alternative pathway of complement activation and that hydroxyl radicals and other hydrogen peroxide-related species such as hypochlorite are most likely involved in hydrogen peroxide-mediated complement activation. Complement activation by oxygen radicals produced by activated neutrophils may be one of the mechanisms by which complement is activated in human immune complex diseases.

Polymorphisms of serum proteins in Japanese patients with vascular diseases. I. Factor XIIIB, plasminogen and complement types in primary varicose veins.

The polymorphisms of the B subunit of coagulation factor XIII (F13B), plasminogen (PLG), complement C6, C7, factor B (BF) and factor I (IF) were studied among 21 unrelated Japanese patients with primary varicose veins (PVV) by isoelectric focusing followed by immunoblotting. The allele frequencies for F13B*2 and IF*A in PVV patients were significantly higher (F13B*2, p = 0.0047; IF*A, p = 0.0006) than those in healthy controls (n = 60). Significant associations of F13B 2 allotype [p = 0.0220, relative risk (RR) = 13.9] and IF A allotype (p = 0.0006, RR = 10.0) with PVV were observed; however, no significant association of PLG, C6, C7 or BF allotype with the disease was found.

Complement allotypes in Japanese patients with pemphigus and bullous pemphigoid.

The allotypes of C6, C7, factor B (BF) and factor I (IF) of the human complement system were studied in 11 Japanese patients with pemphigus (5 with pemphigus vulgaris and 6 with pemphigus foliaceus) and 17 with bullous pemphigoid (BP) to investigate the genetic background of these diseases. The allotypes were detected by using isoelectric focusing and immunoblotting. The frequency for IF*A allele in the pemphigus patients was significantly higher (p = 0.009) than that in healthy controls (n = 60). A significant association of IF A allotype with pemphigus was also observed (p = 0.027), with a relative risk of 6.3. There was no association between the C6, C7, BF or IF allotypes and BP. These data suggest that IF A allotype may be an etiological genetic factor in the development of pemphigus.

A new complement factor B variant (BF S075) in Japanese.

A new slow-moving variant of the complement factor B, named BF S075, was found in a Japanese patient with cerebral thrombosis and urticaria. The variant was inherited in a codominant manner. The protein concentration and functional hemolytic activity of the complement factor B in the patient's serum were within normal limits. The BF S075 is the fourth rare BF variant found in the Japanese population.

C6 reference typing report.

Various C6 protein allotypes were examined using polyacrylamide gel isoelectric focusing followed by immunoblotting or hemolytic overlay. For several 'difficult' allotypes, neuraminidase treatment of samples and long-distance isoelectric focusing gels were applied. Nineteen different allotypes were distinguished besides the two common allotypes C6 A and C6 B. They were designated basically according to the previous statement on C6 nomenclature [Mauff et al., 1980].

Factor I reference typing report.

Complement factor I (IF) reference typing has been examined using polyacrylamide gel isoelectric focusing followed by electroblotting with enzyme immunoassay. The submitted samples could be classified into three common and two new variants, and these were controlled by two common and two rare alleles. Two common alleles were IF*B (the most common allele) and IF*A, and two new rare alleles were designated IF*A1 and IF*B1, respectively.

Complement C6 and C7 polymorphisms in Japanese patients with chronic glomerulonephritis.

C6 and C7 types were studied in 158 Japanese patients with different types of chronic glomerulonephritis: 75 patients with IgA nephropathy (IgA-N); 49 patients with idiopathic membranous nephropathy (IMN), and 34 patients with minimal-change nephrotic syndrome (MCNS). There were significant differences in the C6 and C7 allele and phenotype frequencies between the patient groups and controls. A strong association was found between IgA-N and C7 5 phenotype (p less than 0.001, RR = 12.71), and between MCNS and C7 5 phenotype (p less than 0.001, RR = 14.20). A significant association between MCNS and C6 B2 phenotype (p less than 0.05, RR = 2.42) was also found. In the IMN patient group, a significant association with C7 4 phenotype (p less than 0.05, RR = 2.42) was observed. Thus, C6 and C7 phenotypes may be causative factors in the development of chronic glomerulonephritis.

Activation of human complement by rat peritoneal mast cells and its inhibition by a rat serum factor.

The effects of normal sera from humans, rats, and guinea pigs on unsensitized rat peritoneal mast cells were studied in vitro. Five to 20% fresh human sera induced mast cell death and substantial histamine release. The factor was heat labile. Neither hereditary C3-deficient sera nor experimentally C1q-depleted sera showed cytotoxicity. The CH50 activity of human serum was decreased to about one half after a 15-min incubation with 2 X 10(6) mast cells/ml at 37 degrees C. The cytotoxic activity and CH50 reduction were completely eliminated by an addition of 10 mM Mg-EGTA to the serum. These data demonstrated that unsensitized rat mast cells served as both the initiator and target of complement activity when human serum was used as a complement source. Requirements of both Ca++ and C1q suggested the activation of the classical pathway of complement. Fresh 5-20% sera from rats and guinea pigs, on the other hand, showed neither cytotoxicity nor CH50 reduction. Furthermore, these sera strongly inhibited the human serum-induced reaction. The latter results indicated the presence of a modulating factor in rat and guinea pig sera, which inhibits mast cell associated complement activation.

Three Japanese families with members carrying C7 silent allele (C7*Q0). Possibility for an association between C7*Q0 and C6*B.

Three Japanese families with members carrying C7 silent allele(s) (C7*Q0) are presented. C6 types in the family members were also examined, and it was found that C7*Q0 was transmitted from a parent to offsprings as a haplotype, C7*Q0-C6*B. In another study of C6 types in sera from 3 volunteer blood donors with homozygous C7 deficiencies, the C6 phenotypes were found to be C6 B (homozygote). It seems remarkable that C7*Q0 can be associated with C6*B.

Factor B subtypes in Japanese patients with IgA nephropathy and with idiopathic membranous nephropathy.

The present paper describes a method of subtyping of properdin factor B (BF) using polyacrylamide gel isoelectric focusing followed by immunoblotting, and the distribution of BF subtypes in Japanese patients with IgA nephropathy or idiopathic membranous nephropathy (IMN) along with controls. BF*F allele was splitted into two suballeles, named BF*FA and BF*FB. There were significant differences in the BF suballele frequencies between the IMN patient group and the control group. A significant association of IMN with the BF FA subtype (p less than 0.05) and with the BF FB subtype (p less than 0.001) was also found. BF*FB may be a susceptibility suballele to IMN rather than BF*FA.