Variable correction of Artemis deficiency by I-Sce1-meganuclease-assisted homologous recombination in murine hematopoietic stem cells.

The correction of genetic mutations by homologous recombination is an attractive approach to gene therapy. We used the DNA double-strand breaks introduced by the site-specific endonuclease I-Sce1 as a means of increasing homologous recombination of an exogenous DNA template in murine hematopoietic stem cells (mHSCs). To develop this approach, we chose an Artemis knockout (Art(-/-)) mouse in which exon 12 of the Artemis gene had been replaced by an I-Sce1 recognition site. The I-Sce1 enzyme and the Artemis correction template were each delivered by a self-inactivating (SIN)-integrase-defective lentiviral vector (SIN-IDLV-CMV-ISce1 and SIN-IDLV-Art, respectively). Transduction of Art(-/-) mHSCs with the two vectors successfully reverted the Art(-/-) phenotype in 2 of our 10 experiments. Even though the potential for genotoxicity has yet to be evaluated, this new approach to gene editing appears to be promising. Improving the efficacy of this approach will require further technical work.

Characterization of a monoclonal antibody reacting with antigen-4 domain of gp900 in Cryptosporidium parvum invasive stages.

Cryptosporidium parvum (Protozoa, Apicomplexa) infects the apical surface of intestinal epithelial cells, where it grows and divides within a membrane-bound parasitophorous vacuole. gp900, an abundant glycoprotein of C. parvum merozoites and sporozoites, is localized in micronemes and at the surface of invasive stages and participates in the invasion process. Here, we describe a new monoclonal antibody (mAb) against gp900. As shown by immunofluorescence of excysted parasites and immunoelectron microscopy of infected tissues, the mAb reacted with micronemes present in the apical pole of invasive stages. In immunoprecipitation experiments, the mAb was shown to react with a high molecular weight antigen co-migrating with gp900. Finally, three reactive clones were selected upon screening of a C. parvum genomic expression library with the mAb; and sequencing of the insert from one of them showed a 596 bp sequence identical to the DNA region encoding a domain of gp900 identified as antigen 4.

Effect of Chlamydia trachomatis infection and subsequent tumor necrosis factor alpha secretion on apoptosis in the murine genital tract.

The pathology observed during Chlamydia infection is due initially to localized tissue damage caused by the infection itself, followed by deleterious host inflammatory responses that lead to permanent scarring. We have recently reported that the infection by Chlamydia in vitro results in apoptosis of epithelial cells and macrophages and that infected monocytes secrete the proinflammatory cytokine interleukin-1beta. At the same time, proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) can also trigger apoptosis of susceptible cells. To study the possible relationship between Chlamydia trachomatis infection and apoptosis in vivo, we used the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling technique to determine whether infection may cause apoptosis in the genital tract of mice and, conversely, whether cytokines produced during the inflammatory response may modulate the level of apoptosis. Our results demonstrate that infected cells in the endocervix at day 2 or 7 after infection are sometimes apoptotic, although there was not a statistically significant change in the number of apoptotic cells in the endocervix. However, large clumps of apoptotic infected cells were observed in the lumen, suggesting that apoptotic cells may be shed from the endocervix. Moreover, there was a large increase in the number of apoptotic cells in the uterine horns and oviducts after 2 or 7 days of infection, which was accompanied by obvious signs of upper tract pathology. Interestingly, depletion of TNF-alpha led to a decrease in the level of apoptosis in the uterine horns and oviducts of animals infected for 7 days, suggesting that the inflammatory cytokines may exert part of their pathological effect via apoptosis in infected tissues.

Apoptosis of epithelial cells and macrophages due to infection with the obligate intracellular pathogen Chlamydia psittaci.

We have characterized the cytotoxic activity of the obligate intracellular bacterium Chlamydia psittaci, which resides within a membrane-bound vacuole during the 2-day infection cycle. We have established that infected epithelial cells and macrophages die through apoptosis, which is measurable within 1 day of infection and requires productive infection by the bacteria. Inhibition of host cell protein synthesis has no effect on cell death, but blocking bacterial entry or bacterial protein synthesis prevents apoptosis, implying that bacterial growth is required for death of the host cell. Apoptosis was confirmed through the use of electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, gel agarose electrophoresis of fragmented DNA, and propidium-iodide labeling of host cell nuclei. Although infected cells died preferentially, both infected and uninfected cells became apoptotic, suggesting that the infected cells may secrete proapoptotic factors. Inhibition of either of two proapoptotic enzymes, caspase-1 or caspase-3, did not significantly affect Chlamydia-induced apoptosis. These results suggest that, as in the case of apoptosis due to Bax expression or oncogene dysregulation, which initiate the apoptotic program within the cell interior, the Chlamydia infection may trigger an apoptotic pathway that is independent of known caspases. As apoptotic cells secrete proinflammatory cytokines, Chlamydia-induced apoptosis may contribute to the inflammatory response of the host.

High incidence of Coxiella burnetii markers in a rural population in France.

Since Coxiella burnetii, the causative agent of Q fever, is often transmitted from goats and sheep to humans through aerosols, we examined the sera from 168 persons involved in goat breeding in the Centre region of France and 40 members of veterinary and medical staff from the same region for the presence of antibodies against C. burnetii. An immunofluorescence assay was used to detect the presence of antibodies of the IgG isotope against epitopes from phase II of C. burnetii, which are the first antibodies to appear in infected people, and from phase I, which reflect more chronic stages of the infection. Our serological survey showed that most of the tested sera were positive for C. burnetii markers, indicating at least an encounter with the bacterium. In the overall population of 208 subjects, 71% of the sera had antibodies against phase II epitopes (titres > or = 1:40). Among the goat farmers and their immediate families, 78% had antibodies against phase II and 33% against phase I (titres > or = 1:40). Considering only high titres (> or = 1:320), though, only 37% of the farmers had antibodies against phase II and 15% against phase I. Only 3 out of 12 veterinarians working in the field had high titres of antibodies against phase II and phase I, while none of 28 members of veterinary and medical laboratories had significant levels of antibodies. These results emphasize the need for closer surveillance of populations at risk for Q fever, to prevent the infection by C. burnetii from reaching chronic stages of the disease.

Choosing highly specific primers for the polymerase chain reaction using the octomer frequency disparity method: application to Chlamydia trachomatis.

The eight-nucleotide sequence (octomer) at the 3' end of PCR primers is important to PCR specificity. We describe a correlation between the specificity of PCR primers used with human DNA and the frequency of the 3' octomer in a human database. We therefore applied a methodology (OFD) based on octomer frequency disparity to identify 16 PCR targets in the chromosome of the intracellular bacterium, Chlamydia trachomatis (Ct). In addition, the 16 sets of primers were tested with a standard procedure. All the primer pairs were highly specific for Ct and did not lead to non-specific amplification when used with human DNA. This work shows that the choice of specific PCR primers is possible using a method based on the statistical representativeness of octomers in genomes.