ABSTRACT
The Dombrock blood group system consists of five distinct antigens: two antithetical antigens, Doa and Dob, and three high-frequency antigens:Gya,Hy, and Joa. Although the prevalence of Doa and Dob in different populations makes them useful as genetic markers, the scarcity of reliable antibodies to these antigens has prevented this potential from being realized. The gene (DO;ART4) encoding the Dombrock glycoprotein has been cloned and sequenced, and the molecular bases of the various Dombrock phenotypes have been determined. The purpose of this study was to perform DNA-based assays on the DO homolog in non-human primates to determine the degree of conservation in the DO gene. Murine MoAbs to Dombrock protein were developed by standard hybridoma technologies and used to test RBCs from non-human primates by hemagglutination. PCR-RFLP analysis for the six single-nucleotide polymorphisms (SNPs) that have been defined in human alleles were performed on DNA extracted from fresh or frozen blood samples from numerous non-human primates. Hemagglutination tests with six MoAbs to the Dombrock glycoprotein revealed distinct epitopes on RBCs from the non-human primates. The gorillas and orangutans had the same PCR-RFLP digestion pattern for the six SNPs studied as chimpanzees. Old world monkeys (macaques) were identical at nucleotides (nt) 323, 350, 624, and 793 with the chimpanzees, and at nt 898 the digestion pattern was the same as for the HY1 allele in humans. For the new world monkeys (tamarins and squirrel monkeys) the digestion pattern was conserved for nt 793 but different for nt 624; the other SNPs could not be determined because there was no amplification. The presence of epitopes recognized by the MoAbs and PCR-RFLP results among the non-human primates shows considerable conservation of the DO gene. The difficulties we encountered with the amplification of DNA from the non-human primates lower in the phylogenetic tree are probably due to divergence in sequence.
ABSTRACT
BACKGROUND: The Sl(a) (Knops system) located on complement receptor 1 (CR1) has been associated with malarial rosetting, a process associated with severe malarial infections. Moreover, the long homologous repeats (LHRs) B and C of CR1 were implicated in rosette formation. As a step toward mapping the location of Knops system antigens, truncated CR1 proteins have been expressed and their ability to inhibit antibodies to the high-incidence Knops system antigens was assessed. STUDY DESIGN AND METHODS: Individual LHRs (A, B, C, and D) of CR1 of the common CR1*1 (F) allotype were expressed as secreted forms in 293T cells. Their abilities to specifically neutralize Knops system antibodies were tested by both hemagglutination and flow cytometry. RESULTS: Three examples of anti-Kn(a) (n = 6) were almost completely inhibited by LHR-C and three by LHR-D. Two examples of anti-McC(a) (n = 2) and seven examples of anti-Sl(a) (n = 8) were inhibited by LHR-D. Both examples of anti-Yk(a) (n = 2) were partially inhibited by LHR-D. CONCLUSION: The high-incidence Knops system antigens reside within LHR-D and to a lesser extent within LHR-C. Because of the role of Sl(a) antigen in malaria rosetting, these results indicate that LHR-D may represent an additional malaria interaction region in CR1.
Subject(s)
Blood Group Antigens/immunology , Chromosome Mapping , Isoantigens/genetics , Receptors, Complement 3b/genetics , Antibodies, Monoclonal/drug effects , Antibodies, Monoclonal/pharmacology , Cell Line , Erythrocytes/drug effects , Erythrocytes/immunology , Gene Expression , Humans , Isoantibodies/drug effects , Isoantibodies/immunology , Isoantigens/immunology , Peptide Fragments/pharmacology , Receptors, Complement 3b/antagonists & inhibitors , Receptors, Complement 3b/chemistry , Repetitive Sequences, Amino Acid , Sequence Homology, Amino Acid , SolubilityABSTRACT
BACKGROUND: Cytokines stimulate the expression of the adhesion molecule VCAM-1 in renal tubular epithelial cells. We have recently shown that VCAM-1 can also be upregulated by low molecular weight breakdown products of the matrix constituent hyaluronan (HA) (J Immunol 1998; 161: 3431-3437). The mechanisms of VCAM-I expression in response to HA remain to be defined. METHODS: Using a defined mouse cortical tubular (MCT) cell line we investigated the effect of protein kinase C (PKC) and tyrosine kinase (TK) inhibition on the HA-stimulated VCAM-1 expression by cell ELISA and RT PCR or Northern blotting. Furthermore, we examined the effect of PKC and TK inhibition on NF-kappaB. RESULTS: We found that the PKC inhibitor GF109203X (acting on conventional, novel and atypical isoforms) inhibited the HA-stimulated VCAM-1 expression in MCT cells dose-dependently up to 90%, whereas chelerythrine (acting on conventional and novel isoforms) had no effect. Downregulation of PKC with PMA did not prevent the HA-stimulated VCAM-1 expression, suggesting that Ca2+- and diacylglycerol-independent (atypical) isoforms of PKC are involved. The TK inhibitor genistein also inhibited the HA-stimulated VCAM-1 expression at the mRNA and protein level up to 70%. Interestingly, the HA-stimulated nuclear translocation of NF-kappaB could not be prevented with GF109203X and genistein. CONCLUSION: These data demonstrate that the HA-stimulated VCAM-1 expression in MCT cells involves PKC and TK pathways. The absence of an effect of PKC and TK inhibitors on the nuclear translocation of NF-kappaB suggests that additional transcription factors are involved for VCAM-1 expression.
