Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants

Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.     

CX3CR1 Is Expressed by Human B Lymphocytes and Meditates CX3CL1 Driven Chemotaxis of Tonsil Centrocytes

Background

Fractalkine/CX₃CL1, a surface chemokine, binds to CX₃CR1 expressed by different lymphocyte subsets. Since CX₃CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX₃CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood.

Methodology/Principal Findings

We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX₃CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX₃CR1 but only germinal centre B cells were attracted by soluble CX₃CL1 in a transwell assay. CX₃CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX₃CR1⁺ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX₃CL1. ELISA assay showed that soluble CX₃CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX₃CL1 did not attract spleen B cells from wild type mice. OVA immunized CX₃CR1⁻/⁻ or CX₃CL1⁻/⁻ mice showed significantly decreased specific IgG production compared to wild type mice.

Conclusion/Significance

We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX₃CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.     

The Problem of Adult Play Fighting: A Comparative Analysis of Play and Courtship in Primates

Although play fighting, like play generally, is predominantly a feature of the juvenile phase, such behavior persists in the adults of many species. There are two major contexts in which adults engage in play fighting – with juveniles and with other adults. The least attention has been given to adult–adult play. However, one pattern that has been noted by several authors is that the most commonly occurring context of adult–adult play fighting is during courtship, and that this is more likely to occur in solitary species. Supposedly, such play could function to overcome the aggressiveness of potential pairmates unfamiliar with one another, or as a means of evaluating mate quality by one or both partners. By contrasting the presence and degree of play fighting during courtship with the degree of male–female familiarity, the hypothesis that the former is influenced by the latter is tested. Data on 35 species of primates, from 15 families, were compiled from the literature and compared using a method of independent contrasts that incorporates information on phylogenetic relationships. A significant regression was found, with the degree of male–female familiarity accounting for 40% of the variance in courtship play. Therefore, our data support the hypothesis that play fighting in courtship is influenced by male–female patterns of association. However, the data also indicate that other factors must influence the occurrence of play fighting amongst adults, not only during courtship, but also in nonsexual contexts. The broader context of adult–adult play in mammals is discussed.     

Doubled-haploid production in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): role of stress treatments

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15–20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4°C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50–400 V with 25 μF capacitance and 25 Ω resistance, (3) anthers were centrifuged at 168–1,509g for 2–15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15–20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1–3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10–30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.     

Heterologous protein production and delivery systems for Lactococcus lactis

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed.     

Non-equilibrium allele frequency spectra via spectral methods

A major challenge in the analysis of population genomics data consists of isolating signatures of natural selection from background noise caused by random drift and gene flow. Analyses of massive amounts of data from many related populations require high-performance algorithms to determine the likelihood of different demographic scenarios that could have shaped the observed neutral single nucleotide polymorphism (SNP) allele frequency spectrum. In many areas of applied mathematics, Fourier Transforms and Spectral Methods are firmly established tools to analyze spectra of signals and model their dynamics as solutions of certain Partial Differential Equations (PDEs). When spectral methods are applicable, they have excellent error properties and are the fastest possible in high dimension; see Press et al. (2007). In this paper we present an explicit numerical solution, using spectral methods, to the forward Kolmogorov equations for a Wright–Fisher process with migration of K populations, influx of mutations, and multiple population splitting events.     

Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis

An intracellular bacterium originally isolated from hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was identified by sequences of the small and large subunit ribosomal (16S and 23S) DNA and the internal transcribed spacer (ITS) as Piscirickettsia salmonis. Considering all rDNA sequences compared, the white seabass isolate (WSB-98) had a 96.3 to 98.7% homology with 4 previously described strains of P. salmonis isolated from salmon in Chile, Norway, and British Columbia, Canada. Experimental infections induced by intraperitoneal injections of juvenile white seabass with WSB-98 resulted in disease and mortality similar to that observed in P. salmonis infections in salmon. After 60 d, the cumulative mortality among P. salmonis-injected white seabass was 82 and 40%, respectively, following a high (1.99 x 10(4) TCID50) or low (3.98 x 10(2) TCID50) dose-challenge with WSB-98. The bacterium was recovered by isolation in cell culture or was observed in stains from tissues of injected white seabass but not from control fish. There were no external signs of infection. Internally, the most common gross lesion was a mottled appearance of the liver, sometimes with distinct nodules. Microscopic lesions were evident in both the capsule and parenchyma of the liver and were characterized by multifocal necrosis, often with infiltration of mononuclear leukocytes. Macrophages filled with bacteria were present at tissue sites exhibiting focal necrosis. Foreign body-type granulomas were prevalent in livers of experimentally infected white seabass, but not in control fish. Similar granulomatous lesions were observed in the spleen, kidney, intestine and gills, but these organs were considered secondary sites of infection, with significantly fewer and less severe histologic lesions compared to the liver. The results from this study clearly indicate that infections with P. salmonis are not restricted to salmonid fishes and that the bacterium can cause a disease similar to piscirickettsiosis in nonsalmonid hosts.     

High Diversity of vacA and cagA Helicobacter pylori Genotypes in Patients with and without Gastric Cancer

Background

Helicobacter pylori is associated with chronic gastritis, peptic ulcers, and gastric cancer. The aim of this study was to assess the topographical distribution of H. pylori in the stomach as well as the vacA and cagA genotypes in patients with and without gastric cancer.

Methodology/Principal Findings

Three gastric biopsies, from predetermined regions, were evaluated in 16 patients with gastric cancer and 14 patients with dyspeptic symptoms. From cancer patients, additional biopsy specimens were obtained from tumor centers and margins; among these samples, the presence of H. pylori vacA and cagA genotypes was evaluated. Positive H. pylori was 38% and 26% in biopsies obtained from the gastric cancer and non-cancer groups, respectively (p = 0.008), and 36% in tumor sites. In cancer patients, we found a preferential distribution of H. pylori in the fundus and corpus, whereas, in the non-cancer group, the distribution was uniform (p = 0.003). A majority of the biopsies were simultaneously cagA gene-positive and -negative. The fundus and corpus demonstrated a higher positivity rate for the cagA gene in the non-cancer group (p = 0.036). A mixture of cagA gene sizes was also significantly more frequent in this group (p = 0.003). Ninety-two percent of all the subjects showed more than one vacA gene genotype; s1b and m1 vacA genotypes were predominantly found in the gastric cancer group. The highest vacA-genotype signal-sequence diversity was found in the corpus and 5 cm from tumor margins.

Conclusion/Significance

High H. pylori colonization diversity, along with the cagA gene, was found predominantly in the fundus and corpus of patients with gastric cancer. The genotype diversity observed across systematic whole-organ and tumor sampling was remarkable. We find that there is insufficient evidence to support the association of one isolate with a specific disease, due to the multistrain nature of H. pylori infection shown in this work.