γ-Herpesvirus reactivation differentially stimulates epitope-specific CD8 T cell responses

The γ-herpesviruses are characterized by their ability to establish lifelong latency. Subsequent immune suppression leads to viral reactivation from latency and the onset of a variety of pathologies, including lymphoproliferative disease and cancers. CD8 T cells play a key role in preventing reactivation of latent virus. Therefore, to develop effective therapeutic immune strategies, it is essential to understand the maintenance of CD8 T cell responses during latency. Because the γ-herpesviruses are highly species-specific and mice cannot be infected with the human pathogens, EBV or Kaposi's sarcoma-associated herpesvirus, we have used a natural rodent γ-herpesvirus experimental infection model, γ-herpesvirus-68. In this report, we show that during long-term latent infection, naive CD8 T cells are recruited into the ongoing immune response in an epitope-specific manner. When virus reactivation is induced in vivo, the recruitment of CD8 T cells for some, but not all, epitopes is enhanced. The variation in recruitment is not due to differences in epitope presentation. We also show that CD8 T cells that are newly stimulated during reactivation are functionally impaired compared with acutely stimulated cells in terms of cytokine production. Thus, our results demonstrate unexpected complexity in the response of CD8 T cells specific for different viral epitopes that were stimulated during acute infection, quiescent latency, and reactivation.     

Correction to: Predator‑based selection and the impact of edge sympatry on components of coral snake mimicry

Evolutionary Ecology -     

Regulatory effect of IFN-kappa,a novel type I IFN,on cytokine production by cells of the innate immune system

IFN-kappa is a recently identified type I IFN that exhibits both structural and functional homology with the other type I IFN subclasses. In this study, we have investigated the effect of IFN-kappa on cells of the innate immune system by comparing cytokine release following treatment of human cells with either IFN-kappa or two recombinant IFN subtypes, IFN-beta and IFN-alpha2a. Although IFN-alpha2a failed to stimulate monocyte cytokine secretion, IFN-kappa, like IFN-beta, induced the release of several cytokines from both monocytes and dendritic cells, without the requirement of a costimulatory signal. IFN-kappa was particularly effective in inhibiting inducible IL-12 release from monocytes. Unlike IFN-beta, IFN-kappa did not induce release of IFN-gamma by PBL. Expression of the IFN-kappa mRNA was observed in resting dendritic cells and monocytes, and it was up-regulated by IFN-gamma stimulation in monocytes, while IFN-beta mRNA was minimally detectable under the same conditions. Monocyte and dendritic cell expression of IFN-kappa was also confirmed in vivo in chronic lesions of psoriasis vulgaris and atopic dermatitis. Finally, biosensor-based binding kinetic analysis revealed that IFN-kappa, like IFN-beta, binds strongly to heparin (K(d): 2.1 nM), suggesting that the cytokine can be retained close to the local site of production. The pattern of cytokines induced by IFN-kappa in monocytes, coupled with the unique induction of IFN-kappa mRNA by IFN-gamma, indicates a potential role for IFN-kappa in the regulation of immune cell functions.     

Constraints to growth of annual nettle (Urtica urens) in an elevated CO2 atmosphere: Decreased leaf area ratio and tissue N cannot be explained by ontogenetic drift or mineral N supply

Plant sterols differ from cholesterol in having an alkyl group at Δ-24, and, in the case of stigmasterol, also a Δ-22 double bond. The effects of 10 mol% of three plant sterols (campesterol, β -sitosterol, stigmasterol) and cholesterol on the molecular dynamics and phase behavior in multilamellar liposomes made from different phosphatidylcholine (PC) molecular species have been compared, utilizing the fluorescent probe Laurdan (2-dimethyl-amino-6-laurylnaphthalene). Laurdan reports the molecular mobility in the hydrophilic/hydrophobic interface of the membrane by determining the rate of dipolar relaxation of water molecules close to the glycerol backbone of PC. Our results showed that the Δ-24 alkyl group of plant sterols did not affect their ability to reduce molecular mobility in this region of the PC membranes. However, the plant sterols had a decreased capacity compared to cholesterol to inhibit formation of co-existing domains of gel and liquid-crystalline phases in membranes composed of equimolar dilauroyl-PC and dipalmitoyl-PC. The Δ-22 double bond present in stigmasterol decreased the ability of this sterol, compared to the other phytosterols, to reduce the molecular mobility at the hydrophobic/hydrophilic interface in membranes made of a saturated PC molecular species. However, in membranes made from 16:0/18:2-PC, a lipid species common in plant plasma membranes, stigmasterol was as efficient as other sterols in affecting the polarity and molecular mobility at the hydrophilic/hydrophobic interface of the membrane at 25°C, but was, in contrast to the other sterols, without effect at 0°C. Our results thus confirm as well as contradict the results of previous studies of the interactions between saturated PC and sterols, where other membrane regions were probed. The physiological relevance of the findings is discussed.     

Development and validation of a liquid chromatography-tandem mass spectrometric method for the determination of alpha-methyltyrosine in human plasma

A sensitive and selective LC-MS-MS method for the isolation and quantification of alpha-methyltyrosine (AMT) from human plasma is described. The method employs a simple protein precipitation using zinc sulfate and sodium hydroxide. This precipitation procedure produced samples with high aqueous content that could be directly injected into a LC-MS-MS system without compromising reverse-phase chromatographic performance. Chromatographic separation was performed on a MetaChem MonoChrom C(18) column (2.0 mm x 50 mm; 5 microm) at a flow rate of 1 mL/min. Compounds were eluted using a gradient mixture of water-acetic acid (100:0.1, v/v) and acetonitrile-acetic acid (100:0.1, v/v). The structural analog alpha-hydroxymethyltyrosine was used as the internal standard. Mass spectrometric detection was carried out with a triple quadrupole mass spectrometer. The method was validated and used to determine human plasma AMT concentrations, and has been implemented to derive pharmacokinetic parameters.     

A Phylogenomic Approach Based on PCR Target Enrichment and High Throughput Sequencing: Resolving the Diversity within the South American Species of Bartsia L. (Orobanchaceae)

Advances in high-throughput sequencing (HTS) have allowed researchers to obtain large amounts of biological sequence information at speeds and costs unimaginable only a decade ago. Phylogenetics, and the study of evolution in general, is quickly migrating towards using HTS to generate larger and more complex molecular datasets. In this paper, we present a method that utilizes microfluidic PCR and HTS to generate large amounts of sequence data suitable for phylogenetic analyses. The approach uses the Fluidigm Access Array System (Fluidigm, San Francisco, CA, USA) and two sets of PCR primers to simultaneously amplify 48 target regions across 48 samples, incorporating sample-specific barcodes and HTS adapters (2,304 unique amplicons per Access Array). The final product is a pooled set of amplicons ready to be sequenced, and thus, there is no need to construct separate, costly genomic libraries for each sample. Further, we present a bioinformatics pipeline to process the raw HTS reads to either generate consensus sequences (with or without ambiguities) for every locus in every sample or—more importantly—recover the separate alleles from heterozygous target regions in each sample. This is important because it adds allelic information that is well suited for coalescent-based phylogenetic analyses that are becoming very common in conservation and evolutionary biology. To test our approach and bioinformatics pipeline, we sequenced 576 samples across 96 target regions belonging to the South American clade of the genus Bartsia L. in the plant family Orobanchaceae. After sequencing cleanup and alignment, the experiment resulted in ~25,300bp across 486 samples for a set of 48 primer pairs targeting the plastome, and ~13,500bp for 363 samples for a set of primers targeting regions in the nuclear genome. Finally, we constructed a combined concatenated matrix from all 96 primer combinations, resulting in a combined aligned length of ~40,500bp for 349 samples.