Comparative analysis of whole plant, flower and root extracts of Chamomilla recutita L. and characteristic pure compounds reveals differential anti-inflammatory effects on human T cells.

Introduction: Chronic inflammation is a hallmark of chronic wounds and inflammatory skin diseases. Due to a hyperactive and prolonged inflammation triggered by proinflammatory immune cells, transitioning to the repair and healing phase is halted. T cells may exacerbate the proinflammatory milieu by secreting proinflammatory cytokines. Chamomilla recutita L. (chamomile) has been suggested for use in several inflammatory diseases, implying a capability to modulate T cells. Here, we have characterized and compared the effects of differently prepared chamomile extracts and characteristic pure compounds on the T cell redox milieu as well as on the migration, activation, proliferation, and cytokine production of primary human T cells. Methods: Phytochemical analysis of the extracts was carried out by LC-MS/MS. Primary human T cells from peripheral blood (PBTs) were pretreated with aqueous or hydroethanolic chamomile extracts or pure compounds. Subsequently, the effects on intracellular ROS levels, SDF-1α induced T cell migration, T cell activation, proliferation, and cytokine production after TCR/CD3 and CD28 costimulation were determined. Gene expression profiling was performed using nCounter analysis, followed by ingenuity pathway analysis, and validation at protein levels. Results: The tested chamomile extracts and pure compounds differentially affected intracellular ROS levels, migration, and activation of T cells. Three out of five differently prepared extracts and two out of three pure compounds diminished T cell proliferation. In line with these findings, LC-MS/MS analysis revealed high heterogeneity of phytochemicals among the different extracts. nCounter based gene expression profiling identified several genes related to T cell functions associated with activation and differentiation to be downregulated. Most prominently, apigenin significantly reduced granzyme B induction and cytotoxic T cell activity. Conclusion: Our results demonstrate an anti-inflammatory effect of chamomile- derived products on primary human T cells. These findings provide molecular explanations for the observed anti-inflammatory action of chamomile and imply a broader use of chamomile extracts in T cell driven chronic inflammatory diseases such as chronic wounds and inflammatory skin diseases. Importantly, the mode of extract preparation needs to be considered as the resulting different phytochemicals can result in differential effects on T cells.

Keratinocyte-induced costimulation of human T cells through CD6 - but not CD2 - activates mTOR and prevents oxidative stress.

In psoriasis and other inflammatory skin diseases, keratinocytes (KCs) secrete chemokines that attract T cells, which, in turn, cause epidermal hyperplasia by secreting proinflammatory cytokines. To date, it remains unclear whether skin-homing T cells, particularly memory T cells, can also be activated by direct cell contact with KCs. In this study, we demonstrated the ability of primary human KCs to activate human memory T cells directly by transmitting costimulatory signals through the CD6/CD166/CD318 axis. Interestingly, despite being negative for CD80/CD86, KCs initiate a metabolic shift within T cells. Blockade of the CD6/CD166/CD318 axis prevents mammalian target of rapamycin activation and T cell proliferation but promotes oxidative stress and aerobic glycolysis. In addition, it diminishes formation of central memory T cells. Importantly, although KC-mediated costimulation by CD2/CD58 also activates T cells, it cannot compensate for the lack of CD6 costimulation. Therefore, KCs likely differentially regulate T cell functions in the skin through two distinct costimulatory receptors: CD6 and CD2. This may at least in part explain the divergent effects observed when treating inflammatory skin diseases with antibodies to CD6 versus CD2. Moreover, our findings may provide a molecular basis for selective interference with either CD6/CD166/CD318, or CD2/CD58, or both to specifically treat different types of inflammatory skin diseases.

Complementary medicine usage in surgery: a cross-sectional survey in Germany.

BACKGROUND: Complementary medicine (CM) is frequently used by patients, but little is known about the usage of CM in surgical patients. The study aimed to elucidate the relevance of CM in surgery. METHODS: This cross-sectional, multi-center survey utilized a paper-based questionnaire consisting of 21 questions to capture CM usage and interest as well as CM communication in visceral and thoracic surgical patients being hospitalized at the corresponding departments of surgery at the University Medical Centers in Freiburg, Heidelberg und Ulm, Germany. RESULTS: Overall, 151 patients consented to the survey. On average, current CM usage was stated by 44% of patients. Most frequently used CM approaches were physical exercise (63%), nutritional supplements (59%) and herbal medicine (56%). Strong interest in CM counselling was stated by 51% of patients. Almost 80% of patients wanted to be treated in a holistic manner and desired for reliable information about CM as well as CM informed physicians. Only 12% of patients communicated CM usage and interest with their attending physician. Review of literature revealed similar results showing an overall CM usage of 43%, preferring nutritional supplements and herbal medicine. CONCLUSION: The results of our cross-sectional study indicate a high percentage of CM users and a strong interest in CM among surgical patients. Indeed, the current communication about CM between patients and surgeons is poor. With respect to safety and quality reasons, but also to pay attention to patients' demands, physicians should be aware of patients' CM usage in surgery. TRIAL REGISTRATION: German Clinical Trial register (DRKS00015445).

Inflammation induces pro-NETotic neutrophils via TNFR2 signaling.

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.

Expression of TRX1 optimizes the antitumor functions of human CAR T cells and confers resistance to a pro-oxidative tumor microenvironment.

Use of chimeric antigen receptor (CAR) T cells to treat B cell lymphoma and leukemia has been remarkably successful. Unfortunately, the therapeutic efficacy of CAR T cells against solid tumors is very limited, with immunosuppression by the pro-oxidative tumor microenvironment (TME) a major contributing factor. High levels of reactive oxygen species are well-tolerated by tumor cells due to their elevated expression of antioxidant proteins; however, this is not the case for T cells, which consequently become hypo-responsive. The aim of this study was to improve CAR T cell efficacy in solid tumors by empowering the antioxidant capacity of CAR T cells against the pro-oxidative TME. To this end, HER2-specific human CAR T cells stably expressing two antioxidant systems: thioredoxin-1 (TRX1), and glutaredoxin-1 (GRX1) were generated and characterized. Thereafter, antitumor functions of CAR T cells were evaluated under control or pro-oxidative conditions. To provide insights into the role of antioxidant systems, gene expression profiles as well as global protein oxidation were analyzed. Our results highlight that TRX1 is pivotal for T cell redox homeostasis. TRX1 expression allows CAR T cells to retain their cytolytic immune synapse formation, cytokine release, proliferation, and tumor cell-killing properties under pro-oxidative conditions. Evaluation of differentially expressed genes and the first comprehensive redoxosome analysis of T cells by mass spectrometry further clarified the underlying mechanisms. Taken together, enhancement of the key antioxidant TRX1 in human T cells opens possibilities to increase the efficacy of CAR T cell treatment against solid tumors.

Complementary medicine in Germany: a multi-centre cross-sectional survey on the usage by and the needs of patients hospitalized in university medical centers.

BACKGROUND: The results of recent surveys indicate that more than 50% of the German population has experience with complementary and alternative medicine (CAM) or uses CAM regularly. This study investigated the CAM usage and CAM-related needs of hospitalized patients at university medical centres in the state of Baden-Württemberg, Germany. METHODS: A multi-centre, paper-based, pseudonymous survey was carried out by the members of the Academic Centre for Complementary and Integrative Medicine. Patients of all ages, regardless of sex, diagnosis and treatment, who were hospitalized in the Department of Cardiology, Gastroenterology, Oncology, Gynaecology or Surgery at the university medical centres in Freiburg, Heidelberg, Tübingen and Ulm were eligible for inclusion. RESULTS: Of the 1275 eligible patients, 67% (n = 854) consented to participate in the survey. Forty-eight percent of the study participants stated that they were currently using CAM. The most frequently used therapies were exercise (63%), herbal medicine (54%) and dietary supplements (53%). Only 16% of the patients discussed CAM usage with their attending physician. Half of the patients (48%) were interested in CAM consultations. More than 80% of the patients desired reliable CAM information and stated that physicians should be better informed about CAM. CONCLUSIONS: The frequency of CAM usage and the need for CAM counselling among hospitalized patients at university medical centres in Baden-Württemberg are high. To better meet patients' needs, CAM research and physician education should be intensified. TRIAL REGISTRATION: German Clinical Trial register ( DRKS00015445 ).

Hijacked Immune Cells in the Tumor Microenvironment: Molecular Mechanisms of Immunosuppression and Cues to Improve T Cell-Based Immunotherapy of Solid Tumors.

The understanding of the tumor microenvironment (TME) has been expanding in recent years in the context of interactions among different cell types, through direct cell-cell communication as well as through soluble factors. It has become evident that the development of a successful antitumor response depends on several TME factors. In this context, the number, type, and subsets of immune cells, as well as the functionality, memory, and exhaustion state of leukocytes are key factors of the TME. Both the presence and functionality of immune cells, in particular T cells, are regulated by cellular and soluble factors of the TME. In this regard, one fundamental reason for failure of antitumor responses is hijacked immune cells, which contribute to the immunosuppressive TME in multiple ways. Specifically, reactive oxygen species (ROS), metabolites, and anti-inflammatory cytokines have central roles in generating an immunosuppressive TME. In this review, we focused on recent developments in the immune cell constituents of the TME, and the micromilieu control of antitumor responses. Furthermore, we highlighted the current challenges of T cell-based immunotherapies and potential future strategies to consider for strengthening their effectiveness.

LFA-1 cluster formation in T-cells depends on L-plastin phosphorylation regulated by P90RSK and PP2A.

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-p90RSK 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.

Reactive Oxidative Species-Modulated Ca2+ Release Regulates ß2 Integrin Activation on CD4+ CD28null T Cells of Acute Coronary Syndrome Patients.

The number and activity of T cell subsets in the atherosclerotic plaques are critical for the prognosis of patients with acute coronary syndrome. ß2 Integrin activation is pivotal for T cell recruitment and correlates with future cardiac events. Despite this knowledge, differential regulation of adhesiveness in T cell subsets has not been explored yet. In this study, we show that in human T cells, SDF-1α-mediated ß2 integrin activation is driven by a, so far, not-described reactive oxidative species (ROS)-regulated calcium influx. Furthermore, we show that CD4+CD28null T cells represent a highly reactive subset showing 25-fold stronger ß2 integrin activation upon SDF-1α stimulation compared with CD28+ T cells. Interestingly, ROS-dependent Ca release was much more prevalent in the pathogenetically pivotal CD28null subset compared with the CD28+ T cells, whereas the established mediators of the classical pathways for ß2 integrin activation (PKC, PI3K, and PLC) were similarly activated in both T cell subsets. Thus, interference with the calcium flux attenuates spontaneous adhesion of CD28null T cells from acute coronary syndrome patients, and calcium ionophores abolished the observed differences in the adhesion properties between CD28+ and CD28null T cells. Likewise, the adhesion of these T cell subsets was indistinguishable in the presence of exogenous ROS/H2O2 Together, these data provide a molecular explanation of the role of ROS in pathogenesis of plaque destabilization.

Devilishly radical NETwork in COVID-19: Oxidative stress, neutrophil extracellular traps (NETs), and T cell suppression.

Pandemic coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and poses an unprecedented challenge to healthcare systems due to the lack of a vaccine and specific treatment options. Accordingly, there is an urgent need to understand precisely the pathogenic mechanisms underlying this multifaceted disease. There is increasing evidence that the immune system reacts insufficiently to SARS-CoV-2 and thus contributes to organ damage and to lethality. In this review, we suggest that the overwhelming production of reactive oxygen species (ROS) resulting in oxidative stress is a major cause of local or systemic tissue damage that leads to severe COVID-19. It increases the formation of neutrophil extracellular traps (NETs) and suppresses the adaptive arm of the immune system, i.e. T cells that are necessary to kill virus-infected cells. This creates a vicious cycle that prevents a specific immune response against SARS-CoV-2. The key role of oxidative stress in the pathogenesis of severe COVID-19 implies that therapeutic counterbalancing of ROS by antioxidants such as vitamin C or NAC and/or by antagonizing ROS production by cells of the mononuclear phagocyte system (MPS) and neutrophil granulocytes and/or by blocking of TNF-α can prevent COVID-19 from becoming severe. Controlled clinical trials and preclinical models of COVID-19 are needed to evaluate this hypothesis.

Acupuncture in persons with an increased stress level-Results from a randomized-controlled pilot trial.

BACKGROUND: In today's Western societies a high percentage of people experience increased or chronic stress. Acupuncture could serve as treatment for persons affected adversely by the increased stress. METHODS: The AkuRest study was a two-centre randomized controlled pilot study in adult persons with increased stress levels. Participants were randomly allocated to one of three groups: verum acupuncture treatment, sham acupuncture, and a waiting control group. The feasibility of the study was assessed. In addition, effects on stress level (measured by the Perceived Stress Questionnaire (PSQ-20)) and other variables were assessed at the end of treatment and a 3-month follow-up. RESULTS: Altogether, N = 70 persons were included in the study. At the end of the treatment 15.7% were lost to follow-up. The adherence to the protocol was good: 82.9% of the participants completed 100% of their treatment. The stress level of the participants was high at baseline (mean PSQ-20 score 75.5, SD = 8.2). Effect sizes (ES) at T1 showed that verum and sham acupuncture were superior to the waiting condition in reducing stress (ES (verum) = -1.39, 95%-CI = [-2.11; -0.67]: ES (sham) = -1.12, CI = [-1.78;-0.44]). At follow-up, effect sizes were in favour of the verum group (as compared to sham). However, confidence intervals and t-tests showed that these differences were not significant. CONCLUSION: The pilot study demonstrated the feasibility of the acupuncture RCT in persons with increased stress levels. Estimated parameters can be used to design a larger RCT to prove the-here indicated-efficacy of verum acupuncture to decrease stress. TRIAL REGISTRATION NUMBER: ISRCTN15259166.

Piperlongumine Acts as an Immunosuppressant by Exerting Prooxidative Effects in Human T Cells Resulting in Diminished TH17 but Enhanced Treg Differentiation.

Piperlongumine (PL), a natural small molecule derived from the Piper longum Linn plant, has received growing interest as a prooxidative drug with promising anticancer properties. Yet, the influence of PL on primary human T cells remained elusive. Knowledge of this is of crucial importance, however, since T cells in particular play a critical role in tumor control. Therefore, we investigated the effects of PL on the survival and function of primary human peripheral blood T cells (PBTs). While PL was not cytotoxic to PBTs, it interfered with several stages of T cell activation as it inhibited T cell/APC immune synapse formation, co-stimulation-induced upregulation of CD69 and CD25, T cell proliferation and the secretion of proinflammatory cytokines. PL-induced immune suppression was prevented in the presence of thiol-containing antioxidants. In line with this finding, PL increased the levels of intracellular reactive oxygen species and decreased glutathione in PBTs. Diminished intracellular glutathione was accompanied by a decrease in S-glutathionylation on actin suggesting a global alteration of the antioxidant response. Gene expression analysis demonstrated that TH17-related genes were predominantly inhibited by PL. Consistently, the polarization of primary human naïve CD4+ T cells into TH17 subsets was significantly diminished while differentiation into Treg cells was substantially increased upon PL treatment. This opposed consequence for TH17 and Treg cells was again abolished by thiol-containing antioxidants. Taken together, PL may act as a promising agent for therapeutic immunosuppression by exerting prooxidative effects in human T cells resulting in a diminished TH17 but enhanced Treg cell differentiation.

Vegan diet reduces neutrophils, monocytes and platelets related to branched-chain amino acids - A randomized, controlled trial.

BACKGROUND: Vegan diet (VD) has improved inflammatory activity in patients with rheumatoid arthritis (RA) in several small controlled trials. The underlying mechanism remains widely unclear. We investigated the effect of a VD in comparison to a meat-rich diet (MD) on markers of inflammation (which have been shown to be relevant in patients with RA) in healthy volunteers. METHODS: 53 healthy, omnivore subjects were randomized to a controlled VD (n = 26) or MD (n = 27) for 4 weeks following a pre-treatment phase of a one week controlled mixed diet. Primary parameters of interest were sialylation of immunoglobulins, percentage of regulatory T-cells and level of interleukin 10 (IL10). Usual care immune parameters used in patients with RA and amino acid serum levels as well as granulocytes and monocytes colony stimulating factor (GM-CSF) serum levels were secondary parameters. RESULTS: In the VD group, total leukocyte, neutrophil, monocyte and platelet counts decreased and after four weeks they were significantly lower compared to the MD group (ANCOVA: leukocytes p = 0.003, neutrophils p = 0.001, monocytes p = 0.032, platelets p = 0.004). Leukocytes, neutrophils, monocytes, and platelets correlated with each other and likewise conform with serum levels of branched-chain amino acids, which were significantly lower in the VD compared to the MD group. The primary parameters did not differ between the groups and BMI remained stable in the two groups. CONCLUSION: Four weeks of a controlled VD affected the number of neutrophils, monocytes and platelets but not the number or function of lymphocytes. The relation with branched-chain amino acids and GM-CSF suggests a mode of action via the mTOR signaling pathway. REGISTERED AT: http://www.drks.de (German Clinical Trial register) at DRKS00011963.

Redox Regulation of the Actin Cytoskeleton in Cell Migration and Adhesion: On the Way to a Spatiotemporal View.

The actin cytoskeleton of eukaryotic cells is a dynamic, fibrous network that is regulated by the concerted action of actin-binding proteins (ABPs). In particular, rapid polarization of cells in response to internal and external stimuli is fundamental to cell migration and invasion. Various isoforms of ABPs in different tissues equip cells with variable degrees of migratory and adhesive capacities. In addition, regulation of ABPs by posttranslational modifications (PTM) is pivotal to the rapid responsiveness of cells. In this context, phosphorylation of ABPs and its functional consequences have been studied extensively. However, the study of reduction/oxidation (redox) modifications of oxidation-sensitive cysteine and methionine residues of actin, ABPs, adhesion molecules, and signaling proteins regulating actin cytoskeletal dynamics has only recently emerged as a field. The relevance of such protein oxidations to cellular physiology and pathophysiology has remained largely elusive. Importantly, studying protein oxidation spatiotemporally can provide novel insights into localized redox regulation of cellular functions. In this review, we focus on the redox regulation of the actin cytoskeleton, its challenges, and recently developed tools to study its physiological and pathophysiological consequences.

Keratinocytes costimulate naive human T cells via CD2: a potential target to prevent the development of proinflammatory Th1 cells in the skin.

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.

Vitamin B12 Status Upon Short-Term Intervention with a Vegan Diet-A Randomized Controlled Trial in Healthy Participants.

Vegans are at an increased risk for certain micronutrient deficiencies, foremost of vitamin B12. Little is known about the short-term effects of dietary change to plant-based nutrition on vitamin B12 metabolism. Systemic biomarkers of vitamin B12 status, namely, serum vitamin B12 and holotranscobalamin, may respond quickly to a reduced intake of vitamin B12. To test this hypothesis, 53 healthy omnivore subjects were randomized to a controlled unsupplemented vegan diet (VD, n = 26) or meat-rich diet (MD, n = 27) for 4 weeks. Vitamin B12 status was examined by measurement of serum vitamin B12, holotranscobalamin (holo-TC), methylmalonic acid (MMA) and total plasma homocysteine (tHcy). Holo-TC decreased significantly in the VD compared to the MD group after four weeks of intervention, whereas metabolites MMA and tHcy were unaffected. Body weight remained stable in both groups. VD intervention led to a significant reduction of cholesterol intake, and adequate profiles of nutrient and micronutrient status. Lower intake of vitamin B12 was observed in VD, which was mirrored by a lower concentration of serum vitamin B12 and reduced holo-TC after 4 weeks. Plasma holo-TC may be a fast-responding biomarker to monitor adequate supply of vitamin B12 in plant-based individuals.

3D Cellular Architecture Modulates Tyrosine Kinase Activity, Thereby Switching CD95-Mediated Apoptosis to Survival.

The death receptor CD95 is expressed in every cancer cell, thus providing a promising tool to target cancer. Activation of CD95 can, however, lead to apoptosis or proliferation. Yet the molecular determinants of CD95's mode of action remain unclear. Here, we identify an optimal distance between CD95Ligand molecules that enables specific clustering of receptor-ligand pairs, leading to efficient CD95 activation. Surprisingly, efficient CD95 activation leads to apoptosis in cancer cells in vitro and increased tumor growth in vivo. We show that allowing a 3D aggregation of cancer cells in vitro switches the apoptotic response to proliferation. Indeed, we demonstrate that the absence or presence of cell-cell contacts dictates the cell response to CD95. Cell contacts increase global levels of phosphorylated tyrosines, including CD95's tyrosine. A tyrosine-to-alanine CD95 mutant blocks proliferation in cells in contact. Our study sheds light into the regulatory mechanism of CD95 activation that can be further explored for anti-cancer therapies.

Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells.

Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.

Postnatal human enteric neurospheres show a remarkable molecular complexity.

BACKGROUND: The enteric nervous system (ENS), a complex network of neurons and glial cells, coordinates major gastrointestinal functions. Impaired development or secondary aberrations cause severe enteric neuropathies. Neural crest-derived stem cells as well as enteric neuronal progenitor cells, which form enteric neurospheres, represent a promising tool to unravel molecular pathomechanisms and to develop novel therapy options. However, so far little is known about the detailed cellular composition and the proportional distribution of enteric neurospheres. Comprehensive knowledge will not only be essential for basic research but also for prospective cell replacement therapies to restore or to improve enteric neuronal dysfunction. METHODS: Human enteric neurospheres were generated from three individuals with varying age. For detailed molecular characterization, nCounter target gene expression analyses focusing on stem, progenitor, neuronal, glial, muscular, and epithelial cell markers were performed. Corresponding archived paraffin-embedded individuals' specimens were analyzed accordingly. KEY RESULTS: Our data revealed a remarkable molecular complexity of enteric neurospheres and archived specimens. Amongst the expression of multipotent stem cell, progenitor cell, neuronal, glial, muscle and epithelial cell markers, moderate levels for the pluripotency marker POU5F1 were observed. Furthermore, besides the interindividual variability, we identified highly distinct intraindividual expression profiles. CONCLUSIONS & INFERENCES: Our results emphasize the assessment of molecular signatures to be essential for standardized use, optimization of experimental approaches, and elimination of potential risk factors, as the formation of tumors. Our study pipeline may serve as a blueprint implemented into the characterization procedure of enteric neurospheres for various future applications.