Microfiltration method of removal of bacterial contaminants and their monitoring by nitric oxide and Limulus assays

Zı́dek, Zdeňek; Kmoníčková, Eva; Kostecká, Petra; Jansa, Petr

doi:10.1016/j.niox.2012.08.078

Cited by 9 publications

(5 citation statements)

References 37 publications

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“…Detoxi-gel is considered as a tool for removing the effects of LPS and the Limulus amebocyte lysate assay is widely used for endotoxin detection [45], [46]. Our study demonstrated that purified PEP-1-PON1 protein was further purified using Detoxi-gel endotoxin removing gel in order to eliminate endotoxin in bacteria (Fig.…”

Section: Discussionmentioning

confidence: 79%

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PEP-1-PON1 Protein Regulates Inflammatory Response in Raw 264.7 Macrophages and Ameliorates Inflammation in a TPA-Induced Animal Model

et al. 2014

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Paraoxonase 1 (PON1) is an antioxidant enzyme which plays a central role in various diseases. However, the mechanism and function of PON1 protein in inflammation are poorly understood. Since PON1 protein alone cannot be delivered into cells, we generated a cell permeable PEP-1-PON1 protein using protein transduction domains, and examined whether it can protect against cell death in lipopolysaccharide (LPS) or hydrogen peroxide (H2O2)-treated Raw 264.7 cells as well as mice with 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced skin inflammation. We demonstrated that PEP-1-PON1 protein transduced into Raw 264.7 cells and markedly protected against LPS or H2O2-induced cell death by inhibiting cellular reactive oxygen species (ROS) levels, the inflammatory mediator’s expression, activation of mitogen-activated protein kinases (MAPKs) and cellular apoptosis. Furthermore, topically applied PEP-1-PON1 protein ameliorates TPA-treated mice skin inflammation via a reduction of inflammatory response. Our results indicate that PEP-1-PON1 protein plays a key role in inflammation and oxidative stress in vitro and in vivo. Therefore, we suggest that PEP-1-PON1 protein may provide a potential protein therapy against oxidative stress and inflammation.

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Section: Discussionmentioning

confidence: 79%

“…After purification, endotoxin levels were below the detection limit (<0.1 EU/ml) as tested by the Limulus amebocyte lysate assay (BioWhitaker, Walkersville, MD, USA). In addition, recently reports suggest that the microfiltration is a reliable and highly advantageous tool for the decontamination of samples [45], [46].…”

Section: Discussionmentioning

confidence: 99%

PEP-1-PON1 Protein Regulates Inflammatory Response in Raw 264.7 Macrophages and Ameliorates Inflammation in a TPA-Induced Animal Model

et al. 2014

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“…The samples that we bought were actually not ultrapure and already gave immunostimulatory signals as of the first step. The Pronova alginate is the most commonly applied alginate [14,24,27,32–34]. In our test sample, it was found to contain peptidoglycan (PG) and lipoteichoic acid (LTA).…”

Section: Discussionmentioning

confidence: 90%

A Technology Platform to Test the Efficacy of Purification of Alginate

et al. 2014

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Alginates are widely used in tissue engineering technologies, e.g., in cell encapsulation, in drug delivery and various immobilization procedures. The success rates of these studies are highly variable due to different degrees of tissue response. A cause for this variation in success is, among other factors, its content of inflammatory components. There is an urgent need for a technology to test the inflammatory capacity of alginates. Recently, it has been shown that pathogen-associated molecular patterns (PAMPs) in alginate are potent immunostimulatories. In this article, we present the design and evaluation of a technology platform to assess (i) the immunostimulatory capacity of alginate or its contaminants, (ii) where in the purification process PAMPs are removed, and (iii) which Toll-like receptors (TLRs) and ligands are involved. A THP1 cell-line expressing pattern recognition receptors (PRRs) and the co-signaling molecules CD14 and MD2 was used to assess immune activation of alginates during the different steps of purification of alginate. To determine if this activation was mediated by TLRs, a THP1-defMyD88 cell-line was applied. This cell-line possesses a non-functional MyD88 coupling protein, necessary for activating NF-κB via TLRs. To identify the specific TLRs being activated by the PAMPs, we use different human embryonic kidney (HEK) cell-line that expresses only one specific TLR. Finally, specific enzyme-linked immunosorbent assays (ELISAs) were applied to identify the specific PAMP. By applying this three-step procedure, we can screen alginate in a manner, which is both labor and cost efficient. The efficacy of the platform was evaluated with an alginate that did not pass our quality control. We demonstrate that this alginate was immunostimulatory, even after purification due to reintroduction of the TLR5 activating flagellin. In addition, we tested two commercially available purified alginates. Our experiments show that these commercial alginates contained peptidoglycan, lipoteichoic acid, flagellin, and even lipopolysaccharides (LPS). The platform presented here can be used to evaluate the efficacy of purification procedures in removing PAMPs from alginates in a cost-efficient manner.

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“…Activation of the LAL enzymatic system by LAp120 amounted to approximately 0.4 EU, and the addition of Poly B did not change this value ( Figure 7 ). As the ability to activate LAL is also shown by other organic components potentially contained in PM, such as glucans [ 48 ], lipoteichoic acid and peptidoglycan [ 49 ], only the part of the LAL activity inhibited by Poly B can be considered as the endotoxin content, since Poly B specifically neutralizes lipopolysaccharides of Gram-negative bacteria [ 45 ]. For NIST1648a at a concentration of 100 µg/mL, it would correspond to approximately 0.28 EU/mL, while LAp120 seemed to not contain any LAL-detectable amounts of endotoxin.…”

Section: Resultsmentioning

confidence: 99%

Gene Expression Changes Induced by Exposure of RAW 264.7 Macrophages to Particulate Matter of Air Pollution: The Role of Endotoxins

et al. 2022

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Despite the variable chemical and physical characteristics of particulate air pollutants, inflammation and oxidative stress have been identified as common mechanisms for cell damage and negative health influences. These effects are produced by organic components, especially by endotoxins. This study analyzed the gene expression profile after exposure of RAW 264.7 cells to the standard particulate matter (PM) material, NIST1648a, and PM with a reduced organic matter content, LAp120, in comparison to the effects of lipopolysaccharide (LPS). The selected parameters of cell viability, cell cycle progression, and metabolic and inflammatory activity were also investigated. Both forms of PM negatively influenced the parameters of cell activity. These results were generally reflected in the gene expression profile. Only NIST1648a, excluding LAp120, contained endotoxins and showed small but statistically significant pro-inflammatory activity. However, the gene expression profiling revealed strong pro-inflammatory cell activation induced by NIST1648a that was close to the effects of LPS. Changes in gene expression triggered by LAp120 were relatively small. The observed differences in the effects of NIST1648a and LAp120 were related to the content of organic matter in which bacterial endotoxins play an important role. However, other organic compounds and their interactions with other PM components also appear to be of significant importance.

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Microfiltration method of removal of bacterial contaminants and their monitoring by nitric oxide and Limulus assays

Cited by 9 publications

References 37 publications

PEP-1-PON1 Protein Regulates Inflammatory Response in Raw 264.7 Macrophages and Ameliorates Inflammation in a TPA-Induced Animal Model

PEP-1-PON1 Protein Regulates Inflammatory Response in Raw 264.7 Macrophages and Ameliorates Inflammation in a TPA-Induced Animal Model

A Technology Platform to Test the Efficacy of Purification of Alginate

Gene Expression Changes Induced by Exposure of RAW 264.7 Macrophages to Particulate Matter of Air Pollution: The Role of Endotoxins

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