The role of IL-5 in IgA B cell differentiation

IL-5 enhances secretion of IgA by B cells. The stage of B cell differentiation at which IL-5 enhances IgA secretion and the mechanism by which it exerts this effect are unknown. We examined these issues by separating Peyer's patch (PP) B cells into membrane IgA (mIgA)-positive and mIgA-negative cells with panning or cell sorting. When LPS was used to activate these cells, mIgA-positive PP B cells were induced by IL-5 (either as crude T cell supernatant or rIL-5 to secrete large amounts of IgA. In contrast mIgA-negative PP B cells showed no significant amount of IgA secretion with IL-5. In addition, rIL-5 did not cause expression of mIgA by mIgM-bearing B cells. The mechanism involved in enhancement of IgA secretion was evaluated by utilizing an ELISPOT assay to quantitate IgA secreting cells. Both unsorted PP B cells and mIgA-positive PP B cells, when incubated with IL-5, showed an increase in the number of IgA-secreting cells that was proportional to the increase in total secreted IgA. However, LPS-activated PP mIgA-positive B cells, when incubated with rIL-5, showed no increase in proliferation, as measured by [3H]thymidine incorporation indicating that the increase in IgA-secreting cells after incubation with IL-5 occurred not as a result of proliferation but rather through promotion of terminal differentiation. Thus, IL-5 acts as a differentiation factor on B cells which have already undergone isotype switch to IgA B cells, promoting differentiation into IgA-secreting cells with resultant increased IgA secretion.     

Harnessing Gene Conversion in Chicken B Cells to Create a Human Antibody Sequence Repertoire

Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken V_L and V_H loci were knocked out in DT40 cells and replaced with human V_K and V_H genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human V_K and V_H regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens.     

Herbicide-resistant, transgenic zonal pelargoniums produced by step-down glyphosate selection and plantlet recovery in the presence of aromatic amino acids

A genetic transformation protocol was developed using the transfer of a synthetic CP4 EPSPS transgene, as a conditional positive selectable marker, into commercially relevant zonal pelargoniums using an Agrobacterium tumefaciens strain in combination with a novel step-down glyphosate selection system. The transformation efficiency based on independent T-DNA integration events averaged 1.9?% over 10 experiments. Some 273 independent transformants were produced within an average time of 6?mo from explant inoculation with Agrobacterium to plantlet recovery. For plantlet recovery, three aromatic amino acids were incorporated into the rooting medium to ameliorate the accumulative effects of glyphosate selection. The T-DNA also contained a mutant ethylene receptor (etr1-1) cDNA from Arabidopsis thaliana, under control of the petunia flower-specific, floral-binding protein promoter, to confer ethylene insensitivity. However, delayed flower senescence was not obtained. The transformation protocol provides a reliable method to add herbicide resistance and other traits to zonal pelargonium.     

Cell adhesion antagonists: synthesis and evaluation of a novel series of phenylalanine based inhibitors

Several phenylalanine based inhibitors were synthesized as antagonists of the leukocyte cell adhesion process that is mediated through the interactions of the mucosal addressin cell adhesion molecule (MAdCAM) and the integrin alpha4beta7. Analogues 20, 21, 22 and 24 displayed inhibition of adhesion in a cell based assay in the low micromolar range.     

Seasonal patterns in reproductive success of temperate‐breeding birds: Experimental tests of the date and quality hypotheses

For organisms in seasonal environments, individuals that breed earlier in the season regularly attain higher fitness than their late‐breeding counterparts. Two primary hypotheses have been proposed to explain these patterns: The quality hypothesis contends that early breeders are of better phenotypic quality or breed on higher quality territories, whereas the date hypothesis predicts that seasonally declining reproductive success is a response to a seasonal deterioration in environmental quality. In birds, food availability is thought to drive deteriorating environmental conditions, but few experimental studies have demonstrated its importance while also controlling for parental quality. We tested predictions of the date hypothesis in tree swallows (Tachycineta bicolor) over two breeding seasons and in two locations within their breeding range in Canada. Nests were paired by clutch initiation date to control for parental quality, and we delayed the hatching date of one nest within each pair. Subsequently, brood sizes were manipulated to mimic changes in per capita food abundance, and we examined the effects of manipulations, as well as indices of environmental and parental quality, on nestling quality, fledging success, and return rates. Reduced reproductive success of late‐breeding individuals was causally related to a seasonal decline in environmental quality. Declining insect biomass and enlarged brood sizes resulted in nestlings that were lighter, in poorer body condition, structurally smaller, had shorter and slower growing flight feathers and were less likely to survive to fledge. Our results provide evidence for the importance of food resources in mediating seasonal declines in offspring quality and survival.     

Trends in Dissolved Organic Carbon in UK Rivers and Lakes

Several studies have highlighted an increase in DOC concentration in streams and lakes of UK upland catchments though the causal mechanisms controlling the increase have yet to be fully explained. This study, compiles a comprehensive data set of DOC concentration records for UK catchments to evaluate trends and test whether observed increases are ubiquitous over time and space. The study analysed monthly DOC time series from 198 sites, including 29 lakes, 8 water supply reservoirs and 161 rivers. The records vary in length from 8 to 42 years going back as far as 1961. Of the 198 sites, 153 (77%) show an upward trend in DOC concentration significant at the 95% level, the remaining 45 (23%) show no significant trend and no sites show a significant decrease in DOC concentration. The average annual increase in DOC concentration was 0.17 mg C/l/year. The dataset shows: (i) a spatial consistent upward trend in the DOC concentration independent of regional effects of rainfall, acid and nitrogen deposition, and local effects of land-use change; (ii) a temporally consistent increase in DOC concentration for period back as far as the 1960s; (iii) the increase in DOC concentration means an estimated DOC flux from the UK as 0.86 Mt C for the year 2002 and is increasing at 0.02 Mt C/year. Possible reasons for the increasing DOC concentration are discussed.