Distinct kinetics of memory B-cell and plasma-cell responses in peripheral blood following a blood-stage Plasmodium chabaudi infection in mice

B cell and plasma cell responses take place in lymphoid organs, but because of the inaccessibility of these organs, analyses of human responses are largely performed using peripheral blood mononuclear cells (PBMC). To determine whether PBMC are a useful source of memory B cells and plasma cells in malaria, and whether they reflect Plasmodium-specific B cell responses in spleen or bone marrow, we have investigated these components of the humoral response in PBMC using a model of Plasmodium chabaudi blood-stage infections in C57BL/6 mice. We detected memory B cells, defined as isotype-switched IgD(-) IgM(-) CD19(+) B cells, and low numbers of Plasmodium chabaudi Merozoite Surface Protein-1 (MSP1)-specific memory B cells, in PBMC at all time points sampled for up to 90 days following primary or secondary infection. By contrast, we only detected CD138(+) plasma cells and MSP1-specific antibody-secreting cells within a narrow time frame following primary (days 10 to 25) or secondary (day 10) infection. CD138(+) plasma cells in PBMC at these times expressed CD19, B220 and MHC class II, suggesting that they were not dislodged bone-marrow long-lived plasma cells, but newly differentiated migratory plasmablasts migrating to the bone marrow; thus reflective of an ongoing or developing immune response. Our data indicates that PBMC can be a useful source for malaria-specific memory B cells and plasma cells, but extrapolation of the results to human malaria infections suggests that timing of sampling, particularly for plasma cells, may be critical. Studies should therefore include multiple sampling points, and at times of infection/immunisation when the B-cell phenotypes of interest are likely to be found in peripheral blood.     

A systematic study of the development of the airway (bronchial) system of the avian lung from days 3 to 26 of embryogenesis: a transmission electron microscopic study on the domestic fowl, Gallus gallus variant domesticus

In the embryo of the domestic fowl, Gallus gallus variant domesticus, the lung buds become evident on day 3 of development. After fusing on the ventral midline, the single entity divides into left and right primordial lungs that elongate caudally while diverging and shifting towards the dorsolateral aspects of the coelomic cavity. On reaching their definitive topographical locations, the lungs rotate along a longitudinal axis, attach, and begin to slide into the ribs. First appearing as a solid cord of epithelial cells that runs in the proximal-distal axis of the developing lung, progressively, the intrapulmonary primary bronchus begins to canalize. In quick succession, secondary bronchi sprout from it in a craniocaudal sequence and radiate outwards. On reaching the periphery of the lung, parabronchi (tertiary bronchi) bud from the secondary bronchi and project into the surrounding mesenchymal cell mass. The parabronchi canalize, lengthen, increase in diameter, anastomose, and ultimately connect the secondary bronchi. The luminal aspect of the formative parabronchi is initially lined by a composite epithelium of which the peripheral cells attach onto the basement membrane while the apical ones project prominently into the lumen. The epithelium transforms to a simple columnar type in which the cells connect through arm-like extensions and prominently large intercellular spaces form. The atria are conspicuous on day 15, the infundibulae on day 16, and air capillaries on day 18. At hatching (day 21), the air and blood capillaries have anastomosed profusely and the blood-gas barrier become remarkably thin. The lung is well developed and potentially functionally competent at the end of the embryonic life. Thereafter, at least upto day 26, no further consequential structures form. The mechanisms by which the airways in the avian lung develop fundamentally differ from those that occur in the mammalian one. Compared with the blind-ended bronchial system that inaugurates in the mammalian lung, an elaborate, continuous system of air conduits develops in the avian one. Further studies are necessary to underpin the specific molecular factors and genetic processes that direct the morphogenesis of an exceptionally complex and efficient respiratory organ.     

Met signaling mutants as tools for developmental studies

The Met receptor is widely expressed in embryonic and adult epithelial tissues; its ligand (hepatocyte growth factor/scatter factor, HGF/SF) is expressed in the mesenchymal component of various organs. The generation of hgf and met null mice has revealed an essential role for this ligand-receptor pair in the development of the placenta, liver, and limb muscles. However the early lethality of the null mutants has precluded analysis of Met function in late development. To extend the possible observation period, we generated mutant metalleles of different severity. This was done by impairing the ability of the receptor to transduce the HGF/SF signal, via mutation of consensus sequences in the multifunctional docking site present in the C-terminal tail of the receptor. Mice expressing a Met mutant still active as a kinase, but unable to recruit its effectors, died in mid-gestation with the same phenotype as the metknockout, proving the importance of phosphotyrosine-SH2 interactions in vivo. Mice expressing a Met receptor with partial loss of signaling function survived until birth and revealed novel aspects of HGF/SF-Met function during muscle development.     

The branchial arches and HGF are growth-promoting and chemoattractant for cranial motor axons

During development, cranial motor neurons extend their axons along distinct pathways into the periphery. For example, branchiomotor axons extend dorsally to leave the hindbrain via large dorsal exit points. They then grow in association with sensory ganglia, to their targets, the muscles of the branchial arches. We have investigated the possibility that pathway tissues might secrete diffusible chemorepellents or chemoattractants that guide cranial motor axons, using co-cultures in collagen gels. We found that explants of dorsal neural tube or hindbrain roof plate chemorepelled cranial motor axons, while explants of cranial sensory ganglia were weakly chemoattractive. Explants of branchial arch mesenchyme were strongly growth-promoting and chemoattractive for cranial motor axons. Enhanced and oriented axon outgrowth was also elicited by beads loaded with Hepatocyte Growth Factor (HGF); antibodies to this protein largely blocked the outgrowth and orientation effects of the branchial arch on motor axons. HGF was expressed in the branchial arches, whilst Met, which encodes an HGF receptor, was expressed by subpopulations of cranial motor neurons. Mice with targetted disruptions of HGF or Met showed defects in the navigation of hypoglossal motor axons into the branchial region. Branchial arch tissue may thus act as a target-derived factor that guides motor axons during development. This influence is likely to be mediated partly by Hepatocyte Growth Factor, although a component of branchial arch-mediated growth promotion and chemoattraction was not blocked by anti-HGF antibodies.     

Explosive Lineage-Specific Expansion of the Orphan Nuclear Receptor HNF4 in Nematodes

The nuclear receptor superfamily expanded in at least two episodes: one early in metazoan evolution, the second within the vertebrate lineage. An exception to this pattern is the genome of the nematode Caenorhabditis elegans, which encodes more than 270 nuclear receptors, most of them highly divergent. We generated 128 cDNA sequences for 76 C. elegans nuclear receptors, confirming that these are active genes. Among these numerous receptors are 13 orthologues of nuclear receptors found in arthropods and/or vertebrates. We show that the supplementary nuclear receptors (supnrs) originated from an explosive burst of duplications of a unique orphan receptor, HNF4. This origin has specific implications for the role of ligand binding in the function and evolution of the nematode supplementary nuclear receptors. Moreover, the supplementary nuclear receptors include a group of very rapidly evolving genes found primarily on chromosome V. We propose a model of lineage-specific duplications from a chromosome on which duplication and substitution rates are highly increased. Our results provide a framework to study nuclear receptors in nematodes, as well as to consider the functional and evolutionary consequences of lineage-specific duplications.Reviewing Editor: Dr. Nicolos Galtier     

Novel cyclized Pifithrin-alpha p53 inactivators: synthesis and biological studies

Starting from various cyclic or bicyclic ketones, we have synthesized novel Pifithrin-alpha analogues bearing different methyl substituted phenyl ketone groups at the N3-position of the 2-iminothiazole heterocycle. From stability studies in a biological medium as well as under specific chemical conditions, we have shown by NMR techniques that through a dehydration process, some derivatives can generate their corresponding cyclized analogues. All of the new analogues, Pifithrin-like and polycyclic dehydrated derivatives were assessed for their p53 inactivation potency by measuring survival of cortical neurons, whose death was induced by the DNA-damaging agent etoposide. Pifithrin-alpha like 2f as well as the cyclic dehydrated 6b analogue were found to be one log more potent p53 inactivators than reference compound Pft-alpha, with EC50 values ranging around 30 nM. These results support the finding that p53 inactivation by Pft-alpha analogues could be also due to the presence of the cyclic dehydrated Pft-alpha forms, generated in situ in the biological assay incubation medium.