Thierry Tabary

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

UNLABELLED 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.

Spontaneous retinopathy in HLA-A29 transgenic mice

Humans who have inherited the class I major histocompatibility allele HLA-A29 have a markedly increased relative risk of developing the eye disease termed birdshot chorioretinopathy. This disease affecting adults is characterized by symmetrically scattered, small, cream-colored spots in the fundus associated with retinal vasculopathy and inflammatory signs causing damage to the ocular structures, leading regularly to visual loss. To investigate the role of HLA-A29 in this disease, we introduced the HLA-A29 gene into mice. Aging HLA-A29 transgenic mice spontaneously developed retinopathy, showing a striking resemblance to the HLA-A29-associated chorioretinopathy. These results strongly suggest that HLA-A29 is involved in the pathogenesis of this disease. Elucidation of the role of HLA-A29 should be assisted by this transgenic model.

Homogeneous phase pyrophosphate (PPi) measurement (H3PIM) A non-radioactive, quantitative detection system for nucleic acid specific hybridization methodologies including gene amplification

A one-step, non-radioactive, homogeneous phase revelation system designed to detect and quantify nucleic acid hybridization is described. The principle of the procedure, termed homogeneous phase pyrophosphate (PPi) measurement (H3PIM), is to detect and quantify the release of PPi from nucleotides, which occurs stoichiometrically when nucleic acids are synthesized. The method does not require any special reagents before the H3PIM revelation step. H3PIM is particularly adapted to quantitative measurement of gene amplification or cDNA gene expression using PCR, but can also be used following random priming or simultaneous multi-step nucleic acid amplification. This rapid, sensitive, liquid phase procedure permits the design of low-cost, fully automated devices for gene detection and quantification.

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 α-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.

Recurrent IgA nephropathy is predicted by altered glycosylated IgA, autoantibodies and soluble CD89 complexes

IgA nephropathy (IgAN), the most common primary glomerulonephritis worldwide, frequently leads to end-stage renal disease and kidney transplantation. However, disease recurrence often occurs after transplantation. Here we evaluated the predictive value of three markers for IgAN recurrence: the presence of galactose-deficient IgA1, IgG anti-IgA autoantibodies, and IgA-soluble (s) CD89 complexes. This was analyzed in 38 kidney transplant recipients with IgAN recurrence and compared with 22 patients transplanted for IgAN but without recurrence and with 17 healthy controls. Pre-transplantation galactose-deficient IgA1 serum levels were significantly higher in the recurrence compared with the no recurrence or control groups. IgA-IgG complexes were significantly elevated in the recurrence group. Both the recurrence and no recurrence groups had increased values of IgA-sCD89 complexes compared with healthy controls, but values were significantly lower in patients with recurrence compared with no recurrence. Areas under the receiver operating curve of the markers in pre-transplantation sera were 0.86 for galactose-deficient-IgA, 0.82 for IgA-IgG, and 0.78 for sCD89-IgA; all significant. Disease recurrence was associated with decreased serum galactose-deficient IgA1 and appearance of mesangial-galactose-deficient IgA1 deposits, whereas increased serum IgA-sCD89 complexes were associated with mesangial sCD89 deposits. Thus, galactose-deficient-IgA1, IgG autoantibodies, and IgA-sCD89 complexes are valuable biomarkers to predict disease recurrence, highlighting major pathogenic mechanisms in IgAN.

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.

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.

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.

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.

Deletion of Receptor for Advanced Glycation End Products Exacerbates Lymphoproliferative Syndrome and Lupus Nephritis in B6-MRL Fas lpr/j Mice

The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor that interacts with advanced glycation end products, but also with C3a, CpG DNA oligonucleotides, and alarmin molecules such as HMGB1 to initiate a proinflammatory reaction. Systemic lupus erythematosus is an autoimmune disorder associated with the accumulation of RAGE ligands. We generated mice invalidated for RAGE in the lupus-prone B6-MRL Fas lpr/j background to determine the role of RAGE in the pathogenesis of systemic lupus erythematosus. We compared the phenotype of these mice with that of their wild-type and B6-MRL Fas lpr/j littermates. Lymphoproliferative syndrome, production of anti-dsDNA Abs, lupus nephritis, and accumulation of CD3+B220+CD4−CD8− autoreactive T cells (in the peripheral blood and the spleen) were significantly increased in B6-MRL Fas lpr/j RAGE−/− mice compared with B6-MRL Fas lpr/j mice (respectively p < 0.005, p < 0.05, p < 0.001, and p < 0.001). A large proportion of autoreactive T cells from B6-MRL Fas lpr/j mice expressed RAGE at their surface. Time course studies of annexin V expression revealed that autoreactive T cells in the spleen of B6-MRL Fas lpr/j-RAGE−/− mice exhibited a delay in apoptosis and expressed significantly less activated caspase 3 (39.5 ± 4.3%) than T cells in B6-MRL Fas lpr/j mice (65.5 ± 5.2%) or wild-type mice (75.3 ± 2.64%) (p = 0.02). We conclude that the deletion of RAGE in B6-MRL Fas lpr/j mice promotes the accumulation of autoreactive CD3+B220+CD4−CD8− T cells, therefore exacerbating lymphoproliferative syndrome, autoimmunity, and organ injury. This suggests that RAGE rescues the apoptosis of T lymphocytes when the death receptor Fas/CD95 is dysfunctional.