Instytut Podstawowych Problemów Techniki

Streszczenie:

Human Respiratory Syncytial Virus (hRSV), a prevalent respiratory pathogen affecting various age groups, can trigger prolonged and intense inflammation in humans. The severity and outcome of hRSV infection correlate with elevated levels of pro-inflammatory agents, yet the underlying reasons for this immune system overstimulation remain elusive. We focused on the impact of hRSV non-structural proteins, NS1 and NS2, on immune response within epithelial cells. Available data indicates that these proteins impair the interferon pathway. We reinforce that NS1 and NS2 induce heightened secretion of the pro-inflammatory cytokines IL-6 and CXCL8. We also indicate that hRSV non-structural proteins provoke differential gene expression of human host FosB and long non-coding RNAs (MALAT1, RP11-510N19.5). It suggests an impact of NS molecules beyond IFN pathways. Thus, new light is shed on the interplay between hRSV and host cells, uncovering unexplored avenues of viral interference, especially the NS2 role in cytokine expression and immune modulation.

Słowa kluczowe:

Human respiratory syncytial virus, Non-structural protein 1, Non-structural protein 2, Cytokines, MALAT1 long non-coding RNA

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With the rapid development and commercial interest in the organ-on-a-chip (OoC) field, there is a need for materials addressing key experimental demands and enabling both prototyping and large-scale production. Here, we utilized the gas-permeable, thermoplastic material polymethylpentene (PMP). Three methods were tested to prototype transparent PMP films suitable for transmission light microscopy: hot-press molding, extrusion, and polishing of a commercial, hazy extruded film. The transparent films (thickness 20, 125, 133, 356, and 653 µm) were assembled as the cell-adhering layer in sealed culture chamber devices, to assess resulting oxygen concentration after 4 days of A549 cell culture (cancerous lung epithelial cells). Oxygen concentrations stabilized between 15.6% and 11.6%, where the thicker the film, the lower the oxygen concentration. Cell adherence, proliferation, and viability were comparable to glass for all PMP films (coated with poly-L-lysine), and transparency was adequate for transmission light microscopy of adherent cells. Hot-press molding was concluded as the preferred film prototyping method, due to excellent and reproducible film transparency, the possibility to easily vary film thickness, and the equipment being commonly available. The molecular orientation in the PMP films was characterized by IR dichroism. As expected, the extruded films showed clear orientation, but a novel result was that hot-press molding may also induce some orientation. It has been reported that orientation affects the permeability, but with the films in this study, we conclude that the orientation is not a critical factor. With the obtained results, we find it likely that OoC models with relevant in vivo oxygen concentrations may be facilitated by PMP. Combined with established large-scale production methods for thermoplastics, we foresee a useful role for PMP within the OoC field.

Słowa kluczowe:

polymethylpentene, gas permeability, organ-on-a-chip, prototyping thermoplastics, microfluidic device, microscopy

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Streszczenie:

Type I interferons (IFNs) are key coordinators of the innate immune response to viral infection, which, through activation of the transcriptional regulators STAT1 and STAT2 (STAT1/2) in bystander cells, induce the expression of IFN-stimulated genes (ISGs). Here, we showed that in cells transfected with poly(I:C), an analog of viral RNA, the transcriptional activity of STAT1/2 was terminated because of depletion of the interferon-β (IFN-β) receptor, IFNAR. Activation of RNase L and PKR, products of two ISGs, not only hindered the replenishment of IFNAR but also suppressed negative regulators of IRF3 and NF-κB, consequently promoting IFNB transcription. We incorporated these findings into a mathematical model of innate immunity. By coupling signaling through the IRF3–NF-κB and STAT1/2 pathways with the activities of RNase L and PKR, the model explains how poly(I:C) switches the transcriptional program from being STAT1/2 induced to being IRF3 and NF-κB induced, which converts IFN-β–responding cells to IFN-β–secreting cells.

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Streszczenie:

When infected with a virus, cells may secrete interferons (IFNs) that prompt nearby cells to prepare for upcoming infection. Reciprocally, viral proteins often interfere with IFN synthesis and IFN-induced signaling. We modeled the crosstalk between the propagating virus and the innate immune response using an agent-based stochastic approach. By analyzing immunofluorescence microscopy images we observed that the mutual antagonism between the respiratory syncytial virus (RSV) and infected A549 cells leads to dichotomous responses at the single-cell level and complex spatial patterns of cell signaling states. Our analysis indicates that RSV blocks innate responses at three levels: by inhibition of IRF3 activation, inhibition of IFN synthesis, and inhibition of STAT1/2 activation. In turn, proteins coded by IFN-stimulated (STAT1/2-activated) genes inhibit the synthesis of viral RNA and viral proteins. The striking consequence of these inhibitions is a lack of coincidence of viral proteins and IFN expression within single cells. The model enables investigation of the impact of immunostimulatory defective viral particles and signaling network perturbations that could potentially facilitate containment or clearance of the viral infection.

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Streszczenie:

The applicability of a gas-permeable, thermoplastic material polymethylpentene (PMP) was investigated, experimentally and analytically, for organ-on-a-chip (OoC) and long-term on-a-chip cell cultivation applications. Using a sealed culture chamber device fitted with oxygen sensors, we tested and compared PMP to commonly used glass and polydimethylsiloxane (PDMS). We show that PMP and PDMS have comparable performance for oxygen supply during 4 days culture of epithelial (A549) cells with oxygen concentration stabilizing at 16%, compared with glass control where it decreases to 3%. For the first time, transmission light images of cells growing on PMP were obtained, demonstrating that the optical properties of PMP are suitable for non-fluorescent, live cell imaging. Following the combined transmission light imaging and calcein-AM staining, cell adherence, proliferation, morphology, and viability of A549 cells were shown to be similar on PMP and glass coated with poly-L-lysine. In contrast to PDMS, we demonstrate that a film of PMP as thin as 0.125 mm is compatible with high-resolution confocal microscopy due to its excellent optical properties and mechanical stiffness. PMP was also found to be fully compatible with device sterilization, cell fixation, cell permeabilization and fluorescent staining. We envision this material to extend the range of possible microfluidic applications beyond the current state-of-the-art, due to its beneficial physical properties and suitability for prototyping by different methods. The integrated device and measurement methodology demonstrated in this work are transferrable to other cell-based studies and life-sciences applications.

Słowa kluczowe:

polymethylpentene (PMP), cell culture, oxygen control, microfluidic device, organ-on-a-chip

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Streszczenie:

We observed the interference between two prevalent respiratory viruses, respiratory syncytial virus (RSV) and influenza A virus (IAV) (H1N1), and characterized its molecular underpinnings in alveolar epithelial cells (A549). We found that RSV induces higher levels of interferon beta (IFN-β) production than IAV and that IFN-β priming confers higher-level protection against infection with IAV than with RSV. Consequently, we focused on the sequential infection scheme of RSV and then IAV. Using A549 wild-type (WT), IFNAR1 knockout (KO), IFNLR1 KO, and IFNAR1-IFNLR1 double-KO cell lines, we found that both IFN-β and IFN-λ are necessary for maximum protection against subsequent infection. Immunostaining revealed that preinfection with RSV partitions the cell population into a subpopulation susceptible to subsequent infection with IAV and an IAV-proof subpopulation. Strikingly, the susceptible cells turned out to be those already compromised and efficiently expressing RSV, whereas the bystander, interferon-primed cells are resistant to IAV infection. Thus, virus-virus exclusion at the cell population level is not realized through direct competition for a shared ecological niche (single cell) but rather is achieved with the involvement of specific cytokines induced by the host’s innate immune response.

Słowa kluczowe:

influenza A virus,innate immunity,interferon beta,interferon lambda,respiratory syncytial virus,single cell,viral interference

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Streszczenie:

This paper presents the development and comparison of label-free electrochemical immunosensors based on screen-printed gold and glassy carbon (GC) disc electrodes for efficient and rapid detection of respiratory syncytial virus (RSV). Briefly, the antibody specific to the F protein of RSV was successfully immobilized on modified electrodes. Antibody coupling on the Au surface was conducted via 4-aminothiophenol (4-ATP) and glutaraldehyde (GA). The GC surface was modified with poly-L-lysine (PLL) for direct anti-RSV conjugation after EDC/NHS (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide) activation. Electrochemical characterizations of the immunosensors were carried out by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). GC-based immunosensors show a dynamic range of antigen detection from 1.0 × 10^5 PFU/mL to 1.5×10^7 PFU/mL, more than 1.0 × 10^5 PFU/mL to 1.0 × 10^7 PFU/mL for the Au-based sensor. However, the GC platform is less sensitive and shows a higher detection limit (LOD) for RSV. The limit of detection of the Au immunosensor is 1.1 × 10^3 PFU/mL, three orders of magnitude lower than 2.85 × 10^6 PFU/mL for GC. Thus, the Au-based immunosensor has better analytical performance for virus detection than a carbon-based platform due to high sensitivity and very low RSV detection, obtained with good reproducibility.

Słowa kluczowe:

respiratory syncytial virus, cyclic voltammetry, electrochemical impedance spectroscopy, sensor, gold electrode, glassy carbon

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Streszczenie:

The innate immune system processes pathogen-induced signals into cell fate decisions. How information is turned to decision remains unknown. By combining stochastic mathematical modelling and experimentation, we demonstrate that feedback interactions between the IRF3, NF-κB and STAT pathways lead to switch-like responses to a viral analogue, poly(I:C), in contrast to pulse-like responses to bacterial LPS. Poly(I:C) activates both IRF3 and NF-κB, a requirement for induction of IFNβ expression. Autocrine IFNβ initiates a JAK/STAT-mediated positive-feedback stabilising nuclear IRF3 and NF-κB in first responder cells. Paracrine IFNβ, in turn, sensitises second responder cells through a JAK/STAT-mediated positive feedforward pathway that upregulates the positive-feedback components: RIG-I, PKR and OAS1A. In these sensitised cells, the 'live-or-die' decision phase following poly(I:C) exposure is shorter—they rapidly produce antiviral responses and commit to apoptosis. The interlinked positive feedback and feedforward signalling is key for coordinating cell fate decisions in cellular populations restricting pathogen spread.

Słowa kluczowe:

cellular signalling networks, innate immunity, regulatory networks, stochastic modelling

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Streszczenie:

The NF-κB pathway is known to transmit merely 1 bit of information about stimulus level. We combined experimentation with mathematical modeling to elucidate how information about TNF concentration is turned into a binary decision. Using Kolmogorov-Smirnov distance, we quantified the cell's ability to discern 8 TNF concentrations at each step of the NF-κB pathway, to find that input discernibility decreases as signal propagates along the pathway. Discernibility of low TNF concentrations is restricted by noise at the TNF receptor level, whereas discernibility of high TNF concentrations it is restricted by saturation/depletion of downstream signaling components. Consequently, signal discernibility is highest between 0.03 and 1 ng/ml TNF. Simultaneous exposure to TNF or LPS and a translation inhibitor, cycloheximide, leads to prolonged NF-κB activation and a marked increase of transcript levels of NF-κB inhibitors, IκBα and A20. The impact of cycloheximide becomes apparent after the first peak of nuclear NF-κB translocation, meaning that the NF-κB network not only relays 1 bit of information to coordinate the all-or-nothing expression of early genes, but also over a longer time course integrates information about other stimuli. The NF-κB system should be thus perceived as a feedback-controlled decision-making module rather than a simple information transmission channel.

Słowa kluczowe:

cellular signaling networks, innate immunity, stress signaling

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Streszczenie:

Electrospinning of chitosan blends is a reasonable idea to prepare fibre mats for biomedical applications. Synthetic and natural components provide, for example, appropriate mechanical strength and biocompatibility, respectively. However, solvent characteristics and the polyelectrolyte nature of chitosan influence the spinnability of these blends. In order to compare the effect of solvent on polycaprolactone/chitosan fibres, two types of the most commonly used solvent systems were chosen, namely 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and acetic acid (AA)/formic acid (FA). Results obtained by various experimental methods clearly indicated the effect of the solvent system on the structure and properties of electrospun polycaprolactone/chitosan fibres. Viscosity measurements confirmed different polymer–solvent interactions. Various molecular interactions resulting in different macromolecular conformations of chitosan influenced its spinnability and properties. HFIP enabled fibres to be obtained whose average diameter was less than 250 nm while maintaining the brittle and hydrophilic character of the nonwoven, typical for the chitosan component. Spectroscopy studies revealed the formation of chitosan salts in the case of the AA/FA solvent system. Chitosan salts visibly influenced the structure and properties of the prepared fibre mats. The use of AA/FA caused a reduction of Young's modulus and wettability of the proposed blends. It was confirmed that wettability, mechanical properties and the antibacterial effect of polycaprolactone/chitosan fibres may be tailored by selecting an appropriate solvent system. The MTT cell proliferation assay revealed an increase of cytotoxicity to mouse fibroblasts in the case of 25% w/w of chitosan in electrospun nonwovens.

Słowa kluczowe:

chitosan, electrospinning, PCL/chitosan fibres, solvent system, chitosan salts

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Streszczenie:

BACKGROUND:
Importins and exportins influence gene expression by enabling nucleocytoplasmic shuttling of transcription factors. A key transcription factor of innate immunity, NF-κB, is sequestered in the cytoplasm by its inhibitor, IκBα, which masks nuclear localization sequence of NF-κB. In response to TNFα or LPS, IκBα is degraded, which allows importins to bind NF-κB and shepherd it across nuclear pores. NF-κB nuclear activity is terminated when newly synthesized IκBα enters the nucleus, binds NF-κB and exportin which directs the complex to the cytoplasm. Although importins/exportins are known to regulate spatiotemporal kinetics of NF-κB and other transcription factors governing innate immunity, the mechanistic details of these interactions have not been elucidated and mathematically modelled.
RESULTS:
Based on our quantitative experimental data, we pursue NF-κB system modelling by explicitly including NF-κB-importin and IκBα-exportin binding to show that the competition between importins and IκBα enables NF-κB nuclear translocation despite high levels of IκBα. These interactions reduce the effective relaxation time and allow the NF-κB regulatory pathway to respond to recurrent TNFα pulses of 45-min period, which is about twice shorter than the characteristic period of NF-κB oscillations. By stochastic simulations of model dynamics we demonstrate that randomly appearing, short TNFα pulses can be converted to essentially digital pulses of NF-κB activity, provided that intervals between input pulses are not shorter than 1 h.
CONCLUSIONS:
By including interactions involving importin-α and exportin we bring the modelling of spatiotemporal kinetics of transcription factors to a more mechanistic level. Basing on the analysis of the pursued model we estimated the information transmission rate of the NF-κB pathway as 1 bit per hour.

Słowa kluczowe:

Karyopherins, Nucleocytoplasmic transport, Negative feedback, Channel information capacity, Mathematical modelling

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Streszczenie:

Lipopolysaccharide (LPS) activates macrophages by binding to the TLR4/MD-2 complex and triggers two pro-inflammatory signaling pathways: one relies on MyD88 at the plasma membrane, and the other one depends on TRIF in endosomes. When present in high doses, LPS is internalized and undergoes detoxification. We found that the uptake of a high concentration of LPS (1000 ng/ml) in macrophage-like J774 cells was upregulated upon inhibition of clathrin- and dynamin-mediated endocytosis which, on the other hand, strongly reduced the production of pro-inflammatory mediators TNF-α and RANTES. The binding and internalization of high amounts of LPS was mediated by scavenger receptor A (SR-A) with participation of CD14 without an engagement of TLR4. Occupation of SR-A by dextran sulfate or anti-SR-A antibodies enhanced LPS-induced production of TNF-α and RANTES by about 70%, with CD14 as a limiting factor. Dextran sulfate also elevated the cell surface levels of TLR4 and CD14, which could have contributed to the upregulation of the pro-inflammatory responses. Silencing of SR-A expression inhibited the LPS-triggered TNF-α production whereas RANTES release was unchanged. These data indicate that SR-A is required for maximal production of TNF-α in cells stimulated with LPS, possibly by modulating the cell surface levels of TLR4 and CD14.

Słowa kluczowe:

CD14, CTX-FITC, Endocytosis, HEPES-buffered saline, LBP, LPS conjugated with Alexa Fluor 488, LPS-AF488, LPS-binding protein, Lipopolysaccharide, PD buffer, SR-A, Scavenger receptor, TLR, poly(I:C), polyinosinic–polycytidylic acid, scavenger receptor A, subunit B of cholera toxin conjugated with FITC, transferrin conjugated with Alexa Fluor 647, transferrin-AF647

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Streszczenie:

Mechanizmy wrodzonej odpowiedzi odpornościowej uruchamiane są w wyniku rozpoznania elementów budulcowych mikroorganizmów o strukturze zachowanej w toku ewolucji, nazywanych wzorcami molekularnymi. W ich wiązaniu uczestniczą wyspecjalizowane receptory inicjujące kaskady sygnałów, które prowadzą do produkcji białek o charakterze prozapalnym oraz do aktywacji i regulacji mechanizmów nabytej odpowiedzi odpornościowej. Do receptorów tego typu należą receptory Toll-podobne (TLR), rozpoznające elementy ścian komórkowych, aparatu ruchu, jedno- i dwuniciowe RNA oraz motywy DNA typowe dla mikroorganizmów. Wszystkie receptory TLR zbudowane są z domeny wiążącej ligandy, która zawiera liczne motywy bogate w leucynę, pojedynczej domeny transbłonowej oraz domeny sygnałowej TIR. Pomimo znacznego strukturalnego zróżnicowania ligandów ich związanie prowadzi do dimeryzacji receptorów TLR, co z kolei umożliwia interakcję domeny TIR z białkami adaptorowymi i zapoczątkowuje kaskady sygnałowe. Receptory TLR angażują cztery wspólne białka adaptorowe, około dziesięciu kinaz o charakterze sygnałowym oraz aktywują docelowo kilka czynników transkrypcyjnych, w tym NFκB, IRF i AP-1. Szczególną uwagę poświęca się receptorowi TLR4 aktywowanemu przez lipopolisacharyd (LPS), budulec błony zewnętrznej bakterii Gram-ujemnych, gdyż uruchomiona przez LPS reakcja zapalna może prowadzić do potencjalnie śmiertelnego w skutkach szoku septycznego. Badania ostatnich lat znacznie rozszerzyły naszą wiedzę na temat współdziałania szeregu białek, w tym CD14, kompleksu TLR4/MD-2 oraz receptorów zmiataczy w modulacji odpowiedzi komórek na LPS. Ugruntowały też przekonanie o dychotomii ścieżek sygnałowych receptora TLR4, które zależą od udziału białek adaptorowych MyD88 i TRIF, oraz prowadzą do ekspresji genów kodujących cytokiny prozapalne i interferony typu I. Kluczowym wydarzeniem regulującym generację sygnału na ścieżce zależnej od TRIF jest internalizacja receptora TLR4

Słowa kluczowe:

receptory Toll-podobne, przekazywanie sygnału, lipopolisacharyd, sepsa, wrodzona odpowiedź odpornościowa

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Streszczenie:

Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro-inflammatory mediators. We examined mechanisms of LPS-stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage-like J774 cells. A wound-healing assay revealed that LPS also activated directed cell movement that was followed by TNF-α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5-bisphosphate. Fractionation of Triton X-100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin-regulatory proteins, paxillin on tyrosine 118 by 80% and N-WASP on serine 484/485 by 20%, and these events preceded activation of NF-κB. LPS-induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N-WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS-stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF-α production suggesting that LPS-induced cell motility is not required for TNF-α release.

Słowa kluczowe:

ACTIN CYTOSKELETON, CELL MOTILITY, LPS, N-WASP, PAXILLIN, SRC KINASES

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Streszczenie:

Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) of macrophages triggering production of pro-inflammatory mediators. One of the factors determining the magnitude of responses to LPS, which may even lead to life-threatening septic shock, is the cell surface abundance of TLR4. However, quantitation of the surface TLR4 is difficult due to the low level of receptor expression. To develop a method of TLR4 assessment, we labeled the receptor on the cell surface with a rabbit antibody followed by either anti-rabbit immunoglobulin G–fluorescein isothiocyanate (IgG–FITC) for flow cytometry or with anti-rabbit IgG–peroxidase for a cellular enzyme-linked immunosorbent assay (ELISA). Alternatively, the anti-TLR4 antibody was detected by anti-rabbit IgG labeled with 125I. Flow cytometry did not allow detection of TLR4 on the surface of J774 cells or human macrophages. In contrast, application of cellular ELISA or the radiolabeling technique combined with effective blockage of nonspecific binding of antibodies provided TLR4-specific signals. The level of TLR4 on the surface of J774 cells did not change on treatment with 1–100 ng/ml LPS; however, it was reduced by approximately 30–40% after 2 h of treatment with 1 μg/ml LPS. These data indicate that down-regulation of surface TLR4 can serve as a means of negative regulation of cell responses toward high doses of LPS.

Słowa kluczowe:

Lipopolysaccharide, Toll-like receptor 4, ELISA, Radiolabeling, Flow cytometry

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Streszczenie:

LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1-2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation

Słowa kluczowe:

lipopolysaccharide, TNF alpha, MIP-2, ceramide. ceramide-1-phosphate, sphingomyelinase, J774, macrophages

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Słowa kluczowe:

dżuma, ewolucja, genomika, patogeneza, Yersinia

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