Elimination of negative feedback in TLR signalling allows rapid and hypersensitive detection of microbial contaminants

AbstractThe exquisite specificity of Toll-like receptors (TLRs) to sense microbial molecular signatures is used as a powerful tool to pinpoint microbial contaminants. Various cellular systems, from native human blood cells to transfected cell lines exploit TLRs as pyrogen detectors in biological preparations. However, slow cellular responses and limited sensitivity have hampered the replacement of animal-based tests such as the rabbit pyrogen test or lipopolysaccharide detection by Limulus amoebocyte lysate. Here, we report a novel human cell-based approach to boost detection of microbial contaminants by TLR-expressing cells. By genetic and pharmacologic elimination of negative control circuits, TLR-initiated cellular responses to bacterial molecular patterns were accelerated and significantly elevated. Combining depletion of protein phosphatase PP2ACA and pharmacological inhibition of PP1 in the optimized reporter cells further enhanced the sensitivity to allow detection of bacterial lipoprotein at 30 picogram/ml. Such next-generation cellular monitoring is poised to replace animal-based testing for microbial contaminants.

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A conserved regulatory module regulates receptor kinase signaling in immunity and development

10.1101/2021.01.19.427293 ◽

2021 ◽

Author(s):

Thomas A. DeFalco ◽

Pauline Anne ◽

Sean R. James ◽

Andrew Willoughby ◽

Oliver Johanndrees ◽

...

Keyword(s):

Cell Surface ◽

Cellular Responses ◽

Receptor Signaling ◽

Receptor Kinase ◽

Surface Receptors ◽

Receptor Complexes ◽

Regulatory Circuit ◽

Regulatory Module ◽

Molecular Patterns ◽

Receptor Kinase Signaling

ABSTRACTLigand recognition by cell-surface receptors underlies development and immunity in both animals and plants. Modulating receptor signaling is critical for appropriate cellular responses but the mechanisms ensuring this are poorly understood. Here, we show that signaling by plant receptors for pathogen-associated molecular patterns (PAMPs) in immunity and CLAVATA3/EMBRYO SURROUNDING REGION-RELATED peptides (CLEp) in development employ a similar regulatory module. In the absence of ligand, signaling is dampened through association with specific type-2C protein phosphatases (PP2Cs). Upon activation, PAMP and CLEp receptors phosphorylate divergent cytosolic kinases, which, in turn, phosphorylate the phosphatases, thereby promoting their release from the receptor complexes. Our work reveals a regulatory circuit shared between immune and developmental receptor signaling, which may have broader important implications for plant receptor kinase-mediated signaling in general.

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A Highly Selective Chemical Probe for Activin Receptor-like Kinases ALK4 and ALK5

10.1101/2020.01.23.916502 ◽

2020 ◽

Author(s):

Thomas Hanke ◽

Jong Fu Wong ◽

Benedict-Tilmann Berger ◽

Ismahan Abdi ◽

Lena Marie Berger ◽

...

Keyword(s):

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Heart Diseases ◽

Negative Control ◽

Cellular Systems ◽

Receptor Protein ◽

Chemical Probe ◽

Activin Receptor ◽

Control Compound

AbstractThe transforming growth factor beta-receptor I/activin receptor-like kinase 5 (TGFBR1/ALK5) and its close homologue ALK4 are receptor protein kinases associated with the development of diverse diseases, including cancer, fibrosis, heart diseases and dysfunctional immune response. Therefore, ALK4/5 are among the most studied kinases and several inhibitors have been developed. However, current commercially available inhibitors either lack selectivity or have not been comprehensively characterized, limiting their value for studying ALK4/5 function in cellular systems. To this end, we report the characterization of the 2-oxo-imidazopyridine, TP-008, a potent chemical probe with dual activity for ALK4 and ALK5 as well as the development of a matching negative control compound. TP-008 has excellent cellular potency and strongly abrogates phosphorylation of the substrate SMAD2 (mothers against decapentaplegic homolog 2). Thus, this chemical probe offers an excellent tool for mechanistic studies on the ALK4/5 signaling pathway and the contribution of these targets to disease.

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An Extract of Sericea Lespedeza Modulates Production of Inflammatory Markers in Pathogen Associated Molecular Pattern (PAMP) Activated Ruminant Blood

Journal of Agricultural Science ◽

10.5539/jas.v8n9p1 ◽

2016 ◽

Vol 8 (9) ◽

pp. 1 ◽

Cited By ~ 2

Author(s):

Emmanuel K Asiamah ◽

Sarah Adjei-Fremah ◽

Bertha Osei ◽

Kingsley Ekwemalor ◽

Mulumebet Worku

Keyword(s):

Gene Activation ◽

Wnt Signaling Pathway ◽

Water Extract ◽

Negative Control ◽

Immune Gene ◽

Sericea Lespedeza ◽

Molecular Pattern ◽

Holstein Friesian ◽

Boer Goats ◽

Molecular Patterns

<p>Programs based on antibiotics are failing to control diseases due to increase in resistance of pathogens to antibiotics. Food safety, animal welfare and public health concerns have fueled interest in the use of plant-based alternatives. This study was conducted to evaluate the effect of a plant (Sericea Lespedeza, SL), and pathogen associated molecular patterns (PAMPs) (Lipopolysaccharide (LPS) and peptidoglycan (PGN)) on gene activation in ruminant blood. A water extract of SL, was used as a source of plant-derived tannins. Blood was collected from Holstein-Friesian cows (N = 4), Spanish × Boer goats (N = 4), St Croix sheep (N = 4) and incubated with 100 ng/mL of SL in the presence or absence of LPS or PGN. Samples maintained in Phosphate-buffered saline (PBS) served as negative control. The total protein concentration, WNT5a, and prostaglandin E2 in plasma were determined. Total RNA was isolated, reverse transcribed and Real time-PCR was performed using gene specific primers for TLR2, TLR4, WNT5a, and FZD. TLR2 and FZD were up-regulated in response to PAMPs. WNT5a and TLR4 genes were undetected in PAMP treated blood. SL regulated protein and prostaglandin concentration in all species. SL reduced PGE2 in sheep and cow blood. WNT5a was only secreted in LPS treated cow blood. Transcription and translation of genes involved in innate and adaptive immunity and the WNT signaling pathway in ruminant blood were responsive to diverse PAMPS, and can be modulated by SL. This suggests that dietary tannins may promote the health of ruminants. Further studies are needed to determine the significance of these changes in immune gene expression on ruminant health.</p>

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DAMPening Inflammation by Modulating TLR Signalling

Mediators of Inflammation ◽

10.1155/2010/672395 ◽

2010 ◽

Vol 2010 ◽

pp. 1-21 ◽

Cited By ~ 452

Author(s):

A. M. Piccinini ◽

K. S. Midwood

Keyword(s):

Immune System ◽

Tissue Damage ◽

Tissue Repair ◽

Inflammatory Responses ◽

Current Knowledge ◽

Feedback Loops ◽

Inflammatory Gene Expression ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns ◽

Tlr Signalling

Damage-associated molecular patterns (DAMPs) include endogenous intracellular molecules released by activated or necrotic cells and extracellular matrix (ECM) molecules that are upregulated upon injury or degraded following tissue damage. DAMPs are vital danger signals that alert our immune system to tissue damage upon both infectious and sterile insult. DAMP activation of Toll-like receptors (TLRs) induces inflammatory gene expression to mediate tissue repair. However, DAMPs have also been implicated in diseases where excessive inflammation plays a key role in pathogenesis, including rheumatoid arthritis (RA), cancer, and atherosclerosis. TLR activation by DAMPs may initiate positive feedback loops where increasing tissue damage perpetuates pro-inflammatory responses leading to chronic inflammation. Here we explore the current knowledge about distinct signalling cascades resulting from self TLR activation. We also discuss the involvement of endogenous TLR activators in disease and highlight how specifically targeting DAMPs may yield therapies that do not globally suppress the immune system.

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TLR 4 / MD2 Complex inhibitors potential therapeutic prospective for asthma and asthma exacerbation

Research Journal of Biotechnology ◽

10.25303/1611rjbt164176 ◽

2021 ◽

Vol 16 (11) ◽

pp. 164-176

Author(s):

Swamita Arora ◽

Tanveer Naved ◽

Sanjar Alam ◽

Bhupendra Chauhan ◽

Harikesh Kalonia

Keyword(s):

Initial Step ◽

Cardiovascular Disorder ◽

The Body ◽

Innate And Adaptive Immunity ◽

Cytokines And Chemokines ◽

Asthma Patients ◽

Tlr4 Activation ◽

Molecular Patterns ◽

Tlr Signalling ◽

Inflammatory Effect

The feedback of our body to foreign particles is initially initiated by the innate immunity where the likes Toll- Like Receptors (TLRs), play an important role in the identification process. The identification of the trouble through the interaction of the receptor is the initial step to propagate the protective agents throughout the body. These interact action with the attacking pathogens like viruses and bacteria (PAMPs, pathogen-associated molecular patterns) or substances produced cells of the body which are injured (DAMPs, danger-associated molecular patterns) helping in the identification. However, the innate and adaptive immunity of both of them gets stimulated through the release of various substances such as cytokines and chemokines due to TLR activation. In part of the progress, many reports have been identified as the activation of the TLR4 complex, a receptor of the innate immune system that may underpin the pathophysiology of many human diseases including asthma, cardiovascular disorder, diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory and psychiatric disorders. Substances of synthetic and natural origin have been discussed here and have been found to produce the anti-inflammatory effect by targeting pathways of TLR4 activation. In the present review, our focus is to develop a conclusion about the TL4 complex inhibition for the betterment of asthma patients and combine the reports about the progressing of TLR signalling pathway modulators.

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Bovine intestinal cellular responses following primary and challenge infections with Calicophoron microbothrium metacercariae

Onderstepoort Journal of Veterinary Research ◽

10.4102/ojvr.v75i2.9 ◽

2008 ◽

Vol 75 (2) ◽

Cited By ~ 6

Author(s):

N. Mavenyengwa ◽

S. Mukaratirwa ◽

M. Obwolo ◽

J. Monrad

Keyword(s):

Mast Cells ◽

Cellular Responses ◽

Negative Control Group ◽

Positive Control ◽

Negative Control ◽

Group Iv ◽

Control Group ◽

Post Challenge ◽

Group Iii ◽

Positive Controls

This studyw as carried out to establish whether cattle can develop resistance to re-infectionby Calicophoron microbothrium by assessing the response of intestinal mucosal globule leukocytese, osinophils, mast cells and basophils, and the establishment of the parasite in the host. A total of 241-year old Tuli steers were randomly divided into four groups of six animals each and infected with C. microbothriumm etacercariae. On the first day of the study, animals in Groups I and II were immunized with 5000 metacercariae and then challenged with 15000 metacercariae on Day 150 post immunization. Animals in Group III were immunized with 15000 metacercariae at the same time that Groups I and II animals were challenged to act as a positive control group Animals in Group IV were left uninfected and acted as a negative control group. Three animals from each group were slaughtered on Day 28 post-challenge and the remainder were slaughtered on Day 42 post-challenge. The established amphistomes were recovered and histopathological and cytological examinations were done on the jejunum, duodenuma, bomasum and the rumen. The establishment rates of the challenge infection in the immunized and challenged groups were lower and ranged from 0 to 0.2% as compared to 6% from naive animals infected as positive controls. Animals immunized and then challenged with C. microbothrium had significantly higher eosinophil, mast cell and globule leukocytes counts in the intestinal mucosa (P < 0.05) as compared to those of the control group. The study indicates that cattle can develop resistance to C. microbothrium re-infection and that eosinophils and mast cells may be important cells in the rejection of the parasite.

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Squeegee: de-novo identification of reagent and laboratory induced microbial contaminants in low biomass microbiomes

10.1101/2021.05.06.442815 ◽

2021 ◽

Author(s):

Yunxi Liu ◽

R. A. Leo Elworth ◽

Michael D. Jochum ◽

Kjersti M. Aagaard ◽

Todd J. Treangen

Keyword(s):

Computational Analysis ◽

De Novo ◽

Human Microbiome ◽

Human Microbiome Project ◽

Negative Control ◽

Control Data ◽

Detection Tool ◽

Health And Disease ◽

Microbial Contaminants ◽

Negative Controls

Computational analysis of host-associated microbiomes has opened the door to numerous discoveries relevant to human health and disease. However, contaminant sequences in metagenomic samples can potentially impact the interpretation of findings reported in microbiome studies, especially in low biomass environments. Our hypothesis is that contamination from DNA extraction kits or sampling lab environments will leave taxonomic bread crumbs across multiple distinct sample types, allowing for the detection of microbial contaminants when negative controls are unavailable. To test this hypothesis we implemented Squeegee, a de novo contamination detection tool. We tested Squeegee on simulated and real low biomass metagenomic datasets. On the low biomass samples, we compared Squeegee predictions to experimental negative control data and show that Squeegee accurately recovers known contaminants. We also analyzed 749 metagenomic datasets from the Human Microbiome Project and identified likely previously unreported kit contamination. Collectively, our results highlight that Squeegee can identify microbial contaminants with high precision. Squeegee is open-source and available at: https://gitlab.com/treangenlab/squeegee

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A Synthetic Influenza Virus Vaccine Induces a Cellular Immune Response That Correlates with Reduction in Symptomatology and Virus Shedding in a Randomized Phase Ib Live-Virus Challenge in Humans

Clinical and Vaccine Immunology ◽

10.1128/cvi.00098-15 ◽

2015 ◽

Vol 22 (7) ◽

pp. 828-835 ◽

Cited By ~ 36

Author(s):

Olga Pleguezuelos ◽

Stuart Robinson ◽

Ana Fernández ◽

Gregory A. Stoloff ◽

Alex Mann ◽

...

Keyword(s):

Influenza Virus ◽

Cellular Immunity ◽

Cellular Responses ◽

Negative Control ◽

Live Virus ◽

Virus Shedding ◽

Phase Ib ◽

Control Value ◽

Symptom Scores ◽

Ifn Γ

ABSTRACTCurrent influenza vaccines elicit primarily antibody-based immunity. They require yearly revaccination and cannot be manufactured until the identification of the circulating viral strain(s). These issues remain to be addressed. Here we report a phase Ib trial of a vaccine candidate (FLU-v) eliciting cellular immunity. Thirty-two males seronegative for the challenge virus by hemagglutination inhibition assay participated in this single-center, randomized, double-blind study. Volunteers received one dose of either the adjuvant alone (placebo,n= 16) or FLU-v (500 μg) and the adjuvant (n= 16), both in saline. Twenty-one days later, FLU-v (n= 15) and placebo (n= 13) volunteers were challenged with influenza virus A/Wisconsin/67/2005 (H3N2) and monitored for 7 days. Safety, tolerability, and cellular responses were assessed pre- and postvaccination. Virus shedding and clinical signs were assessed postchallenge. FLU-v was safe and well tolerated. No difference in the prevaccination FLU-v-specific gamma interferon (IFN-γ) response was seen between groups (average ± the standard error of the mean [SEM] for the placebo and FLU-v, respectively, 1.4-fold ± 0.2-fold and 1.6-fold ± 0.5-fold higher than the negative-control value). Nineteen days postvaccination, the FLU-v group, but not the placebo group, developed FLU-v-specific IFN-γ responses (8.2-fold ± 3.9-fold versus 1.3-fold ± 0.1-fold higher than the negative-control value [average ± SEM] for FLU-v versus the placebo [P= 0.0005]). FLU-v-specific cellular responses also correlated with reductions in both viral titers (P= 0.01) and symptom scores (P= 0.02) postchallenge. Increased cellular immunity specific to FLU-v correlates with reductions in both symptom scores and virus loads. (This study has been registered at ClinicalTrials.gov under registration no. NCT01226758 and at hra.nhs.uk under EudraCT no. 2009-014716-35.)

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Enhanced Activity of P4503A4 and UGT1A10 Induced by Acridinone Derivatives C-1305 and C-1311 in MCF-7 and HCT116 Cancer Cells: Consequences for the Drugs’ Cytotoxicity, Metabolism and Cellular Response

International Journal of Molecular Sciences ◽

10.3390/ijms21113954 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3954

Author(s):

Monika Pawłowska ◽

Anna Kwaśniewska ◽

Zofia Mazerska ◽

Ewa Augustin

Keyword(s):

Drug Metabolism ◽

Cancer Cells ◽

Anticancer Drugs ◽

Cellular Response ◽

Antitumor Agent ◽

Cellular Responses ◽

Cellular Systems ◽

Metabolism Enzymes ◽

Hct116 Cells ◽

Mcf 7

Activity modulation of drug metabolism enzymes can change the biotransformation of chemotherapeutics and cellular responses induced by them. As a result, drug-drug interactions can be modified. Acridinone derivatives, represented here by C-1305 and C-1311, are potent anticancer drugs. Previous studies in non-cellular systems showed that they are mechanism-based inhibitors of cytochrome P4503A4 and undergo glucuronidation via UDP-glucuronosyltranspherase 1A10 isoenzyme (UGT1A10). Therefore, we investigated the potency of these compounds to modulate P4503A4 and UGT1A10 activity in breast MCF-7 and colon HCT116 cancer cells and their influence on cytotoxicity and cellular response in cells with different expression levels of studied isoenzymes. We show that C-1305 and C-1311 are inducers of not only P4503A4 but also UGT1A10 activity. MCF-7 and HCT116 cells with high P4503A4 activity are more sensitive to acridinone derivatives and undergo apoptosis/necrosis to a greater extent. UGT1A10 was demonstrated to be responsible for C-1305 and C-1311 glucuronidation in cancer cells and glucuronide products were excreted outside the cell very fast. Finally, we show that glucuronidation of C-1305 antitumor agent enhances its pro-apoptotic properties in HCT116 cells, while the cytotoxicity and cellular response induced by C-1311 did not change after drug glucuronidation in both cell lines.

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Targeting Toll like Receptors in Cancer: Role of TLR Natural and Synthetic Modulators

Current Pharmaceutical Design ◽

10.2174/1381612826666200720235058 ◽

2020 ◽

Vol 26 (39) ◽

pp. 5040-5053 ◽

Cited By ~ 2

Author(s):

Arunaksharan Narayanankutty ◽

Aswathi Sasidharan ◽

Joice T. Job

Keyword(s):

Immune Response ◽

Web Of Science ◽

Broad Class ◽

The Body ◽

Toll Like Receptors ◽

Transmembrane Receptors ◽

Molecular Patterns ◽

Tlr Signalling ◽

Detailed Outline

Background: Toll like receptors (TLRs) are a group of transmembrane receptors belonging to the broad class pattern recognition receptors (PRR), involved in recognition of Pathogen Associated Molecular Patterns (PAMPs) thereby inducing an immune response. Apart from these exogenous PAMPs, numerous endogenous PAMPs are also ligands for various TLRs thereby activating the TLR dependent immune response, subsequently leading to the onset of an inflammatory response. Prolonged activation of TLR by these endogenous PAMPs leads to chronic inflammatory insults to the body and which in turn alters the proliferative patterns of the cells, which ultimately leads to the development of cancer. Objectives: The present review aims to provide a detailed outline of the differential roles of various TLRs in cancer and the possible use of them as a therapeutic target. Methods: Data were collected from PubMed/Sciencedirect/Web of Science database and sorted; the latest literature on TLRs was incorporated in the review. Results: Among the different TLRs, few are reported to be anti-neoplastic, which controls the cell growth and multiplication in response to the endogenous signals. On the contrary, numerous studies have reported the procarcinogenic potentials of TLRs. Hence, TLRs have emerged as a potential target for the prevention and treatment of various types of cancers. Several molecules, such as monoclonal antibodies, small molecule inhibitors and natural products have shown promising anticancer potential by effectively modulating the TLR signalling. Conclusion: Toll-like receptors play vital roles in the process of carcinogenesis, hence TLR targeting is a promising approach for cancer prevention.

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