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.     

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.     

Transport Rankings of Non-Steroidal Antiinflammatory Drugs across Blood-Brain Barrier In Vitro Models

The aim of this work was to conduct a comprehensive study about the transport properties of NSAIDs across the blood-brain barrier (BBB) in vitro. Transport studies with celecoxib, diclofenac, ibuprofen, meloxicam, piroxicam and tenoxicam were accomplished across Transwell models based on cell line PBMEC/C1-2, ECV304 or primary rat brain endothelial cells. Single as well as group substance studies were carried out. In group studies substance group compositions, transport medium and serum content were varied, transport inhibitors verapamil and probenecid were added. Resulted permeability coefficients were compared and normalized to internal standards diazepam and carboxyfluorescein. Transport rankings of NSAIDs across each model were obtained. Single substance studies showed similar rankings as corresponding group studies across PBMEC/C1-2 or ECV304 cell layers. Serum content, glioma conditioned medium and inhibitors probenecid and verapamil influenced resulted permeability significantly. Basic differences of transport properties of the investigated NSAIDs were similar comparing all three in vitro BBB models. Different substance combinations in the group studies and addition of probenecid and verapamil suggested that transporter proteins are involved in the transport of every tested NSAID. Results especially underlined the importance of same experimental conditions (transport medium, serum content, species origin, cell line) for proper data comparison.     

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.     

Investigation of ubiquinol formation in isolated photosynthetic reaction centers by rapid-scan Fourier transform IR spectroscopy

Light-induced formation of ubiquinol-10 in Rhodobacter sphaeroides reaction centers was followed by rapid-scan Fourier transform IR difference spectroscopy, a technique that allows the course of the reaction to be monitored, providing simultaneously information on the redox states of cofactors and on protein response. The spectrum recorded between 4 and 29 ms after the second flash showed bands at 1,470 and 1,707 cm(-1), possibly due to a QH(-) intermediate state. Spectra recorded at longer delay times showed a different shape, with bands at 1,388 (+) and 1,433 (+) cm(-1) characteristic of ubiquinol. These spectra reflect the location of the ubiquinol molecule outside the Q(B) binding site. This was confirmed by Fourier transform IR difference spectra recorded during and after continuous illumination in the presence of an excess of exogenous ubiquinone molecules, which revealed the process of ubiquinol formation, of ubiquinone/ubiquinol exchange at the Q(B) site and between detergent micelles, and of Q(B)(-) and QH(2) reoxidation by external redox mediators. Kinetics analysis of the IR bands allowed us to estimate the ubiquinone/ubiquinol exchange rate between detergent micelles to approximately 1 s. The reoxidation rate of Q(B)(-) by external donors was found to be much lower than that of QH(2), most probably reflecting a stabilizing/protecting effect of the protein for the semiquinone form. A transient band at 1,707 cm(-1) observed in the first scan (4-29 ms) after both the first and the second flash possibly reflects transient protonation of the side chain of a carboxylic amino acid involved in proton transfer from the cytoplasm towards the Q(B) site.     

Long term effects of manure, charcoal and mineral fertilization on crop production and fertility on a highly weathered Central Amazonian upland soil

Application of organic fertilizers and charcoal increase nutrient stocks in the rooting zone of crops, reduce nutrient leaching and thus improve crop production on acid and highly weathered tropical soils. In a field trial near Manaus (Brazil) 15 different amendment combinations based on equal amounts of carbon (C) applied through chicken manure (CM), compost, charcoal, and forest litter were tested during four cropping cycles with rice (Oryza sativa L.) and sorghum (Sorghum bicolor L.) in five replicates. CM amendments resulted in the highest (P < 0.05) cumulative crop yield (12.4 Mg ha⁻¹) over four seasons. Most importantly, surface soil pH, phosphorus (P), calcium (Ca), and magnesium (Mg) were significantly enhanced by CM. A single compost application produced fourfold more grain yield (P < 0.05) than plots mineral fertilized in split applications. Charcoal significantly improved plant growth and doubled grain production if fertilized with NPK in comparison to the NPK-fertilizer without charcoal (P < 0.05). The higher yields caused a significantly greater nutrient export in charcoal-amended fields, but available nutrients did not decrease to the same extent as on just mineral fertilized plots. Exchangeable soil aluminum (Al) was further reduced if mineral fertilizer was applied with charcoal (from 4.7 to 0 mg kg⁻¹). The resilience of soil organic matter (SOM) in charcoal amended plots (8 and 4% soil C loss, mineral fertilized or not fertilized, respectively) indicates the refractory nature of charcoal in comparison to SOM losses over 20 months in CM (27%), compost amended (27%), and control plots (25% loss).     

Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.     

Identification of tissue-specific microRNAs from mouse

MicroRNAs (miRNAs) are a new class of noncoding RNAs, which are encoded as short inverted repeats in the genomes of invertebrates and vertebrates. It is believed that miRNAs are modulators of target mRNA translation and stability, although most target mRNAs remain to be identified. Here we describe the identification of 34 novel miRNAs by tissue-specific cloning of approximately 21-nucleotide RNAs from mouse. Almost all identified miRNAs are conserved in the human genome and are also frequently found in nonmammalian vertebrate genomes, such as pufferfish. In heart, liver, or brain, it is found that a single, tissue-specifically expressed miRNA dominates the population of expressed miRNAs and suggests a role for these miRNAs in tissue specification or cell lineage decisions. Finally, a miRNA was identified that appears to be the fruitfly and mammalian ortholog of C. elegans lin-4 stRNA.