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.     

The Soluble Periplasmic Domains of Escherichia coli Cell Division Proteins FtsQ/FtsB/FtsL Form a Trimeric Complex with Submicromolar Affinity

Cell division in Escherichia coli involves a set of essential proteins that assembles at midcell to form the so-called divisome. The divisome regulates the invagination of the inner membrane, cell wall synthesis, and inward growth of the outer membrane. One of the divisome proteins, FtsQ, plays a central but enigmatic role in cell division. This protein associates with FtsB and FtsL, which, like FtsQ, are bitopic inner membrane proteins with a large periplasmic domain (denoted FtsQ_p, FtsB_p, and FtsL_p) that is indispensable for the function of each protein. Considering the vital nature and accessible location of the FtsQBL complex, it is an attractive target for protein-protein interaction inhibitors intended to block bacterial cell division. In this study, we expressed FtsQ_p, FtsB_p, and FtsL_p individually and in combination. Upon co-expression, FtsQ_p was co-purified with FtsB_p and FtsL_p from E. coli extracts as a stable trimeric complex. FtsB_p was also shown to interact with FtsQ_p in the absence of FtsL_p albeit with lower affinity. Interactions were mapped at the C terminus of the respective domains by site-specific cross-linking. The binding affinity and 1:1:1 stoichiometry of the FtsQ_pB_pL_p complex and the FtsQ_pB_p subcomplex were determined in complementary surface plasmon resonance, analytical ultracentrifugation, and native mass spectrometry experiments.     

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).     

Herbicide effects on planktonic systems of different complexity

Bioassays of different complexity were compared with respect to their capability to predict the environmental impact of the herbicide atrazine in aquatic systems. Acute toxicity tests with Daphnia did not yield meaningful results. Sublethal tests with Daphnia (feeding inhibition, reduction of growth and reproduction) were more sensitive, but effective concentrations of atrazine were still rather high (2 mg/L). A relatively complicated artificial food chain system that incorporated direct and indirect effects on Daphnia yielded significant reduction of daphnid population growth at 0.1 mg/L. Enclosure experiments with natural communities were by far the most sensitive tools. Community responses could be measured at concentrations as low as 1 µg/L and 0.1 µg atrazine/L. At the lowest concentration, however, communities recovered after three weeks. We conclude that in complex systems indirect effects can be more important than direct effects, so that, contrary to the conditions in simple tests, non-target organisms may be the better indicators of herbicide stress to natural communities.     

Isolation and characterization of rtcs, a maize mutant deficient in the formation of nodal roots

The root system of maize consists of the primary root and a variable number of lateral seminal-, crown- and brace roots. Except for the primary root and some minor roots forming at the mesocotyl, all other roots grow out of nodal regions, namely, the embryogenic scutellar node and the underground—as well as the lower above-ground stem nodes. Besides their role in water and nutrient uptake, some of these roots (crown- and brace roots) are essential for the lodging resistance of the plants. This property of the crown roots has now been successfully used for screening a segregating F₂ population of a cross between a flint inbred line and an En transposon line. Two allelic root-deficient mutants have been isolated and have been designated rtcs-1 and rtcs-2 for their complete lack of formation of c rown- and lateral s eminal roots. They survive by the ability of the primary root to support the growth of the developing plant. The monogenic and recessive mutants appear to be affected in an early root-forming function since no primordia are formed either in the case of embryo-borne lateral seminal or stem-derived crown roots. The Rtcs locus could be mapped to the short arm of chromosome 1 with the help of a co-segregating RAPD marker. The effect of the mutation seems to be highly specific since no pleiotropic effects on other parts of the plants have been observed. The formation of adventitious roots can, however, still be induced in the mesocotyl region of the mutant.     

The EndoPredict Gene-Expression Assay in Clinical Practice - Performance and Impact on Clinical Decisions

The validated EndoPredict assay is a novel tool to predict the risk of metastases of patients with estrogen receptor positive, HER2 negative breast cancer treated with endocrine therapy alone. It has been designed to integrate genomic and clinical information and includes clinico-pathological factors such as tumor size and nodal status. The test is feasible in a decentral setting in molecular pathology laboratories. In this project, we investigated the performance of this test in clinical practice, and performed a retrospective evaluation of its impact on treatment decisions in breast cancer. During one year, EndoPredict assays from 167 patients could be successfully performed. For retrospective evaluation of treatment decisions, a questionnaire was sent to the clinical partner. Regarding the molecular EP class, samples from 56 patients (33.5%) had a low-risk, whereas 111 patients (66.5%) showed a high-risk gene profile. After integration of the clinicopathological factors the combined clinical and molecular score (EPclin) resulted in a low-risk group of 77 patients (46.4%), while 89 (53.6%) had a high risk EPclin score. The EPclin-based estimated median 10-year-risk for metastases with endocrine therapy alone was 11% for the whole cohort. The median handling time averaged three days (range: 0 to 11 days), 59.3% of the tests could be performed in three or less than three days. Comparison of pre- and post-test therapy decisions showed a change of therapy in 37.7% of patients. 16 patients (12.3%) had a change to an additional chemotherapy while 25.4% of patients (n = 33) changed to an endocrine therapy alone. In 73 patients (56.2%) no change of therapy resulted. In 6.1% of patients (n = 8), the patients did not agree to the recommendation of the tumor board. Our results show that the EndoPredict assay could be routinely performed in decentral molecular pathology laboratories and the results markedly change treatment decisions.     

Balanced Hydroxyethylstarch (HES 130/0.4) Impairs Kidney Function In-Vivo without Inflammation

Volume therapy is a standard procedure in daily perioperative care, and there is an ongoing discussion about the benefits of colloid resuscitation with hydroxyethylstarch (HES). In sepsis HES should be avoided due to a higher risk for acute kidney injury (AKI). Results of the usage of HES in patients without sepsis are controversial. Therefore we conducted an animal study to evaluate the impact of 6% HES 130/0.4 on kidney integrity with sepsis or under healthy conditions Sepsis was induced by standardized Colon Ascendens Stent Peritonitis (sCASP). sCASP-group as well as control group (C) remained untreated for 24 h. After 18 h sCASP+HES group (sCASP+VOL) and control+HES (C+VOL) received 50 ml/KG balanced 6% HES (VOL) 130/0.4 over 6h. After 24h kidney function was measured via Inulin- and PAH-Clearance in re-anesthetized rats, and serum urea, creatinine (crea), cystatin C and Neutrophil gelatinase-associated lipocalin (NGAL) as well as histopathology were analysed. In vitro human proximal tubule cells (PTC) were cultured +/- lipopolysaccharid (LPS) and with 0.1–4.0% VOL. Cell viability was measured with XTT-, cell toxicity with LDH-test. sCASP induced severe septic AKI demonstrated divergent results regarding renal function by clearance or creatinine measure focusing on VOL. Soleley HES (C+VOL) deteriorated renal function without sCASP. Histopathology revealed significantly derangements in all HES groups compared to control. In vitro LPS did not worsen the HES induced reduction of cell viability in PTC cells. For the first time, we demonstrated, that application of 50 ml/KG 6% HES 130/0.4 over 6 hours induced AKI without inflammation in vivo. Severity of sCASP induced septic AKI might be no longer susceptible to the way of volume expansion.     

A complex composed of at least two HeLa nuclear proteins protects preferentially one DNA strand of the simple (gt)n(ga)m containing region of intron 2 in HLA-DRB-genes

Electrophoretic mobility shift assays reveal that HeLa neuclear proteins bind fast and with measurable affinity to target DNAs containing mixed simple repetitive (gt)_n(ga)_m stretches. Preincubation of the proteins at elevated temperature prevents the formation of the major DNA/protein complex in favour of several distinct assemblies. A similar pattern of retarded bands was observed employing higher salt concentrations in binding reaction. Thus conformational changes of different proteins appear to influence the complex rather than alternating DNA structures. Separation of the total nuclear extract into a water soluble and an insoluble protein fraction leads to a complete loss of target DNA bindinlg capability of the fractions. The binding capacity is restored by combining the two fractions suggesting that at least two protein components are necessary to form a complex with the target sequence. The proteins can be differentiated into head sensitive, water soluble and temporary stable, water insoluble, respectively. Furthermore, specifically binding polypeptides are not detectable by Southwestern analyses, probably because the essential components are separated during electrophoresis. DNase 1 footpoint analyses yield four different protein binding regions only on the (gt)_n(ga)_m harbouring strand. The footprints cover larger portions of the mixed simple repeat in addition to a portion 5′ of the (gt)_n part. Hence at lealst two nuclear protein components of unknown biological function have to be present simultaneously to protect preferentially the (gt)_n(ga)_m-containing strand intron 2 in HLA-DRB genes     

The mglB sequence of Salmonella typhimurium LT2; promoter analysis by gene fusions and evidence for a divergently oriented gene coding for the mgl repressor

Summary The mglB gene of Salmonella typhimurium LT2 coding for the galactose-binding protein (GBP) was sequenced. We compared the deduced amino acid sequence with the GBP sequence of Escherichia coli K12. The mature proteins differ in only 19 of 309 amino acid residues, corresponding to 94% homology. Analysis of the mglB control region by promoter-probe vectors revealed that two promoters, P1 and P2, constitute the mgl control region (P _mgl ). P1 and P2 function in a synergistic way. P1 is the main promoter of the operon; its activity is 20 times the activity of P2. Both promoters are activated by the cyclic adenosine monophosphate catabolite activator protein (cAMP/CAP) complex. While P1 is inactive in the absence of the cAMP/CAP complex, there is residual activity of P2 under these conditions. Studies on the inducibility of the mglBAEC operon using multicopy plasmid promoter-probe vectors were hampered by the titration of the mgl repressor resulting in a partially constitutive expression of the mgl operon. The results indicate that only P1 is responding to induction by D-fucose. A weak promoter, P _D , within the P1 region but divergent to it was found. P _D is neither stimulated by the cAMP/CAP complex nor by D-fucose. We cloned the gene located downstream to P _D and found it to strongly repress the expression of the mgl operon. We termed this gene mglD. The presence of D-fucose abolished the repression caused by the plasmid-encoded mglD gene product.Abbreviations IPTG isopropyl-1-thic--D-galatopyranoside - ONPG 2-nitrophenyl--D-galatopyranoside - XG 5-bromo-4-chloro-3-indolyl--D-galatopyranoside - Kan^r Kanamycin resistance     

Oscillations in epidemic models with spread of awareness

Journal of Mathematical Biology - We study ODE models of epidemic spreading with a preventive behavioral response that is triggered by awareness of the infection. Previous studies of such models...