A novel method to screen genomic libraries that combines genomic immunization with the prime-boost strategy

We employed a prime-boost regimen in combination with the expression library immunization protocol to improve the protective effectiveness of a genomic library used as immunogen. To demonstrate the feasibility of this novel strategy, we used as a prime a serogroup B Neisseria meningitidis random genomic library constructed in a eukaryotic expression vector. Mice immunized with different fractions of this library and boosted with a single dose of meningococcal outer membrane vesicles elicited higher bactericidal antibody titers compared with mice primed with the empty vector. After the boost, passive administration of sera from mice primed with two of these fractions significantly reduced the number of viable bacteria in the blood of infant rats challenged with live N. meningitidis. The method proposed could be applied to the identification of subimmunogenic antigens during vaccine candidate screening by employing expression library immunization.     

Rapid recycling of ClC‐2 chloride channels between plasma membrane and endosomes: Role of a tyrosine endocytosis motif in surface retrieval

ClC‐2 chloride channel is present in the brain and some transporting epithelia where its function is poorly understood. We have now demonstrated that the surface channels are rapidly internalised and approximately the 70% of the surface membrane protein recycles after 4‐ to 8‐min internalisation. Endocytosis of ClC‐2 was dependent upon tyrosine 179 located within an endocytic motif. Rapid recycling accompanied by an even faster internalisation could account for the abundant presence of ClC‐2 in intracellular membranous structures. At least a proportion of ClC‐2 resides in lipid rafts. Use of β‐cyclodextrin led to an increase in cell surface channel, but, surprisingly, a decrease in functionally active channels. We suggest that ClC‐2 requires residing in β‐cyclodextrin sensitive clusters with other molecules in order to remain active. Regulation of ClC‐2 trafficking to and within the membrane could be a means of modulating its activity. J. Cell. Physiol. 221: 650–657, 2009. © 2009 Wiley‐Liss, Inc.     

Mucus secretion from single submucosal glands of pig. Stimulation by carbachol and vasoactive intestinal peptide

Secretion rates of >700 individual glands in isolated tracheal mucosa from 56 adult pigs were monitored optically. "Basal" secretion of 0.7 +/- 0.1 nl x min(-1) gland(-1) was observed 1-9 h post-harvest but was near zero on day 2. Secretion to carbachol (10 microm) peaked at 2-3 min and then declined to a sustained phase. Peak secretion was 12.4 +/- 1.1 nl x min(-1) gland(-1); sustained secretion was approximately one-third of peak secretion. Thapsigargin (1 microm) increased secretion from 0.1 +/- 0.05 to 0.7 +/- 0.2 nl x min(-1) gland(-1); thapsigargin did not cause contraction of the trachealis muscles. Isoproterenol and phenylephrine (10 microm each) were ineffective, but vasoactive intestinal peptide (1 microm) and forskolin (10 microm) each produced sustained secretion of 1.0 +/- 0.5 and 1.7 +/- 0.2 nl x min(-1) gland(-1), respectively. The density of actively secreting glands was 1.3/mm(2). Secretion to either carbachol or forskolin was inhibited (approximately 50%) by either bumetanide or HCO(3)(-) removal and inhibited approximately 90% by the combined treatments. Mucus secreted in response to carbachol or forskolin was acidic by approximately 0.2 pH units relative to the bath and remained acidic by approximately 0.1 pH units after bumetanide. The strong secretory response to vasoactive intestinal peptide, the acidity of [cAMP](i)-stimulated mucus, and its inhibition by bumetanide were unexpected.     

Continuous fractional component Monte Carlo simulations of high-density adsorption in metal–organic frameworks

The continuous fractional component Monte Carlo method, which was designed to overcome difficulties with insertions and deletions of molecules, is modified to include configurational bias Monte Carlo methods and is further extended to binary systems. The modified method is shown to correctly predict adsorption of Ar in silicalite, Xe and Kr in HKUST-1, and enantiomers in a homochiral metal–organic framework. The modified method is also found to be approximately an order of magnitude more efficient in inserting and deleting molecules than traditional configurational bias grand canonical Monte Carlo simulations in dense systems.     

Transmigration of Trypanosoma cruzi trypomastigotes through 3D cultures resembling a physiological environment

To disseminate and colonise tissues in the mammalian host, Trypanosoma cruzi trypomastogotes should cross several biological barriers. How this process occurs or its impact in the outcome of the disease is largely speculative. We examined the in vitro transmigration of trypomastigotes through three‐dimensional cultures (spheroids) to understand the tissular dissemination of different T. cruzi strains. Virulent strains were highly invasive: trypomastigotes deeply transmigrate up to 50 μm inside spheroids and were evenly distributed at the spheroid surface. Parasites inside spheroids were systematically observed in the space between cells suggesting a paracellular route of transmigration. On the contrary, poorly virulent strains presented a weak migratory capacity and remained in the external layers of spheroids with a patch‐like distribution pattern. The invasiveness—understood as the ability to transmigrate deep into spheroids—was not a transferable feature between strains, neither by soluble or secreted factors nor by co‐cultivation of trypomastigotes from invasive and non‐invasive strains. Besides, we demonstrated that T. cruzi isolates from children that were born congenitally infected presented a highly migrant phenotype while an isolate from an infected mother (that never transmitted the infection to any of her children) presented significantly less migration. In brief, we demonstrated that in a 3D microenvironment each strain presents a characteristic migration pattern that can be associated to their in vivo behaviour. Altogether, data presented here repositionate spheroids as a valuable tool to study host–pathogen interactions.     

A combined approach of VNTR and MLST analysis: improving molecular typing of Argentinean isolates of Leptospira interrogans

Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.