Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway ...using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/β3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.
The integrin LFA-1 is crucial for T-cell/ APC interactions and sensitive recognition of antigens. Precise nanoscale organization and valency regulation of LFA-1 are mandatory for an appropriate ...function of the immune system. While the inside-out signals regulating the LFA-1 affinity are well described, the molecular mechanisms controlling LFA-1 avidity are still not fully understood. Here, we show that activation of the actin-bundling protein
l
-plastin (LPL) through phosphorylation at serine-5 enables the formation of clusters containing LFA-1 in high-affinity conformation. Phosphorylation of LPL is induced by an nPKC–MEK–p90
RSK
pathway and counter-regulated by the serine–threonine phosphatase PP2A. Interestingly, recruitment of LFA-1 into the T-cell/APC contact zone is not affected by LPL phosphorylation. Instead, for this process, activation of the actin-remodeling protein cofilin through dephosphorylation is essential. Together, this study reveals a dichotomic spatial regulation of LFA-1 clustering and microscale movement in T-cells by two different actin-binding proteins, LPL and cofilin.
The interplay between keratinocytes and immune cells, especially T cells, plays an important role in the pathogenesis of chronic inflammatory skin diseases. During psoriasis, keratinocytes attract T ...cells by releasing chemokines, while skin-infiltrating self-reactive T cells secrete proinflammatory cytokines, e.g., IFNγ and IL-17A, that cause epidermal hyperplasia. Similarly, in chronic graft-versus-host disease, allogenic IFNγ-producing Th1/Tc1 and IL-17-producing Th17/Tc17 cells are recruited by keratinocyte-derived chemokines and accumulate in the skin. However, whether keratinocytes act as nonprofessional antigen-presenting cells to directly activate naive human T cells in the epidermis remains unknown. Here, we demonstrate that under proinflammatory conditions, primary human keratinocytes indeed activate naive human T cells. This activation required cell contact and costimulatory signaling via CD58/CD2 and CD54/LFA-1. Naive T cells costimulated by keratinocytes selectively differentiated into Th1 and Th17 cells. In particular, keratinocyte-initiated Th1 differentiation was dependent on costimulation through CD58/CD2. The latter molecule initiated STAT1 signaling and IFNγ production in T cells. Costimulation of T cells by keratinocytes resulting in Th1 and Th17 differentiation represents a new explanation for the local enrichment of Th1 and Th17 cells in the skin of patients with a chronic inflammatory skin disease. Consequently, local interference with T cell-keratinocyte interactions may represent a novel strategy for the treatment of Th1 and Th17 cell-driven skin diseases.
The International Federation of Associations of Anatomists (IFAA) recommended in 2012 that only donated bodies be used for anatomy teaching and research. However, in many countries around the world, ...anatomists still depend on bodies that do not stem from voluntary donations by the deceased, but rather are “unclaimed.” A broad search of the literature was conducted to produce a baseline overview of the sources of cadavers used for anatomy teaching in undergraduate medical curricula on a global scale. Information from the literature search was supplemented with data from a 2016–2017 survey of selected senior local anatomists. Of 165 countries with medical schools, information was gathered for 71. In 22 (32%) of the 68 countries that use cadavers for anatomy teaching, body donation is the exclusive source of bodies. However, in most other countries, unclaimed bodies remain the main (n = 18, 26%) or exclusive (n = 21, 31%) source. Some countries import cadavers from abroad, mainly from the United States or India. In one country, bodies of executed persons are given to anatomy departments. The heterogeneous geographical distribution of body sources cannot easily be accounted for, but religion, culture, and folk beliefs about what should happen to bodies after death seem to play a role. Implementation of the IFAA recommendations still has a long way to go, but it is encouraging that functioning body donation programs exist on all continents and that there are examples of recent rises in donations and of anatomists initiating new donation programs.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
: The promise of mesotherapy is maintenance and/or recovery of a youthful skin with a firm, bright and moisturized texture. Currently applied medications employ microinjections of hyaluronic acid, ...vitamins, minerals and amino acids into the superficial layer of the skin. However, the molecular and cellular processes underlying mesotherapy are still elusive. Here we analysed the effect of five distinct medication formulas on pivotal parameters involved in skin ageing, that is collagen expression, cell proliferation and morphological changes using normal human skin fibroblast cultures in vitro. Whereas in the presence of hyaluronic acid, NCTF135® and NCTF135HA®, cell proliferation was comparable to control cultures; however, with higher expression of collagen type‐1, matrix metalloproteinase‐1 and tissue inhibitor of matrix metalloproteinase‐1, addition of Soluvit® N and Meso‐BK led to apoptosis and/or necrosis of human fibroblasts. The data indicate that bioactive reagents currently applied for skin rejuvenation elicit strikingly divergent physiological processes in human skin fibroblast in vitro.
Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti‐host defense factors, including the immunosuppressive cysteine‐rich ...secretory glycoprotein Salp15 fromIxodes scapularis ticks and orthologs like Iric‐1 fromIxodes
ricinus. All tick‐borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti‐tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low‐yield eukaryotic systems, we investigated cytoplasmic expression inEscherichia coli, even though this would not result in glycosylation. His‐tagged Salp15 was efficiently expressed but insoluble. Among the various solubility‐enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric‐1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross‐react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric‐1 from protease‐cleavable fusions, despite limited solubility, allowed the recording of1H–15N 2D NMR spectra, suggesting partial folding of the wild‐type proteins but not of Cys‐free variants. Fusion to the NMR‐compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in13C/15N double‐labeled Iric‐1. Together,E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.
Tick saliva proteins Salp15 and Iric‐1 promote tick feeding and pathogen transmission.
We established the first bacterial expression system for soluble Salp15 and Iric‐1.
Using this system we mapped monoclonal antibody epitopes on Salp15 and Iric‐1.
We defined the interaction sites with Borrelia outer surface protein C (OspC).
We elucidated first secondary structure features in Iric‐1 by NMR.
Many pathogenic microorganisms express fibronectin-binding molecules that facilitate their adherence to the extracellular matrix and/or entry into mammalian cells. We have previously described a ...Borrelia recurrentis gene, cihC that encodes a 40-kDa surface receptor for both, fibronectin and the complement inhibitors C4bp and C1-Inh. We now provide evidence for the expression of a group of highly homologues surface proteins, termed FbpA, in three B. hermsii isolates and two tick-borne relapsing fever spirochetes, B. parkeri and B. turicatae. When expressed in Escherichia coli or B. burgdorferi, four out of five proteins were shown to selectively bind fibronectin, whereas none of five proteins were able to bind the human complement regulators, C4bp and C1-Inh. By applying deletion mutants of the B. hermsii fibronectin-binding proteins a putative high-affinity binding site for fibronectin was mapped to its central region. In addition, the fibronectin-binding proteins of B. hermsii were found to share sequence homology with BBK32 of the Lyme disease spirochete B. burgdorferi with similar function suggesting its involvement in persistence and/or virulence of relapsing fever spirochetes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
New therapeutic targets for advanced colorectal cancer (CRC) are critically needed. Our laboratory recently performed an insertional mutagenesis screen in mice to identify novel CRC driver genes and, ...thus, potential drug targets. Here, we define Transmembrane 9 Superfamily 2 (TM9SF2) as a novel CRC oncogene. TM9SF2 is an understudied protein, belonging to a well conserved protein family characterized by their nine putative transmembrane domains. Based on our transposon screen we found that TM9SF2 is a candidate progression driver in digestive tract tumors. Analysis of The Cancer Genome Atlas (TCGA) data revealed that approximately 35% of CRC patients have elevated levels of TM9SF2 mRNA, data we validated using an independent set of CRC samples. RNAi silencing of TM9SF2 reduced CRC cell growth in an anchorage-independent manner, a hallmark of cancer. Furthermore, CRISPR/Cas9 knockout of TM9SF2 substantially diminished CRC tumor fitness in vitro and in vivo. Transcriptome analysis of TM9SF2 knockout cells revealed a potential role for TM9SF2 in cell cycle progression, oxidative phosphorylation, and ceramide signaling. Lastly, we report that increased TM9SF2 expression correlates with disease stage and low TM9SF2 expression correlate with a more favorable relapse-free survival. Taken together, this study provides evidence that TM9SF2 is a novel CRC oncogene.
Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMNs). Here, we describe the development of a subgroup of human PMNs ...expressing CCR5, termed CCR5+ PMNs. Auto- and paracrine tumor necrosis factor (TNF) signaling increases intracellular neutrophil elastase (ELANE) abundance and induces neutrophil extracellular traps formation (NETosis) in CCR5+ PMNs, and triggering of CCR5 amplifies NETosis. Membranous TNF (mTNF) outside-in signaling induces the formation of reactive oxygen species, known activators of NETosis. In vivo, we find an increased number of CCR5+ PMNs in the peripheral blood and inflamed lamina propria of patients with ulcerative colitis (UC). Notably, failure of anti-TNF therapy is associated with higher frequencies of CCR5+ PMNs. In conclusion, we identify a phenotype of pro-NETotic, CCR5+ PMNs present in inflamed tissue in vivo and inducible in vitro. These cells may reflect an important component of tissue damage during chronic inflammation and could be of diagnostic value.
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•Priming induces neutrophil diversification into CCR5− and CCR5+ cells•TNFR2 signaling renders CCR5+ neutrophils pro-NETotic by ELANE upregulation•CCR5+ neutrophils appear in the lamina propria of ulcerative colitis patients•Retrograde TNF signaling induces NETosis of CCR5+ neutrophils
Neuenfeldt et al. show that neutrophil priming induces dichotomous expression of CCR5. TNFR2 signaling increases ELANE expression in CCR5+ neutrophils. CCR5 triggering or reverse signaling of membrane-bound TNF activates ELANE, leading to enhanced NETosis. Pro-NETotic CCR5+ neutrophils in the lamina propria of ulcerative colitis patients may affect the success of anti-TNF therapies.
Intensive forms of outreach mental health care (IOC) such as crisis resolution or home treatment teams are increasingly implemented as alternatives to inpatient admission, providing recovery-oriented ...treatment at home at comparable costs and outcomes. However, one issue with IOC is the lack of continuity regarding staff members who provide home visits, complicating relationship building and meaningful therapeutic exchange. The aim of this study is to validate existing primarily qualitative findings using performance data and to explore a possible correlation between the number of staff involved within IOC treatment and the service users' length of stay (LOS).
Routine data from an IOC team in a catchment area in Eastern Germany were analyzed. Basic parameters of service delivery were calculated and an in-depth descriptive analysis regarding staff continuity was performed. Further, an exploratory single case analysis was conducted, presenting the exact sequence of all treatment contacts for one case with low and one with high staff continuity.
We analyzed 10.598 face-to-face treatment contacts based on 178 IOC users. The mean LOS was 30.99 days. About 75% of all home visits were conducted by two or more staff members simultaneously. Service users saw an average of 10.24 different staff per treatment episode. On 11% of the care days, only unknown staff, and on 34% of the care days at least one unknown staff member conducted the home visit. 83% of the contacts were performed by the same three staff members and 51% were made by one and the same staff member. A significant positive correlation (
= 0.0007) was found between the number of different practitioners seen by a service user in the first seven days of care and the LOS.
Our results suggest that a high number of different staff in the early period of IOC episodes correlates with an extended LOS. Future research must clarify the exact mechanisms of this correlation. Furthermore, it should be investigated how the multiple professions within IOC teams influence the LOS and the quality of treatment and what quality indicators may be suitable to ensure treatment processes.
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