Real-time PCR quantification of human complement C4A and C4B genes

BACKGROUND: The fourth component of human complement (C4), an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B) in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes. RESULTS: A novel quantitative real-time PCR (qPCR) technique was developed for rapid and accurate quantification of the C4A and C4B genes applying a duplex, TaqMan based methodology. The reliable, single-step analysis provides the determination of the copy number of the C4A and C4B genes applying a wide range of DNA template concentration (0.3–300 ng genomic DNA). The developed qPCR was applied to determine C4A and C4B gene dosages in a healthy Hungarian population (N = 118). The obtained data were compared to the results of an earlier study of the same population. Moreover a set of 33 samples were analyzed by two independent methods. No significant difference was observed between the gene dosages determined by the employed techniques demonstrating the reliability of the novel qPCR methodology. A Microsoft Excel worksheet and a DOS executable are also provided for simple and automated evaluation of the measured data. CONCLUSION: This report describes a novel real-time PCR method for single-step quantification of C4A and C4B genes. The developed technique could facilitate studies investigating disease association of different C4 isotypes

Autoimmune-associated HLA-B8-DR3 haplotypes in Asian Indians are unique in C4 complement gene copy numbers and HSP-2 1267A/G

The classical AH8.1 (HLA-A1-B8-DR3-DQ2) is the most common Caucasian haplotype, associated with several autoimmune diseases, immunologic hyperreactivity and rapid progression to the acquired immunodeficiency syndrome. However, in Asian Indians, there are multiple unique B8-DR3 haplotypes that are associated with autoimmunity and differ significantly from the common Caucasian AH8.1. The Indian HLA-A1-B8-DR3 is therefore referred to as an AH8.1 variant. The aims of this study were to compare C4A and C4B copy numbers and to identify alleles in HSP70-2 and LTA in these haplotypes. The Indian B8-DR3 haplotypes differ from the Caucasian AH8.1 at C4A and HSP70-2 loci. The Indian B8-DR3 haplotypes have 1 copy each at C4A and C4B, while the Caucasian AH8.1 has 1 copy at C4B but no C4A gene. Moreover, the Indian and Caucasian B8-DR3 haplotypes had HSP70-2 1267 *A, and *G alleles, respectively. By contrast, the LTA 252 *G allele occurred both in the Indian and Caucasian haplotypes. The Indian haplotypes also contained Bf*F and TNF-308*G that were different from the Caucasian equivalents Bf*S and TNF-308*A. These differences and previous studies support the hypothesis that B8-DR3-DQ2 haplotypes in Asian Indian population might have originated independently of Caucasian AH8.1 selectively through recombination and mutations. Because autoimmune disease associations are shared among these otherwise diverse haplotypes, these data strongly suggest that some shared component(s) of all these associated haplotypes may be playing a key role in such associations

Log rolling [picture].

Condition: good.; "Pasquin - Feby. 8th 1868"-- l.r corner.; Cartoon is related to the poem "Log-Rolling" which appears in Pasquin, Saturday, February 8, 1868, p354.Pasquin (Adelaide, S.A.

Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells

Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin beta(3), a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin beta(3), to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin beta(3) and Rac1. In the absence of TG2, integrin beta(3), cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals. The Journal of Immunology, 2009, 182: 2084-2092