Lorna Proudfoot

The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line

This study investigated the uptake, kinetics and cellular distribution of different surface coated quantum dots (QDs) before relating this to their toxicity. J774.A1 cells were treated with organic, COOH and NH2 (PEG) surface coated QDs (40 nM). Model 20 nm and 200 nm COOH-modified coated polystyrene beads (PBs) were also examined (50 microg ml(-1)). The potential for uptake of QDs was examined by both fixed and live cell confocal microscopy as well as by flow cytometry over 2 h. Both the COOH 20 nm and 200 nm PBs were clearly and rapidly taken up by the J774.A1 cells, with uptake of 20 nm PBs being relatively quicker and more extensive. Similarly, COOH QDs were clearly taken up by the macrophages. Uptake of NH2 (PEG) QDs was not detectable by live cell imaging however, was observed following 3D reconstruction of fixed cells, as well as by flow cytometry. Cells treated with organic QDs, monitored by live cell imaging, showed only a small amount of uptake in a relatively small number of cells. This uptake was insufficient to be detected by flow cytometry. Imaging of fixed cells was not possible due to a loss in cell integrity related to cytotoxicity. A significant reduction (p<0.05) in the fluorescent intensity in a cell-free environment was found with organic QDs, NH2 (PEG) QDs, 20 nm and 200 nm PBs at pH 4.0 (indicative of an endosome) after 2 h, suggesting reduced stability. No evidence of exocytosis was found over 2 h. These findings confirm that surface coating has a significant influence on the mode of NP interaction with cells, as well as the subsequent consequences of that interaction.

Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos.

The potential toxicity of carbon nanotubes (CNTs) has been compared to pathogenic fibres such as asbestos. It is important to test this hypothesis to ascertain safe methods for CNT production, handling and disposal. In this study aspects reported to contribute to CNT toxicity were assessed: length, aspect ratio, iron content and crystallinity; with responses compared to industrially produced MWCNTs and toxicologically relevant materials such as asbestos. The impacts of these particles on a range of macrophage models in vitro were assessed due to the key role of macrophages in particle clearance and particle/fibre-induced disease. Industrially produced and long MWCNTs were cytotoxic to cells, and were potent in inducing pro-inflammatory and pro-fibrotic immune responses. Short CNTs did not induce any cytotoxicity. Frustrated phagocytosis was most evident in response to long CNTs, as was respiratory burst and reduction in phagocytic ability. Short CNTs, metal content and crystallinity had less or no influence on these endpoints, suggesting that many responses were fibre-length dependent. This study demonstrates that CNTs are potentially pathogenic, as they were routinely found to induce detrimental responses in macrophages greater than those induced by asbestos at the same mass-based dose.

Zinc oxide nanoparticles and monocytes: Impact of size, charge and solubility on activation status

Zinc oxide (ZnO) particle induced cytotoxicity was dependent on size, charge and solubility, factors which at sublethal concentrations may influence the activation of the human monocytic cell line THP1. ZnO nanoparticles (NP; average diameter 70nm) were more toxic than the bulk form (<44μm mesh) and a positive charge enhanced cytotoxicity of the NP despite their relatively high dissolution. A positive charge of the particles has been shown in other studies to have an influence on cell viability. Centrifugal filtration using a cut off of 5kDa and Zn element analysis by atomic absorption spectroscopy confirmed that exposure of the ZnO particles and NP to 10% foetal bovine serum resulted in a strong association of the Zn(2+) ion with protein. This association with protein may influence interaction of the ZnO particles and NP with THP1 cells. After 24h exposure to the ZnO particles and NP at sublethal concentrations there was little effect on immunological markers of inflammation such as HLA DR and CD14, although they may induce a modest increase in the adhesion molecule CD11b. The cytokine TNFα is normally associated with proinflammatory immune responses but was not induced by the ZnO particles and NP. There was also no effect on LPS stimulated TNFα production. These results suggest that ZnO particles and NP do not have a classical proinflammatory effect on THP1 cells.

Expression of C-Reactive Protein in Human Lung Epithelial Cells and Upregulation by Cytokines and Carbon Particles

C-reactive protein (CRP) is the prototypic human acute-phase protein and is found at increased levels in the blood during episodes of inflammation. CRP was generally thought to be produced only by hepatocytes; however, several studies have shown extrahepatic synthesis of CRP. A previous study showed that PM10 and ultrafine carbon black (ufCB) were able to induce CRP expression in A549 cells. This study aims to examine the factors that lead to the production of CRP in A549 cells. A549 human lung epithelial cells were treated with cytokines (interleukin 6, tumor necrosis factor α, interferon γ, or interleukin 1β) or carbon particles (CB and ufCB) for 18 h. It was found that CRP could be expressed within the cells and that CRP was secreted from the cells particularly with tumor necrosis factor α, CB and ufCB treatments. It was also found that this expression of CRP with CB and ufCB treatments was dependent on nuclear factor kappa B (NFκB). The expression of CRP in A549 cells may indicate an important role for CRP expression and secretion from lung epithelial cells in response to inflammatory stimuli.

Anti‐inflammatory responses and oxidative stress in Nippostrongylus brasiliensis‐induced pulmonary inflammation

Migration of L3 larvae of Nippostrongylus brasiliensis through the lungs of the rat, during primary infection, was studied at 24 h, 72 h and 8 days. At 24 h p.i., there was evidence of damage to lung epithelial cells and microvasculature, with increased protein and γ‐glutamyl transpeptidase in the bronchoalveolar lavage (BAL) fluid. However, there was little evidence of inflammatory cell recruitment. At 24 h p.i., there was a significant reduction in the inflammatory cytokine tumour necrosis factor α. Superoxide (O2−·) production was also reduced, accompanied by an increase in superoxide dismutase activity. Lipid peroxidation was reduced at 24 h p.i. and L3 larvae were shown to possess high levels of glutathione compared to host lung tissue. Nitric oxide, detected as nitrite, was produced in BAL fluid, and inducible nitric oxide synthase protein was increased by 72 h p.i. There was evidence of peroxynitrite production throughout the infection period with specific protein bands nitrosylated at 75, 30 and 25 kDa. It appears that despite early evidence of lung damage, the inflammation was reduced in response to L3 larvae of N. brasiliensis.

Inhibition of neutrophil recruitment by ES of Nippostrongylus brasiliensis.

It has been reported that excretory–secretory (ES) material from the parasitic nematode Nippostrongylus brasiliensis has potential modulatory effects on the hosts immune system. We observed that intratracheal instillation of ES from the L3 stage of the parasite reduced neutrophil numbers in LPS‐induced inflammation as assessed by bronchoalveolar lavage.

Cationic host defense peptides; novel antimicrobial therapeutics against Category A pathogens and emerging infections.

Cationic Host Defense Peptides (HDP, also known as antimicrobial peptides) are crucial components of the innate immune system and possess broad-spectrum antibacterial, antiviral, and immunomodulatory activities. They can contribute to the rapid clearance of biological agents through direct killing of the organisms, inhibition of pro-inflammatory mediators such as lipopolysaccharide, and by modulating the inflammatory response to infection. Category A biological agents and materials, as classified by the United States National Institutes for Health, the US Centers for Disease Control and Prevention, and the US Department of Homeland Security, carry the most severe threat in terms of human health, transmissibility, and preparedness. As such, there is a pressing need for novel frontline approaches for prevention and treatment of diseases caused by these organisms, and exploiting the broad antimicrobial activity exhibited by cationic host defense peptides represents an exciting priority area for clinical research. This review will summarize what is known about the antimicrobial and antiviral effects of the two main families of cationic host defense peptides, cathelicidins, and defensins in the context of Category A biological agents which include, but are not limited to; anthrax (Bacillus anthracis), plague (Yersinia pestis), smallpox (Variola major), tularemia (Francisella tularensis). In addition, we highlight priority areas, particularly emerging viral infections, where more extensive research is urgently required.

Effect of Nippostrongylus brasiliensis L3 ES on inflammatory mediator gene transcription in lipopolysaccharide lung inflammation.

The anti‐inflammatory properties of parasitic helminths have been largely linked to their excretory–secretory (ES) products. Some studies have noted a lack of TNF‐α production and limited recruitment of neutrophils into the lungs after Nippostrongylus brasiliensis infection. We previously reported that instillation of ES from L3 larvae of N. brasiliensis to the lungs could inhibit the recruitment of neutrophils on a background of LPS‐induced inflammation. A similar reduction in neutrophil recruitment was observed in this study. This reduction was associated with the significant inhibition in gene transcription of the adhesion molecule, ICAM‐1, and the chemokine, MIP‐2 in bronchoalveolar lavage (BAL) cells. The LPS‐stimulated gene transcription of the pro‐inflammatory cytokines TNF‐α and IL‐1β was also significantly reduced by L3 ES. Inducible nitric oxide synthase (iNOS) is normally elevated in classically activated macrophages, however, in this case gene transcription of iNOS was inhibited by L3 ES and may suggest a phenotype change to anti‐inflammatory. The general inhibition of pro‐inflammatory mediators observed in this study suggests that infective stage L3 larvae excrete and/or secrete inhibitory products capable of modifying the normally potent LPS inflammatory response.

Parasitic helminths tip the balance: potential anti-inflammatory therapies

Parasitic helminths are worms that are classified within the phyla Nematoda (roundworms) and Platyhelminthes (flatworms) (see Table 1). Some nematode species, Filariae being a notable example, are able to coexist with their human host for decades. Interestingly, although millions suffer severe morbidity, a lower incidence of allergy and autoimmune disease has been reported in infected individuals.1,2 Viral and bacterial infections, and autoimmune diseases such as type I diabetes and multiple sclerosis typically induce a T helper type 1 (Th1) pro-inflammatory response producing cytokines such as interleukin-12 (IL-12), interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α). Chronic parasitic helminth infection is associated with high levels of immunoglobulin E (IgE), eosinophilia, mastocytosis and a predominantly Th2 immune response. This Th2 response is characterized by production of IL-4, IL-5, IL-10 and IL-13,3 a cytokine pattern that is also linked to an anti-inflammatory phenotype. There is evidence that a Th2 response is protective in the case of gastrointestinal nematodes.4 However, there is also overwhelming evidence from other helminth infections that a Th2 response may provide an anti-inflammatory regulatory environment.1,5 The counter-regulatory effects of Th1 and Th2 cytokines can be seen in many immune responses and there is strong evidence that the Th2 response generated by helminths can down-regulate Th1 responsiveness to other infections.

Cathelicidins display conserved direct antiviral activity towards rhinovirus

&NA; Human rhinoviruses (HRVs) are the most common cause of viral respiratory tract infections, and are associated with significant morbidity and mortality in immunocompromised individuals and patients with pre‐existing pulmonary conditions. The therapeutic options available are extremely limited and therefore novel therapeutics for HRV infections are of significant interest. Cathelicidins have been shown to have potent antiviral activity against a range of pathogens and are known to be key immunomodulatory mediators during infection. We therefore assessed the antiviral potential of cathelicidins from humans and other mammalian species against HRV, together with the potential for the human cathelicidin to modulate apoptotic pathways and alter cell viability during HRV infection. We demonstrate that LL‐37, the porcine cathelicidin Protegrin‐1, and the ovine cathelicidin SMAP‐29 display potent antiviral activity towards HRV and that this activity is visible when either the virus is exposed to the peptides prior to cell infection or after cells have been infected. We further demonstrate that, in contrast to established findings with bacterial infection models, LL‐37 does not induce apoptosis or necrosis in HRV‐infected lung epithelial cells at physiological or superphysiological concentrations, but does reduce the metabolic activity of infected cells compared to uninfected cells treated with similar peptide concentrations. Collectively, the findings from this study demonstrate that the mechanism of action of cathelicidins against rhinovirus is by directly affecting the virus and we propose that the delivery of exogenous cathelicidins, or novel synthetic analogues, represent an exciting and novel therapeutic strategy for rhinovirus infection. HighlightsThe host defence peptide LL‐37 can reduce Human Rhinovirus 1B (HRV1B) replication in airway epithelial cells.The antiviral activity of LL‐37 is sequence specific.LL‐37 reduces the metabolic activity of cells infected with HRV1B without inducing substantial apoptotic or necrotic cell death.The antiviral activity of cathelicidin peptides towards HRV1B is conserved in peptides from other mammalian species including pig and sheep.