Impact of HLA-B alleles, epitope binding affinity, functional avidity, and viral coinfection on the immunodominance of virus-specific CTL responses

Immunodominance is variably used to describe either the most frequently detectable response among tested individuals or the strongest response within a single individual, yet factors determining either inter- or intraindividual immunodominance are still poorly understood. More than 90 individuals were tested against 184 HIV- and 92 EBV-derived, previously defined CTL epitopes. The data show that HLA-B-restricted epitopes were significantly more frequently recognized than HLA-A- or HLA-C-restricted epitopes. HLA-B-restricted epitopes also induced responses of higher magnitude than did either HLA-A- or HLA-C-restricted epitopes, although this comparison only reached statistical significance for EBV epitopes. For both viruses, the magnitude and frequency of recognition were correlated with each other, but not with the epitope binding affinity to the restricting HLA allele. The presence or absence of HIV coinfection did not impact EBV epitope immunodominance patterns significantly. Peptide titration studies showed that the magnitude of responses was associated with high functional avidity, requiring low concentration of cognate peptide to respond in in vitro assays. The data support the important role of HLA-B alleles in antiviral immunity and afford a better understanding of the factors contributing to inter- and intraindividual immunodominance.     

Phylogenetic analyses of morphological evolution in the gametophyte and sporophyte generations of the moss order Hookeriales (Bryopsida)

Morphological characters from the gametophyte and sporophyte generations have been used in land plants to infer relationships and construct classifications, but sporophytes provide the vast majority of data for the systematics of vascular plants. In bryophytes both generations are well developed and characters from both are commonly used to classify these organisms. However, because morphological traits of gametophytes and sporophytes can have different genetic bases and experience different selective pressures, taxonomic emphasis on one generation or the other may yield incongruent classifications. The moss order Hookeriales has a controversial taxonomic history because previous classifications have focused almost exclusively on either gametophytes or sporophytes. The Hookeriales provide a model for comparing morphological evolution in gametophytes and sporophytes, and its impact on alternative classification systems. In this study we reconstruct relationships among mosses that are or have been included in the Hookeriales based on sequences from five gene regions, and reconstruct morphological evolution of six sporophyte and gametophyte traits that have been used to differentiate families and genera. We found that the Hookeriales, as currently circumscribed, are monophyletic and that both sporophyte and gametophyte characters are labile. We documented parallel changes and reversals in traits from both generations. This study addresses the general issue of morphological reversals to ancestral states, and resolves novel relationships in the Hookeriales.     

Nuclear magnetic resonance imaging of mummified corpses

Clinical applications of nuclear magnetic resonance (NMR) imaging have demonstrated its great potential for noninvasive examinations of soft tissues. Here preliminary experiments are reported on mummified human tissue by means of NMR cross-sectional and transmission imaging. For this purpose the natural viscosity of the tissues has been lowered by rehydration with an aqueous solution of 20% acetone.     

Effects of glyphosate residues and different concentrate feed proportions on performance,energy metabolism and health characteristics in lactating dairy cows

The aim of this study was to examine the influence of glyphosate (GL) residues in feedstuffs on performance, energy balance and health-related characteristics of lactating dairy cows fed diets with different concentrate feed proportions. After an adaption period, 64 German Holstein cows (207 ± 49 d in milk; mean ± SD) were assigned to either groups receiving a GL contaminated total mixed ration (TMR) (GL groups) or an uncontaminated TMR (CON groups) during a 16 weeks trial. Contaminated feedstuffs used were legally GL-treated peas and wheat (straw and grain). GL and CON groups were subdivided into a “low concentrate” group (LC) fed on dry matter (DM) basis of 21% maize silage, 42% grass silage, 7% straw and 30% concentrate and a “high concentrate” group (HC) composed of 11% maize silage, 22% grass silage, 7% straw and 60% concentrate for ad libitum consumption. Body condition score, body weight, DM intake and milk performance parameters were recorded. In blood serum, β-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA) and glucose were measured and energy balance was calculated. Milk was analysed for GL residues. At week 0, 7 and 15, general health status was evaluated by a modified clinical score. The average individual GL intake amounted for Groups CON_LC, CON_HC, GL_LC and GL_HC to 0.8, 0.8, 73.8 and 84.5 mg/d, respectively. No GL residues were detected in milk. GL contamination did not affect body condition score, body weight, DM intake, nutrient digestibility, net energy intake, net energy balance or BHB, glucose, NEFA and milk performance parameters; whereas concentrate feed proportion and time did affect most parameters. The clinical examination showed no adverse effect of GL-contaminated feedstuffs on cows’ health condition. In the present study, GL-contaminated feedstuffs showed no influence on performance and energy balance of lactating dairy cows, irrespective of feed concentrate proportion.     

HLA-B57/B*5801 Human Immunodeficiency Virus Type 1 Elite Controllers Select for Rare Gag Variants Associated with Reduced Viral Replication Capacity and Strong Cytotoxic T-Lymphotye Recognition

Human immunodeficiency virus type 1 (HIV-1) elite controllers (EC) maintain viremia below the limit of commercial assay detection (<50 RNA copies/ml) in the absence of antiviral therapy, but the mechanisms of control remain unclear. HLA-B57 and the closely related allele B*5801 are particularly associated with enhanced control and recognize the same Gag_240-249 TW10 epitope. The typical escape mutation (T242N) within this epitope diminishes viral replication capacity in chronically infected persons; however, little is known about TW10 epitope sequences in residual replicating viruses in B57/B*5801 EC and the extent to which mutations within this epitope may influence steady-state viremia. Here we analyzed TW10 in a total of 50 B57/B*5801-positive subjects (23 EC and 27 viremic subjects). Autologous plasma viral sequences from both EC and viremic subjects frequently harbored the typical cytotoxic T-lymphocyte (CTL)-selected mutation T242N (15/23 sequences [65.2%] versus 23/27 sequences [85.1%], respectively; P = 0.18). However, other unique mutants were identified in HIV controllers, both within and flanking TW10, that were associated with an even greater reduction in viral replication capacity in vitro. In addition, strong CTL responses to many of these unique TW10 variants were detected by gamma interferon-specific enzyme-linked immunospot assay. These data suggest a dual mechanism for durable control of HIV replication, consisting of viral fitness loss resulting from CTL escape mutations together with strong CD8 T-cell immune responses to the arising variant epitopes.A subset of human immunodeficiency virus type 1 (HIV-1)-infected persons who control viremia to below the limit of detection (<50 RNA copies/ml plasma) without antiviral therapy has been termed elite controllers/suppressors (EC) (2, 3, 6, 13, 32). Some of these individuals have been infected in excess of 30 years, indicating prolonged containment of HIV replication, but the mechanisms associated with this extreme viremia control remain elusive (13). Among EC, certain HLA class I alleles are overrepresented, in particular HLA-B57, strongly suggesting that HIV-1-specific cytotoxic T-lymphocyte (CTL) responses restricted by these alleles may be crucial for viremia control (16, 29, 32). However, to date, there has been no clear explanation as to why some subjects can control viremia but others cannot, even when carrying the same allegedly protective HLA alleles. Moreover, the characteristics of virus-specific immune responses as well as the impact of viral escape mutations on in vitro replicative fitness in persons with different disease outcomes remain unclear.Growing numbers of studies suggest that CTL targeting Gag, particularly the p24 capsid protein, play an important role in controlling viremia (7, 15, 22, 26, 32, 33, 38). Indeed, the most protective HLA class I allele, B57, which is present in over 40% of EC (32), restricts four immunodominant CTL epitopes in the p24 capsid protein. Previous studies have failed to find differences in the recognition of Gag epitopes or in gamma interferon (IFN-γ) responses to HIV proteins between B57-positive (B57⁺) long-term nonprogressors and B57⁺ progressors (28). Other studies have shown differences in the frequency of polyfunctional CD8⁺ T cells between B57⁺ EC and B57⁺ progressors (5); likewise, differences in the frequency of IFN-γ/interleukin-2-producing CD8⁺ T cells between controllers and progressors with protective HLA alleles were reported (16). Recently, Bailey et al. reported that plasma viruses in B57⁺ EC can harbor CTL escape mutations in the Gag protein, and in some cases these autologous variants were recognized by CTL (3). However, since there were no comparisons to progressors, it is unclear whether the viral variants that were detected or the apparent de novo CTL responses to the variant viruses are characteristic features among B57⁺ persons who maintain persistent control.Of the four immunodominant Gag CTL epitopes restricted by HLA-B57, TW10 (TSTLQEQIGW [Gag residues 240 to 249]) is known to be the earliest target in acute infection (1, 11, 36), therefore likely playing an important role in defining the plasma viral load set point. This epitope is also known to be presented by the closely related B*5801 allele, which is also associated with viral control (21). One of the most frequently detected mutations within this epitope, T242N, is known to occur rapidly and almost universally after acute infection in persons expressing HLA-B57/B*5801 (11, 17, 23). The same mutation has been shown to have a negative impact on viral replication capacity (VRC) by both clinical observation and in vitro experiments (8, 23, 25). Moreover, as plasma viral load increases, compensatory mutations accumulate, restoring VRC to some extent (8). Additional studies, predominantly with children, indicated that some TW10 escape variants may be targeted by specific immune responses (17). Together, these data suggest a hypothesis to explain the diverse disease courses among B57⁺ subjects, namely, that a combination of fitness cost by CTL escape from the TW10 response, variable accumulation of compensatory mutations, and variable generation of specific CTL responses to the new variant influence plasma viral loads.In this study, we investigated plasma viral sequences and IFN-γ-specific enzyme-linked immunospot (ELISPOT) assay responses to autologous Gag TW10 sequences in HLA-B57/B*5801-positive EC and compared these data to those obtained from persons with detectable viremia. Our results indicate that the TW10 T242N mutation does not differentiate HLA-B57/B*5801 EC from those with viremia and that CTL responses to this variant epitope are frequently detected in both viremic and aviremic subjects. However, some rare variants within and flanking this epitope were observed exclusively in HIV controllers, most of which not only reduced VRC but also were recognized by specific CTL at a high magnitude. These data suggest that the additive effects of both CTL-mediated selection for less fit viral variants and CD8 T-cell responses to the variant viruses contribute to strict viremia control in HLA-B57/B*5801-positive controllers.     

Comparison of linkage disequilibrium and haplotype diversity on macro- and microchromosomes in chicken

Background

Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.

Results

The study presents association between TLR gene mutations and increased susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further in silico analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.

Conclusion

The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^th amino acid position in LRR motif (TLR1–LRR10) and 4^th residue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.     

Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.