Darren W. Sexton

Single-cell gene expression analysis reveals genetic associations masked in whole-tissue experiments

Gene expression in multiple individual cells from a tissue or culture sample varies according to cell-cycle, genetic, epigenetic and stochastic differences between the cells. However, single-cell differences have been largely neglected in the analysis of the functional consequences of genetic variation. Here we measure the expression of 92 genes affected by Wnt signaling in 1,440 single cells from 15 individuals to associate single-nucleotide polymorphisms (SNPs) with gene-expression phenotypes, while accounting for stochastic and cell-cycle differences between cells. We provide evidence that many heritable variations in gene function--such as burst size, burst frequency, cell cycle-specific expression and expression correlation/noise between cells--are masked when expression is averaged over many cells. Our results demonstrate how single-cell analyses provide insights into the mechanistic and network effects of genetic variability, with improved statistical power to model these effects on gene expression.

Methods for qPCR gene expression profiling applied to 1440 lymphoblastoid single cells

Highlights ► Microfluidic arrays enable analysis of 96 qPCR assays on 1440 single cells. ► Detailed methods on obtaining qPCR data and performing preliminary data processing. ► Data from sufficient cells to address noise inherent in single-cell transcription. ► Methods used for conventional qPCR do not necessarily apply to single-cell qPCR.

Cetirizine and levocetirizine inhibit eotaxin-induced eosinophil transendothelial migration through human dermal or lung microvascular endothelial cells

Background Several second‐generation antihistamines have documented anti‐inflammatory effects which appear independent of H1‐receptor blockade. We investigated the inhibitory effect of cetirizine and its active enantiomer levocetirizine on eosinophil transendothelial migration (TEM) through monolayers of normal human dermal microvascular endothelial cells (HMVEC‐d) or human lung microvascular endothelial cells (HMVEC‐l).

Phagocytosis of apoptotic eosinophils but not neutrophils by bronchial epithelial cells

Background We have previously demonstrated that human bronchial epithelial cells engulf apoptotic eosinophils.

Trypanosoma brucei:chemical evidence that cathepsin L is essential for survival and a relevant drug target

The protozoan parasite causing human African trypanosomiasis, Trypanosoma brucei, displays cysteine peptidase activity, the chemical inhibition of which is lethal to the parasite. This activity comprises a cathepsin B (TbCATB) and a cathepsin L (TbCATL). Previous RNA interference (RNAi) data suggest that TbCATB rather than TbCATL is essential to survival even though silencing of the latter was incomplete. Also, chemical evidence supporting the essentiality of either enzyme which would facilitate a target-based drug development programme is lacking. Using specific peptidyl inhibitors and substrates, we quantified the contributions of TbCATB and TbCATL to the survival of T. brucei. At 100 μM, the minimal inhibitory concentration that kills all parasites in culture, the non-specific cathepsin inhibitors, benzyloxycarbonyl-phenylalanyl-arginyl-diazomethyl ketone (Z-FA-diazomethyl ketone) and (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline methyl ester (CA-074Me) inhibited TbCATL and TbCATB by >99%. The cathepsin L (CATL)-specific inhibitor, ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) (CAA0225), killed parasites with >99% inhibition of TbCATL but only 70% inhibition of TbCATB. Conversely, the cathepsin B (CATB)-specific inhibitor, (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074), did not affect survival even though TbCATB inhibition at >95% was statistically indistinguishable from the complete inhibition by Z-FA-diazomethyl ketone and CA-074Me. The observed inhibition of TbCATL by CA-074 and CA-074Me was shown to be facilitated by the reducing intracellular environment. All inhibitors, except the CATB-specific inhibitor, CA-074, blockaded lysosomal hydrolysis prior to death. The results suggest that TbCATL, rather than TbCATB, is essential to the survival of T. brucei and an appropriate drug target.

Ligation of CD45 and the isoforms CD45RA and CD45RB accelerates the rate of constitutive apoptosis in human eosinophils

BACKGROUND Eosinophils are important effector cells in asthma pathogenesis, and an understanding of the mechanisms involved in eosinophil apoptosis induction might thus be relevant to the resolution of asthmatic inflammation. OBJECTIVE Our aim was to determine the role of the common leukocyte antigen CD45 and the isoforms CD45RA, CD45RB, and CD45RO in human eosinophil apoptosis induction. METHODS Immmunostaining and flow cytometry were used to assess CD45 and CD45 isoform expression by eosinophils purified with use of density gradients and immunomagnetic negative selection. Apoptosis induction was measured by binding of fluorescein isothiocyanate-labeled annexin V to eosinophils cultured for 20 hours alone or with saturating quantities of mAb against CD45, CD45RA, CD45RB, CD45RO, CD9, CD11b, and isotype-matched controls in the presence or absence of GM-CSF. RESULTS Freshly isolated eosinophils had high expression of CD45 and CD45RO, modest expression of CD45RB, and low expression of CD45RA. Eosinophils cultured alone for 20 hours were found to be approximately 20% to 25% apoptotic. Incubation with mAb against CD45, CD45RA, and CD45RB resulted in significant (P <.005) enhancement (>100%) of their constitutive rate of apoptosis. Incubation with CD45RO, CD11b, CD9 mAb, or isotype controls had no significant effect on the rate of eosinophil constitutive apoptosis. The addition of GM-CSF inhibited eosinophil apoptosis but did not prevent CD45, CD45RA, or CD45RB mAb-dependent apoptosis induction. CONCLUSION These data indicate that ligation of CD45, CD45RA, or CD45RB represents a novel pathway for the induction of apoptosis in human eosinophils.

Effectiveness of common household cleaning agents in reducing the viability of human influenza A/H1N1

Background In the event of an influenza pandemic, the majority of people infected will be nursed at home. It is therefore important to determine simple methods for limiting the spread of the virus within the home. The purpose of this work was to test a representative range of common household cleaning agents for their effectiveness at killing or reducing the viability of influenza A virus. Methodology/Principal Findings Plaque assays provided a robust and reproducible method for determining virus viability after disinfection, while a National Standard influenza virus RT-PCR assay (VSOP 25, www.hpa-standardmethods.org.uk) was adapted to detect viral genome, and a British Standard (BS:EN 14476:2005) was modified to determine virus killing. Conclusions/Significance Active ingredients in a number of the cleaning agents, wipes, and tissues tested were able to rapidly render influenza virus nonviable, as determined by plaque assay. Commercially available wipes with a claimed antiviral or antibacterial effect killed or reduced virus infectivity, while nonmicrobiocidal wipes and those containing only low concentrations (<5%) of surfactants showed lower anti-influenza activity. Importantly, however, our findings indicate that it is possible to use common, low-technology agents such as 1% bleach, 10% malt vinegar, or 0.01% washing-up liquid to rapidly and completely inactivate influenza virus. Thus, in the context of the ongoing pandemic, and especially in low-resource settings, the public does not need to source specialized cleaning products, but can rapidly disinfect potentially contaminated surfaces with agents readily available in most homes.

The Serum Opsonin L-ficolin Is Detected in Lungs of Human Transplant Recipients Following Fungal Infections and Modulates Inflammation and Killing of Aspergillus fumigatus

BACKGROUND Invasive aspergillosis (IA) is a life-threatening systemic fungal infection in immunocompromised individuals that is caused by Aspergillus fumigatus. The human serum opsonin, L-ficolin, has been observed to recognize A. fumigatus and could participate in fungal defense. METHODS Using lung epithelial cells, primary human monocyte-derived macrophages (MDMs), and neutrophils from healthy donors, we assessed phagocytosis and killing of L-ficolin-opsonized live A. fumigatus conidia by flow cytometry and microscopy. Additionally, cytokines were measured by cytometric bead array, and L-ficolin was measured in bronchoalveolar lavage (BAL) fluid from lung transplant recipients by enzyme-linked immunosorbent assay. RESULTS L-ficolin opsonization increased conidial uptake and enhanced killing of A. fumigatus by MDMs and neutrophils. Opsonization was also shown to manifest an increase in interleukin 8 release from A549 lung epithelial cells but decreased interleukin 1β, interleukin 6, interleukin 8, interleukin 10, and tumor necrosis factor α release from MDMs and neutrophils 24 hours after infection. The concentration of L-ficolin in BAL fluid from patients with fungal infection was significantly higher than that for control subjects (P = .00087), and receiving operating characteristic curve analysis highlighted the diagnostic potential of L-ficolin for lung infection (area under the curve, 0.842; P < .0001). CONCLUSIONS L-ficolin modulates the immune response to A. fumigatus. Additionally, for the first time, L-ficolin has been demonstrated to be present in human lungs.

H‐ficolin binds Aspergillus fumigatus leading to activation of the lectin complement pathway and modulation of lung epithelial immune responses

Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H‐Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H‐ficolin in fungal defence. Additionally, we investigated the presence of H‐ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H‐Ficolin exhibited demonstrable binding to A. fumigatus conidia via l‐fucose, d‐mannose and N‐acetylglucosamine residues in a calcium‐ and pH‐dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H‐ficolin opsonization manifested an increase in interleukin‐8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen‐activated protein kinase MAPK kinase 1/2, p38 MAPK and c‐Jun N‐terminal kinase. Finally, H‐ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H‐ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H‐ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus–host interactions and modulated immune responses.

Control of Eosinophil Toxicity in the Lung

The inappropriate accumulation of eosinophils and the subsequent release of their potent pro-inflammatory mediator arsenal are thought to be important contributors to the pathogenesis of asthma and other allergic diseases. It is also becoming apparent that eosinophils may play a role in the orchestration of immune responses in the asthmatic lung. There is therefore much interest in the development of strategies to limit or prevent eosinophil-induced toxicity. The mechanisms by which eosinophils accumulate in the peribronchial tissues of the lung are complex and include enhanced differentiation and release from the bone marrow, selective adhesion and transendothelial migration, directed movement in response to specific chemotactic mediators and finally prolonged survival as a consequence of delayed apoptosis. Thus it can be appreciated that there are many points at which the toxicity of eosinophils can be limited or even prevented. Important areas for potential advances in glucocorticoid (GC) development include efforts to dissociate their anti-inflammatory effects from unwanted side effects. Other areas include the development of humanized monoclonal antibodies against IL-4, IL-13 and IL-5 together with the inhibition of adhesion pathways and/or chemokines responsible for eosinophil accumulation in the asthmatic lung. Several avenues of research are currently underway in an attempt to define mechanisms by which pro-inflammatory cells such as eosinophils can be safely removed from the asthmatic lung through apoptosis induction and their subsequent ingestion by phagocytes. This review will discuss both the potential and shortcomings of these diverse approaches to limit eosinophil toxicity in the asthmatic lung.