Discovery of natural bispecific antibodies: Is psoriasis induced by a toxigenic Corynebacterium simulans and maintained by CIDAMPs as autoantigens?

The high abundance of Corynebacterium simulans in psoriasis skin suggests a contribution to the psoriasis aetiology. This hypothesis was tested in an exploratory study, where western blot (WB) analyses with extracts of heat-treated C. simulans and psoriasis serum-derived IgG exhibited a single 16 kDa-WB-band. Proteomic analyses revealed ribosomal proteins as candidate C. s.-antigens. A peptidomic analysis unexpectedly showed that psoriasis serum-derived IgG already contained 31 immunopeptides of Corynebacteria ssp., suggesting the presence of natural bispecific antibodies (BsAbs). Moreover, peptidomic analyses gave 372 DECOY-peptides with similarity to virus- and phage proteins, including Corynebacterium diphtheriae phage, and similarity to diphtheria toxin. Strikingly, a peptidomic analysis for human peptides revealed 64 epitopes of major psoriasis autoantigens such as the spacer region of filaggrin, hornerin repeats and others. Most identified immunopeptides represent potential cationic intrinsically disordered antimicrobial peptides (CIDAMPs), which are generated within the epidermis. These may form complexes with bacterial disordered protein regions, representing chimeric antigens containing discontinuous epitopes. In addition, among 128 low-abundance immunopeptides, 48 are putatively psoriasis-relevant such as epitope peptides of PGE2-, vitamin D3- and IL-10-receptors. Further, 47 immunopeptides originated from tumour antigens, and the endogenous retrovirus HERV-K. I propose that persistent infection with a toxigenic C. simulans initiates psoriasis, which is exacerbated as an autoimmune disease by CIDAMPs as autoantigens. The discovery of natural BsAbs allows the identification of antigen epitopes from microbes, viruses, autoantigens and tumour-antigens, and may help to develop epitope-specific peptide-vaccines and therapeutic approaches with antigen-specific regulatory T cells to improve immune tolerance in an autoimmune disease-specific-manner.

Revealing the Achilles heel of bacterial toxins.

In addition to having antimicrobial properties, defensins inactivate various structurally unrelated bacterial toxins by a yet unknown manner. In this issue of Immunity, Kudryashova et al. (2014b) provide insights into mechanisms by which human ?-defensins destabilize and inactivate bacterial toxins.

Evolution of innate defense in human skin.

More often as compared to other barrier systems (gastrointestinal, urogenital, and respiratory linings) human skin over millions of years has been subject to fundamental changes in structure and function. When life on land started, the first changes consisted in the formation of a coherent impermeable stratum corneum. Two-legged locomotion was followed by loss of body hair and formation of sweat glands. Major changes took place after the agricultural revolution, investigating settlements with domestication of animals and plants. Living together after giving up nomadic life, hairless skin became a battlefield for pathogens, members of the skin microbiome, and arthropod visits. Human skin became exceptional in showing a boosted, highly developed immune system which is much more complex as compared to the "skins" of other species. A recently found skin disinfection system ("Cationic Intrinsically Disordered Antimicrobial Peptides, CIDAMPs") dates back to the origins of life and still is active in present-day integuments. As a skin-restricted and effective principle, keratinocyte- myeloid synergy (KMS) is recognized. As a consequence of such highly developed immune defense, the basic contributions of KMS - cells (keratinocytes, neutrophils, macrophages) in regulating innate immunity is emphasized. Antimicrobial peptides and chemokines became major keratinocyte products. The formation of impermeable str. corneum membrane has enabled KMS - cells to accumulate within upper skin levels and cause a special group of human skin diseases, pustular dermatoses.

Microbial and transcriptional differences elucidate atopic dermatitis heterogeneity across skin sites.

It is well established that different sites in healthy human skin are colonized by distinct microbial communities due to different physiological conditions. However, few studies have explored microbial heterogeneity between skin sites in diseased skin, such as atopic dermatitis (AD) lesions. To address this issue, we carried out deep analysis of the microbiome and transcriptome in the skin of a large cohort of AD patients and healthy volunteers, comparing two physiologically different sites: upper back and posterior thigh. Microbiome samples and biopsies were obtained from both lesional and nonlesional skin to identify changes related to the disease process. Transcriptome analysis revealed distinct disease-related gene expression profiles depending on anatomical location, with keratinization dominating the transcriptomic signatures in posterior thigh, and lipid metabolism in the upper back. Moreover, we show that relative abundance of Staphylococcus aureus is associated with disease severity in the posterior thigh, but not in the upper back. Our results suggest that AD may select for similar microbes in different anatomical locations-an "AD-like microbiome," but distinct microbial dynamics can still be observed when comparing posterior thigh to upper back. This study highlights the importance of considering the variability across skin sites when studying the development of skin inflammation.

Intubation, tracheostomy, and decannulation in patients with Guillain-Barré-syndrome-does dysphagia matter?

INTRODUCTION: Although patients with Guillain-Barré syndrome frequently require orotracheal intubation and tracheostomy, the incidence and relevance of neurogenic dysphagia prior to intubation and risk factors for prolonged requirement for a tracheal cannula have not yet been identified. METHODS: Retrospective analysis of the medical records of 88 patients was performed. Clinical characteristics were compared between intubated and nonintubated patients and between immediately decannulated and not immediately decannulated patients. RESULTS: Thirty-five (39.7%) patients required tracheostomy. Neuromuscular weakness and related respiratory insufficiency were the main reasons for intubation. In the subgroup of tracheotomized patients, immediate decannulation after completed respiratory weaning was possible in 14 (40%) patients. The severity of dysphagia, in particular pharyngolaryngeal hypesthesia, was related to the length of cannulation. DISCUSSION: Respiratory muscle weakness is the main reason for intubation, whereas neurogenic dysphagia is the main risk factor for persisting cannulation. Dysphagia after weaning is most frequently characterized by severe laryngeal sensory deficit. Muscle Nerve 59:194-200, 2019.

The Diversity of Plant Small RNAs Silencing Mechanisms.

Small RNAs gene regulation was first discovered about 20 years ago. It represents a conserve gene regulation mechanism across eukaryotes and is associated to key regulatory processes. In plants, small RNAs tightly regulate development, but also maintain genome stability and protect the plant against pathogens. Small RNA gene regulation in plants can be divided in two canonical pathways: Post-transcriptional Gene Silencing (PTGS) that results in transcript degradation and/or translational inhibition or Transcriptional Gene Silencing (TGS) that results in DNA methylation. In this review, we will focus on the model plant Arabidopsis thaliana. We will provide a brief overview of the molecular mechanisms involved in canonical small RNA pathways as well as introducing more atypical pathways recently discovered.

Introducing a Virtual Lesion Model of Dysphagia Resulting from Pharyngeal Sensory Impairment.

BACKGROUND/AIMS: Performing neurophysiological and functional imaging studies in severely affected patients to investigate novel neurostimulation techniques for the treatment of neurogenic dysphagia is difficult. Therefore, basic research needs to be conducted in healthy subjects. Swallowing is a motor function highly dependent on sensory afferent input. Here we propose a virtual peripheral sensory lesion model to mimic pharyngeal sensory impairment, which is known as a major contributor to dysphagia in neurological disease. METHODS: In this randomized crossover study on 11 healthy volunteers, cortical activation during pneumatic pharyngeal stimulation was measured applying magnetoencephalography in two separate sessions, with and without pharyngeal surface anesthesia. RESULTS: Stimulation evoked bilateral event-related desynchronization (ERD) mainly in the caudolateral pericentral cortex. In comparison to the no-anesthesia condition, topical anesthesia led to a reduction of ERD in beta (13-30 Hz) and low gamma (30-60 Hz) frequency ranges (p<0.05) in sensory but also motor cortical areas. CONCLUSIONS: Withdrawal of sensory afferent information by topical anesthesia leads to reduced response to pneumatic pharyngeal stimulation in a distributed cortical sensorimotor network in healthy subjects. The proposed paradigm may serve to investigate the effect of neuromodulatory treatments specifically on pharyngeal sensory impairment as relevant cause of neurogenic dysphagia.

The Effect of Improved Dysphagia Care on Outcome in Patients with Acute Stroke: Trends from 8-Year Data of a Large Stroke Register.

BACKGROUND: Early dysphagia screening and appropriate management are recommended by current guidelines to reduce complications and case fatality in acute stroke. However, data on the potential benefit of changes in dysphagia care on patient outcome are limited. Our objective was to assess the degree of implementation of dysphagia guidelines and determine the impact of modifications in dysphagia screening and treatment practices on disease complications and outcome in stroke patients over time. METHODS: In this prospective register-based study ("Stroke Register of Northwestern Germany"), all adult stroke patients admitted to 157 participating hospitals between January, 2008 and December, 2015 were included (n = 674,423). Dysphagia incidence upon admission, the proportion of patients receiving a standardized swallowing screening, and the percentage of dysphagic patients being referred to a speech language therapist (SLT) for treatment were obtained per year. Pneumonia rate, modified Rankin Scale (mRS) at discharge, and in-hospital mortality were compared between groups of dysphagic vs. non-dysphagic patients over time. RESULTS: Screening proportions continuously increased from 47.2% in 2008 to 86.6% in 2015. But the proportion diagnosed with dysphagia remained stable with about 19%. The number of dysphagic patients receiving SLT treatment grew from 81.6 up to 87.0%. Pneumonia incidence was higher in dysphagic stroke cases (adjusted OR 5.4 [5.2-5.5], p < 0.001), accompanied by a worse mRS at discharge (adjusted OR for mRS ≥3: 3.1 [3.0-3.1], p < 0.001) and higher mortality (adjusted OR 3.1 [3.0-3.2], p < 0.001). The order of magnitude of these end points did not change over time. CONCLUSION: Although advances have been made in dysphagia care, prevalent screening and treatment practices remain insufficient to reduce pneumonia rate, improve functional outcome, and decrease case fatality in dysphagic stroke patients. More research is urgently needed to develop more effective swallowing therapies.

Skin-Derived C-Terminal Filaggrin-2 Fragments Are Pseudomonas aeruginosa-Directed Antimicrobials Targeting Bacterial Replication.

Soil- and waterborne bacteria such as Pseudomonas aeruginosa are constantly challenging body surfaces. Since infections of healthy skin are unexpectedly rare, we hypothesized that the outermost epidermis, the stratum corneum, and sweat glands directly control the growth of P. aeruginosa by surface-provided antimicrobials. Due to its high abundance in the upper epidermis and eccrine sweat glands, filaggrin-2 (FLG2), a water-insoluble 248 kDa S100 fused-type protein, might possess these innate effector functions. Indeed, recombinant FLG2 C-terminal protein fragments display potent antimicrobial activity against P. aeruginosa and other Pseudomonads. Moreover, upon cultivation on stratum corneum, P. aeruginosa release FLG2 C-terminus-containing FLG2 fragments from insoluble material, indicating liberation of antimicrobially active FLG2 fragments by the bacteria themselves. Analyses of the underlying antimicrobial mechanism reveal that FLG2 C-terminal fragments do not induce pore formation, as known for many other antimicrobial peptides, but membrane blebbing, suggesting an alternative mode of action. The association of the FLG2 fragment with the inner membrane of treated bacteria and its DNA-binding implicated an interference with the bacterial replication that was confirmed by in vitro and in vivo replication assays. Probably through in situ-activation by soil- and waterborne bacteria such as Pseudomonads, FLG2 interferes with the bacterial replication, terminates their growth on skin surface and thus may contributes to the skin's antimicrobial defense shield. The apparent absence of FLG2 at certain body surfaces, as in the lung or of burned skin, would explain their higher susceptibility towards Pseudomonas infections and make FLG2 C-terminal fragments and their derivatives candidates for new Pseudomonas-targeting antimicrobials.

Increase of Substance P Concentration in Saliva after Pharyngeal Electrical Stimulation in Severely Dysphagic Stroke Patients - an Indicator of Decannulation Success?

BACKGROUND/AIMS: Substance P (SP) is a neuropeptide, likely acting as a neurotransmitter in the pharyngeal mucosa enhancing the swallow and cough reflex. Pharyngeal Electrical Stimulation (PES) induces a temporary increase of salivary SP levels in healthy adults. Previous evidence suggests that post-stroke dysphagia is related to reduced SP levels. Here, we investigated the effects of PES on SP levels in severely dysphagic stroke patients and a possible link between increase of SP and treatment success. METHODS: 23 tracheotomized stroke patients who could not be decannulated due to severe and persisting dysphagia according to endoscopic evaluation received PES for 10 minutes a day over three consecutive days in this prospective single-center study. If initial treatment failed, repetitive stimulation cycles were provided. Saliva samples were collected before and directly after each PES. RESULTS: 61% of participants were decannulated after the first treatment cycle. Increase of SP levels post-stimulation was closely related to treatment success, i.e. decannulation with 79% of successfully treated patients showing increase of SP, whereas 89% of unsuccessfully treated patients had stable or decreased SP levels. Applying logistic regression analysis, increase of SP level remained the only independent predictor of decannulation after PES. All 3 repetitively treated patients showed increased SP levels when progressing from the 1st to the 2nd cycle, two of whom were decannulated hereafter. CONCLUSIONS: The physiological mechanism of PES may consist in restoration of sensory feedback, which is known to be crucial for the execution of a safe swallow. SP possibly acts as a biomarker for indicating response to PES.

Cationic antimicrobial peptides in psoriatic skin cooperate to break innate tolerance to self-DNA.

Psoriasis is a T-cell-mediated skin autoimmune disease characterized by the aberrant activation of dermal dendritic cells (DCs) and the sustained epidermal expression of antimicrobial peptides. We have previously identified a link between these two events by showing that the cathelicidin antimicrobial peptide LL37 has the ability to trigger self-nucleic acid mediated activation of plasmacytoid DCs (pDCs) in psoriatic skin. Whether other cationic antimicrobial peptides exert similar activities is unknown. By analyzing heparin-binding HPLC fractions of psoriatic scales, we found that human beta-defensin (hBD)2, hBD3, and lysozyme are additional triggers of pDC activation in psoriatic skin lesions. Like LL37, hBD2, hBD3, and lysozyme are able to condense self-DNA into particles that are endocytosed by pDCs, leading to activation of TLR9. In contrast, other antimicrobial peptides expressed in psoriatic skin including elafin, hBD1, and psoriasin (S100A7) did not show similar activities. hBD2, hBD3, and lysozyme were detected in psoriatic skin lesions in the vicinity of pDCs and found to cooperate with LL37 to induce high levels of IFN production by pDCs, suggesting their concerted role in the pathogenesis of psoriasis.

Focal targeting by human ß-defensin 2 disrupts localized virulence factor assembly sites in Enterococcus faecalis.

Virulence factor secretion and assembly occurs at spatially restricted foci in some Gram-positive bacteria. Given the essentiality of the general secretion pathway in bacteria and the contribution of virulence factors to disease progression, the foci that coordinate these processes are attractive antimicrobial targets. In this study, we show in Enterococcus faecalis that SecA and Sortase A, required for the attachment of virulence factors to the cell wall, localize to discrete domains near the septum or nascent septal site as the bacteria proceed through the cell cycle. We also demonstrate that cationic human ß-defensins interact with E. faecalis at discrete septal foci, and this exposure disrupts sites of localized secretion and sorting. Modification of anionic lipids by multiple peptide resistance factor, a protein that confers antimicrobial peptide resistance by electrostatic repulsion, renders E. faecalis more resistant to killing by defensins and less susceptible to focal targeting by the cationic antimicrobial peptides. These data suggest a paradigm in which focal targeting by antimicrobial peptides is linked to their killing efficiency and to disruption of virulence factor assembly.

The skin microbiome of caspase-14-deficient mice shows mild dysbiosis.

Caspase-14, an important proteinase involved in filaggrin catabolism, is mainly active in terminally differentiating keratinocytes, where it is required for the generation of skin natural moisturizing factors (NMFs). Consequently, caspase-14 deficient epidermis is characterized by reduced levels of NMFs such as urocanic acid and 2-pyrrolidone-5-carboxylic acid. Patients suffering from filaggrin deficiency are prone to develop atopic dermatitis, which is accompanied with increased microbial burden. Among several reasons, this effect could be due to a decrease in filaggrin breakdown products. In this study, we found that caspase-14(-/-) mice show enhanced antibacterial response compared to wild-type mice when challenged with bacteria. Therefore, we compared the microbial communities between wild-type and caspase-14(-/-) mice by sequencing of bacterial 16S ribosomal RNA genes. We observed that caspase-14 ablation leads to an increase in bacterial richness and diversity during steady-state conditions. Although both wild-type and caspase-14(-/-) skin were dominated by the Firmicutes phylum, the Staphylococcaceae family was reduced in caspase-14(-/-) mice. Altogether, our data demonstrated that caspase-14 deficiency causes the imbalance of the skin-resident bacterial communities.

Fungal protein MGL_1304 in sweat is an allergen for atopic dermatitis patients.

BACKGROUND: Sweat is a major aggravating factor of atopic dermatitis (AD) and approximately 80% of patients with AD show type I hypersensitivity against sweat. OBJECTIVE: To identify and characterize an antigen in sweat that induces histamine release from basophils of patients with AD. METHODS: Basophil histamine-releasing activity in sweat was purified by a combination of chromatographies, and proteins were analyzed with mass spectrometry. Recombinant proteins of the sweat antigen were generated, and their biological characteristics were studied by immunoblots, histamine release tests, and neutralization assays. RESULTS: We identified a fungal protein, MGL_1304, derived from Malassezia globosa (M globosa) in the purified sweat antigen. Recombinant MGL_1304 induced histamine release from basophils of most of the patients with AD, in accordance with the semi-purified sweat antigen. Moreover, recombinant MGL_1304 abolished the binding of serum IgE of patients with AD to the semi-purified sweat antigen, or vice versa in immunoblot analysis, and attenuated the sensitization of RBL-48 mast cells expressing human FcɛRI by serum IgE. Studies of truncated mutants of MGL_1304 indicated that IgE of patients with AD recognized the conformational structure of MGL_1304 rather than short peptide sequences. Western blot analysis of the whole lysate, the culture supernatant of M globosa, and the semi-purified sweat antigen showed that MGL_1304 was produced as a minor immunological antigen of M globosa with posttranslational modification, cleaved, and secreted as a 17-kDa major histamine-releasing sweat antigen. CONCLUSION: MGL_1304 is a major allergen in human sweat and could cause type I allergy in patients with AD.

Revealing legacy effects of extreme droughts on tree growth of oaks across the Northern Hemisphere.

Forests are undergoing increasing risks of drought-induced tree mortality. Species replacement patterns following mortality may have a significant impact on the global carbon cycle. Among major hardwoods, deciduous oaks (Quercus spp.) are increasingly reported as replacing dying conifers across the Northern Hemisphere. Yet, our knowledge on the growth responses of these oaks to drought is incomplete, especially regarding post-drought legacy effects. The objectives of this study were to determine the occurrence, duration, and magnitude of legacy effects of extreme droughts and how that vary across species, sites, and drought characteristics. The legacy effects were quantified by the deviation of observed from expected radial growth indices in the period 1940-2016. We used stand-level chronologies from 458 sites and 21 oak species primarily from Europe, north-eastern America, and eastern Asia. We found that legacy effects of droughts could last from 1 to 5 years after the drought and were more prolonged in dry sites. Negative legacy effects (i.e., lower growth than expected) were more prevalent after repetitive droughts in dry sites. The effect of repetitive drought was stronger in Mediterranean oaks especially in Quercus faginea. Species-specific analyses revealed that Q. petraea and Q. macrocarpa from dry sites were more negatively affected by the droughts while growth of several oak species from mesic sites increased during post-drought years. Sites showing positive correlations to winter temperature showed little to no growth depression after drought, whereas sites with a positive correlation to previous summer water balance showed decreased growth. This may indicate that although winter warming favors tree growth during droughts, previous-year summer precipitation may predispose oak trees to current-year extreme droughts. Our results revealed a massive role of repetitive droughts in determining legacy effects and highlighted how growth sensitivity to climate, drought seasonality and species-specific traits drive the legacy effects in deciduous oak species.

The skin surface as antimicrobial barrier: present concepts and future outlooks.

Human skin protects the body as an outer barrier from various environmental threats and has to deal with an immense quantity of microbes comprising members of the own microbiota as well as potential pathogenic microbes. In this viewpoint, we highlight important cutaneous first-line defense barriers from the perspective of a microbe that tries to colonize and invade human skin. We place special emphasis on the role of antimicrobial peptides and the microbiota. In addition, we discuss and try to provide answers to some emerging issues concerning the various defense strategies of the epidermis.

Pseudomonas aeruginosa-derived rhamnolipids subvert the host innate immune response through manipulation of the human beta-defensin-2 expression.

Pseudomonas aeruginosa is a well-known cause of infections especially in compromised patients. To neutralize this pathogen, the expression of antimicrobial factors in epithelial cells is crucial. In particular the human beta-defensin hBD-2 is especially active against P. aeruginosa. In this study, we identified rhamnolipids in P. aeruginosa culture supernatants that are able to prevent the pathogen-induced hBD-2 response in keratinocytes. The presence of rhamnolipids within the host cells and inhibition assays suggest that calcium-regulated pathways and protein kinase C activation are impaired by rhamnolipids. In consequence, the induction of hBD-2 in keratinocytes by P. aeruginosa-derived flagellin as well as the host's own hBD-2 mediator interleukin IL-1ß is inhibited. Strikingly, rhamnolipids did not affect the release of the proinflammatory mediator interleukin IL-8 by flagellin. Thus, in addition to their function in establishment and persistence of P. aeruginosa infections, rhamnolipids can be engaged by P. aeruginosa for a targeted attenuation of the innate immunity to manage its survival and colonization on compromised epithelia.

Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide.

Plasmacytoid dendritic cells (pDCs) sense viral and microbial DNA through endosomal Toll-like receptors to produce type 1 interferons. pDCs do not normally respond to self-DNA, but this restriction seems to break down in human autoimmune disease by an as yet poorly understood mechanism. Here we identify the antimicrobial peptide LL37 (also known as CAMP) as the key factor that mediates pDC activation in psoriasis, a common autoimmune disease of the skin. LL37 converts inert self-DNA into a potent trigger of interferon production by binding the DNA to form aggregated and condensed structures that are delivered to and retained within early endocytic compartments in pDCs to trigger Toll-like receptor 9. Thus, our data uncover a fundamental role of an endogenous antimicrobial peptide in breaking innate tolerance to self-DNA and suggest that this pathway may drive autoimmunity in psoriasis.

Non-cell-autonomous small RNA silencing in Arabidopsis female gametes.

In recent years, small RNA movement has been both hypothesized and shown to be an integral part of the epigenetic DNA methylation reprogramming occurring during plant reproduction.1It was suggested that the release of epigenetic silencing in accessory cell types or tissues is necessary to reinforce epigenetic silencing in the gametes (egg cell and sperm cells), which would in turn ensure the genomic stability of the next generation plant.2,3 In Arabidopsis thaliana, small RNA (sRNA) movement was indeed shown to occur during male gametogenesis.4,5,6 However, the situation within the female gametophyte and in early seed development is mostly unknown. Here, we show that small RNAs can induce non-cell-autonomous silencing from the central cell toward the egg cell but also from the synergids to the egg cell and central cell. Our data show that in addition to the movement of sRNAs during pollen development, hairpin RNAs can have non-cell-autonomous effects in the female gametes.