The African child

No Abstrac

On-demand delivery of single DNA molecules using nanopipettes

Understanding the behavioral properties of single molecules or larger scale populations interacting with single molecules is currently a hotly pursued topic in nanotechnology. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single molecule detection strategies have existed for a number of years, currently lacking are efficient methods for the controllable delivery of single molecules in aqueous environments. In this article we show both experimentally and from simulations that nanopipets in conjunction with asymmetric voltage pulses can be used for label-free detection and delivery of single molecules through the tip of a nanopipet with “on-demand” timing resolution. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA molecules from solutions with concentrations as low as 3 pM

The immune system of the liver: 50 years of strangeness

Editoria

CMV and the Art of Memory Maintenance

The CD8+ T cell responses to CMV gradually increase in magnitude over time—so-called memory “inflation.” In this issue of Immunity, Snyder et al. (2008) examine the dynamics of memory inflation and demonstrate continuous turnover of inflating T cells, drawing on both memory cells and naive cells to replace them

On-Demand Delivery of Single DNA Molecules Using Nanopipets

Understanding the behavioral properties of single molecules or larger scale populations interacting with single molecules is currently a hotly pursued topic in nanotechnology. This arises from the potential such techniques have in relation to applications such as targeted drug delivery, early stage detection of disease, and drug screening. Although label and label-free single molecule detection strategies have existed for a number of years, currently lacking are efficient methods for the controllable delivery of single molecules in aqueous environments. In this article we show both experimentally and from simulations that nanopipets in conjunction with asymmetric voltage pulses can be used for label-free detection and delivery of single molecules through the tip of a nanopipet with “on-demand” timing resolution. This was demonstrated by controllable delivery of 5 kbp and 10 kbp DNA molecules from solutions with concentrations as low as 3 pM

The upper and lower airway microbiome in severe asthma

Introduction: Severe asthma is complex and immunologically heterogenous; airways infection and innate immune dysregulation may confer inhaled corticosteroid resistance and remains clinically challenging. We hypothesise that marked airway microbiome compositional shifts in severe asthma represent a ‘treatable trait’, with dominance of pathogenic species, warranting targeted therapy. Additionally, these changes are thought to be distinct in the paucibacillary lower airway vs. heavily colonised nasopharynx. Methods: We performed Oxford Nanopore metagenomic sequencing, and RT-qPCR of induced sputum (n=67), bronchoalveolar lavage (BAL;n=71) and nasal lavage (NL;n=28 paired with sputum) samples from the Wessex Severe Asthma Cohort encompassing mild/moderate/severe asthma and health. Findings were integrated with clinical and cytokine data. Key Results: A dominant pathogenic organism (H.influenzae, S.pneumoniae, M.catarrhalis) was identified in sputua of 21% of patients with severe asthma and accompanied by sputum neutrophilia and elevated type-1 cytokines (including IL-1β, IL-6, IL-8, TNF;p<0.01, unpaired t-test, Benjamini-Hochberg correction). From BAL, airways infection was identified infrequently (Severe asthma, n=1/21 H.influenzae; Mild-Moderate asthma,n=2/25 T.whipplei). Metagenomic analysis of NL demonstrated a distinct microbiome; presence of pathogenic organisms in the upper airways did not predict concurrent presence in sputum of severe asthmatics. Conclusion: Airways infection is reliably identified in sputum using metagenomic sequencing and is associated with neutrophilic inflammation. The microbiome in the upper and lower airway are distinct and ongoing work is exploring its impact on mucosal immune responses at these sites

A Dominant Role for the Immunoproteasome in CD8+ T Cell Responses to Murine Cytomegalovirus

Murine cytomegalovirus (MCMV) is an important animal model of human cytomegalovirus (HCMV), a β-Herpesvirus that infects the majority of the world's population and causes disease in neonates and immunocompromised adults. CD8+ T cells are a major part of the immune response to MCMV and HCMV. Processing of peptides for presentation to CD8+ T cells may be critically dependent on the immunoproteasome, expression of which is affected by MCMV. However, the overall importance of the immunoproteasome in the generation of immunodominant peptides from MCMV is not known. We therefore examined the role of the immunoproteasome in stimulation of CD8+ T cell responses to MCMV – both conventional memory responses and those undergoing long-term expansion or “inflation”. We infected LMP7−/− and C57BL/6 mice with MCMV or with newly-generated recombinant vaccinia viruses (rVVs) encoding the immunodominant MCMV protein M45 in either full-length or epitope-only minigene form. We analysed CD8+ T cell responses using intracellular cytokine stain (ICS) and MHC Class I tetramer staining for a panel of MCMV-derived epitopes. We showed a critical role for immunoproteasome in MCMV affecting all epitopes studied. Interestingly we found that memory “inflating” epitopes demonstrate reduced immunoproteasome dependence compared to non-inflating epitopes. M45-specific responses induced by rVVs remain immunoproteasome-dependent. These results help to define a critical restriction point for CD8+ T cell epitopes in natural cytomegalovirus (CMV) infection and potentially in vaccine strategies against this and other viruses

Identification of neutralising pembrolizumab anti-drug antibodies in patients with melanoma

Development of anti-drug antibodies (ADAs) can interfere with therapeutic monoclonal antibodies and may lead to drug neutralisation and clinical disease progression. Measurement of circulating drug levels and development of ADAs in the setting of anti-programmed cell death-1 agent pembrolizumab has not been well-studied. Enzyme-linked immunosorbent assays were used to measure pembrolizumab drug level and ADAs in 41 patients with melanoma at baseline, Time-point 1 (3 weeks) and Time-point 2 (21 weeks). Assay results were related to patient demographics and clinical outcome data at 6 months. The median pembrolizumab drug level at 3 weeks was 237 ng/μL and did not correlate with age, sex or body surface area.17/41 patients had an ADA detected at any timepoint, with the highest prevalence at Timepoint 1 (median concentration = 17 ng/μL). The presence of an ADA did not correlate with clinical progression at 6 months. 3/41 (7%) of patients displayed a falling pembrolizumab drug level and rising ADA titre between Timepoint 1 and 2 suggestive of a neutralising ADA. Pembrolizumab drug levels and ADAs can be readily measured. The rates of total and treatment-emergent ADAs may be higher in "real-word" settings than those previously reported. Larger studies are needed to determine effect of neutralising ADAs on long-term clinical outcome