[Embryonic stem cells in pharmacology]. / Les cellules souches embryonnaires et la piharmacologie.

Because of their self-renewal and pluripotency properties, human embryonic stem cells (hES) receive a marked attention from scientists and clinicians for regenerative medicine. The most recent application of hES cells may however reside in their use as a tool in drug development. The currently available cellular models for preclinical testing consist in primary and immortalized cells that display limitations in terms of available amount and likeliness to their in vivo counterparts, respectively. hES cells have the potential to revolutionize drug discovery by providing a physiological model for any human cell type in the desired amount for the earliest steps of drug development, notably for pharmacological, metabolic and toxicity evaluation. This new generation of model may contribute to reduce, refine or replace animal testing and decrease drug attrition.

Uropathogenic Escherichia coli AL511 requires flagellum to enter renal collecting duct cells.

Escherichia coli is the leading cause of urinary tract infections, but the mechanisms governing renal colonization by this bacterium remain poorly understood. We investigated the ability of 13 E. coli strains isolated from the urine of patients with pyelonephritis and cystitis and normal stools to invade collecting duct cells, which constitute the first epithelium encountered by bacteria ascending from the bladder. The AL511 clinical isolate adhered to mouse collecting duct mpkCCD(cl4) cells, used as a model of renal cell invasion, and was able to enter and persist within these cells. Previous studies have shown that bacterial flagella play an important role in host urinary tract colonization, but the role of flagella in the interaction of E. coli with renal epithelial cells remains unclear. An analysis of the ability of E. coli AL511 mutants to invade renal cells showed that flagellin played a key role in bacterial entry. Both flagellum filament assembly and the motor proteins MotA and MotB appeared to be required for E. coli AL511 uptake into collecting duct cells. These findings indicate that pyelonephritis-associated E. coli strains may invade renal collecting duct cells and that flagellin may act as an invasin in this process.

Molecular cloning of the Chlamydophila abortus groEL gene and evaluation of its protective efficacy in a murine model by genetic vaccination.

The immunogenicity and protective effect of a DNA vaccine encoding the heat-shock protein (Hsp) GroEL of Chlamydophila abortus AB7, an obligate intracellular bacterium that causes abortion in sheep, was evaluated in pregnant and non-pregnant mouse models. The C. abortus groEL gene was cloned by screening a genomic library constructed in lambdaFIX II arms with a nucleic acid probe corresponding to the central portion of the groEL gene from C. abortus. Sequence analysis of a positive clone revealed an open reading frame of 1632 bp encoding a 544 amino acid polypeptide with a predicted molecular mass of 58 256 Da and highly similar to GroEL of Chlamydia trachomatis (93 %) and Chlamydophila pneumoniae (94 %). As observed in other sequenced chlamydial genomes, the groEL gene belongs to an operon comprising another gene encoding the Hsp GroES. OF1 outbred mice were immunized intramuscularly with plasmid DNA carrying the groEL gene three times at 3 week intervals and challenged 2 weeks after the last DNA injection. In pregnant mice, no reduction in abortion was observed and the DNA vaccination failed to reduce the bacterial infection in the placenta and spleen of mice. Nevertheless, partial protection of fetuses was obtained. Immunization of non-pregnant mice with the groEL gene resulted in a specific humoral response with the predominant IgG2a isotype, suggesting a Th1-type immune response. The anti-GroEL antibodies showed no neutralizing effect in vitro on C. abortus infectivity. Although the DNA vaccine induced a delayed-type hypersensitivity response, it failed to elicit an efficient cellular immune response since the mice were not protected against bacterial challenge.

DNA vaccination against Chlamydiaceae: current status and perspectives.

DNA vaccination (also called genetic vaccination) recently celebrated its ten years of existence. This new method of immunization presents several advantages, including the induction of both humoral and cellular immune responses. This vaccination strategy has been very successful and has served as a basis for numerous experiments that had the aim of resolving parasitic, viral, and bacterial infections. In particular, DNA vaccination has been evaluated against Chlamydiaceae, small obligate intracellular bacteria, that induce many pathologies in humans and animals. Despite promising protective effects obtained in murine and turkey models with genes encoding outer membrane proteins and heat shock proteins, DNA vaccination against Chlamydiaceae must be optimized by further investigations and could benefit from the genomic sequencing in terms of the identification of new antigens.

Proteic boost enhances humoral response induced by DNA vaccination with the dnaK gene of Chlamydophila abortus but fails to protect pregnant mice against a virulence challenge.

In order to enhance the quantity and the protective properties of the antibodies induced by DNA vaccination with the heat shock protein dnaK gene of Chlamydophila abortus AB7 as well as to elicit an efficient cellular immune response, we vaccinated mice with a DNA prime followed by a boost with the recombinant DnaK protein. In non-pregnant mice, this strategy induced the same predominance of the IgG2a isotype as DNA immunization alone with a substantial increased antibody level. The induced antibodies had no in vitro neutralizing properties on C. abortus infectivity. Moreover, the proteic boost probably failed to elicit an efficient cellular immune response since the pregnant or non-pregnant mice were not protected against the bacterial challenge.

Evaluation of protection against Chlamydophila abortus challenge after DNA immunization with the major outer-membrane protein-encoding gene in pregnant and non-pregnant mice.

The protective effect of DNA vaccination with the gene encoding the major outer-membrane protein (MOMP) of Chlamydophila abortus has been studied in non-pregnant and pregnant mouse models after chlamydial challenge. OF1 outbred mice were vaccinated intramuscularly three times every 3 weeks, mated and challenged with C. abortus 2 weeks after the last injection of DNA. In non-pregnant mice, the MOMP DNA vaccine elicited a specific humoral response with predominantly IgG2a antibodies, suggesting a Th1-type immune response. The induced antibodies showed no in vitro neutralizing effect on C. abortus infectivity. Moreover, immunization with the momp gene showed no reduction in the mean splenic bacterial counts of non-pregnant or pregnant mice or in the mean placental bacterial counts of pregnant mice after the C. abortus challenge. Nevertheless, the MOMP DNA immunization induced a non-specific and partial protection in fetuses against challenge.

Protection evaluation against Chlamydophila abortus challenge by DNA vaccination with a dnaK-encoding plasmid in pregnant and non-pregnant mice.

Mice were intramuscularly immunized with a dnaK-encoding DNA plasmid. The protective effect of DNA immunization against Chlamydophila abortus infection was studied in pregnant and non-pregnant mice models. In non-pregnant mice, the dnaK vaccine induced a specific humoral response with the predominant IgG2a isotype, which failed to have in vitro neutralizing properties. No delayed-type hypersensitivity reaction was observed and the spleens of dnaK vaccinated-mice were not protected against C. abortus challenge. In pregnant mice, the dnaK vaccine induced a non-specific partial protection from abortion. This may be due to the immunogenic properties of the CpG motifs of bacterial DNA present in the vaccinal plasmid backbone. Nevertheless, spleens of dnaK vaccinated-pregnant mice were not protected.