Key role of the number of complement receptor 1 on erythrocytes for binding of Escherichia coli to erythrocytes and for leukocyte phagocytosis and oxidative burst in human whole blood.

AIM: To study the role of complement receptor 1 (CR1) for binding of Escherichia coli (E. coli) to erythrocytes, for leukocyte phagocytosis, oxidative burst and complement activation in human whole blood from a CR1 deficient (CR1D) patient and healthy controls with low, medium and high CR1 numbers. METHODS: Alexa-labelled bacteria were used to quantify erythrocyte-bound bacteria, free bacteria in plasma and phagocytosis using flow cytometry. Complement activation in plasma was measured by enzyme-linked immunosorbent assay. The CR1 numbers as well as C3bc and C4bc deposition on erythrocytes were measured by flow cytometry. Cytokines were measured using multiplex technology, and bacterial growth was measured by colony forming units. CR1 was blocked using the anti-CR1 blocking mAb 3D9. RESULTS: Approximately 85% of E. coli bound to erythrocytes after 15 min incubation in donor blood with high and medium CR1 numbers, 50% in the person with low CR1 numbers and virtually no detectable binding in the CR1D (r2 = 0.87, P < 0.0007). The number of free bacteria in plasma was inversely related to erythrocyte CR1 numbers (r2 = 0.98, P < 0.0001). E. coli-induced phagocytosis and oxidative burst were significantly enhanced by the anti-CR1 mAb 3D9 and in the CR1D and the donor with low CR1 numbers. E. coli-induced complement activation in plasma, C3bc and C4bc deposition on erythrocytes, and bacterial growth were similar in all four cases. CONCLUSIONS: CR1D and low CR1 numbers prevented E. coli binding to erythrocytes, increased free bacteria in plasma, phagocytosis and oxidative burst, but did not affect plasma or surface complement activation and bacterial growth.

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates.

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.

Alzheimer's disease is associated with low density of the long CR1 isoform.

The long complement receptor type 1 (CR1) isoform, CR1*2 (S), has been identified as being associated with Alzheimer's disease (AD) risk. We aimed to analyze the phenotypic structural and expression aspects (length and density) of CR1 in erythrocytes of 135 Caucasian subjects (100 AD and 35 controls). CR1 length polymorphism was assessed at protein and gene levels using Western blot and high-resolution melting, respectively. CR1 sites on erythrocytes were enumerated by flow cytometry. CR1 gene analysis, spotting the rs6656401 and rs3818361 polymorphisms, was performed by pyrosequencing. The CR1 density was significantly lower in AD patients expressing the CR1*2 isoform compared with the controls (p = 0.001), demonstrating lower expression of CR1 in CR1*2 carriers. Our data suggested the existence of silent CR1 alleles. Finally, rs6656401 and rs3818361 were strongly associated with CR1 length polymorphism (p < 0.0001). These observations indicate that AD susceptibility is associated with the long CR1 isoform (CR1*2), albeit at a lower density, suggesting that AD results from insufficient clearance of plaque deposits rather than increased inflammation.

Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes.

Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms. FROM THE CLINICAL EDITOR: The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms.

Advanced procedures for labeling of antibodies with quantum dots.

Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs' advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs' amines with the sulfhydryl groups issued from the reduced Abs' disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD-Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy-light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD-Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals.

Analysis of complement receptor type 1 expression on red blood cells in negative phenotypes of the Knops blood group system, according to CR1 gene allotype polymorphisms.

BACKGROUND: The KN blood group system, which consists of nine antigen specificities, is located on complement receptor Type 1 (CR1/CD35). CR1, a complement regulatory protein, acts as a vehicle for immune complex clearance. CR1 exhibits a red blood cell (RBC) density polymorphism. CR1 sites on RBCs in normal individuals range from 150 to 1200 molecules per cell. CR1 density polymorphism is regulated by HindIII restriction fragment length polymorphism and Q981H and P1786R polymorphisms in Caucasians. Yet, the role of the different polymorphisms in determining the CR1 density on RBCs remains unknown. The "null" serologic KN phenotype, known as Helgeson phenotype, was reported to be related with a very low CR1 density, less than 150 molecules per cell. STUDY DESIGN AND METHODS: The aim of this work was to investigate whether the KN-negative phenotype displayed by 60 individuals was related to the CR1 density by performing the phenotypic and genetic analysis of CR1 and to investigate the molecular background associated with the KN system. RESULTS: We showed that the Helgeson-like phenotype had a prevalence of 12% in this population. The overall genotype/phenotype concordance was 90%. Among individuals with a KN-negative phenotype, the prevalences of Kn(a-), McC(a-), Sl1-negative, Sl3-negative, and KCAM-negative deduced phenotype were 37, 12, 29, 7, and 24%, respectively. CONCLUSION: From our data, we suggest that the definition of the Helgeson phenotype must be revised, since the latter may be due not only to a very low CR1 density on RBCs, but also to the absence of expression of a high-prevalence KN antigen.

A new HLA extended haplotype containing the A*2910 allele in birdshot retinochoroidopathy: susceptibility narrowed to the HLA molecule itself.

PURPOSE: Birdshot retinochoroidopathy (BSRC) is a rare posterior uveitis characterized by distinctive, multiple, hypopigmented choroidal and retinal lesions. Most, if not all, patients are white and share the major histocompatibility antigen HLA-A29. Furthermore, the A*2902 subtype is closely associated with BSRC, and only a very few patients share the A*2901 subtype. Surprisingly, although A*2901 and A*2902 differ only by a single mutation (D102H), studies of microsatellites located near HLA-A have shown that two strong A*2901 and A*2902 extended haplotypes are observed in patients and control subjects. The present study analyzes the HLA-A extended haplotype of two patients who were HLA-A*2910 carriers. METHODS: Among 180 patients who fulfilled internationally defined criteria for the diagnosis of BSRC and who were HLA-A29 subtyped, two patients were found to be HLA-A*2910 carriers. These patients were tested for the microsatellite alleles MOGa, -b, -c, and -e (of the myelin oligodendrocyte glycoprotein [MOG] gene) and D6S265, D6S510, RF, C5_4_5, and D6S105. RESULTS: Although A*2902 and A*2910 differed by only a single mutation, (E177K) a new A*2910 extended haplotype was found to be distinct from the A*2901 and A*2902 extended haplotypes previously described in patients and control subjects. Among all studied microsatellite markers, no allele was shared by these extended haplotypes. CONCLUSIONS: These results suggest that susceptibility to BSRC is linked to the histocompatibility HLA-A29 molecule itself, although the development of the disease also involves inherited or probably acquired factors not linked to the major histocompatibility complex.

Nanocrystal-encoded fluorescent microbeads for proteomics: antibody profiling and diagnostics of autoimmune diseases.

The first application of nanocrystal (NC)-encoded microbeads to clinical proteomics is demonstrated by multiplexed detection of circulating autoantibodies, markers of systemic sclerosis. Two-color complexes, consisting of NC-encoded, antigen-covered beads, anti-antigen antibody or clinical serum samples, and dye-tagged detecting antibodies, were observed using flow cytometry assays and on the surface of single beads. The results of flow cytometry assays correlated with the ELISA technique and provided clear discrimination between the sera samples of healthy donors and patients with autoimmune disease. Microbead fluorescence signals exhibited narrow distribution regardless of their surface antigen staining, without the need of any fluorescence compensation-a parameter determining the limit of sensitivity of flow cytometry assays. In single bead measurements, less than 30 dye-labeled antibodies interacting with the topoI-specific antibodies at the surface of a bead have been detected by the emission of dye excited through the FRET from NCs. In this format, the antibody-bead interaction reaction turns specifically the fluorescence signal from dye label off and on, additionally increasing autoantibody detection sensitivity.

Significant genetic and antigenic variability within the env gene of systemic human immunodeficiency virus type 1 group O populations during the natural course of a heterosexual infection: a pilot study.

We assessed the genetic and the antigenic variability within the env gene of peripheral blood human immunodeficiency virus (HIV) type 1 (HIV-1) group O populations during the natural course of a female heterosexual infection. Our data revealed the existence of a significant increase in amino acid sequence variability within the C2-V3 and gp41 regions (P = 0.023 and P < 0.001, respectively) in association with substitutions within neutralizing epitope sequences usually selected for HIV serological assays. These antigenic variations might significantly decrease the sensitivity of classical HIV enzyme-linked immunosorbent assays with blood samples of subjects heterosexually infected by HIV-1 group O strains. These findings may be of significant use both to devise diagnostic tools and to pursue suitable therapeutic modalities in cases of heterosexual infection by outlier HIV-1 strains.

Different HLA class IA region complotypes for HLA-A29.2 and -A29.1 antigens, identical in birdshot retinochoroidopathy patients or healthy individuals.

PURPOSE: Birdshot retinochoroidopathy (BSCR) is a rare posterior uveitis characterized by distinctive, multiple, hypopigmented choroidal and retinal lesions. At least 96% of patients, if not all, share the major histocompatibility antigen HLA-A29. Although it was hypothesized earlier that more frequently the A*2902 subtype was closely associated with BSCR, new patients were found to share the A*2901 subtype and were further investigated. The present study was designated to check patients' HLA-A*2901 subtyping and the polymorphisms available in the HLA region in patients and control subjects sharing the A*2901 and A*2902 subtypes. METHODS: HLA-A29 was assessed and subtyped by molecular biology. cDNA from one patient (HLA-A*2901) was sequenced. A29.1 antigenic expression on peripheral blood lymphocytes was checked by microlymphocytotoxicity (MLCT). Four homozygous A29.2 and 4 heterozygous A29.2 patients, 3 homozygous A29.2 healthy subjects, 3 heterozygous A29.1 patients, and 11 heterozygous A29.1 healthy subjects were tested for the microsatellite alleles MOGa, -b, -c, and e (of the myelin oligodendrocyte glycoprotein [MOG]gene), D6S265, D6S510, RF, C5_4_5, D6S105, and D6S276 and the mutation H63D of the familial hemochromatosis gene (HFE). RESULTS: The patients' cDNA sequences and MLCT reactivities of HLA-A29.1 subtypes were found to be identical with published data from healthy individuals. Surprisingly, though A*2901 and A*2902 differed only by a single mutation (G376C/ D102H) two strong A*2901 and A*2902 complotypes were observed in patients and control subjects, the polymorphisms being identical at all loci near HLA-A, whereas more distant loci exhibited some diversity. CONCLUSIONS: Susceptibility to BSCR thus appeared to be located between the left and right remote markers C5_4_5 and D6S276, if not relying on the HLA-A29 molecule itself.