Bothrops jararaca Snake Venom Inflammation Induced in Human Whole Blood: Role of the Complement System.

The clinical manifestations of envenomation by Bothrops species are complex and characterized by prominent local effects that can progress to tissue loss, physical disability, or amputation. Systemic signs can also occur, such as hemorrhage, coagulopathy, shock, and acute kidney failure. The rapid development of local clinical manifestations is accompanied by the presence of mediators of the inflammatory process originating from tissues damaged by the bothropic venom. Considering the important role that the complement system plays in the inflammatory response, in this study, we analyzed the action of Bothrops jararaca snake venom on the complement system and cell surface receptors involved in innate immunity using an ex vivo human whole blood model. B. jararaca venom was able to induce activation of the complement system in the human whole blood model and promoted a significant increase in the production of anaphylatoxins C3a/C3a-desArg, C4a/C4a-desArg, C5a/C5a-desArg and sTCC. In leukocytes, the venom of B. jararaca reduced the expression of CD11b, CD14 and C5aR1. Inhibition of the C3 component by Cp40, an inhibitor of C3, resulted in a reduction of C3a/C3a-desArg, C5a/C5a-desArg and sTCC to basal levels in samples stimulated with the venom. Exposure to B. jararaca venom induced the production of inflammatory cytokines and chemokines such as TNF-α, IL-8/CXCL8, MCP-1/CCL2 and MIG/CXCL9 in the human whole blood model. Treatment with Cp40 promoted a significant reduction in the production of TNF-α, IL-8/CXCL8 and MCP-1/CCL2. C5aR1 inhibition with PMX205 also promoted a reduction of TNF-α and IL-8/CXCL8 to basal levels in the samples stimulated with venom. In conclusion, the data presented here suggest that the activation of the complement system promoted by the venom of the snake B. jararaca in the human whole blood model significantly contributes to the inflammatory process. The control of several inflammatory parameters using Cp40, an inhibitor of the C3 component, and PMX205, a C5aR1 antagonist, indicates that complement inhibition may represent a potential therapeutic tool in B. jararaca envenoming.

Discovery and quantification of plastic particle pollution in human blood.

Plastic particles are ubiquitous pollutants in the living environment and food chain but no study to date has reported on the internal exposure of plastic particles in human blood. This study's goal was to develop a robust and sensitive sampling and analytical method with double shot pyrolysis - gas chromatography/mass spectrometry and apply it to measure plastic particles ≥700 nm in human whole blood from 22 healthy volunteers. Four high production volume polymers applied in plastic were identified and quantified for the first time in blood. Polyethylene terephthalate, polyethylene and polymers of styrene (a sum parameter of polystyrene, expanded polystyrene, acetonitrile butadiene styrene etc.) were the most widely encountered, followed by poly(methyl methacrylate). Polypropylene was analysed but values were under the limits of quantification. In this study of a small set of donors, the mean of the sum quantifiable concentration of plastic particles in blood was 1.6 µg/ml, showing a first measurement of the mass concentration of the polymeric component of plastic in human blood. This pioneering human biomonitoring study demonstrated that plastic particles are bioavailable for uptake into the human bloodstream. An understanding of the exposure of these substances in humans and the associated hazard of such exposure is needed to determine whether or not plastic particle exposure is a public health risk.

Plasma Corona Protects Human Immune Cells from Structurally Nanoengineered Antimicrobial Peptide Polymers.

Safe and effective antimicrobials are needed to combat emerging antibiotic-resistant bacteria. Structurally nanoengineered antimicrobial peptide polymers (termed SNAPPs) interact with bacterial cell membranes to potently kill bacteria but may also interact at some level with human cell membranes. We studied the association of four different SNAPPs with six different white blood cells within fresh whole human blood by flow cytometry. In whole human blood, SNAPPs had detectable association with phagocytic cells and B cells, but not natural killer and T cells. However, without plasma proteins and therefore no protein corona on the SNAPPs, a greater marked association of SNAPPs with all white blood cell types was detected, resulting in cytotoxicity against most blood cell components. Thus, the formation of a protein corona around the SNAPPs reduced the association and prevented human blood cell cytotoxicity of the SNAPPs. Understanding the bio-nano interactions of these SNAPPs will be crucial to ensuring that the design of next-generation SNAPPs and other promising antimicrobial nanomaterials continues to display high efficacy toward antibiotic-resistant bacteria while maintaining a low toxicity to primary human cells.

Influence of Poly(ethylene glycol) Molecular Architecture on Particle Assembly and Ex Vivo Particle-Immune Cell Interactions in Human Blood.

Poly(ethylene glycol) (PEG) is widely used in particle assembly to impart biocompatibility and stealth-like properties in vivo for diverse biomedical applications. Previous studies have examined the effect of PEG molecular weight and PEG coating density on the biological fate of various particles; however, there are few studies that detail the fundamental role of PEG molecular architecture in particle engineering and bio-nano interactions. Herein, we engineered PEG particles using a mesoporous silica (MS) templating method and investigated how the PEG building block architecture impacted the physicochemical properties (e.g., surface chemistry and mechanical characteristics) of the PEG particles and subsequently modulated particle-immune cell interactions in human blood. Varying the PEG architecture from 3-arm to 4-arm, 6-arm, and 8-arm generated PEG particles with a denser, stiffer structure, with increasing elastic modulus from 1.5 to 14.9 kPa, inducing an increasing level of immune cell association (from 15% for 3-arm to 45% for 8-arm) with monocytes. In contrast, the precursor PEG particles with the template intact (MS@PEG) were stiffer and generally displayed higher levels of immune cell association but showed the opposite trend-immune cell association decreased with increasing PEG arm numbers. Proteomics analysis demonstrated that the biomolecular corona that formed on the PEG particles minimally influenced particle-immune cell interactions, whereas the MS@PEG particle-cell interactions correlated with the composition of the corona that was abundant in histidine-rich glycoproteins. Our work highlights the role of PEG architecture in the design of stealth PEG-based particles, thus providing a link between the synthetic nature of particles and their biological behavior in blood.

In vitro proinflammatory gene expression changes in human whole blood after contact with plasma-treated implant surfaces.

BACKGROUND: The aim of this in vitro study was to identify changes in gene expression of proinflammatory cytokines in human whole blood after contact with titanium implant surfaces after plasma treatment. MATERIALS AND METHODS: Grade 4 titanium dental implants were conditioned with low-pressure plasma (LPP) and atmospheric-pressure plasma (APP) and submerged in human whole blood in vitro. Unconditioned implants and blood samples without implants served as control and negative control groups, respectively. Sampling was performed at 1, 8, and 24 h. Changes in mRNA expression levels of interleukin 1-beta (IL1-ß) and tumor necrosis factor-alpha (TNF-α) were assessed using RT-qPCR. RESULTS: In the control group, significant increases in IL1-ß and TNF-α expression were observed. Significant decreases in the expression of IL1-ß and TNF-α were identified in blood with implants after plasma treatment. CONCLUSION: Differences in gene expression of proinflammatory cytokines after incubation of plasma-conditioned titanium implants can be assessed using human whole blood. The results of the present study indicate that plasma treatment (APP and LPP) of titanium dental implants leads to downregulation of proinflammatory cytokine gene expression, which might be beneficial in early osseointegration.

The synthetic antimicrobial peptide LTX21 induces inflammatory responses in a human whole blood model and a murine peritoneum model.

The global spread of antimicrobial resistance and the increasing number of immune-compromised patients are major challenges in modern medicine. Targeting bacterial virulence or the human host immune system to increase host defence are important strategies in the search for novel antimicrobial drugs. We investigated the inflammatory response of the synthetic short antimicrobial peptide LTX21 in two model systems: a human whole blood ex vivo model and a murine in vivo peritoneum model - both reflecting early innate immune response. In the whole blood model, LTX21 increased the secretion of a range of different cytokines, decreased the level of tumour necrosis factor (TNF) and activated the complement system. In a haemolysis assay, we found 2.5% haemolysis at a LTX21 concentration of 500 mg/L. In the murine model, increased influx of white blood cells (WBCs) and polymorphonuclear neutrophils (PMNs) in the murine peritoneal cavity was observed after treatment with LTX21. In addition, LTX21 increased monocyte chemoattractant protein-1 (MCP-1). In conclusion, LTX21 affected the inflammatory response; the increase in cytokine secretion, complement activation and WBC influx indicates an activated inflammatory response. The present results indicate the impact of LTX21 on the host-pathogen interplay. Whether this will also affect the course of infection has to be investigated.

Iron oxide nanoparticles enhance Toll-like receptor-induced cytokines in a particle size- and actin-dependent manner in human blood.

Aim: To assess the effects of different-sized iron oxide nanoparticles (IONPs) on inflammatory responses in human whole blood. Materials & methods: Human whole blood with and without 10 and 30 nm IONPs was incubated with Toll-like receptor (TLR) ligands. Cytokine levels, complement activation, reactive oxygen species and viability were determined. Results: The 10 nm IONPs enhanced the TLR2/6, TLR4 and partly TLR8-mediated cytokine production, whereas the 30 nm IONPs partly enhanced TLR2/6 and decreased TLR8-mediated cytokine production. Particle-mediated enhancement of TLR4-induced cytokines could not be explained by complement activation, but was dependent on TLR4/MD2 and CD14, as well as actin polymerization. Conclusion: The IONPs differentially affected the TLR ligand-induced cytokines, which has important implications for biomedical applications of IONPs.

Three-dimensional ionic liquid-ferrite functionalized graphene oxide nanocomposite for pipette-tip solid phase extraction of 16 polycyclic aromatic hydrocarbons in human blood sample.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously found in the environment and have been proved to be prospectively associated with the risk of cancer. In this study, a simple method based on pipette-tip solid phase extraction (PT-SPE) and gas chromatography-mass spectrometry (GC-MS) has been firstly developed for the determination of 16 PAHs in human whole blood. Three-dimensional ionic liquid-ferrite functionalized graphene oxide nanocomposite (3D-IL-Fe3O4-GO) was used as sorbent in PT-SPE. Compared with conventional SPE method, the PT-SPE method was solvent-saving (1.0 mL), reusable (at least 10 times) and required less blood sample (200 µL). Affecting parameters on extraction efficiency were investigated and optimized. Under the optimized conditions, a good linearity was obtained and the recoveries of 16 PAHs at three spiked levels ranged from 85.0% to 115%. The limits of quantification (LOQs) were in the range of 0.007-0.013 µg/L. Furthermore, the developed method was successfully applied to the analysis of 16 PAHs in 14 human blood samples. The results showed that the predominant PAHs in human whole blood was low-molecular-weight PAHs, with the rank order phenanthrene (PHE)> naphthalene (NAP)> fluorene (FLU)> fluoranthene (FLT)> pyrene (PYR). Because of its simplicity, accuracy and reliability, the PT-SPE method combined with GC-MS demonstrated the applicability for clinical analysis and provided more information for PAHs exposure studies.

Efficient determination of six fatty acid ethyl ethers in human whole blood by gas chromatography-mass spectrometry.

Fatty acid ethyl esters (FAEEs) have been widely studied as specific markers of ethanol intake and mediators of ethanol-induced diseases. In the present study, a simple and rapid gas chromatography-mass spectrometry (GC-MS) method was established for the qualitative and quantitative analysis of six fatty acid ethyl esters (FAEEs), including ethyl myristate, ethyl palmitate, ethyl stearate, ethyl oleate, ethyl linoleate, and ethyl arachidonate, in human whole blood. FAEEs were extracted from 200 µL of human whole blood by a modified liquid-liquid extraction, and the hexane layer was injected directly into GC-MS with ethyl heptadecanoate as the internal standard. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 5-50 ng/mL and 15-200 ng/mL, respectively. Linearity ranged up to 10 µg/mL with r2 higher than 0.998. Accuracy was in the range of 90.3-109.7%, while intra-day and inter-day precision were 0.7-9.3% and 3.4-12.5%, respectively. This method was then applied to 38 real samples from forensic cases. Differences in the most common FAEEs between Chinese and Western subjects were discussed. The relationship of FAEE concentrations with age and gender was also investigated.

Iron oxide nanoparticles induce cytokine secretion in a complement-dependent manner in a human whole blood model.

Iron oxide nanoparticles (IONPs) are promising nanomaterials for biomedical applications. However, their inflammatory potential has not been fully established. Here, we used a lepirudin anti-coagulated human whole blood model to evaluate the potential of 10 nm IONPs to activate the complement system and induce cytokine production. Reactive oxygen species and cell death were also assessed. The IONPs activated complement, as measured by C3a, C5a and sC5b-9, and induced the production of pro-inflammatory cytokines in a particle-dose dependent manner, with the strongest response at 10 µg/mL IONPs. Complement inhibitors at C3 (compstatin analog Cp40) and C5 (eculizumab) levels completely inhibited complement activation and secretion of inflammatory mediators induced by the IONPs. Additionally, blockade of complement receptors C3aR and C5aR1 significantly reduced the levels of various cytokines, indicating that the particle-induced secretion of inflammatory mediators is mainly C5a and C3a mediated. The IONPs did not induce cell death or reactive oxygen species, which further suggests that complement activation alone was responsible for most of the particle-induced cytokines. These data suggest that the lepirudin anti-coagulated human whole blood model is a valuable ex vivo system to study the inflammatory potential of IONPs. We conclude that IONPs induce complement-mediated cytokine secretion in human whole blood.

A simple and rapid method of isolation of active polypeptide complex, yolkin, from chicken egg yolk.

A large number of bioactive peptides isolated from natural sources are known to play important physiological roles in the human body. It is possible to use these as alternative therapy agents. One example is yolkin which can be useful as a food supplement, a natural therapeutic agent for preventing and treating cognitive disorders of various origins, preferably in patients with unsatisfactory responses to known therapies. A new simple method of isolation of yolkin based on precipitation with ethanol or acetone was developed. The best precipitation efficiency of both ethanol and acetone was achieved when stirred into the starting material to a final concentration of 70%. These methods preserved the ability of yolkin to stimulate human whole blood cells to release anti-inflammatory cytokines and neurotrophins. At first we indicated that yolkin displayed a potential neuroprotective effect by the ability to stimulate cells to produce pro-survival brain derived neurotrophic factor (BDNF).

Chlorinated Flavonoids Modulate the Inflammatory Process in Human Blood.

Flavonoids are known to react with neutrophil-generated hypochlorous acid (HOCl) at inflammation loci to form stable mono- and dichlorinated products. Some of these products have been shown to retain or even enhance their inflammatory potential, but further information is required in a broader approach to inflammatory mechanisms. In that sense, we performed an integrated evaluation on the anti-inflammatory potential of a panel of novel chlorinated flavonoids and their parent compounds, in several steps of the complex inflammatory cascade, namely, in the activity of cyclooxygenase (COX)-1 and COX-2, and in the production of cytokines [interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)], and the chemokine, IL-8, as well as in the production of reactive species, using human whole blood as a representative in vitro model, establishing, whenever possible, a structure-activity relationship. Although luteolin was the most active compound, chlorinated flavonoids demonstrated a remarkable pattern of activity for the resolution of the inflammatory processes. Our results demonstrated that 6-chloro-3',4',5,7-tetrahydroxyflavone deserves scientific attention due to its ability to modulate the reactive species and cytokines/chemokine production. In this regard, the therapeutic potential of flavonoids' metabolites, and in this particular case the chlorinated flavonoids, should not be neglected.

Ultrasound-assisted dispersive liquid-liquid microextraction for the determination of seven recreational drugs in human whole blood using gas chromatography-mass spectrometry.

Recreational drugs have large impact on public health and security, and to monitor them is of urgent demand. In the present study, ultrasound-assisted dispersive liquid-liquid microextraction combined with the detection of gas chromatography-mass spectrometry was applied to the determination of seven common recreational drugs, including amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, meperidine, methadone and ketamine in 200µL of human whole blood. A series of factors which would affect the extraction efficiency were systematically investigated, including the nature and the volume of extraction and dispersing solvents, ultrasonication time, salting-out effect and pH value. The method consumed small amount of sample. The limits of detection and limits of quantification for each analyte were 10 and 40ng/mL, respectively, and the linearity was in the range of 0.04-25µg/mL (R2 higher than 0.99). Good specificity, precision (1.5-8.2% for the intra-day study and 2.6-12.8% for the inter-day study), satisfactory accuracy (85.0-117.1%) and extraction recovery (77.0-92.4%) were obtained, which makes it a high performance method for the determination of recreational drugs in human whole blood samples.

A high ratio of IC31(®) adjuvant to antigen is necessary for H4 TB vaccine immunomodulation.

A tuberculosis (TB) vaccine consisting of a recombinant fusion protein (H4) and a novel TLR9 adjuvant (IC31) is in clinical development. To better understand the H4-IC31 ratio, we measured the binding capacity of IC31 for H4 protein and immunized mice with formulations that contained limiting to excess ratios of IC31 to H4. An immunomodulated H4-specific IFNγ response was only observed when IC31 was present in excess of H4. Since TLR expression is species-specific and the vaccine is intended to boost BCG-primed immunity, we questioned whether data in mice would translate to humans. To address this question, we used the fresh human Whole Blood (hWB) recovered from BCG-vaccinated subjects to screen H4-IC31 formulations. We found IC31 modulation in hWB to be quite distinct from the TLR4-Adjuvant. Unlike TLR4-Adjuvant, IC31 formulations did not induce the pro-inflammatory cytokine TNFα, but modulated a robust H4-specific IFNγ response after 12 d of culture. We then re-stimulated the fresh hWB of 5 BCG-primed subjects with formulations that had excess or limiting IC31 binding for H4 protein and again found that an immunomodulated H4-specific IFNγ response needed an excess of IC31. Finally, we monitored the zeta (ζ) potential of H4-IC31 formulations and found that the overall charge of H4-IC31 particles changes from negative to positive once IC31 is in greater than 9-fold excess. Using two diverse yet mutually supportive approaches, we confirm the need for an excess of IC31 adjuvant in H4 TB vaccine formulations and suggest surface potential may be an important factor.

Concentration and correlations of perfluoroalkyl substances in whole blood among subjects from three different geographical areas in Korea.

Toxicity and persistence of perfluorinated alkyl substances (PFASs) in human have raised considerable concern and several biomonitoring studies throughout the world reported the widespread occurrence of these compounds in human tissues. However, information regarding influence of geographic, lifestyle and demographic factor on PFAS levels in human blood tissues is limited. In this study, whole blood samples collected in 2006-2007 from 319 donors from suburban Seoul (Suwon and Yongin), Busan and Yeosu in Korea were analyzed for perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorohexane sulfonate (PFHxS) and perfluorooctane sulfonamide (PFOSA). Blood donors classified into seven age groups with ages ranging from 8 to 82 years, and different lifestyles and socio-economic status. PFOS (median=4.15 ng/mL) was found at the highest concentration with a maximum concentration of 59.1 ng/mL. The concentrations of other PFASs were in the decreasing order of; PFOA (median=1.30 ng/mL)>PFNA (median=0.85 ng/mL)>PFHxS (median=0.47 ng/mL)>PFOSA (median=0.12 ng/mL). Geographical differences in the concentrations of five target PFASs were found. Significant positive relationships between PFAS concentrations and the age of the donors were found. Gender-related differences were found in the concentrations of PFOA, PFNA, PFHxS and PFOSA. No association was found between PFAS levels and several lifestyle factors and socio-economic status which included drinking habit, furniture/carpet in an indoor environment and monthly income. Occupation was an important determinant for PFNA and PFHxS concentrations in the whole blood. Except for PFOSA, significant associations were noted between PFASs concentrations and smoking habit. The results of this study provide information for further public health monitoring and safety management for PFASs in Korea.

Isolating epidermal growth factor receptor overexpressing carcinoma cells from human whole blood by bio-ferrography.

BACKGROUND: The epidermal growth factor receptor (EGFR) is overexpressed in carcinoma. In some cases, including in colorectal cancer, it is used as a therapeutic target. Bio-Ferrography is a nondestructive method for isolating magnetized cells and tissues from a fluid onto a glass slide based on their interaction with an external, strong, and focused magnetic field. METHODS: Here, we implement Bio-Ferrography to separate EGFR-positive cancer cells from EGFR-negative noncancer cells, mixed at a ratio of 1 to 1 × 10(6) , from either phosphate-buffered saline or human whole blood. Incubation of the cells with an anti-EGFR antibody and magnetic microbeads coupled to a secondary antibody was used to magnetize the target cells prior to the ferrographic analysis. RESULTS: A procedure was developed for "a proof of concept" isolation. Recovery values as high as 78% for 1 mL phosphate-buffered saline, and 53% for 1 mL human whole blood, with a limit-of-detection of 30 and 100 target cells, respectively, were achieved. CONCLUSIONS: These capture efficiency values are considered significant and, therefore, warrant further study on isolation of real circulating tumor cells from blood samples of patients, aiming at early diagnosis of EGFR-overexpressing tumor types.

Isolating EGFR Overexpressing Carcinoma Cells from Human Whole Blood by Bio-Ferrography.

Background: The epidermal growth factor receptor (EGFR) is overexpressed in carcinoma. In some cases, including in colorectal cancer, it is used as a therapeutic target. Bio-Ferrography is a non-destructive method for isolating magnetized cells and tissues from a fluid onto a glass slide based on their interaction with an external, strong and focused magnetic field. Methods: Here, we implement Bio-Ferrography to separate EGFR positive cancer cells from EGFR negative non-cancer cells, mixed at a ratio of 1 to 1 × 106 , from either PBS or human whole blood (HWB). Incubation of the cells with an anti-EGFR antibody and magnetic microbeads coupled to a secondary antibody was used to magnetize the target cells prior to the ferrographic analysis. Results: A procedure was developed for "a proof of concept" isolation. Recovery values as high as 78% for 1 mL PBS, and 53% for 1 mL HWB, with a limit of detection (LOD) of 30 and 100 target cells, respectively, were achieved. Conclusions: These capture efficiency values are considered significant and therefore warrant further study on isolation of real circulating tumor cells (CTCs) from blood samples of patients, aiming at early diagnosis of EGFR overexpressing tumor types. This article is protected by copyright. All rights reserved.

Gas signatures from Escherichia coli and Escherichia coli-inoculated human whole blood.

BACKGROUND: The gaseous headspace above naïve Escherichia Coli (E. coli) cultures and whole human blood inoculated with E. coli were collected and analyzed for the presence of trace gases that may have the potential to be used as novel, non-invasive markers of infectious disease. METHODS: The naïve E. coli culture, LB broth, and human whole blood or E. coli inoculated whole blood were incubated in hermetically sealable glass bioreactors at 37°C for 24 hrs. LB broth and whole human blood were used as controls for background volatile organic compounds (VOCs). The headspace gases were collected after incubation and analyzed using a gas chromatographic system with multiple column/detector combinations. RESULTS: Six VOCs were observed to be produced by E. coli-infected whole blood while there existed nearly zero to relatively negligible amounts of these gases in the whole blood alone, LB broth, or E. coli-inoculated LB broth. These VOCs included dimethyl sulfide (DMS), carbon disulfide (CS2), ethanol, acetaldehyde, methyl butanoate, and an unidentified gas S. In contrast, there were several VOCs significantly elevated in the headspace above the E. coli in LB broth, but not present in the E. coli/blood mixture. These VOCs included dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), methyl propanoate, 1-propanol, methylcyclohexane, and unidentified gases R2 and Q. CONCLUSIONS: This study demonstrates 1) that cultivated E. coli in LB broth produce distinct gas profiles, 2) for the first time, the ability to modify E. coli-specific gas profiles by the addition of whole human blood, and 3) that E. coli-human whole blood interactions present different gas emission profiles that have the potential to be used as non-invasive volatile biomarkers of E. coli infection.