Major histocompatibility complex (MHC) markers in conservation biology

Human impacts through habitat destruction, introduction of invasive species and climate change are increasing the number of species threatened with extinction. Decreases in population size simultaneously lead to reductions in genetic diversity, ultimately reducing the ability of populations to adapt to a changing environment. In this way, loss of genetic polymorphism is linked with extinction risk. Recent advances in sequencing technologies mean that obtaining measures of genetic diversity at functionally important genes is within reach for conservation programs. A key region of the genome that should be targeted for population genetic studies is the Major Histocompatibility Complex (MHC). MHC genes, found in all jawed vertebrates, are the most polymorphic genes in vertebrate genomes. They play key roles in immune function via immune-recognition and -surveillance and host-parasite interaction. Therefore, measuring levels of polymorphism at these genes can provide indirect measures of the immunological fitness of populations. The MHC has also been linked with mate-choice and pregnancy outcomes and has application for improving mating success in captive breeding programs. The recent discovery that genetic diversity at MHC genes may protect against the spread of contagious cancers provides an added impetus for managing and protecting MHC diversity in wild populations. Here we review the field and focus on the successful applications of MHC-typing for conservation management. We emphasize the importance of using MHC markers when planning and executing wildlife rescue and conservation programs but stress that this should not be done to the detriment of genome-wide diversity.

Security protocols for EPC class-1 Gen-2 RFID multi-tag systems

The objective of the research is to develop security protocols for EPC C1G2 RFID Passive Tags in the areas of ownership transfer and grouping proof.

Expression of the nonclassical HLA-G and HLA-E molecules in laryngeal lesions as biomarkers of tumor invasiveness

Introduction: HLA-G and HLA-E are two nonclassical class I molecules, which have been well recognized as modulators of innate and adaptive immune responses, and the expression of these molecules in virus infected cells has been associated with subversion of the immune response. Objective: In this study we performed a cross-sectional study, systematically comparing the expression of HLA-G and HLA-E in benign, premalignant and malignant laryngeal lesions, correlating with demographic and clinical variables and with the presence of high-risk and low-risk HPV types. Materials and methods: Laryngeal lesions were collected from 109 patients and stratified into 27 laryngeal papillomas, 17 dysplasias, 10 in situ laryngeal carcinomas, 27 laryngeal carcinomas without metastases, 28 laryngeal carcinomas with metastasis along with their respective draining cervical lymph nodes, and 10 normal larynx specimens. The expression of HLA-G and HLA-E molecules was determined by immunohistochemistry. HPV DNA detection and typing was performed using generic and specific primers. Results: HLA nonclassical molecules showed a distinct distribution pattern, according to the larynx lesion grade. HLA-G expression increased in benign and premalignant lesions, and gradually decreased in invasive carcinomas and in respective draining cervical lymph nodes. Conversely, HLA-E expression increased as far as lesion grade increased, including increased molecule expression in the draining lymph nodes of malignant lesions. Only 17 (15.6%) patients were HPV DNA positive. Conclusions: Overexpression of HLA-E and underexpression of HLAG appear to be good markers for malignant larynx lesion.

Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detection of bla(CTX-M)-type genes in complex class 1 integrons carried by Enterobacteriaceae isolated from retail chicken meat in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Disruption of the CD4–major histocompatibility complex class II interaction blocks the development of CD4+ T cells in vivo

The experiments presented in this report were designed to specifically examine the role of CD4–major histocompatibility complex (MHC) class II interactions during T cell development in vivo. We have generated transgenic mice expressing class II molecules that cannot interact with CD4 but that are otherwise competent to present peptides to the T cell receptor. MHC class II expression was reconstituted in Aβ gene knock-out mice by injection of a transgenic construct encoding either the wild-type I-Aβb protein or a construct encoding a mutation designed to specifically disrupt binding to the CD4 molecule. We demonstrate that the mutation, EA137 and VA142 in the β2 domain of I-Ab, is sufficient to disrupt CD4–MHC class II interactions in vivo. Furthermore, we show that this interaction is critical for the efficient selection of a complete repertoire of mature CD4+ T helper cells as evidenced by drastically reduced numbers of conventional CD4+ T cells in animals expressing the EA137/VA142 mutant I-Ab and by the failure to positively select the transgenic AND T cell receptor on the mutated I-Ab. These results underscore the importance of the CD4–class II interaction in the development of mature peripheral CD4+ T cells.

Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope

Vaccinia virus (VV) produces two antigenically and structurally distinct infectious virions, intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Here we have investigated the resistance of EEV and IMV to neutralization by complement in the absence of immune antibodies. When EEV is challenged with complement from the same species as the cells used to grow the virus, EEV is resistant to neutralization by complement, whereas IMV is not. EEV resistance was not a result of EEV protein B5R, despite its similarity to proteins of the regulators of complement activation (RCA) family, or to any of the other EEV proteins tested (A34R, A36R, and A56R gene products). EEV was sensitive to complement when the virus was grown in one species and challenged with complement from a different species, suggesting that complement resistance might be mediated by host RCA incorporated into the EEV outer envelope. This hypothesis was confirmed by several observations: (i) immunoblot analysis revealed that cellular membrane proteins CD46, CD55, CD59, CD71, CD81, and major histocompatibility complex class I antigen were detected in purified EEV but not IMV; (ii) immunoelectron microscopy revealed cellular RCA on the surface of EEV retained on the cell surface; and (iii) EEV derived from rat cells expressing the human RCA CD55 or CD55 and CD59 were more resistant to human complement than EEV derived from control rat cells that expressed neither CD55 nor CD59. These data justify further analysis of the roles of these (and possible other) cellular proteins in EEV biology.

Molecular basis for the recognition of two structurally different major histocompatibility complex/peptide complexes by a single T-cell receptor

2C is a typical alloreactive cytotoxic T lymphocyte clone that recognizes two different ligands. These ligands are adducts of the allo-major histocompatibility complex (MHC) molecule H-2Ld and an endogenous octapeptide, and of the self-MHC molecule H-2Kb and another peptide. MHC-binding and T-cell assays with synthetic peptides in combination with molecular modeling studies were employed to analyze the structural basis for this crossreactivity. The molecular surfaces of the two complexes differ greatly in densities and distributions of positive and negative charges. However, modifications of the peptides that increase similarity decrease the capacities of the resulting MHC peptide complexes to induce T-cell responses. Moreover, the roles of the peptides in ligand recognition are different for self- and allo-MHC-restricted T-cell responses. The self-MHC-restricted T-cell responses were finely tuned to recognition of the peptide. The allo-MHC-restricted responses, on the other hand, largely ignore modifications of the peptide. The results strongly suggest that adaptation of the T-cell receptor to the different ligand structures, rather than molecular mimicry by the ligands, is the basis for the crossreactivity of 2C. This conclusion has important implications for T-cell immunology and for the understanding of immunological disorders.

Interaction between HLA-DM and HLA-DR involves regions that undergo conformational changes at lysosomal pH

Antigenic peptide loading of major histocompatibility complex class II molecules is enhanced by lysosomal pH and catalyzed by the HLA-DM molecule. The physical mechanism behind the catalytic activity of DM was investigated by using time-resolved fluorescence anisotropy (TRFA) and fluorescence binding studies with the dye 8-anilino-1-naphthalenesulfonic acid (ANS). We demonstrate that the conformations of both HLA-DM and HLA-DR3, irrespective of the composition of bound peptide, are pH sensitive. Both complexes reversibly expose more nonpolar regions upon protonation. Interaction of DM with DR shields these hydrophobic domains from the aqueous environment, leading to stabilization of the DM and DR conformations. At lysosomal pH, the uncovering of additional hydrophobic patches leads to a more extensive DM–DR association. We propose that DM catalyzes class II peptide loading by stabilizing the low-pH conformation of DR, favoring peptide exchange. The DM–DR association involves a larger hydrophobic surface area with DR/class II-associated invariant chain peptides (CLIP) than with stable DR/peptide complexes, explaining the preferred association of DM with the former. The data support a release mechanism of DM from the DM–DR complex through reduction of the interactive surface, upon binding of class II molecules with antigenic peptide or upon neutralization of the DM–DR complex at the cell surface.

CD1c molecules broadly survey the endocytic system

The ability of antigen-presenting cells to sample distinct intracellular compartments is crucial for microbe detection. Major histocompatibility complex class I and class II molecules sample the cytosol or the late endocytic compartment, allowing detection of microbial peptide antigens that arise in distinct intracellular compartments. In contrast, CD1a and CD1b molecules mediate the presentation of lipid and glycolipid antigens and differentially sample early recycling endosomes or late endocytic compartments, respectively, that contain distinct sets of lipid antigens. Here, we show that, unlike the other CD1 isoforms or major histocompatibility complex molecules that each sample restricted only intracellular compartments, CD1c is remarkable in that it distributes broadly throughout the endocytic system and is expressed in both recycling endosomes and late endocytic compartments. Further, in contrast to CD1b, which requires an acidic environment to function, antigen presentation by CD1c was able to overcome dependence on vesicular acidification. Because CD1c is expressed on essential antigen-presenting cells, such as epidermal Langerhans cells (in the absence of CD1b), or on B cells (without CD1a or -b), we suggest that CD1c molecules allow a comprehensive survey for lipid antigens throughout the endocytic system even in the absence of other CD1 isoforms.

Murine Nkg2d and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells

Natural killer (NK) cells express C-type lectin-like receptors, encoded in the NK gene complex, that interact with major histocompatibility complex class I and either inhibit or activate functional activity. Human NK cells express heterodimers consisting of CD94 and NKG2 family molecules, whereas murine NK cells express homodimers belonging to the Ly-49 family. The corresponding orthologues for other species, however, have not been described. In this report, we used probes derived from the expressed sequence tag database to clone C57BL/6-derived cDNAs homologous to human NKG2-D and CD94. Among normal tissues, murine NKG2-D and CD94 transcripts are highly expressed only in activated NK cells, including both Ly-49A+ and Ly-49A− subpopulations. Additionally, mNKG2-D is expressed in murine NK cell clones KY-1 and KY-2, whereas mCD94 expression is observed only in KY-1 cells but not KY-2. Last, we have finely mapped the physical location of the Cd94 (centromeric) and Nkg2d (telomeric) genes between Cd69 and the Ly49 cluster in the NK complex. Thus, these data indicate the expanding complexity of the NK complex and the corresponding repertoire of C-type lectin-like receptors on murine NK cells.

Altered thymic positive selection and intracellular signals in Cbl-deficient mice

Cbl is the product of the protooncogene c-cbl and is involved in T cell antigen receptor (TCR)-mediated signaling. To understand the role of Cbl for immune system development and function, we generated a Cbl-deficient mouse strain. In Cbl-deficient mice, positive selection of the thymocytes expressing major histocompatibility complex class II-restricted transgenic TCR was significantly enhanced. Two factors may have contributed to the altered thymic selection. First, Cbl deficiency markedly up-regulated the activity of ZAP-70 and mitogen-activated protein kinases. The mitogen-activated protein kinase pathway was shown previously to be involved in thymic positive selection. Second, Cbl-deficient thymocytes expressed CD3 and CD4 molecules at higher levels, which consequently may increase the avidity of TCR/major histocompatibility complex/coreceptor interaction. Thus, Cbl plays a novel role in modulating TCR-mediated multiple signaling pathways and fine-tunes the signaling threshold for thymic selection.

Negative Factor from SIV Binds to the Catalytic Subunit of the V-ATPase to Internalize CD4 and to Increase Viral Infectivity

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239 for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity.