The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients.

Mutations in Notch3 cause CADASIL (cerebral autosomal dominant adult onset arteriopathy), which leads to stroke and dementia in humans. CADASIL arteriopathy is characterized by major alterations of vascular smooth muscle cells and the presence of specific granular osmiophilic deposits. Patients carry highly stereotyped mutations that lead to an odd number of cysteine residues within EGF-like repeats of the Notch3 receptor extracellular domain. Such mutations may alter the processing or the trafficking of this receptor, or may favor its oligomerization. In this study, we examined the Notch3 expression pattern in normal tissues and investigated the consequences of mutations on Notch3 expression in transfected cells and CADASIL brains. In normal tissues, Notch3 expression is restricted to vascular smooth muscle cells. Notch3 undergoes a proteolytic cleavage leading to a 210-kDa extracellular fragment and a 97-kDa intracellular fragment. In CADASIL brains, we found evidence of a dramatic and selective accumulation of the 210-kDa Notch3 cleavage product. Notch3 accumulates at the cytoplasmic membrane of vascular smooth muscle cells, in close vicinity to but not within the granular osmiophilic material. These results strongly suggest that CADASIL mutations specifically impair the clearance of the Notch3 ectodomain, but not the cytosolic domain, from the cell surface.

Generation of monoclonal antibodies to native human immunodeficiency virus type 1 envelope glycoprotein by immunization of mice with naked RNA.

The Semliki Forest virus (SFV) vector system is a new approach for in vivo expression of heterologous proteins and can also be used to generate specific immune responses in animal models. HIV-1 envelope glycoprotein produced using the SFV expression system is correctly folded, cleaved, transported to the cell surface and exhibits functional activity. We evaluated a recombinant Semliki Forest virus naked RNA-based immunization protocol for generation of monoclonal antibodies against the HIV-1 envelope glycoprotein. In vitro-transcribed RNA encoding for the SFV replicase complex and Env protein of HIV-1 (HXB2 strain) was injected intramuscularly to mice. This approach elicited an Env-specific antibody response in four mice out of five and a monoclonal antibody, 12H2, directed against gp41 was produced. Our results show that recombinant SFV RNA immunization can potentially be used as a quick and direct method to produce monoclonal antibodies, with the particular advantage that vectored RNA, rather than purified antigen, delivers a complex oligomer produced correctly.

HCV core immunodominant region analysis using mouse monoclonal antibodies and human sera: characterization of major epitopes useful for antigen detection.

Monoclonal antibodies (MAbs) were generated by immunizing mice with a truncated recombinant protein corresponding to the immunodominant region (residues 1-120) of hepatitis C virus (HCV) nucleocapsid protein. The specific recognition by either human sera or mouse monoclonal antibodies of overlapping peptides spanning the core region 1-120 as well as the comparison with epitopes described earlier allowed the fine mapping of HCV core. Within the region 1-120, the major antigenic domain could be restricted to the first 45 amino acids. Indeed, the peptide S42G (residues 2-45) allowed the detection of an anti-HCV core response by all anticore-positive human sera examined. According to their epitope localization, three groups of mouse MABs could be evidenced that were directed against different regions of core. Group II MAbs recognized a strictly linear epitope (QDVKF, residues 20-24), whereas group I MABs were directed against a conformational epitope mainly located at the amino acid residues (QIVGG, 29-33). The epitope of group III MABs was also conformational (PRGRRQPI, residues 58-65). These three epitopes appeared close but different from the three major human epitopes RKTKRNTN, VYLLPR, and GRTWAQPGYPWPLY (residues 7-17, 34-39, and 73-86, respectively). Group II MAB 7G12A8 and group I MAB 19D9D6 were used in a sandwich ELISA for the capture and the detection, respectively, of viral core antigen in sera of patients with chronic HCV infection. After treatment of sera with triton x 100 in acidic conditions, amounts of viral antigen as low as 20 pg/ml of sera could be detected.

Monoclonal antibodies targeted to alpha-oligonucleotides. Characterisation and application in nucleic acid detection.

The aim of the present study was to test the antigenicity of alpha-deoxyribonucleotides in order to develop a new tool for the detection of nucleic acid sequences for use in diagnostic applications. We describe four monoclonal antibodies (Mabs) which recognize alpha-deoxyribonucleotides. Two were raised against a poly(alpha-dT) sequence and specifically recognized the alpha-dT nucleotide. Two were raised against a sequence containing all four common nucleotides as alpha-nucleotides and, surprisingly, only recognized the alpha-dG nucleotide. For all four Mabs, no cross reactivity was observed with beta-oligonucleotides. These Mabs were reactive with alpha-oligonucleotide sequences whether these sequences were single-stranded or hybridized to DNA or RNA. The four Mabs were tested in a sandwich hybridization assay that consisted of an alpha-oligonucleotide (for target sequence recognition), one of the four Mabs (for recognition of the hybridized alpha-oligonucleotide), and goat anti-mouse antibody conjugated to horse radish peroxidase (HRP) (for detection). One of the monoclonal antibodies, Mab 2E11D7, was directly conjugated to HRP and used in sandwich hybridization to detect PCR fragments of HPV 18 DNA. The sensitivity of this reaction was 1 pg of plasmid DNA containing the HPV 18 fragment. The specificity of the detection was demonstrated using HPV 6/11 and 16 DNA sequences.

[Selective binding in vitro of a modified form of the C3 component of complement to human erythrocytes]. / Fixation sélective in vitro d'une forme modifiée du composant C3 du complément sur des érythrocytes humains.

C3 was bound to human erythrocytes from autologous plasma or from serum brought to low ionic strength (mu less than or equal to 0.03) and pH between 4.0 and 5.0, then subsequently incubated with erythrocytes (50/1, v/v) for 20 min at 0 degree C. This capacity was preserved up to 72 h by prolonged incubation at 20, 25 or 37 degrees C, whereas it was quickly lost by incubation at 0 degree C. C3 binding did not require complement activation and was not observed with neuraminidase-treated erythrocytes. Crossed immunoelectrophoretic analysis of the pretreated serum or plasma revealed that a fraction having more cathodal migration than that of native C3 was generated upon incubation in the above-mentioned conditions. This fraction appeared able to selectively bind to the erythrocytes. Cell-bound C3 reacted positively to antisera against C3a, C3c, C3d or native C3; they rosetted positively with EAC3b, clearly showing that this C3 binding was not dependent on the proteolysis of C3 and that it concerned the acceptor sites on the cells, since C3b receptors were free. The functional significance of this C3 binding was also investigated: EC3 were not able to lyse through the alternative pathway, whereas lysis clearly increased when C3 was found to AET-treated erythrocytes. This finding, together with the modulation in the capacity of EC3 or E(AET)C3 to form an alternative pathway convertase by antibodies to C3c or C3d, strongly suggests a contribution of bound C3 to such a convertase. In contrast to "C3b-like" C3, this modified C3 was able to bind to acceptor sites on erythrocytes but, like the former, it retained the capacity to form an alternative pathway convertase. In this light, it may represent an intermediate between C3 and "C3b-like" C3.