Toxoplasma gondii and MHC-restricted antigen presentation: on degradation, transport and modulation

Resistance against Toxoplasma gondii, an obligate intracellular protozoan parasite surrounded by a parasitophorous vacuolar membrane, is mediated by the cellular arm of the immune system, namely CD8+ and CD4+ T cells. Thus, priming and activation of these cells by presentation of antigenic peptides in the context of major histocompatibility complex class I and class II molecules have to take place. This is despite the fact that the vacuolar membrane avoids fusion with the endocytic compartment and acts like a molecular sieve, restricting passive diffusion of larger molecules. This raises several cell biological and immunological questions which will be discussed in this review in the context of our current knowledge about major histocompatibility complex-restricted antigen presentation in other systems: (1) By which pathways are parasite-derived antigens presented to T cells? (2) Has the parasite evolved mechanisms to interfere with major histocompatibility complex-restricted antigen presentation in order to avoid immune recognition? (3) To what extent and by which mechanism is antigenic material, originating from the parasite, able to pass through the vacuolar membrane into the cytosol of the infected cell and is it then accessible to the antigen presentation machinery of the infected cell? (4) What are the actual antigen-presenting cells which prime specific T cells in lymphoid organs? An understanding of these mechanisms will not only provide new insights into the pathogenesis of Toxoplasma gondii and possibly other intravacuolar parasites, but will also improve vaccination strategies.     

Memory: enduring traces of perceptual and reflective attention

Attention and memory are typically studied as separate topics, but they are highly intertwined. Here we discuss the relation between memory and two fundamental types of attention: perceptual and reflective. Memory is the persisting consequence of cognitive activities initiated by and/or focused on external information from the environment (perceptual attention) and initiated by and/or focused on internal mental representations (reflective attention). We consider three key questions for advancing a cognitive neuroscience of attention and memory: to what extent do perception and reflection share representational areas? To what extent are the control processes that select, maintain, and manipulate perceptual and reflective information subserved by common areas and networks? During perception and reflection, to what extent are common areas responsible for binding features together to create complex, episodic memories and for reviving them later? Considering similarities and differences in perceptual and reflective attention helps integrate a broad range of findings and raises important unresolved issues.     

What Are Imprinted Genes Doing in the Brain?

As evidence for the existence of brain?expressed imprinted genes accumulates, we need to address exactly what they are doing in this tissue, especially in terms of organizational themes and the major challenges posed by reconciling imprinted gene action in brain with current evolutionary theories attempting to explain the origin and maintenance of genomic imprinting. We are at the beginning of this endeavor and much work remains to be done but already it is clear that imprinted genes have the potential to influence diverse behavioral processes via multiple brain mechanisms. There are also grounds to believe that imprinting may contribute to risk of mental and neurological disease. As well as being a source of basic information about imprinted genes in the brain (e.g., via the newly established website, www.bgg.cardiff.ac.uk/imprinted_tables/index.html), we have used this chapter to identify and focus on a number of key questions. How are brain?expressed imprinted genes organized at the molecular and cellular levels? To what extent does imprinted action depend on neurodevelopmental mechanisms? Do imprinted gene effects interact with other epigenetic influences, especially early on in life? Are imprinted effects on adult behaviors adaptive or just epiphenomena? If they are adaptive, what areas of brain function and behavior might be sensitive to imprinted effects? These are big questions and, as shall become apparent, we need much more data, arising from interactions between behavioral neuroscientists, molecular biologists and evolutionary theorists, if we are to begin to answer them.     

A serologic comparison of Moloney lymphoma cell surface and Moloney oncornavirus antigens.

Moloney lymphomas and Moloney sarcomas share strong tumor antigens. In this report we analyze the cell-surface antigens on a Balb/c Moloney lymphoma, LSTRA, using hyperimmune sarcoma regressor sera (alphaMo) as a primary reagent. We also use heterologous anti-viral p30 and gp70 sera for a direct analysis of virion protein antigens on the LSTRA surface. Using radiolabeled alphaMo-binding assays, we demonstrate that LSTRA tumor antigens detected by these sera are all Moloney viral antigens; approximately 1/3 of these antigenic determinants are expressed on the intact virus, and the other determinants are revealed by detergent lysis of the virus. The major viral antigens expressed on the LSTRA cell surface are viral env gene products, whereas gag gene products are only sparsely represented. We conclude that alphaMo sera detect almost exclusively viral antigens on LSTRA cells, and these antigens are almost exclusively virion env gene products.     

Dissociation of influenza virus hemagglutinin and neuraminidase eliminates their intravirionic antigenic competition.

When presented together on the intact influenza virus particle, the external hemagglutinin (HA) and neuraminidase (NA) antigens are competitive, with HA dominant over NA in both T- and B-cell priming (B. E. Johansson, T. M. Moran, and E. D. Kilbourne, Proc. Natl. Acad. Sci. USA 84:6869-6873, 1987). Dissociation and purification of HA and NA from virus and their injection separately or in combination into BALB/c mice eliminates their antigenic competition as measured by antibody response, confirming that it is their structural association that leads to what we have termed intravirionic antigenic competition. We discuss this phenomenon with respect to previously described intermolecular antigenic competition and with regard to its probable mechanism. Our findings are relevant to contemporary interest in viral vaccine vectors and multicomponent vaccines.     

The Evolutionary Dynamics of Human Influenza B Virus

Despite their close phylogenetic relationship, type A and B influenza viruses exhibit major epidemiological differences in humans, with the latter both less common and less often associated with severe disease. However, it is unclear what processes determine the evolutionary dynamics of influenza B virus, and how influenza viruses A and B interact at the evolutionary scale. To address these questions we inferred the phylogenetic history of human influenza B virus using complete genome sequences for which the date (day) of isolation was available. By comparing the phylogenetic patterns of all eight viral segments we determined the occurrence of segment reassortment over a 30-year sampling period. An analysis of rates of nucleotide substitution and selection pressures revealed sporadic occurrences of adaptive evolution, most notably in the viral hemagglutinin and compatible with the action of antigenic drift, yet lower rates of overall and nonsynonymous nucleotide substitution compared to influenza A virus. Overall, these results led us to propose a model in which evolutionary changes within and between the antigenically distinct 'Yam88' and 'Vic87' lineages of influenza B virus are the result of changes in herd immunity, with reassortment continuously generating novel genetic variation. Additionally, we suggest that the interaction with influenza A virus may be central in shaping the evolutionary dynamics of influenza B virus, facilitating the shift of dominance between the Vic87 and the Yam88 lineages.     

Epistasis between mutations is host-dependent for an RNA virus

How, and to what extent, does the environment influence the way mutations interact? Do environmental changes affect both the sign and the magnitude of epistasis? Are there any correlations between environments in the variability, sign or magnitude of epistasis? Very few studies have tackled these questions. Here, we addressed them in the context of viral emergence. Most emerging viruses are RNA viruses with small genomes, overlapping reading frames and multifunctional proteins for which epistasis is abundant. Understanding the effect of host species in the sign and magnitude of epistasis will provide insights into the evolutionary ecology of infectious diseases and the predictability of viral emergence.     

The enigma of dendritic cell-immunodeficiency virus interplay

A dendritic cell (DC) encountering an immunodeficiency virus should pose a threat to the virus, by efficiently processing and presenting viral antigenic determinants to activate specific anti-viral T and B cell immunity. While this may occur in vivo, it is apparent that DC-entrapped viruses can freely spread between cells, move to distal tissues, and proliferate rapidly particularly upon meeting CD4+ T cells. In fact, the latter is further augmented when the T cells are activated. Thus, it seems that immunodeficiency viruses exploit the unique ability of DCs to survey the periphery and capture incoming pathogens, traffic around the body often targeting the lymphoid tissues, and efficiently communicate with na?ve and memory T cells. Combined with the fact that DCs are likely the first leukocytes interacting with virions crossing the mucosae, these features provide the basis on which the virus maximizes its chance to establish infection even in the face of immune activation. How this is actually achieved by the virus is still an enigma. Herein, we intend to summarize what is known about how distinct DC subsets and immunodeficiency viruses interact, what cellular and viral factors influence these events, and how this drives virus replication versus stimulation of protective immunity. Clarifying these issues is necessary to define the exact role of DCs in the transmission and dissemination of HIV infection, to facilitate the development of methods to improve the immune-activating capacity of DCs as well as the design of strategies to prevent DC-driven infection.     

Molecular determinants of major histocompatibility complex class I complex stability: shaping antigenic features through short and long range electrostatic interactions

A single amino acid exchange between the major histocompatibility complex molecules HLA-B(*)2705 and HLA-B(*)2709 (Asp-116/His) is responsible for the emergence of distinct HLA-B27-restricted T cell repertoires in individuals harboring either of these two subtypes and could correlate with their differential association with the autoimmune disease ankylosing spondylitis. By using fluorescence depolarization and pK(a) calculations, we investigated to what extent electrostatic interactions contribute to shape antigenic differences between these HLA molecules complexed with viral, self, and non-natural peptide ligands. In addition to the established main anchor of peptides binding to HLA-B27, arginine at position 2 (pArg-2), and the secondary anchors at the peptide termini, at least two further determinants contribute to stable peptide accommodation. 1) The interaction of Asp-116 with arginine at peptide position 5, as found in pLMP2 (RRRWRRLTV; viral) and pVIPR (RRKWRRWHL; self), and with lysine in pOmega, as found in gag (KRWIILGLNK; viral), additionally stabilizes the B(*)2705 complexes by approximately 5 and approximately 27 kJ/mol, respectively, in comparison with B(*)2709. 2) The protonation state of the key residues Glu-45 and Glu-63 in the B-pocket, which accommodates pArg-2, affects peptide binding strength in a peptide- and subtype-dependent manner. In B(*)2705/pLMP2, protonation of Glu-45/Glu-63 reduces the interaction energy of pArg-2 by approximately 24 kJ/mol as compared with B(*)2705/pVIPR. B(*)2705/pVIPR is stabilized by a deprotonated Glu-45/Glu-63 pair, evoked by allosteric interactions with pHis-8. The mutual electrostatic interactions of peptide and HLA molecule, including peptide- and subtype-dependent protonation of key residues, modulate complex stability and antigenic features of the respective HLA-B27 subtype.     

Monoclonal antibodies against Aleutian disease virus distinguish virus strains and differentiate sites of virus replication from sites of viral antigen sequestration.

Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low-molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens.     

Antigen specificity of semi-invariant CD1d-restricted T cell receptors: the best of both worlds?

T lymphocytes are characterized by the use of structurally diverse TCR. The discovery of subsets of canonical T cells that have structurally homogeneous TCR presents an enigma: What antigens do these T cells recognize, and how does their antigen specificity relate to their functions? One subset of canonical T cells is restricted by CD1d, a non-classical antigen presenting molecule that presents lipids and glycolipids. Canonical CD1d-restricted T cells have semi-invariant TCR consisting of an invariantly rearranged TCR alpha chain, paired with diversely rearranged TCR beta chains. Most respond strongly to the unusual glycolipid alpha-galactosylceramide (alpha-GalCer), and can also respond to cellular antigens presented by CD1d. Mounting evidence indicates that alpha-GalCer responsive T cells are heterogeneous in their reactivities to cellular antigens, suggesting that an individual semi-invariant TCR may be capable of recognizing more than one ligand. Recent crystal structures of CD1b molecules with three different bound lipids indicate that the antigenic features of lipids may be localized over a smaller area than those of peptides, and that the positioning of the polar head group can vary substantially. A model that explains how CD1d-restricted T cells could possess both conserved and heterogeneous antigen specificities, is that different lipid antigens may interact with distinct areas of a TCR due to differences in the positioning of the polar head group. Hence, canonical CD1d-restricted TCR could recognize conserved antigens via the invariant TCR alpha chain, and have diverse antigen specificities that are conferred by their individual TCR beta chains.     

Assessing the Importance of Intraspecific Variability in Dung Beetle Functional Traits

Functional diversity indices are used to facilitate a mechanistic understanding of many theoretical and applied questions in current ecological research. The use of mean trait values in functional indices assumes that traits are robust, in that greater variability exists between than within species. While the assertion of robust traits has been explored in plants, there exists little information on the source and extent of variability in the functional traits of higher trophic level organisms. Here we investigated variability in two functionally relevant dung beetle traits, measured from individuals collected from three primary forest sites containing distinct beetle communities: body mass and back leg length. In doing so we too addressed the following questions: (i) what is the contribution of intra vs. interspecific differences in trait values; (ii) what sample size is needed to provide representative species mean trait values; and (iii) what impact does omission of intraspecific trait information have on the calculation of functional diversity (FD) indices from naturally assembled communities? At the population level, interspecific differences explained the majority of variability in measured traits (between 94% and 96%). In accordance with this, the error associated with calculating FD without inclusion of intraspecific variability was low, less than 20% in all cases. This suggests that complete sampling to capture intraspecific variance in traits is not necessary even when investigating the FD of small and/or naturally formed communities. To gain an accurate estimation of species mean trait values we encourage the measurement of 30–60 individuals and, where possible, these should be taken from specimens collected from the site of study.     

Gammadelta T cells: functional plasticity and heterogeneity

Gammadelta T cells remain an enigma. They are capable of generating more unique antigen receptors than alphabeta T cells and B cells combined, yet their repertoire of antigen receptors is dominated by specific subsets that recognize a limited number of antigens. A variety of sometimes conflicting effector functions have been ascribed to them, yet their biological function(s) remains unclear. On the basis of studies of gammadelta T cells in infectious and autoimmune diseases, we argue that gammadelta T cells perform different functions according to their tissue distribution, antigen-receptor structure and local microenvironment; we also discuss how and at what stage of the immune response they become activated.     

Identification of major histocompatibility complex class II-restricted antigens and epitopes of the Epstein-Barr virus by a novel bacterial expression cloning approach

Epstein-Barr virus (EBV)-specific T cells have been successfully used to treat or prevent EBV-positive lymphoproliferative disease in hematopoietic stem cell transplant recipients, but the antigens recognized by the infused CD4+ T cells have remained unknown. Here, we describe a simple procedure that permits the identification of viral T-helper (TH)-cell antigens and epitopes. This direct antigen identification method is based on the random expression of viral polypeptides fused to chloramphenicol acetyltransferase (CAT) in bacteria, which are subsequently fed to major histocompatibility complex class II+ antigen-presenting cells and probed with antigen-specific T cells. The fusion of antigenic fragments to CAT offers several advantages. First, chloramphenicol treatment allows the selection of bacteria expressing antigen-CAT fusion proteins in frame, which greatly reduces the number of colonies to be screened. Second, antigenic fragments fused to CAT are expressed at high levels, even when derived from proteins that are toxic to bacteria. Third, the uniformly high expression level of antigen-CAT fusion proteins permits the establishment of large and representative pool sizes. Finally, antigen identification does not require knowledge of the restriction element and often leads directly to the identification of the T-cell epitope. Using this approach, the BALF4 and BNRF1 proteins were identified as targets of the EBV-specific T-helper-cell response, demonstrating that lytic cycle antigens are a relevant component of the EBV-specific TH-cell response.     

Retrovirus-induced spongiform neurodegeneration is mediated by unique central nervous system viral targeting and expression of env alone

Certain murine leukemia viruses (MLVs) can induce progressive noninflammatory spongiform neurodegeneration similar to that caused by prions. The primary MLV determinants responsible have been mapped to within the env gene; however, it has remained unclear how env mediates disease, whether non-Env viral components are required, and what central nervous system (CNS) cells constitute the critical CNS targets. To address these questions, we examined the effect of transplanting engraftable C17.2 neural stem cells engineered to pseudotype, disseminate, and trans-complement neurovirulent (CasBrE, CasE, and CasES) or non-neurovirulent (Friend and SFF-FE) env sequences (SU or SU/TM) within the CNS using either the "non-neurovirulent" amphotropic helper virus, 4070A, or pgag-polgpt (a nonpackaged vector encoding Gag-Pol). These studies revealed that acute MLV-induced spongiosis results from two separable activities of Env. First, Env causes neuropathology through unique viral targeting within the CNS, which was efficiently mediated by ecotropic Envs (CasBrE and Friend), but not 4070A amphotropic Env. Second, Env induces spongiosis through a toxin activity that is MLV-receptor independent and does not require the coexpression of other viral structural proteins. CasBrE and 4070A Envs possess the toxin activity, whereas Friend Env does not. Although the identity of the critical viral target cell(s) remains unresolved, our results appear to exclude microglia and oligodendrocyte lineage cells, while implicating viral entry into susceptible neurons. Thus, MLV-induced disease parallels prionopathies in that a single protein, Env, mediates both the CNS targeting and the toxicity of the infectious agent that manifests itself as progressive vacuolar neurodegeneration.     

Cell death: questions for histochemists concerning the causes of the various cytological changes

Summary The question of cell death is accessible to study by histochemists and many questions remain to be resolved. From a physiological point of view, the most important are the causal relationships. (1) At what phase in cell death is the synthesis of RNA disrupted and at what phase is the rate of degradation of RNA increased? (2) Does the disruption of synthesis result from a direct genetic command, or does it result indirectly from gradual deterioration of energy resources or optimal ionic conditions? (3) What properties, presumably of the substrate organelles, marks them for specific absorption into autophagic vacuoles? (4) What proteases and other hydrolases operate currently undetected in the cytoplasm? How are they controlled and regulated? (5) Why does the physiologically dying cell shrink and appear more dense? To what extent is a cell in this state able to regulate any metabolic parameter? The advent of newer, more sensitive and quantitative techniques, and greater attention to the controls and causes as opposed to the phenomena, should help to resolve these questions.     

A new analysis of allogeneic interactions.

Allogeneic reactions have conventionally been considered as typical immune responses by one population of cells to antigens present on the other. This view is inadequate, since it does not explain many features of these reactions, among which are: (1) reactivity is much higher between different strains within a species than between species, in spite of the much greater antigenic disparity in the second case; (2) a very high proportion of cells may respond to allogeneic stimuli; (3) major histocompatibility differences are not essential for vigorous allogeneic reactions; (4) the responding population need not be immunologically competent to respond to antigens of the stimulating population; (5) the stimulating population must be both metabolically active and immunocompetent. We have tried to produce a model of cell interaction which will account for these and other anomalies, which at the same time explaining both normal antigenic stimulation (through cell-cell cooperation) and allogeneic interactions as examples of the same basic mechanisms. The model is based on the Bretscher-Cohn scheme of cell interaction. An allogeneic reaction is seen as having two stages: (1) Cells come together when antibody receptors on cells of one population combine with antigens on cells of the other. To this extent, our model is the same as the conventional one. It need not be the responding population which has the receptors, however. (2) A species-specific proliferation signal passes between the cells. This is the same signal as is involved in normal antibody induction. Even antigen-receptor bonds which are very weak may result in effective stimulation of one or both partners because of enhancing effect of this signal, and because the antigens involved are probably repeated over the cell surface, enabling multipoint binding. This explains the very proportions of cells which proliferate. The exact outcome of any allogeneic interaction will depend on which of the two populations have antibody receptors for antigens on the other, which can produce the proliferative stimulus, and which can respond to either the proliferative signal alone or to this stimulus plus antigen.     

Immunosenescence, suppression and tumour progression

There are good arguments for suggesting that two seminal papers published 50 years ago can be taken as the beginning of modern tumour immunology. These papers by R. Baldwin, “Immunity to transplanted tumour: the effect of tumour extracts on the growth of homologous tumours in rats” and “Immunity to methylcholanthrene-induced tumours in inbred rats following atrophy and regression of the implanted tumours” (Br J Cancer 9:646–51 and 652–657, 1955) showed that once tumours are established, they and their products can be recognised by the adaptive immune system and rejected. However, the tumour normally co-evolves with immunity, like a parasite, rather than being suddenly introduced as in these, and many other, experimental models. Dynamics of this co-evolution are illustrated by findings that inflammation enhances tumorigenicity, yet is important to enable T cells to respond properly to tumour antigen and exert anti-tumour effects. The important thing is to maintain the balance between effective anti-tumour immunity and tumour escape and/or stimulatory mechanisms. Tumours almost always co-exist with immune defence systems over extended periods and interact chronically with T cells. The effect of this is potentially similar to other situations of chronic antigenic stress, particularly lifelong persistent virus infection, most strikingly, CMV infection. The questions briefly explored in this symposium paper are what happens when T lymphocyte clones are chronically stimulated by antigen which is not or cannot be eliminated? What are the similarities and differences between chronic antigenic stimulation by tumour antigen versus CMV antigen? What can we learn in one system which may illuminate the other?     

Changes in Health between Ages 54 and 65: The Role of Job Characteristics and Socioeconomic Status

We model the relationships between socioeconomic status (SES), the conditions of paid employment, and changes between ages 54 and 65 in a variety of health outcomes: self-reported overall health, musculoskeletal health, and depression. To what extent is SES associated with changes in these health outcomes net of the conditions of paid employment? At the same time, to what extent are the conditions of paid employment independently associated with these outcomes net of SES? To address these questions we use unique data collected from a single cohort of men and women to model changes in these health outcomes between ages 54 and 65. Although results vary across outcomes, it is clear that there are some circumstances in which associations between SES and changes in health can be (at least partly) attributed to working conditions, and that there are other circumstances in which associations between working conditions and changes in health can be (at least partly) attributed to SES. We conclude that the largely disconnected literatures on health disparities (in the social sciences and public health) and job design (in occupational stress and ergonomics) could and should be fruitfully connected.