Yvonne Williams

Probing Cell‐Type‐Specific Intracellular Nanoscale Barriers Using Size‐Tuned Quantum Dots

The compartmentalization of size-tuned luminescent semiconductor nanocrystal quantum dots (QDs) in four distinctive cell lines, which would be representative of the most likely environmental exposure routes to nanoparticles in humans, is studied. The cells are fixed and permeabilized prior to the addition of the QDs, thus eliminating any cell-membrane-associated effects due to active QD uptake mechanisms or to specificity of signaling routes in different cell types, but leaving intact the putative physical subcellular barriers. All quantitative assays are performed using a high content analysis (HCA) platform, thereby obtaining robust data on large cell populations. While smaller QDs 2.1 nm in diameter enter the nuclei and localize to the nucleoli in all cell types, the rate and dynamics of their passage vary depending on the cell origin. As the QD size is increased to 4.4 nm, penetration into the cell is reduced but each cell line displays its own cutoff size thresholds reflecting cell-type-determined cytoplasmic and nuclear pore penetration specificity. These results give rise to important considerations regarding the differential compartmentalization and susceptibility of organs, tissues, and cells to nanoparticles, and may be of prime importance for biomedical imaging and drug-delivery research employing nanoparticle-based probes and systems.

Activation of stress-related signalling pathway in human cells upon SiO2 nanoparticles exposure as an early indicator of cytotoxicity.

BackgroundNanomaterials such as SiO2 nanoparticles (SiO2NP) are finding increasing applications in the biomedical and biotechnological fields such as disease diagnostics, imaging, drug delivery, food, cosmetics and biosensors development. Thus, a mechanistic and systematic evaluation of the potential biological and toxic effects of SiO2NP becomes crucial in order to assess their complete safe applicability limits.ResultsIn this study, human monocytic leukemia cell line THP-1 and human alveolar epithelial cell line A549 were exposed to a range of amorphous SiO2NP of various sizes and concentrations (0.01, 0.1 and 0.5 mg/ml). Key biological indicators of cellular functions including cell population density, cellular morphology, membrane permeability, lysosomal mass/pH and activation of transcription factor-2 (ATF-2) were evaluated utilizing quantitative high content screening (HCS) approach and biochemical techniques. Despite the use of extremely high nanoparticle concentrations, our findings showed a low degree of cytotoxicity within the panel of SiO2NP investigated. However, at these concentrations, we observed the onset of stress-related cellular response induced by SiO2NP. Interestingly, cells exposed to alumina-coated SiO2NP showed low level, and in some cases complete absence, of stress response and this was consistent up to the highest dose of 0.5 mg/ml.ConclusionsThe present study demonstrates and highlights the importance of subtle biological changes downstream of primary membrane and endocytosis-associated phenomena resulting from high dose SiO2NP exposure. Increased activation of transcription factors, such as ATF-2, was quantitatively assessed as a function of i) human cell line specific stress-response, ii) SiO2NP size and iii) concentration. Despite the low level of cytotoxicity detected for the amorphous SiO2NP investigated, these findings prompt an in-depth focus for future SiO2NP-cell/tissue investigations based on the combined analysis of more subtle signalling pathways associated with accumulation mechanisms, which is essential for establishing the bio-safety of existing and new nanomaterials.

Correlation of platelet FcγRIIA polymorphism in refractory idiopathic (immune) thrombocytopenic purpura

FcγRIIA, a low affinity receptor for IgG, is a polymorphic molecule: FcγRIIA‐HH131, FcγRIIA‐HR131 and FcγRIIA‐RR131. This polymorphism influences the efficiency of the receptor to bind with IgG2. Recent reports on altered distribution amongst individuals with heparin‐induced thrombocytopenia (HIT) prompted us to examine the FcγRIIA polymorphism in a cohort of patients with refractory idiopathic (immune) thrombocytopenic purpura (ITP), in whom severe disease had required them to undergo splenectomy. 29 post splenectomy ITP individuals and 61 normal controls were investigated. Polymerase chain reaction (PCR) and a Southern blotting technique were used to determine the FcγRII polymorphism. Although the distribution of the allotypes of FcγRIIA in the control population was similar to that found in other European studies of Caucasian populations (15 (25%) HH131; 35 (57%) HR131; 11(18%) RR131), the patient group was skewed towards the RR131 allotype which has least efficiency for IgG2 binding (3 (10%) HH131; 12 (42%) HR131; 14 (48%) RR131; P < 0.005). These findings suggest that FcγRIIA polymorphisms may be implicated in the pathophysiology of ITP or may be responsible for modulating the immune response in this heterogenous autoimmune disease.

Synthesis, characterisation, and biological studies of CdTe quantum dot-naproxen conjugates.

The first naproxen–QD conjugates have been synthesised and investigated. These conjugates demonstrated interesting photophysical properties, good stability in an aggressive enzymatic medium, and cellular localisation in macrophage (THP-1) cells. These nanocomposites might have the potential to act as drug delivery and cellular imaging agents.

Comparison of three cell fixation methods for high content analysis assays utilizing quantum dots

Semiconductor nanoparticles or quantum dots are being increasingly utilized as fluorescent probes in cell biology both in live and fixed cell assays. Quantum dots possess an immense potential for use in multiplexing assays that can be run on high content screening analysers. Depending on the nature of the biological target under investigation, experiments are frequently required on cells retaining an intact cell membrane or also on those that have been fixed and permeabilized to expose intracellular antigens. Fixation of cell lines before or after the addition of quantum dots may affect their localization, emission properties and stability. Using a high content analysis platform we perform a quantitative comparative analysis of three common fixation techniques in two different cell lines exposed to carboxylic acid stabilized CdTe quantum dots.

Effect of lithium therapy on inflammatory response.

Chemiluminescence produced by normal cells was reduced in response to zymosan which was opsonized with serum from patients on prophylactic lithium therapy, compared to control serum from normal subjects (68±3.1 vs. 93±3.4 mV/5×105 cells). Preincubation of normal cells with serum from patients also resulted in reduced chemiluminescence activity when the cells were stimulated with autologous serum-coated zymosan (47±4.5 vs. 64±6.3 mV/5×105 cells). Spontaneous complement conversion was increased in the serum of patients on lithium therapy (46.3±3.8 vs. 25.3±2.5% conversion). These studies demonstrated that lithium, at safe therapeutic levels (0.4–0.9 mmol/liter), significantly altered complement conversion and had a marked affect on chemiluminescence activity by normal cells.

The use of Cellomics to study enterocyte cytoskeletal proteins in coeliac disease patients

Coeliac disease is characterised by inflammation of small intestinal mucosa accompanied by abnormal villous architecture. It is now accepted that some patients with positive coeliac serology tests may have minor mucosal lesions that may not be apparent on routine histopathological analysis. The aim of the study was to perform detailed examination of enterocyte morphology and cytoskeletal structures using a high content analysis technology. Duodenal biopsies from 14 untreated and 10 treated coeliac patients and from 20 non-coeliac controls were examined. Tissue sections from six patients (study group subjects) before and after the development of gluten-sensitive enteropathy were also investigated. Immunohistochemical studies were performed on paraffin-embedded sections using an anti-α-tubulin antibody. Significant differences in enterocyte morphology and intracellular cytoskeletal structures were demonstrated in patients with proven coeliac disease and in the study group subjects. These changes were present in study group biopsies before evidence of enteropathy, as assessed by routine microscopy. This is the first study to demonstrate detailed characteristics of enterocyte morphology in coeliac patients using a high content analysis approach. The use of this technology allows a quantitative analysis of enterocyte intracellular structures from routine biopsy material and permits detection of subtle changes that precede the characteristic histological lesion.

Type II Hereditary Angioedema—Presenting as Food Allergy

Hereditary angioedema (HAE) is caused by a genetic defect of C1 inhibitor (C1INH), a control protein of the classical complement pathway. In the majority of cases the disease is inherited in an autosomal dominant manner, however, up to 25% of patients may present with a de novo mutation. It is well established that approximately 85% of patients have low or undetectable levels of C1INH (type I HAE), however, 15% of patients can present with normal or increased levels of dysfunctional C1INH (type II HAE). The clinical manifestations of both types of HAE are very similar and are due to uncontrolled activation of the complement pathway. The characteristic clinical presentation is recurrent episodes of angioedema affecting different parts of the body including the face, hands, feet, upper airways, and abdomen. The disease has a prevalence of 1 in 50,000 and can often be misdiagnosed. While most children will have had at least one episode before the teenage years, many are not diagnosed until the second or third decade [1]. A 15-year-old female (S.O.H.) was referred to our clinical service, with a 4-year history of abdominal cramps and vomiting. In the past 2 years this was accompanied by facial swelling which caused breathing difficulties resulting in three separate visits to A&E with suspected anaphylactic

High Content Screening and Analysis with Nanotechnologies

Conventional fluorophors have proven to be invaluable in the arsenal of biomedical scientists for many years. However, they still suffer from many limitations. Commonly featuring a wide emission wavelength spectrum, most fluorophors have their own individual absorbance maxima, thus requiring separate activation sources and making multiparametric analysis difficult and costly.