The negative consequences of biofilms are widely reported. A defining feature of biofilms is the extracellular matrix, a complex mixture of biomacromolecules, termed EPS, which contributes to reduced ...antimicrobial susceptibility. EPS targeting is a promising, but underexploited, approach to biofilm control allowing disruption of the matrix and thereby increasing the susceptibility to antimicrobials. Nanoparticles (NPs) can play a very important role as ’carriers’ of EPS matrix disruptors, and several approaches have recently been proposed. In this review, we discuss the application of nanoparticles as antibiofilm technologies with a special emphasis on the role of the EPS matrix in the physicochemical regulation of the nanoparticle–biofilm interaction. We highlight the use of nanoparticles as a platform for a new generation of antibiofilm approaches.
Self-adhering bacterial communities embedded in a matrix of hydrated macromolecules, known as biofilms, are prevalent and widespread. The matrix offers protection to the bacteria, reducing the cell susceptibility to antimicrobials.Functional nanoparticles are a promising technology to control or eradicate biofilms, providing the ability to enhance antimicrobial transport to the cell vicinity or alternatively to carry matrix dispersion agents.Critical to the development of novel strategies to control biofilm infections is an in-depth knowledge of the biofilm matrix which is still poorly understood due to its spatial and chemical variability and complexityA fundamental understanding of NP–EPS interactions has the potential to improve our ability to design more effective antibiofilm strategies.
The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic ...cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner.
Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways.
CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) ...antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE<E<<L). Moreover, double-knockdown experiments showed that BILF1 and BNLF2a co-operate to further inhibit antigen presentation of L epitopes. Together, these data firstly indicate which potential immune-evasion functions are actually relevant in the context of lytic virus replication, and secondly identify lytic-cycle phase-specific effects that provide mechanistic insight into the immunodominance pattern seen for CD8+ T cell responses to EBV lytic antigens.
The ubiquitous nature of bacterial biofilms combined with the enhanced resistance towards antimicrobials has led to the development of an increasing number of strategies for biofilm eradication. Such ...strategies must take into account the existence of extracellular polymeric substances, which obstruct the diffusion of antibiofilm agents and assists in the maintenance of a well-defended microbial community. Within this context, nanoparticles have been studied for their drug delivery efficacy and easily customised surface. Nevertheless, there usually is a requirement for nanocarriers to be used in association with an antimicrobial agent; the intrinsically antimicrobial nanoparticles are most often made of metals or metal oxides, which is not ideal from ecological and biomedical perspectives. Based on this, the use of polymeric micelles as nanocarriers is appealing as they can be easily prepared using biodegradable organic materials.
In the present work, micelles comprised of poly(lactic-co-glycolic acid) and dextran are prepared and then functionalised with curcumin. The effect of the functionalisation in the micelle's physical properties was elucidated, and the antibacterial and antibiofilm activities were assessed for the prepared polymeric nanoparticles against Pseudomonas spp. cells and biofilms. It was found that the nanoparticles have good penetration into the biofilms, which resulted in enhanced antibacterial activity of the conjugated micelles when compared to free curcumin. Furthermore, the curcumin-functionalised micelles were efficient at disrupting mature biofilms and demonstrated antibacterial activity towards biofilm-embedded cells.
Curcumin-functionalised poly(lactic-co-glycolic acid)-dextran micelles are novel nanostructures with an intrinsic antibacterial activity tested against two Pseudomonas spp. strains that have the potential to be further exploited to deliver a secondary bioactive molecule within its core.
Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV ...immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV-infected cells undergoing lytic replication in vitro, and studies in both humans and mice with reconstituted human immune systems have shown that NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV-infected cells undergoing lytic replication and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as-yet-unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, while cells entering the lytic cycle become sensitized to NK cell killing, we observed that cells in the late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses.
This report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV-infected B cells undergoing lytic replication is dependent upon the phase of the lytic cycle. Induction of the lytic cycle is associated with acquired sensitization to NK cell killing, while progress through the late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, indicating important roles for the BZLF1 immediate early transactivator, the BHRF1 vBcl-2 homologue, and a novel ligand for the DNAM-1 NK cell receptor.
Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha‐fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical ...biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer‐specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity‐optimized T‐cell receptor (TCR) with specificity to AFP/HLA‐A*02+ tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA‐A*02‐restricted AFP158‐166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X‐scan) and testing TCR‐transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR‐transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T‐cell immunotherapy.
Soweto and Lenasia, the most densely populated area of South Africa, is simultaneously a thriving metropolis, with a fair share of people still living in squalor conditions directly dependant on the ...natural resources. Because of industrialisation the populace and environment in this urban area are exposed to various pollutants. The aquatic environment was selected as a proxy to study the effect of industrial pollution in this area. The concentrations, source identification, and various environmental risks of polycyclic aromatic hydrocarbons (PAHs) were determined in sediments of the upper reaches of the Klip River. Composite sediment samples collected in low-flow conditions in 2013 and 2014 ranged from 270–5400 ng/g. The PAHs in this aquatic ecosystem were dominated by 4-ring congeners and could be attributed to combustion of organic fuels by chemical mass balance. Heavy traffic and industrial complexes in the northern part of the study area were responsible for the PAH fingerprints. Probable adverse effects such as toxicity to benthic biota were proven after comparison with international sediment quality guidelines (SQG) both survey years. Toxic equivalence quotients (TEQs) calculated for the sediments using fish potency factors (FPFs) were up to 30 times greater than the Canadian guideline for dioxin-like compounds, indicating high probability of carcinogenic effect to fish mediated through the aryl-hydrocarbon receptor. Finally, sediments in the area posed moderate to high ecological risk, which corroborates the other toxicity assessments. The advantage of investigating multiple risk endpoints, is the comprehensive results obtained that allows for a more realistic representation of the study area. Consequently more aspects are kept into account that results in better conclusions.
We investigated the presence, levels, relationships, and risks of HCHs, DDTs, chlordanes, mirex, PCBs, and brominated flame retardants (BFRs) in terrestrial and aquatic bird eggs from an area in ...South Africa where DDT is used for malaria control. We found one of the highest ΣDDT levels reported this century; 13000ng/gwm (wet mass) in Grey Heron eggs which exceeds critical levels for reproductive success (3000ng/gwm) calculated for Brown Pelicans, with a no-effect level estimated at 500ng/gwm. Even higher ΣDDT levels at 16000ng/gwm were found in House Sparrow eggs (possibly the highest ever recorded for sparrows), with a maximum of 24400ng/gwm. Significant eggshell thinning in Cattle Egrets (33% between thickest and thinnest) was associated with increased levels of p,p′-DDT and p,p′-DDE. There were indications of unknown use of DDT and lindane. Relative to DDT, PCBs and BFRs levels were quite low. Ordinated data showed that different terrestrial pollutant profiles converged to a homogenised aquatic profile. Converging profiles, high levels of DDT in heron and sparrow eggs, and thinning eggs shells, indicate risk and impacts at release, in the aquatic environment, and in between. If characteristic life-strategies of birds in warm areas (e.g. longer-lived and fewer eggs per clutch) increases the risk compared with similar birds living in colder regions when both experience the same environmental pollutant levels, then malaria control using DDT probably has more significant impacts on biota than previously realised. Therefore, risk assessment and modelling without hard data may miss crucial impacts and risks, as the chemical use patterns and ecologies in Africa and elsewhere may differ from the conditions and assumptions of existing risk assessment and modelling parameters. Consideration of other findings associated with DDT from the same area (intersex in fish and urogental birth defects in baby boys), together with the findings of this study (high levels of DDT in bird eggs, eggshell thinning in the Cattle Egrets, and the apparent absence of breeding piscivore birds in the sprayed area) are strongly suggestive of negative impacts from DDT spraying for Malaria control. Our data presents strong arguments for an expedited process of replacing DDT with sustainable methods.
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•POPs data in bird eggs from an area where malaria is controlled with DDT.•We sampled heron, egret, sparrow and weaver eggs.•PCB and BFR levels were low, with high levels of DDT in heron and sparrow eggs.•Eggshell thinning was found in egret eggs.•There is an urgent need to find sustainable alternatives to DDT.
A substantial obstacle to the success of adoptive T cell-based cancer immunotherapy is the sub-optimal affinity of T-cell receptors (TCRs) for most tumor antigens. Genetically engineered TCRs that ...have enhanced affinity for specific tumor peptide-MHC complexes may overcome this barrier. However, this enhancement risks increasing weak TCR cross-reactivity to other antigens expressed by normal tissues, potentially leading to clinical toxicities. To reduce the risk of such adverse clinical outcomes, we have developed an extensive preclinical testing strategy, involving potency testing using 2D and 3D human cell cultures and primary tumor material, and safety testing using human primary cell and cell-line cross-reactivity screening and molecular analysis to predict peptides recognized by the affinity-enhanced TCR. Here, we describe this strategy using a developmental T-cell therapy, ADP-A2M4, which recognizes the HLA-A2-restricted MAGE-A4 peptide GVYDGREHTV. ADP-A2M4 demonstrated potent anti-tumor activity in the absence of major off-target cross-reactivity against a range of human primary cells and cell lines. Identification and characterization of peptides recognized by the affinity-enhanced TCR also revealed no cross-reactivity. These studies demonstrated that this TCR is highly potent and without major safety concerns, and as a result, this TCR is now being investigated in two clinical trials (NCT03132922, NCT04044768).
Biofilms are microbial communities embedded in an extracellular polymeric matrix and display an enhanced tolerance to the action of antimicrobials. The emergence of novel functionalised nanoparticles ...is considered a promising avenue for the development of biofilm-specific antimicrobial technologies. However, there is a gap in the understanding of interactions between nanoparticles and the biofilm matrix. Particularly, questions are raised on how nanoparticle charge and surface groups play a role in aggregation when in contact with biofilm components. Herein we present the synthesis of four types of silica nanoparticles and undertake an analysis of their interactions with Pseudomonas fluorescens biofilm matrix. The effect of the biofilm matrix components on the charge and aggregation of the nanoparticles was assessed. Additionally, the study focused on the role of matrix proteins, with the in-depth characterisation of the protein corona of each nanoparticle by Liquid Chromatography with Tandem Mass Spectrometry experiments. The protein corona composition is dependent on the nanoparticle type; non-functionalised nanoparticles show less protein selectivity, whereas carboxylate-functionalised nanoparticles prefer proteins with a higher isoelectric point. These outcomes provide insights into the field of biofilm-nanoparticle interactions that can be valuable for the design of new nano-based targeting systems in future anti-biofilm applications.
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