Multivariate meta-analysis of the association of G-protein beta 3 gene (GNB3) haplotypes with cardiovascular phenotypes

The objective of the present study was to review previous investigations on the association of haplotypes in the G-protein β3 subunit (GNB3) gene with representative cardiovascular risk factors/phenotypes: hypertension, overweight, and variation in the systolic and diastolic blood pressures (SBP and DBP, respectively) and as well as body mass index (BMI). A comprehensive literature search was undertaken in Pubmed, Web of Science, EMBASE, Biological Abstracts, LILACS and Google Scholar to identify potentially relevant articles published up to April 2011. Six genetic association studies encompassing 16,068 participants were identified. Individual participant data were obtained for all studies. The three most investigated GNB3 polymorphisms (G-350A, C825T and C1429T) were considered. Expectation–maximization and generalized linear models were employed to estimate haplotypic effects from data with uncertain phase while adjusting for covariates. Study-specific results were combined through a random-effects multivariate meta-analysis. After carefully adjustments for relevant confounding factors, our analysis failed to support a role for GNB3 haplotypes in any of the investigated phenotypes. Sensitivity analyses excluding studies violating Hardy–Weinberg expectations, considering gender-specific effects or more extreme phenotypes (e.g. obesity only) as well as a fixed-effects “pooled” analysis also did not disclose a significant influence of GNB3 haplotypes on cardiovascular phenotypes. We conclude that the previous cumulative evidence does not support the proposal that haplotypes formed by common GNB3 polymorphisms might contribute either to the development of hypertension and obesity, or to the variation in the SBP, DBP and BMI.     

Mucosal layers and related nerve fibres in non-chagasic and chagasic human colon—a quantitative immunohistochemical study

Chagasic megacolon is accompanied by extensive myenteric and, simultaneously, moderate submucosal neuron loss. Here, we examined changes of the innervation pattern of the lamina propria (LP) and muscularis mucosae (MM). Two alternating sets of cryosections were taken from seven non-chagasic colonic and seven chagasic megacolonic specimens (the latter included both the dilated megacolonic and the non-dilated transitional oral and anal zones) and were immunohistochemically triple-stained for smooth-muscle actin (SMA), synaptophysin (SYN) and glial acid protein S100 and, alternatively, for SMA, vasoactive intestinal peptide (VIP) and somatostatin (SOM). Subsequent image analysis and statistical evaluation of nervous tissue profile areas revealed that, in LP, the most extreme differences (i.e. increase in thickness or decrease in nerve, glia and muscle tissue profile area, respectively) compared with control values occurred in the dilated megacolonic zone itself. In contrast, the most extreme differences in the MM were in the anal-to-megacolonic zone (except the profile area of muscle tissue, which was lowest in the megacolonic zone). This parallels our previous results in the external muscle coat. A partial and selective survival of VIP-immunoreactive in contrast to SOM-immunoreactive nerve fibres was observed in both mucosal layers investigated. Thus, VIPergic nerve elements might be crucial for the maintenance of the mucosal barrier. The differential changes of neural tissue parameters in LP and MM might reflect a multifactorial rather than a pure neurogenic development of megacolon in chronic Chagas’ disease.     

IL-15 augments antitumoral activity of an ErbB2/HER2 cancer vaccine targeted to professional antigen-presenting cells

Targeted delivery of tumor-associated antigens to professional antigen-presenting cells (APC) is being explored as a strategy to enhance the antitumoral activity of cancer vaccines. Here, we generated a cell-based system for continuous in vivo production of a CTLA-4-ErbB2 fusion protein as a therapeutic vaccine. The chimeric CTLA-4-ErbB2 molecule contains the extracellular domain of CTLA-4 for specific targeting to costimulatory B7 molecules on the surface of APC, genetically fused to residues 1-222 of human ErbB2 (HER2) as an antigenic determinant. In wild-type BALB/c mice, inoculation of syngeneic epithelial cells continuously secreting the CTLA-4-ErbB2 fusion vaccine in the vicinity of subcutaneously growing ErbB2-expressing renal cell carcinomas resulted in the rejection of established tumors, accompanied by the induction of ErbB2-specific antibodies and cytotoxic T cells. In contrast, treatment with CTLA-4-ErbB2 vaccine-secreting producer cells alone was insufficient to induce tumor rejection in ErbB2-transgenic WAP-Her-2 F1 mice, which are characterized by pronounced immunological tolerance to the human self-antigen. When CTLA-4-ErbB2 producer cells were modified to additionally secrete interleukin (IL)-15, antigen-specific antitumoral activity of the vaccine in WAP-Her-2 F1 mice was restored, documented by an increase in survival, and marked inhibition of the growth of established ErbB2-expressing, but not antigen-negative tumors. Our results demonstrate that continuous in vivo expression of an APC-targeted ErbB2 fusion protein results in antigen-specific immune responses and antitumoral activity in tumor-bearing hosts, which is augmented by the pleiotropic cytokine IL-15. This provides a rationale for further development of this approach for specific cancer immunotherapy.     

Functional binding of hexanucleotides to 3C protease of hepatitis A virus

Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C^pro). Inhibition assays in vitro identified the hexanucleotide 5′-GGGGGT-3′ (G₅T) as a 3C^pro protease inhibitor. Using ¹H NMR spectroscopy, G₅T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of ¹H, ¹⁵N-HSQC experiments the binding site for G₅T was located to the C-terminal β-barrel of HAV 3C^pro. Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G₅T-binding site, nor does G₅T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid–protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.     

Pain-induced skin autoimmunity

A recent paper published in Nature reports sensory nerve fibers in the skin that give local immune cells important instructions for the organization of an immune response; in this particular case the cooperation between the nervous and immune systems had disastrous consequences, namely an auto-destruction of the skin.The relationship between the immune and nervous systems has been traditionally viewed as neutral at best: the systems “ignore” each other. This is highlighted by the anatomical structure of the blood-brain barrier, a border between the bloodstream and nervous tissue that nearly completely inhibits the entry of immune cells and soluble factors from the periphery into the central nervous system (CNS). When this border is breached, for example after trauma, stroke or (autoimmune) inflammation, the influx of immune cells can have dramatic consequences for the integrity of the CNS. However, there is also another, “social” side to the relationship between the immune and nervous systems. Immune cells in the area surrounding the CNS (the meninges and the perivascular space) effectively shield the nervous tissue from pathogenic intruders, helping the nervous system to function undisturbed. Activated T cells can even overcome the blood-brain barrier and patrol the nervous tissue for damage without causing perceptible problems. Immune cells also play an active role in neuronal function. For example, microglial cells, recognized as brain-resident immune cells, contribute to synaptic pruning and maturation by phagocytizing discarded synapses. A disturbance in the microglia function leads to severe neurological deficits¹.Conversely, the nervous system exerts a regulatory influence on the immune system. For example, the activation of the hypothalamic-pituitary-adrenal axis induces the release of glucocorticoids, which can strongly modulate immune reactivity². In addition, the CNS can directly act on the immune system through its widely distributed nerve fiber network: in the inflammatory reflex, a prototypical neural-immune reflex, vegetative nerves innervating the visceral organs upon activation secrete neurotransmitters that act directly on T cells and macrophages. A hyper-activation of these neuronal inputs, e.g. occuring after stroke, can bring drastic down-modulations in the immune function with life-threatening infectious consequences³. Interestingly, not only vegetative nerve fibers but also unmyelinated temperature and pain fibers participate in the cross-talk between CNS and immune system. Upon closer scrutiny, a synergy between innervatory pain fibers and immune cells is plausible: 1) These fibers are present in all types of tissue communicating with the outside; 2) Pain stimuli often occur together with tissue damage that requires an immune response; 3) Pain fibers express danger and damage receptors (e.g., TLRs and purine receptors) and thereby can directly react to immune-relevant stimuli; 4) Although pain fibers are among the slowest conducting fibers, electrical conduction is still much faster than immune cell mobilization; and 5) Pain fibers can conduct signals not only orthodromically (from periphery to CNS), but also antidromically (from CNS to periphery), inducing the release of neural mediators that can act locally on immune cells.In their Nature paper, von Andrian and colleagues shed light on a specific facet of this synergy between the immune and nervous systems, namely how unmyelinated pain and temperature fibers in the skin affect the reaction of local dendritic cells and thereby contribute to a psoriasis-like autoimmune reaction⁴. Based on the clinical observation that psoriasis skin lesions are regularly accompanied by symptoms of irritation such as itchiness and pain and that the surgical or pharmacological impairment of peripheral nerve function in psoriatic lesions results in an amelioration of skin inflammation, the authors investigated how nerve fibers influence skin immune reactions in a mouse model of psoriasis induced by topical application of imiquimod (IMQ), an immune stimulating compound. This was achieved by an elegant complementary approach including pharmacologic, genetic and imaging tools. The authors showed that a specific subset of sensory fibers co-expressing the cation channel TRPV1 and the sodium channel Na_v1.8 and functionally responsible for the sensation of heat and pain, initiated the cutaneous immune response by inducing IL23 release by dermal dendritic cells (DDCs). IL23 then acted directly on resident γ/δ T cells, the main skin cell population expressing IL23 receptor, to induce IL17 and IL22 secretion with subsequent recruitment of immune infiltrates and thus the initiation of a pathogenic inflammatory response (Figure 1). This chain of events was thoroughly tested. The essential role of the unmyelinated sensory fibers was demonstrated by pharmacological silencing of their neuronal function by application of resiniferatoxin, a kind of ultrapotent chili pepper. This chemical completely suppressed IMQ-induced effects, which was verified by genetic ablation of the sensory nerves. In contrast, a sympathetic fiber pharmacological blockage by the catecholaminergic neurotoxin 6 hydroxy-dopamine did not change the IMQ-mediated inflammation. Of note, when IL23 was injected topically after sensory denervation, the inflammatory response was restored, pointing to a crucial effect of sensory neurons on the DDCs. The interactions between DDCs and sensory nerve fibers were corroborated by eye-catching imaging data. Intravital two-photon microscopy of intact skin in transgenic mice in which both the cutaneous nerves and the dendritic cells could be visualized elucidated the strategic location of the DDCs: around 75% of DDCs established close interactions with the sensory fibers along the entire length of the nerves.Open in a separate windowFigure 1Schematic drawing of intact skin. Afferent nerve (red); efferent nerve (blue). Magnification of the chain of events that drives the formation of the psoriatic lesions upon activation of the TRPV1⁺ and Na_v1.8⁺ sensory neurons.By its very nature this impressive insight into such a complex immune-neuronal interaction raises numerous questions. For example, which signals regulate the communication between dendritic cells and nerve fibers? This also applies to the initial simulation of pain fibers, still completely unclear for human disease and also not fully understood for the IMQ model. IMQ exerts a stimulatory effect via the TLR signal cascade. Apart from DDCs, pain fibers also express TLRs. Are these fibers stimulated directly by IMQ or by factors from the DDCs or by other stromal or immune cells? Moreover, which neuronal signals stimulate the dendritic cells to release IL23? Do neuropeptides play a role that may be assumed from studies on “neurogenic inflammation”⁵? Neuropeptides like the CGRP, vasointestinal peptide or substance P have been associated with the regulation of skin Langerhans cells⁶ and experimental inflammation in joints⁷ and liver⁸. Although von Andrian and colleagues did not observe an effect of CGRP antagonists on skin inflammation, a role for CGRP cannot be completely ruled out, due to its complex modular structure and manifold biological effects. The observation that DDCs seem to be in direct physical contact with axons also suggests that electrical impulses or transmitter-type substances released at the contact points might influence DDC function. It is very likely that neuronal signals do not exclusively contribute to DDC activation but also control their “quiescence state”. In this respect, neurotrophins such as NGF have been shown to downregulate the immune reactivity of microglia within the CNS⁹. A forced neuronal inactivity causes a reduction in neurotrophin secretion and an upregulation of MHC molecules. Interestingly, in psoriasis NGF has been assigned an active role in the induction of skin lesions via stimulation of epidermis epithelial cells, T cells and pain fibers¹⁰. Ultimately this interesting work by von Andrian and colleagues will surely inspire researchers to examine the stimulation mode of sensory fibers more closely, such as to name the receptors involved and to find out the extent to which electrical stimulation of sensory fibers play a role and whether the catalyst for the release of stimulatory nerve factors is an axonal reflex or local stimulus.     

Liposome-based Systems for Anti-tumor Vaccination: Influence of Lipopeptide Adjuvants

We have developed liposome-based synthetic constructs incorporating peptide epitope(s) (ErbB2 p63-67 CTL which is overexpressed in many tumors and/or HA 307-319 T-helper) and lipopeptide adjuvants (Pam3CysSerSer, Pam3CysAlaGly) in order to elicit an anti-tumor immune response. The epitopes, derivatized with a linker containing a cysteine residue, were conjugated on preformed vesicles (dia. ～ 100 nm) containing lipopeptides functionalized with thiol reactive groups (maleimide or bromoacetyl). The therapeutic efficacy of these constructs was evaluated on a Balb/c mice tumor model inoculated with syngenic murine renal carcinoma (Renca) cells expressing human ErbB2 (Her2/neu) receptor. A successful therapeutic vaccination was obtained which was antigen specific. Furthermore, it appeared that the nature of the polar head group of the lipopeptide adjuvant and also its type of functionalization influence the efficacy of the construct. In our study, the best results were obtained with formulations containing a Pam₃CSS anchor in association with the CTL and Th epitopes. Considering these promising results studies are in progress with a new generation of liposomes that incorporate a neutral lipid – lacking adjuvant properties – that serves as anchor of the peptide epitopes and new adjuvants synthesized in our laboratory, which are screened for their antitumour activity in a therapeutic setting.     

Diagnosis, clinical features, and self-reported morbidity of Strongyloides stercoralis and hookworm infection in a Co-endemic setting

Background

Infections with Strongyloides stercoralis and other helminths represent important, yet often neglected issues in developing countries. Indeed, strongyloidiasis can be fatal, but only a few studies provide information regarding its health relevance in Africa. Moreover, clinical data on symptomatology and typical recognition patterns mainly originate from Western travel clinics.

Methodology

A cross-sectional epidemiological survey was carried out in a rural part of south-central Côte d''Ivoire. Stool samples from 292 randomly selected individuals were examined for intestinal helminths, using a suite of diagnostic techniques (i.e., Kato-Katz, Baermann funnel, and Koga agar plate). Participants were interviewed with a pre-tested questionnaire and clinically examined. Multivariate logistic regression analysis was done to relate perceived morbidity and clinical findings to helminth infection status.

Principal Findings

The prevalence of hookworm and S. stercoralis was 51.0% and 12.7%, respectively. Both infections were strongly associated with each other (adjusted odds ratio, 6.73; P<0.001) and higher prevalences were observed with age. S. stercoralis-infected individuals expressed self-reported morbidity considerably more often than those with hookworm infection. Clinical examination identified high prevalences of various pathologies and detected tendencies to worse health conditions in helminth-infected subjects.

Conclusions/Significance

The use of multiple diagnostic tools showed that S. stercoralis and hookworm are co-endemic in rural Côte d''Ivoire and that each infection causes clinical symptoms and sequelae. Our findings are important for (re-)estimating the burden of helminth infections, and highlight the need for integrating epidemiological surveys, rigorous diagnostic approaches, and clinical assessments in the developing world.     

Human TCR transgenic Bet v 1-specific Th1 cells suppress the effector function of Bet v 1-specific Th2 cells

Pollinosis to birch pollen is a common type I allergy in the Northern Hemisphere. Moreover, birch pollen-allergic individuals sensitized to the major birch pollen allergen Bet v 1 frequently develop allergic reactions to stone fruits, hazelnuts, and certain vegetables due to immunological cross-reactivity. The major T cell epitope Bet v 1(142-153) plays an important role in cross-reactivity between the respiratory allergen Bet v 1 and its homologous food allergens. In this study, we cloned and functionally analyzed a human αβ TCR specific for the immunodominant epitope Bet v 1(142-153). cDNAs encoding TCR α- and β-chains were amplified from a Bet v 1(142-153)-specific T cell clone, introduced into Jurkat T cells and peripheral blood T lymphocytes of allergic and nonallergic individuals, and evaluated functionally. The resulting TCR transgenic (TCRtg) T cells responded in an allergen-specific and costimulation-dependent manner to APCs either pulsed with Bet v 1(142-153) peptide or coexpressing invariant chain::Bet v 1(142-153) fusion proteins. TCRtg T cells responded to Bet v 1-related food and tree pollen allergens that were processed and presented by monocyte-derived dendritic cells. Bet v 1(142-153)-presenting but not Bet v 1(4-15)-presenting artificial APCs coexpressing membrane-bound IL-12 polarized allergen-specific TCRtg T cells toward a Th1 phenotype, producing high levels of IFN-γ. Coculture of such Th1-polarized T cells with allergen-specific Th2-differentiated T cells significantly suppressed Th2 effector cytokine production. These data suggest that human allergen-specific TCR can transfer the fine specificity of the original T cell clone to heterologous T cells, which in turn can be instructed to modulate the effector function of the disease initiating/perpetuating allergen-specific Th2-differentiated T cells.     

A functional-structural model of rice linking quantitative genetic information with morphological development and physiological processes

Background and Aims

Although quantitative trait loci (QTL) analysis of yield-related traits for rice has developed rapidly, crop models using genotype information have been proposed only relatively recently. As a first step towards a generic genotype–phenotype model, we present here a three-dimensional functional–structural plant model (FSPM) of rice, in which some model parameters are controlled by functions describing the effect of main-effect and epistatic QTLs.

Methods

The model simulates the growth and development of rice based on selected ecophysiological processes, such as photosynthesis (source process) and organ formation, growth and extension (sink processes). It was devised using GroIMP, an interactive modelling platform based on the Relational Growth Grammar formalism (RGG). RGG rules describe the course of organ initiation and extension resulting in final morphology. The link between the phenotype (as represented by the simulated rice plant) and the QTL genotype was implemented via a data interface between the rice FSPM and the QTLNetwork software, which computes predictions of QTLs from map data and measured trait data.

Key Results

Using plant height and grain yield, it is shown how QTL information for a given trait can be used in an FSPM, computing and visualizing the phenotypes of different lines of a mapping population. Furthermore, we demonstrate how modification of a particular trait feeds back on the entire plant phenotype via the physiological processes considered.

Conclusions

We linked a rice FSPM to a quantitative genetic model, thereby employing QTL information to refine model parameters and visualizing the dynamics of development of the entire phenotype as a result of ecophysiological processes, including the trait(s) for which genetic information is available. Possibilities for further extension of the model, for example for the purposes of ideotype breeding, are discussed.Key words: Functional–structural plant model, ecophysiology, QTL analysis, plant modelling, quantitative genetics