Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile

In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of Clostridium difficile also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT. We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected C. difficile-associated diarrhea or colitis. Twenty-two strains (a prevalence of 6%) harbored both genes. When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of C. perfringens (anti-Ia and anti-Ib). Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains. Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis. All binary toxin-positive strains also produced TcdB and/or TcdA. However, they had significant changes in the tcdA and tcdB genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII. We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated.     

Cerebro-osseous-digital syndrome: four new cases of a lethal skeletal dysplasia--distinct from Neu-Laxova Syndrome

Neu-Laxova Syndrome (NLS) is a severe disorder with intrauterine growth retardation, edema, and characteristic face (including microcephaly with receding forehead, protuberant eyes, a flattened nose, deformed ears, cleft palate, and micrognathia). Ichthyosis is often present. Limb anomalies include hypoplastic fingers and syndactyly of fingers and toes. Patients are usually stillborn or die shortly after birth. We report five unrelated patients--four with atypical NLS and one with typical NLS. All five patients were stillbirths. Clinically, the atypical NLS patients showed a large skull; rhizo-, meso-, and acromelia; and hypoplasia of the metacarpals and phalanges. The feet were similarly affected. Radiographically, the atypical patients showed interpediculate narrowing and hypoplastic vertebral bodies. The long bones were stick-like, showing diaphyseal widening that spared the metaphyses and was more pronounced in the lower extremities. The ilia had a half-moon configuration with widening of the sacrosciatic notches. The ischia were vertical and the pubic bone was absent. The typical NLS patient showed microcephaly, normal vertebral body, and long bone ossification, but a pelvic configuration similar to that of the atypical NLS patients. The common and distinguishing clinical and radiographic features are reviewed. Scott et al. [1981: Am J Med Genet 9:165-175] described two patients with NLS with radiographic and clinical findings similar to patients 1-4 reported here. Patients 1-4 of this report lack the typical findings of NLS and likely represent a distinct lethal skeletal dysplasia.     

IgE-mediated anaphylaxis caused by bites of the pigeon tick Argas reflexus: cloning and expression of the major allergen Arg r 1

BACKGROUND: Anaphylactic reactions caused by bites of the European pigeon tick Argas reflexus are repeatedly reported. This soft-backed tick is a parasite of wild pigeons colonizing urban buildings and houses. Occasionally the ticks can bite human beings, inducing anaphylactic reactions in sensitized patients. OBJECTIVE: Our aim was to characterize the major allergen implicated in a series of anaphylactic reactions caused by Argas bites and to produce the allergen as recombinant protein for diagnostic purposes. METHODS: Protein extracts were prepared from whole A reflexus bodies, and IgE immunoblots were performed with sera from 13 patients who had an anaphylactic reaction with pigeon tick bites. A cDNA expression library was constructed from whole ticks and screened with a polyclonal rabbit antiserum raised against the major allergen. RESULTS: The cDNA coding for the dominant allergen Arg r 1 could be isolated. It encodes a protein belonging to the lipocalin family. Allergenicity of the recombinant Arg r 1 was confirmed by immunoblot, ELISA, and intradermal skin tests. CONCLUSION: The dominant allergen of A reflexus has been isolated and the corresponding cDNA cloned. The recombinant protein, a lipocalin, was expressed in Escherichia coli and was shown to be immunoreactive in vitro and in vivo. Recombinant Arg r 1 was used as a diagnostic tool in a series of anaphylactic reactions caused by pigeon tick bites.     

Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes

PCR-restriction fragment length polymorphism analysis of heat shock protein 70 genes discriminates most neotropical Leishmania species, as well as Trypanosoma cruzi. The assay, combined with capillary electrophoresis in a microchip device, may be applied directly on clinical samples with a high sensitivity, hence supporting clinical and epidemiological monitoring of leishmaniasis.     

Distinctive alterations of invasiveness, drug resistance and cell-cell organization in 3D-cultures of MCF-7, a human breast cancer cell line, and its multidrug resistant variant

Growth of human tumor cells as three-dimensional (3D) multicellular spheroids modifies their invasive properties. Here we study the differences in the biological features of MCF-7, a human breast cancer cell line, and its multidrug resistant variant (MDR-MCF-7) cultured as spheroids or as monolayers. Three-dimensional culture decreased the proliferative rate of both cell lines, reduced the drug sensitivity of MCF-7 cells and did not affect the resistance of MDR-MCF-7 cells. Transmission electron microscopic studies and intercellular junctions labeling showed that MCF-7 spheroids had a junctional system involving E-cadherin, tight-junctions and desmosomes. In MDR-MCF-7 cell spheroids, cell cohesion was mostly due to membrane interdigitations. MDR-MCF-7 cells, but not their parental counterpart, displayed a higher invasive potential when cultured as spheroids, as shown in the Boyden chamber assay. 3D-induced invasiveness was correlated with serine protease and plasminogen activator (PA) secretion. MCF-7 cells did not show any tendency to invade, whatever the mode of culture. These results show that 3D-cultures as spheroids distinctively altered structural features of parental and MDR-MCF-7 cells. In MCF-7 cells, 3D-culture increased cell-cell contacts and drug resistance; in MDR-MCF-7 cells, it induced invasive properties.     

Factors involved in the sensitivity of different hematopoietic cell lines to infection by subgroup C adenovirus: implication for gene therapy of human lymphocytic malignancies

Gene transfer approaches using viruses such human adenovirus (HAdV) may provide an alternative treatment for diseases involving hematopoietic cells. Better understanding of the cellular mechanisms by which the HAdV introduces DNA into these cells should help in vector design. We examined HAdV intracellular delivery in several cell lines including B and T lymphocytes. We demonstrated that HAdV resistance in most B lymphocytes is the result of moderate HAdV uptake. In contrast, high levels of coxsackie and HAdV receptor (hCAR) are expressed on the surface of HSB2 (T cells), allowing efficient binding and uptake but no transgene expression, probably because of deficient endosomolysis and subsequent exocytose. This work demonstrates the existence of hCAR-dependent and -independent endocytic route in hematopoietic cells. Moreover, it precises the intracellular barriers to be overcome by HAdV in such cells to be infectious and gives previous information's to design new vectors for gene transfer.     

Synthèse par voie radicalaire de polymères à extrémités hydroxylées, 8. Synthèse de polybutadiènes: Etude de différents paramètres influençant la polymolécularité

It was shown that radical polymerization of butadiene in 1-butanol and 2-butanol, initiated by hydrogen peroxide cleaved into two hydroxyl radicals by heat, leads to three types of polymers according to their respective polydispersity. It was found that the formation of these products — oligomers, polymers, and high polymers — can be influenced by modifying the experimental conditions. Thus, it is possible to obtain selectively oligomers free of polymers and polymers free of low molecular weight oligomers.     

Monomères acryliques à fonction carbonate cyclique, 2 Modification chimique de copolymères à groupements carbonate cyclique lateraux

Acrylic monomers with a cyclic carbonate function, their synthesis having been described previously, are used to synthesize copolymers with pendent cyclic carbonate side groups. Their chemical modification by ring opening addition reactions provides a convenient method for preparing functional polymers. The study of reactions with amines, on model compounds and on polymers, shows the possibility of obtaining a macromolecular network with crosslinking groups of carbamate type.