Predicting Pulmonary Function From the Analysis of Voice: A Machine Learning Approach.

INTRODUCTION: To self-monitor asthma symptoms, existing methods (e.g. peak flow metre, smart spirometer) require special equipment and are not always used by the patients. Voice recording has the potential to generate surrogate measures of lung function and this study aims to apply machine learning approaches to predict lung function and severity of abnormal lung function from recorded voice for asthma patients. METHODS: A threshold-based mechanism was designed to separate speech and breathing from 323 recordings. Features extracted from these were combined with biological factors to predict lung function. Three predictive models were developed using Random Forest (RF), Support Vector Machine (SVM), and linear regression algorithms: (a) regression models to predict lung function, (b) multi-class classification models to predict severity of lung function abnormality, and (c) binary classification models to predict lung function abnormality. Training and test samples were separated (70%:30%, using balanced portioning), features were normalised, 10-fold cross-validation was used and model performances were evaluated on the test samples. RESULTS: The RF-based regression model performed better with the lowest root mean square error of 10·86. To predict severity of lung function impairment, the SVM-based model performed best in multi-class classification (accuracy = 73.20%), whereas the RF-based model performed best in binary classification models for predicting abnormal lung function (accuracy = 85%). CONCLUSION: Our machine learning approaches can predict lung function, from recorded voice files, better than published approaches. This technique could be used to develop future telehealth solutions including smartphone-based applications which have potential to aid decision making and self-monitoring in asthma.

Monolithically-integrated cytometer for measuring particle diameter in the extracellular vesicle size range using multi-angle scattering.

Size measurement of extracellular vesicles is hampered by the high cost and measurement uncertainty of conventional flow cytometers which is mainly due to the use of non-specialised free space optics. Integrated cytometry, where the optics and fluidics are embedded in a monolithic chip shows promise for the production of low cost, micro-flow cytometers dedicated for extracellular vesicle (EV) analysis with improved size measurement accuracy and precision. This research demonstrates a unique integrated cytometer for sub-micron particle size measurement using multi-angle scattering analysis. A combination of three technologies is used: (i) Dean-based hydrodynamic focussing to deliver a tight sample core stream to the analysis region, (ii) integrated waveguides with multimode interference devices to focus a narrow excitation beam onto the sample stream, and (iii) an angular array of collection waveguides to measure particle scattering distribution and calculate diameter. Low index 200 nm liposomes could be detected and polystyrene size standards as small as 400 nm diameter could be measured with an uncertainty of ±21 nm (1/2 IQR) demonstrating a first step on the path to high performance integrated cytometry of EVs.

FCMPASS Software Aids Extracellular Vesicle Light Scatter Standardization.

The study of extracellular vesicles (EVs) is a rapidly growing field due to their great potential in many areas of clinical medicine including diagnostics, prognostics, theranostics, and therapeutics. Flow cytometry is currently one of the most popular methods of analyzing EVs due to it being a high-throughput, multiparametric technique, that is readily available in the majority of research labs. Despite its wide use, few commercial flow cytometers are designed specifically for the detection of EVs. Many flow cytometers used for EV analysis are working at their detection limits and are unable to detect the majority of EVs. Currently, very little standardization exists for EV flow cytometry, which is an issue because flow cytometers vary considerably in the way they collect scattered or fluorescent light from particles being interrogated. This makes published research hard to interpret, compare, and in some cases, impossible to reproduce. Here we demonstrate a method of flow cytometer light scatter standardization, utilizing flow cytometer postacquisition analysis software (FCMPASS ). FCMPASS is built upon Mie theory and enables the approximation of flow cytometer geometric parameters either by analyzing beads of known diameter and refractive index or by inputting the collection angle if known. The software is then able to create a scatter-diameter curve and scatter-refractive index curve that enables researchers to convert scattering data and instrument sensitivity into standardized units. Furthermore, with the correct controls, light scatter data can be converted to diameter distributions or refractive index distributions. FCMPASS therefore offers a freely available and ergonomic method of standardizing and further extending EV characterization using flow cytometry.

Upskilling healthcare professionals to manage clinical allergy.

It has long been recognised that given the high prevalence and considerable impact of allergic disease globally, there needs to be a focus on appropriate training for clinical professionals. The health-economic consequences of allergic disease are significant, with both direct healthcare costs (doctor, nurse and dietitian consultations, hospital admissions and prescribed medications) and indirect costs (lost school and work time, reduced productivity and over-the-counter medications). There is also a well-recognised impairment of quality of life, with less tangible costs including anxiety, distress, discomfort, disability and, occasionally, death. To help to mitigate these effects, there is a need to upskill the professional workforce at all levels, and also to equip those trained with the skills to become future healthare professional trainers. Upskilling the workforce from the grass-roots of undergraduate study in Medical, Nursing and Allied Health Professionals (AHP) through the entirety of training to senior consultant levels could have a major beneficial impact on the patient and their families, lead to a reduction in emergency use of clinical service, and help increase economic productivity.

The impact of a General Practitioner-led community paediatric allergy clinic: A service evaluation.

BACKGROUND: The NHS is not meeting the nation's allergy needs. There are insufficient allergy specialists, with variable care across the country. General practitioners (GPs) are lacking in allergy training. London's Whittington Hospital created a GP with Special Interest (GPwSI) community paediatric allergy clinic, running alongside pre-existing hospital clinics, to address local unmet needs, aiming to provide equity for patients, improve patient experience and decrease secondary care burden. OBJECTIVES: To establish whether improvements have occurred within the service by introducing a GPwSI-led community paediatric allergy clinic alongside providing GP education and referral pathways. This study asks: (a) Have allergy-related hospital attendances decreased with the provision of the community service? (b) Are patients seen in the appropriate clinic? (c) What proportion of patients require GPwSI follow-up? (d) Is there good patient satisfaction? (e) Have allergy clinic waiting times changed? METHODS: Numbers of allergy-related hospital attendances and waiting times in 2013, 2014 and 2016 were assessed. Data were analysed regarding proportions of patients requiring GPwSI follow-up or referral from the GPwSI community clinic to hospital. Patient satisfaction was assessed. RESULTS: Since introducing the GPwSI community service the burden on secondary care has decreased, with reduced hospital attendances for allergy clinic patients, although waiting times have increased. In 2013, 65% of allergy clinic patients attended other hospital services for allergy-related complaints prior to their first allergy clinic appointment. This was reduced to 27.3% (community) and 36.9% (hospital) in 2014 and maintained in 2016 (27.5% community and 37.5% hospital), P < 0.01. Patient satisfaction in the hospital and community clinics is very high. CLINICAL RELEVANCE: This integrated, multidisciplinary Paediatric Allergy Service could provide a model to improve the unmet allergy need both in the UK and beyond. This GPwSI model could also be applied to other chronic diseases in both adults and children, improving care beyond allergy.

Extracellular Vesicle Flow Cytometry Analysis and Standardization.

The term extracellular vesicles (EVs) describes membranous vesicles derived from cells, ranging in diameter from 30 to 1,000 nm with the majority thought to be in the region of 100-150 nm. Due to their small diameter and complex and variable composition, conventional techniques have struggled to accurately count and phenotype EVs. Currently, EV characterization using high-resolution flow cytometry is the most promising method when compared to other currently available techniques, due to it being a high-throughput, single particle, multi-parameter analysis technique capable of analyzing a large range of particle diameters. Whilst high resolution flow cytometry promises detection of the full EV diameter range, standardization of light scattering and fluorescence data between different flow cytometers remains an problem. In this mini review, we will discuss the advances in high-resolution flow cytometry development and future direction of EV scatter and fluorescence standardization. Standardization and therefore reproducibility between research groups and instrumentation is lacking, hindering the validation of EVs use as diagnostic biomarkers and therapeutics.

Paper-based colorimetric enzyme linked immunosorbent assay fabricated by laser induced forward transfer.

We report the Laser Induced Forward Transfer (LIFT) of antibodies from a liquid donor film onto paper receivers for application as point-of-care diagnostic sensors. To minimise the loss of functionality of the active biomolecules during transfer, a dynamic release layer was employed to shield the biomaterial from direct exposure to the pulsed laser source. Cellulose paper was chosen as the ideal receiver because of its inherent bio-compatibility, liquid transport properties, wide availability and low cost, all of which make it an efficient and suitable platform for point-of-care diagnostic sensors. Both enzyme-tagged and untagged IgG antibodies were LIFT-printed and their functionality was confirmed via a colorimetric enzyme-linked immunosorbent assay. Localisation of the printed antibodies was exhibited, which can allow the creation of complex 2-d patterns such as QR codes or letters for use in a final working device. Finally, a calibration curve was determined that related the intensity of the colour obtained to the concentration of active antibodies to enable quantitative assessment of the device performance. The motivation for this work was to implement a laser-based procedure for manufacturing low-cost, point-of-care diagnostic devices on paper.

Comparison of venous and capillary differential leukocyte counts using a standard hematology analyzer and a novel microfluidic impedance cytometer.

Capillary blood sampling has been identified as a potentially suitable technique for use in diagnostic testing of the full blood count (FBC) at the point-of-care (POC), for which a recent need has been highlighted. In this study we assess the accuracy of capillary blood counts and evaluate the potential of a miniaturized cytometer developed for POC testing. Differential leukocyte counts in the normal clinical range from fingerprick (capillary) and venous blood samples were measured and compared using a standard hematology analyzer. The accuracy of our novel microfluidic impedance cytometer (MIC) was then tested by comparing same-site measurements to those obtained with the standard analyzer. The concordance between measurements of fingerprick and venous blood samples using the standard hematology analyzer was high, with no clinically relevant differences observed between the mean differential leukocyte counts. Concordance data between the MIC and the standard analyzer on same-site measurements presented significantly lower leukocyte counts determined by the MIC. This systematic undercount was consistent across the measured (normal) concentration range, suggesting that an internal correction factor could be applied. Differential leukocyte counts obtained from fingerprick samples accurately reflect those from venous blood, which confirms the potential of capillary blood sampling for POC testing of the FBC. Furthermore, the MIC device demonstrated here presents a realistic technology for the future development of FBC and related tests for use at the site of patient care.

What factors affect the carriage of epinephrine auto-injectors by teenagers?

BACKGROUND: Teenagers with allergies are at particular risk of severe and fatal reactions, but epinephrine auto-injectors are not always carried as prescribed. We investigated barriers to carriage. METHODS: Patients aged 12-18 years old under a specialist allergy clinic, who had previously been prescribed an auto-injector were invited to participate. Semi-structured interviews explored the factors that positively or negatively impacted on carriage. RESULTS: Twenty teenagers with food or venom allergies were interviewed. Only two patients had used their auto-injector in the community, although several had been treated for severe reactions in hospital. Most teenagers made complex risk assessments to determine whether to carry the auto-injector. Most but not all decisions were rational and were at least partially informed by knowledge. Factors affecting carriage included location, who else would be present, the attitudes of others and physical features of the auto-injector. Teenagers made frequent risk assessments when deciding whether to carry their auto-injectors, and generally wanted to remain safe. Their decisions were complex, multi-faceted and highly individualised. CONCLUSIONS: Rather than aiming for 100% carriage of auto-injectors, which remains an ambitious ideal, personalised education packages should aim to empower teenagers to make and act upon informed risk assessments.

On-chip epithelial barrier function assays using electrical impedance spectroscopy.

A bio-impedance chip has been developed for real-time monitoring of the kinetics of epithelial cell monolayers in vitro. The human bronchial epithelial cell line (16-HBE 14o-) was cultured in Transwells creating a sustainable and interactive model of the airway epithelium. Conducting polymer polypyrrole (PPy) doped with polystyrene sulfonate (PSS) was electrochemically deposited onto the surface of gold-plated electrodes to reduce the influence of the electrical double layer on the impedance measurements. Finite element and equivalent circuit models were used to model and determine the electrical properties of the epithelial cell monolayer from the impedance spectra. Electrically tight, confluent monolayers of 16 HBE 14o- cells were treated with increasing concentrations of either Triton X-100 to solubilize cell membranes or ethylene glycol-bis(2-aminoethyl-ether)-N,N,N'N'-tetraacetic acid (EGTA) to disrupt cell-cell adhesion. Experimental impedance data showed that disruption of epithelial barrier function in response to Triton X-100 and EGTA can be successfully measured by the bio-impedance chip. The results were consistent with the conventional hand-held trans-epithelial electrical resistance measurements. Immunofluorescent staining of the ZO-1 tight junction protein in the untreated and treated 16HBEs was performed to verify the disruption of the tight junctions by EGTA.

ADAM33 expression in atherosclerotic lesions and relationship of ADAM33 gene variation with atherosclerosis.

A-disintegrin-and-metalloproteinase-domains (ADAMs) are membrane-anchored glycoproteins involved in cell adhesion, cell migration and proteolysis. ADAM15 has been implicated in atherosclerosis, with an effect on vascular smooth muscle cell migration. We investigated whether ADAM33, which is evolutionally closely related to ADAM15, was expressed in atheromas and whether it had an effect on vascular smooth muscle migration. We also tested whether ADAM33 gene variation had an influence on the extent of atherosclerosis in patients with coronary artery disease. Immunohistochemical analyses showed that ADAM33 was expressed in smooth muscle cells in the arterial wall and that the expression was increased in smooth muscle cells in atheromas. ADAM33 immunostaining on inflammatory cells in atheromas was also observed. Primary vascular smooth muscle cells in culture were also found to express ADAM33. Boyden chamber assays showed that a neutralising antibody against ADAM33 increased the ability of arterial smooth muscle cells to migrate through a reconstituted basement membrane, suggesting that ADAM33 has an inhibitory effect on vascular smooth muscle migration. Moreover, we detected an association between ADAM33 genotype and the extent of atherosclerosis in a large cohort of coronary artery disease patients. These findings suggest that ADAM33 is implicated in the pathogenesis of atherosclerosis.

Human mid-gestation amniotic fluid contains interleukin-16 bioactivity.

CD4-positive cells are detectable in the human fetal gastrointestinal tract from 11 weeks of gestation. Interleukin-16 (IL-16) is a chemoattractant for CD4(+) cells and, via fetal swallowing of amniotic fluid, could mediate the influx of CD4(+) cells into the fetal gut. We have shown that IL-16 was detectable in human amniotic fluid at 16-18 weeks of gestation (mid-pregnancy) but was not detectable at term (late pregnancy; > 37 weeks of gestation). Similarly, mid-pregnancy, but not late pregnancy, amniotic fluid contained chemotactic activity for CD4(+) T cells, this activity was reduced by 58% in the presence of a neutralizing anti-IL-16 antibody. The levels of IL-16 in fetal plasma at 16-24 weeks of gestation were very high, and decreased significantly by 25-36 weeks but at > 37 weeks remained significantly higher than adult levels. IL-16 transcripts were detectable in whole tissue extracts of fetal gut, skin and placenta but not in amniocytes, and IL-16 immunoreactivity was detectable in cells within the lamina propria of the fetal gut and within the skin, where it was associated with the basement membrane. Neither IL-16 levels nor chemotactic activity for CD4(+) T cells in mid-pregnancy amniotic fluid was related to atopic outcomes at 1 year of age. IL-16 might have an important role in the early development of the human immune system and/or in regulating fetal and maternal immunological responsiveness during pregnancy.

Phenotypic analysis of circulating dendritic cells during the second half of human gestation.

Dendritic cells (DCs) have been characterized as having an immature phenotype in infants when compared with adults; but it is unclear whether the phenotype or function of these populations changes during human intrauterine development. Three-colour flow cytometry was used to phenotype fetal/neonatal circulating DCs during the second half (>20-wk gestation) of pregnancy, (n = 34) and adults (n = 9). DCs were identified from peripheral blood mononuclear cells (PBMCs) or cord blood mononuclear cells (CBMCs) as staining brightly for HLA-DR but negative for T cell, B cell, monocyte, and NK cell lineage markers. The surface molecule of interest was detected in a third colour. During gestation CD34, a marker of immaturity was significantly higher, and CD4, a differentiation marker, was significantly lower than adult levels. The percentage of CD11c+ cells did not differ significantly at any age, although a trend to reduced intensity of expression at earlier stages of gestation was observed. Significantly fewer DCs expressed the IgG receptors CD32 and CD64 at all gestations. The percentage of HLA-DR+/lin- cells expressing CD40 was lowest at 20-23 wks and was always significantly lower on DCs from cord blood vs. adult blood. Similarly, the percentage of CD86+ and CD54+ DCs was significantly lower than adults throughout gestation. Thus, immaturity of cord blood DCs is likely to arise as a consequence of decreased ability to take up antigen (at least via IgG-mediated mechanisms) and reduced provision of co-stimulation.

Phenotypic characterization of CD3-7+ cells in developing human intestine and an analysis of their ability to differentiate into T cells.

We have identified a large population of CD3(-)7(+) cells in human fetal gut. Three- and four-color flow cytometry revealed a distinct surface Ag profile on this population; the majority were negative for CD4 and CD8, whereas most of the remainder expressed the CD8alphaalpha homodimer. In contrast about half of CD3(+) cells expressed CD4 and half expressed CD8alpha. A large proportion of CD3(-)7(+) cells expressed CD56, CD94, and CD161, and whereas CD3(+) T cells also expressed CD161, they only rarely expressed CD56 or CD94. Further studies were conducted to determine whether the CD3(-)7(+) cells have the potential to differentiate into CD3(+) cells. About half of CD3(-)7(+) cells contain intracellular CD3epsilon. Rearranged TCR gamma-chains were detected in highly purified CD3(-)7(+) cells as an early molecular sign of T cell commitment, and the pattern of rearrangement with V regions spliced to the most 5' Jgamma segment is reminiscent of early thymocyte differentiation. In reaggregate thymic organ cultures, CD3(-)7(+) cells also gave rise to CD3(+) T cells. Thus, we demonstrate that the CD3(-)7(+) cells present in the human fetal gut display a distinct phenotype and are able to develop into CD3(+) T cells.

Impaired immunity to intestinal bacterial infection in stromelysin-1 (matrix metalloproteinase-3)-deficient mice.

Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. Matrix metalloproteinases (MMPs) have been shown to mediate matrix remodeling and cell migration during tissue injury and repair in the intestine. We have previously shown enhanced pathology in infected TNFRp55-/-, IL-12p40-/-, and IFN-gamma-/- mice, and here we show that this is associated with an increase in stromelysin-1 (MMP3) transcripts in colonic tissues. We have therefore investigated the role of MMP3 in colonic mucosal hyperplasia and the local Th1 responses using MMP3-/- mice. In MMP3-/- mice, similar mucosal thickening was observed after infection as in wild-type (WT) mice. Colonic tissues from MMP3-/- mice showed a compensatory increase in the expression of other MMP transcripts, such as MMP7 and MMP12. However, MMP3-/- mice showed delayed clearance of bacteria and delayed appearance of CD4+ T lymphocytes into intestinal lamina propria. CSFE-labeled mesenteric lymph node CD4+ T lymphocytes from infected WT mice migrated in fewer numbers into the mesenteric lymph nodes and colon of MMP3-/- mice than into those of WT mice. These studies show that mucosal remodeling can occur in the absence of MMP3, but that MMP3 plays a role in the migration of CD4+ T lymphocytes to the intestinal mucosa.

The effect of labor on neonatal T-cell phenotype and function.

With increasing interest in the role of fetal programming in child and adulthood diseases, and therefore interest in the measurement of various factors at birth, it is essential to ascertain whether the factors of interest show any gestation- or parturition-associated changes. We have investigated whether mode of delivery influenced T-cell phenotype and function (CD4+) as has been described for monocytes and neutrophils. Interferon-gamma production in response to either the mitogen phytohemagglutinin or anti-CD2/CD3/CD28 F(ab')3 was significantly reduced by neonatal mononuclear cells compared with adult cells but did not differ with mode of delivery at term (normal vaginal delivery versus elective lower-segment cesarean section). Likewise, anti-CD2/CD3/CD28-stimulated IL-2 production by the neonate was lower than adult levels but did not differ with mode of delivery. The expression of common T-cell activation markers (CD25, MHC class II, CD69, CD62L, CD11a, CD44, and CD49d) was examined. Only CD62L (L-selectin) expression was significantly different, with fewer adult T cells expressing this surface antigen compared with neonatal T cells (p < 0.0003), and significantly more T cells from lower-segment cesarean section than normal vaginal delivery were positive for CD62L (p = 0.012). sCD62L levels were significantly lower in cord plasma compared with adult plasma but did not differ with mode of delivery. Thus the phenotype and function of cord blood T cells did not differ greatly with mode of delivery, but possible differences for the marker of interest should always be assessed. Furthermore, although there was no significant difference with mode of delivery for all markers, except CD62L, the variation in the normal vaginal delivery samples, as for the adults, was greater than in the lower-segment cesarean section samples, indicating that the effects of length of labor and stress at delivery may well be relevant.

Expression of CD21 and CD23 during human fetal development.

Neonates produce lower levels of IgE compared with adults. Diminished IL-4 production and impaired up-regulation of CD40L by neonatal T cells could explain this, however other regulators of IgE production, such as CD21 and CD23, could contribute to reduced circulating IgE levels during fetal development. Heparinized blood samples were collected from adults and from the umbilical cord at premature and term births. Whole blood flow cytometry was used to assess the percentage of T (CD3(+)) and B (CD19(+)) lymphocytes expressing CD21 and/or CD23 at 26-29 (n = 3), 30-33 (n = 7), 34-37 (n = 5), and >37 (n = 11) wk of gestation, as well as in adults (n = 15). Plasma-soluble CD21 was also measured. At term, the percentage of CD21(+) and CD23(+) B cells was comparable to the adult, however, the percentage of cells positive for each of these surface antigens was decreased significantly before term. The percentage of T cells expressing CD21 from all gestations was significantly higher than the adult and the percentage positive decreased with increasing gestational age. Conversely, soluble CD21 levels increased with increasing gestation to be comparable to the adult by term. Thus, it is unlikely that altered expression of CD21 and CD23 on B cells contributes to the low level of IgE in the neonatal circulation unless functional differences occur or a lack of processing to the soluble form is important in regulating IgE production. However the abundance of CD21-positive T cells could alter the T- and B-cell interaction necessary for IgE switching by B cells and, thereby, especially with impaired IL-4 production, limit IgE production.

Immunoregulatory molecules during pregnancy and at birth.

Regulation of the maternal immune response to the fetal allograft is essential for the success of pregnancy and delivery of a well-developed neonate. Numerous mechanisms have been postulated to mediate this. We hypothesised that the potent immunosuppressive molecules TGF-beta1 and IL-10 could contribute to this regulation in the mother and neonate during gestation. In comparison to non-pregnant women, TGF-beta1 and cortisol levels were increased significantly in mid (16-18 weeks) and late pregnancy (>37 weeks, no labour), with levels of both highest in late gestation. In contrast, IL-10 levels were significantly lower in maternal plasma in mid-gestation compared with that from late pregnancy and from non-pregnant women. TGF-beta1, IL-10 and cortisol were all detectable in umbilical cord blood plasma with TGF-beta1 levels significantly decreased in association with labour in contrast to cortisol levels that increased with labour. IL-10 levels in cord plasma were comparable to those of adults and did not change with mode of delivery. Elevated levels of TGF-beta1, but not IL-10, in the maternal and neonatal circulation could have a role in immunoregulation of the maternal response to the fetal allograft as well as growth and development of the fetus.