Vaccine strategies against cystic fibrosis pathogens

A great number of cystic fibrosis (CF) pathogens such as Pseudomonas aeruginosa, the Burkholderia cepacia and the Mycobacterium abscessus complex raised difficult therapeutic problems due to their intrinsic multi-resistance to numerous antibiotics. Vaccine strategies represent one of the key weapons against these multi-resistant bacteria in a number of clinical settings like CF. Different strategies are considered in order to develop such vaccines, linked either to priming the host response, or by exploiting genomic data derived from the bacterium. Interestingly, virulence factors synthesized by various pathogens might serve as targets for vaccine development and have been, for example, evaluated in the context of CF

Human brain-specific L-proline transporter: molecular cloning, functional expression, and chromosomal localization of the gene in human and mouse genomes

L-Proline fulfills several of the classic criteria used to identify amino acid neurotransmitters, including the presence of a high affinity, Na(+)- (and Cl-)-dependent synaptosomal transport process and the Ca(2+)-dependent release of exogenously loaded radiolabeled L-proline from brain slices and synaptosomes after K(+)-induced depolarization. However, studies to define the role of L-proline in discrete pathways in the mammalian brain have been precluded by the inability to block its biosynthesis or high affinity transport in nervous tissue. We report the molecular cloning, functional expression, and chromosomal localization of a human brain-specific high affinity L-proline transporter (hPROT). The pharmacological specificity, kinetic properties, and ionic requirements of hPROT clearly distinguish this carrier from the other Na(+)-dependent plasma membrane carriers that transport L-proline. Multiple tissue Northern blot analysis revealed a prominent approximately 4-kb mRNA transcript in human brain tissue, whereas no specific hybridizing species were detected in peripheral tissue. An antipeptide antiserum directed against the carboxy-terminus of the predicted hPROT protein identified a single, broad immunoreactive protein of 68 kDa on immunoblots of synaptosomal membranes from various human brain regions. In contrast, no specific labeling was detected on immunoblots of membranes from human liver, kidney, or heart. A differential distribution of hPROT mRNA and protein was observed in the human corpus striatum, consistent with the hypothesis that the hPROT protein is synthesized in neuronal cell bodies in an extrastriatal location and axonally transported to the corpus striatum. These findings warrant the consideration of a synaptic regulatory role for this transporter and its presumed natural substrate, L-proline, in the mammalian central nervous system

A novel nonopioid action of enkephalins: competitive inhibition of the mammalian brain high affinity L-proline transporter

The high affinity L-proline transporter (PROT) is a member of the family of Na+ (and Cl-)-dependent plasma membrane transport proteins that comprises transporters for several neurotransmitters, osmolytes, and metabolites. The brain-specific expression of PROT in a subset of putative glutamatergic pathways implies a specialized function for this novel transporter and its presumed natural substrate L-proline in excitatory synaptic transmission. However, definitive studies of the physiological role(s) of high affinity L-proline uptake have been precluded by the lack of specific uptake inhibitors. Here, we report that Leu- and Met-enkephalin and their des-tyrosyl derivatives potently and selectively inhibited high affinity L-proline uptake in rat hippocampal synaptosomes and in PROT-transfected HeLa cells. High concentrations of the opiate receptor antagonist naltrexone did not block the inhibitory actions of these peptides, arguing against an involvement of opioid receptors. Des-tyrosyl-Leu-enkephalin elevated the apparent K(m) of L-proline transport in transfected HeLa cells without altering the V(max). PROT-transfected HeLa cells did not accumulate [3H]Leu-enkephalin above background levels, demonstrating that enkephalins are not substrates for PROT. These findings indicate that enkephalins competitively inhibit mammalian brain PROT through a direct interaction with the transporter protein at or near the L-proline binding site. The high potency and specificity of des-tyrosyl-Leu-enkephalin make this compound a useful tool for elucidating the structure-function properties and physiological role(s) of PROT

The combination of plasmid interleukin-12 with a single DNA vaccine is more effective than Mycobacterium bovis (bacille Calmette–Guèrin) in protecting against systemic Mycobacterim avium infection

Sub-unit vaccines utilizing purified mycobacterial proteins or DNA vaccines induce partial protection against mycobacterial infections. For example, immunization with DNA vaccines expressing the gene for the immunodominant 35 000 MW protein, common to Mycobacterium avium and Mycobacterium leprae but absent from the Mycobacterium tuberculosis complex, conferred significant protection against infection with either virulent M. avium or M. leprae in mice. However, the level of protection was equivalent to that obtained with the viable, attenuated vaccine, Mycobacterium bovis, bacille Calmette–Guèrin (BCG). The cytokine, interleukin (IL)-12, is essential for priming naïve CD4(+) T lymphocytes to differentiate into interferon-γ (IFN-γ)-secreting T cells. We have used a novel self-splicing vector expressing both chains of murine IL-12 to determine if plasmid IL-12 would increase the efficacy of a vaccine expressing the M. avium 35 000 MW protein (DNA-Av35). Co-immunization with p2AIL-12 and DNA-Av35 led to a significant increase in the number of antigen-specific IFN-γ secreting cells and total amount of IFN-γ released, but a concomitant fall in the antibody response to the 35 000 MW protein. This pattern of response was associated with enhanced clearance of M. avium from the liver and spleen of coimmunized mice, and was significantly more effective than BCG or DNA-Av35. alone. Following M. avium challenge there was significant increase in the expansion of the 35 000 MW antigen-reactive T cells in the coimmunized mice. Therefore, plasmid-delivered IL-12 acts as an effective adjuvant to increase the protective efficacy of a single DNA vaccine against M. avium infection above that achieved by BCG, and this strategy may improve the efficacy of subunit vaccines against M. leprae and M. tuberculosis