New HLA-DRB1*07 allele identified from microlymphocytotoxicity/SSP discrepancies.

A HLA-DRB1*07 variant allele has been identified in a cadaver kidney donor. Serological typing using monoclonal antibodies detected HLA-DR4 and HLA-DR7. HLA class II DNA typing using sequence-specific primer (PCR-SSP) polymerase chain reaction only detected DRB1*04, while sequence-specific oligonucleotide (PCR-SSO) polymerase chain reaction confirmed the presence of both DRB1*04 and DRB1*07 alleles, although two extra reactions were also found. Exon 2 of the HLA-DRB1*07 was isolated using allele-specific PCR, then cloned and sequenced. Four mutations, at positions 170 (T --> C), 171 (C --> T), 174 (C --> G), and 179 (C --> A), were observed. These mutations changed codons 57 and 60 (V --> A; S --> Y, respectively). This amino acid sequence at position 56-61 is only found in DRB1*0811.

A soluble recombinant multimeric anti-Rh(D) single-chain Fv/CR1 molecule restores the immune complex binding ability of CR1-deficient erythrocytes.

CR1 (CD35, the C3b/C4b receptor) is a widely distributed membrane glycoprotein with a unique cluster conformation on the surface of erythrocytes (E). CR1 on E is responsible for the transport of immune complexes (IC) to liver and spleen. As a cofactor of the C3b cleavage by factor I, CR1 is also a potent inhibitor of C activation and inflammation. In some diseases (systemic lupus erythematosus, hemolytic anemia, AIDS, etc.) an acquired low level of CR1 on E has been observed, leading to an impaired clearance of IC. The aim of this study was to design a heterofunctional molecule that will bind to E and restore a normal or a supranormal CR1 density on E that could mimic the unique distribution pattern of CR1 on normal E. For that purpose a new multimerizing system based on the properties of the C-terminal part of the alpha-chain of the C4 binding protein (C4bp) was used. We first produced a multimeric soluble CR1 that proved to be a better inhibitor of in vitro C activation than the monomeric form of CR1, then a heteromultimeric molecule made of CR1 and single-chain Fv anti-Rh(D) valences able to attach E and providing E with as much as a 10-fold increase in CR1 density with the same CR1 distribution pattern as native E. CR1/single-chain Fv anti-Rh(D)-treated E were able in vitro to attach as many opsonized IC as native E. These data open the way for future use of multimeric and heteromultimeric forms of soluble recombinant CR1 as therapy of IC diseases.

No quantitative relationship between CR1 and Lutheran expression on erythrocytes: In(Lu) gene product is not a common regulator of CR1 expression on erythrocytes.

The density of CR1, the C3b/C4b receptor (CD35), on erythrocytes (E) (CR1/E) is genetically determined. However, the broad distribution of CR1/E within a given genotype suggests that other genetic elements might contribute to the regulation of CR1/E. In some pathological conditions, including systemic lupus erythematosus (SLE), AIDS and hemolytic anemia, CR1 deficiency parallels the severity of the disease. When compared to healthy individuals, an accelerated decrease in CR1/E in these patients has been demonstrated, but other mechanisms interfering with CR1 density regulation during erythropoiesis might also contribute. In exceptional circumstances, CR1/E can be dramatically decreased in healthy individuals by the effect of a regulatory gene, In(Lu), that switches off various surface molecules on E, the structure genes of which are located on four different chromosomes, suggesting a transcription regulatory role for In(Lu) gene products. The hypothesis that products of this gene could physiologically regulate the surface density of all these molecules has been tested by determining Lub density on E (Lub/E) using quantitative flow cytometry. Lub antigenic sites were then compared to CR1/E among healthy individuals of the different CR1 density phenotypes, SLE patients with and without CR1 deficiency, and an exceptional SLE patient totally lacking CR1/E and reticulocytes. No quantitative relationship was found between CR1 and Lub expression in either normal or pathological conditions. These data establish that In(Lu) products are not involved in normal or pathological CR1 density regulation.

A recombinant human scFv anti-Rh(D) antibody with multiple valences using a C-terminal fragment of C4-binding protein.

Monomeric recombinant molecules prove generally unsatisfactory for in vivo use. Most biological systems are indeed multivalent either structurally, associating different chains, or functionally, when cross-linked by their ligands. Mimicking natural molecules for immune intervention implies the need for multimerizing systems to create multivalent molecules capable of interfering with physiological processing. A multivalent anti-Rh(D) recombinant protein has been designed by reconstructing the antibody binding site of a human monoclonal anti-Rh(D) antibody as a single chain Fv mini antibody, then multimerizing it by inserting at its C-terminal end the C-terminal part of the C4 binding protein (C4bp) alpha chain, which is responsible for the octamer multimerization of that molecule. This soluble multivalent recombinant molecule was functional, bound red blood cells (RBCs), agglutinated them, and did not activate complement. This demonstration model opens the way for future in vivo use of multivalent molecules associating antibody valences and other functional molecules for cell targeting, imaging, or removal of cells such as Rh(D)-positive RBCs for preventing Rh alloimmunization.

Serum complement activation of SLE patients during plasmapheresis.

The erythrocyte complement receptor 1 (ECR1)-immune complex binding assay is a sensitive method for the determination of complement fragments which can be activated by bovine serum albumin (BSA)-anti-BSA in vitro. When the C3b/C4b containing bovine serum albumin (BSA)-anti-BSA was formed in the presence of the serum of patients with systemic lupus erythematosus (SLE) its binding to ECR1 was found to be lower than that formed in sera of normal volunteers. The plasmapheresis of SLE patients homozygous for the CR1/E high density allele displays a beneficial effect on the formation of C3b/C4b containing BSA-anti-BSA and its binding to ECR1. There was no significant correlation between the serum C3/C4 level and the percentage of C3b/C4b containing BSA-anti-BSA binding to the ECR1 of SLE patients during plasmapheresis. At the same time, there was an inverse correlation between the serum immune complex level and the ECR1 binding, which was significant in 3 of 5 cases. These data suggest that, besides the determination of different components of complement activation, the functional assay of complement activation might be useful in monitoring the effect of plasmapheresis in SLE.

Catabolism of the human erythrocyte C3b/C4b receptor (CR1, CD35): vesiculation and/or proteolysis?

Human erythrocytes (E) react by exocytosis of membrane vesicles to various stresses including the fixation of the membrane attack complex of Complement. E from normal individuals loose a notable proportion of their initial number of surface CR1 molecules during the ageing process. An acquired decrease of CR1 on E also occurs in pathological conditions such as Systemic Lupus Erythematosus or AIDS. The present study investigated whether calcium ionophore A23187 (Ca-ion) induced vesicle formation of human E in vitro is responsible for a preferential loss of CR1 as well as whether CR1 molecules at the surface of Ca-ion treated E or vesicles are: (i) functional, (ii) native or protease degraded, or (iii) more clustered than CR1 on native E. A study of E from 137 normal individuals showed that a one-hour Ca-ion induced vesicle formation preferentially removed one third of E surface CR1. Kinetic experiments suggested that all surface CR1 could be removed from E upon longer incubation times. CR1 molecules on vesicles were still able to inhibit Complement activation, and were found in larger clusters than on native E. These data suggest that a significant part of surface CR1 molecules may be removed from E by vesicle formation during the life of E in normal individuals. This phenomenon could be exacerbated in pathological conditions.

Genomic determination of the CR1 (CD35) density polymorphism on erythrocytes using polymerase chain reaction amplification and HindIII restriction enzyme digestion.

The density of CR1 (the C3b receptor, CD35) on erythrocytes from normal individuals is determined by a codominant bi-allelic system associated with a single base mutation within an intron of the CR1 structural gene, leading to an additional polymorphic HindIII endonuclease site. The CR1 genotype is determined by HindIII digestion of genomic DNA and Southern blotting. We have developed a method based on polymerase chain reaction (PCR) amplification of the genomic DNA fragment of interest followed by HindIII endonuclease digestion and agarose gel electrophoresis which permits a more rapid and reliable determination of the CR1 genotype. The method is suitable for large scale clinical studies in diseases with altered expression of CR1 on erythrocytes.

The Niagara River: A water quality management overview.

The Niagara River constitutes part of the Laurentian Great Lakes and St. Lawrence River system which represents approximately 80% of North America's supply of surface fresh water. The river is a major source of water for industry, municipalities, recreation and power generation and is the link between Lakes Erie and Ontario. The river forms part of the Canada-U.S. border and falls under the jurisdiction of both countries.The massive industrialization of the region surrounding the river has led to a typical resource use conflict situation in which pollution of the river continues to be a major public concern.A number of constitutional, institutional and jurisdictional factors make the management of the Niagara River an involved and complicated matter. The interests, intent, philosophies, laws and regulations are not necessarily the same among the numerous jurisdiction involved. Despite these differences, however, Canada and the United States have succeeded in developing and implementing a model cooperative international management plan for the river. An overview of the main international aspects relating to the development and implementation of this plan, the Niagara River Toxics Management Plan, is presented.