Baseline levels of influenza-specific CD4 memory T-cells affect T-cell responses to influenza vaccines

Background

Factors affecting immune responses to influenza vaccines have not been studied systematically. We hypothesized that T-cell and antibody responses to the vaccines are functions of pre-existing host immunity against influenza antigens.

Methodology/Principal Findings

During the 2004 and 2005 influenza seasons, we have collected data on cellular and humoral immune reactivity to influenza virus in blood samples collected before and after immunization with inactivated or live attenuated influenza vaccines in healthy children and adults. We first used cross-validated lasso regression on the 2004 dataset to identify a group of candidate baseline correlates with T-cell and antibody responses to vaccines, defined as fold-increase in influenza-specific T-cells and serum HAI titer after vaccination. The following baseline parameters were examined: percentages of influenza-reactive IFN-γ⁺ cells in T and NK cell subsets, percentages of influenza-specific memory B-cells, HAI titer, age, and type of vaccine. The candidate baseline correlates were then tested with the independent 2005 dataset. Baseline percentage of influenza-specific IFN-γ⁺ CD4 T-cells was identified as a significant correlate of CD4 and CD8 T-cell responses, with lower baseline levels associated with larger T-cell responses. Baseline HAI titer and vaccine type were identified as significant correlates for HAI response, with lower baseline levels and the inactivated vaccine associated with larger HAI responses. Previously we reported that baseline levels of CD56^dim NK reactivity against influenza virus inversely correlated with the immediate T-cell response to vaccination, and that NK reactivity induced by influenza virus depended on IL-2 produced by influenza-specific memory T-cells. Taken together these results suggest a novel mechanism for the homeostasis of virus-specific T-cells, which involves interaction between memory helper T-cells, CD56^dim NK and DC.

Significance

These results demonstrate that assessment of baseline biomarkers may predict immunologic outcome of influenza vaccination and may reveal some of the mechanisms responsible for variable immune responses following vaccination and natural infection.     

The blood-brain barrier significantly limits eflornithine entry into Trypanosoma brucei brucei infected mouse brain

Drugs to treat African trypanosomiasis are toxic, expensive and subject to parasite resistance. New drugs are urgently being sought. Although the existing drug, eflornithine, is assumed to reach the brain in high concentrations, little is known about how it crosses the healthy and infected blood-brain barrier. This information is essential for the design of drug combinations and new drugs. This study used novel combinations of animal models to address these omissions. Eflornithine crossed the healthy blood-CNS interfaces poorly, but this could be improved by co-administering suramin, but not nifurtimox, pentamidine or melarsoprol. Work using a murine model of sleeping sickness demonstrated that Trypanosoma brucei brucei crossed the blood-CNS interfaces, which remained functional, early in the course of infection. Concentrations of brain parasites increased during the infection and this resulted in detectable blood-brain barrier, but not choroid plexus, dysfunction at day 28 post-infection with resultant increases in eflornithine brain delivery. Barrier integrity was never restored and the animals died at day 37.9 +/- 1.2. This study indicates why an intensive treatment regimen of eflornithine is required (poor blood-brain barrier penetration) and suggests a possible remedy (combining eflornithine with suramin). The blood-brain barrier retains functionality until a late, possibly terminal stage, of trypanosoma infection.     

RNA based vaccines

The recognition that CD8(+) T-cell mediated Th1 immune responses were necessary to produce immunity to intracellular and transformed self pathogens led to intense interest in the delivery of nucleic acids, DNA, or RNA encoding candidate antigens, as vaccines. Antigen presenting cells (APC) encounter most protein and vaccine immunogens as extracellular proteins and, thus, present them on major histocompatibility complex (MHC) class II molecules leading to the activation of CD4(+) T cells. Protein antigens encoded by nucleic acids delivered to dendritic cell (DC) are produced inside the cell and, thus, can stimulate MHC class I mediated activation of CD8(+) T-cell immune responses. Unfortunately, DCs are not readily transfected with DNA (Akbari et al., 1999) resulting in the requirement for high concentrations of DNA and repeated immunizations to achieved immune responses. RNA, on the other hand, is readily taken up and expressed by DC, making it an alternative vaccine candidate. In this article, we will discuss immune responses developed, interactions between APC and RNA that activate and dictate DC activation, and preliminary studies using RNA in vivo and in vitro to develop protective immunity.     

Membrane biochips

Membrane-bound proteins represent the single most important class of drug targets. This article discusses the issues surrounding fabrication of membrane-protein microarrays by conventional robotic pin printing techniques. Ligand binding selectivity and specificity to G protein-coupled receptor (GPCR) microarrays are presented. The potential applications of these arrays for drug screening are discussed.     

Intrastrain and interstrain genetic variation within a paralogous gene family in Chlamydia pneumoniae

Background

Chlamydia pneumoniae causes human respiratory diseases and has recently been associated with atherosclerosis. Analysis of the three recently published C. pneumoniae genomes has led to the identification of a new gene family (the Cpn 1054 family) that consists of 11 predicted genes and gene fragments. Each member encodes a polypeptide with a hydrophobic domain characteristic of proteins localized to the inclusion membrane.

Results

Comparative analysis of this gene family within the published genome sequences provided evidence that multiple levels of genetic variation are evident within this single collection of paralogous genes. Frameshift mutations are found that result in both truncated gene products and pseudogenes that vary among isolates. Several genes in this family contain polycytosine (polyC) tracts either upstream or within the terminal 5' end of the predicted coding sequence. The length of the polyC stretch varies between paralogous genes and within single genes in the three genomes. Sequence analysis of genomic DNA from a collection of 12 C. pneumoniae clinical isolates was used to determine the extent of the variation in the Cpn 1054 gene family.

Conclusions

These studies demonstrate that sequence variability is present both among strains and within strains at several of the loci. In particular, changes in the length of the polyC tract associated with the different Cpn 1054 gene family members are common within each tested C. pneumoniae isolate. The variability identified within this newly described gene family may modulate either phase or antigenic variation and subsequent physiologic diversity within a C. pneumoniae population.     

The consequences of rewardlessness in orchids: reward-supplementation experiments with Anacamptis morio (Orchidaceae)

Pollinators are expected to respond to low reward availability in an inflorescence by visiting fewer flowers before departure, thus potentially causing reduced visitation, but also reduced geitonogamous selfing. I tested this hypothesis using Anacamptis morio, an orchid that does not reward its pollinators. Supplementation of inflorescences with artificial nectar did not result in an increase in fruit set on supplemented inflorescences compared to control inflorescences and tended to reduce pollinia removal. Supplementation resulted in reduced fruit quality, but there was no evidence that this was as a result of inbreeding depression. Behavioral experiments showed that pollinating bumble bees, as predicted, visited more flowers on supplemented inflorescences. Bumble bees also deposited more self-pollen on supplemented inflorescences, but this was marginally significant. Bumble bee queens removed significantly more pollinia from control inflorescences, while Bombus terrestris and B. lucorum workers did not. I conclude that while pollinators behaved as predicted, there was weak evidence that pollinia removal, pollen deposition, and fruit set followed the predictions of the hypothesis. I argue that this was probably because some pollinators were more efficient at removing and depositing pollen on control inflorescences, while others were not.     

Biogenesis of water splitting by photosystem II during de‐etiolation of barley (Hordeum vulgare L.)

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de‐etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll‐binding protein complexes and development of water splitting via O₂ production in plastids (etiochloroplasts) isolated during de‐etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light‐harvesting antenna [PSII‐light‐harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de‐etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de‐etiolation, etiochloroplasts revealed the same water‐splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de‐etiolation precedes assembly of the PSII‐LHCII supercomplexes. Taken together, data show a rapid establishment of water‐splitting activity during etioplast‐to‐chloroplast transition and emphasize that assembly of the functional water‐splitting site of PSII is not the rate‐limiting step in the formation of photoactive thylakoid membranes.     

Lectin binding defines and differentiates M-cells in mouse small intestine and caecum

M-cell surface glycoconjugate expression was investigated by applying a panel of lectins to whole fixed mouse Peyer's and caecal patches. While the majority of lectins failed to identify mouse M-cells, the lectinEuonymus europaeus differentially stained the surface of M-cells in both mouse Peyer's and caecal patches, and the lectinsUlex europaeus II andBandeiraea simplicifolia I isolectin B₄ identified M-cells in the Peyer's and caecal patch follicle associated epithelium, respectively. These three mouse M-cell markers failed to identify rat and rabbit Peyer's patch M-cells, although bothEuonymus europaeus andUlex europaeus II differentially stained M-cells in the periphery of rabbit caecal patch domes. These site and species related variations in M-cell surface glycoconjugate expression may reflect the local microorganism populations and will have important implications if orally delivered vaccines and drugs are to be targeted to M-cells via their surface glycoconjugates.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Global changes may be promoting a rise in select cyanobacteria in nutrient‐poor northern lakes

The interacting effects of global changes—including increased temperature, altered precipitation, reduced acidification and increased dissolved organic matter loads to lakes—are anticipated to create favourable environmental conditions for cyanobacteria in northern lakes. However, responses of cyanobacteria to these global changes are complex, if not contradictory. We hypothesized that absolute and relative biovolumes of cyanobacteria (both total and specific genera) are increasing in Swedish nutrient‐poor lakes and that these increases are associated with global changes. We tested these hypotheses using data from 28 nutrient‐poor Swedish lakes over 16 years (1998–2013). Increases in cyanobacteria relative biovolume were identified in 21% of the study sites, primarily in the southeastern region of Sweden, and were composed mostly of increases from three specific genera: Merismopedia, Chroococcus and Dolichospermum. Taxon‐specific changes were related to different environmental stressors; that is, increased surface water temperature favoured higher Merismopedia relative biovolume in low pH lakes with high nitrogen to phosphorus ratios, whereas acidification recovery was statistically related to increased relative biovolumes of Chroococcus and Dolichospermum. In addition, enhanced dissolved organic matter loads were identified as potential determinants of Chroococcus suppression and Dolichospermum promotion. Our findings highlight that specific genera of cyanobacteria benefit from different environmental changes. Our ability to predict the risk of cyanobacteria prevalence requires consideration of the environmental condition of a lake and the sensitivities of the cyanobacteria genera within the lake. Regional patterns may emerge due to spatial autocorrelations within and among lake history, rates and direction of environmental change and the niche space occupied by specific cyanobacteria.