The milk proteome and the acute phase response during bovine mastitis

The principal aims of the work presented in this thesis were to further investigate the pathophysiology of the acute phase proteins in bovine mastitis and to document the changes in the protein composition of milk during mastitis using modem proteomic methods. Mastitis is one of the most important diseases affecting cattle worldwide. It has adverse effects on the economics of milk production through reducing the quality and quantity of milk. Acute phase proteins (APPs) are valuable markers of inflammatory lesions and are widely used in the veterinary field for disease diagnosis. This study further investigated the presence of APPs, haptoglobin (Hp) 1 I and mammary- associated serum amyloid A3 (M-SAA3) in the milk of cows with mastitis and evaluated the possibility of using these proteins to detect mastitis. An Enzyme linked immunosorbent assay (ELISA) was developed and validated for the measurement of milk Hp. A commercially available ELISA was used for the detection ofM-SAA3 in milk. Both Hp and M-SAA3 showed similar kinetics to somatic cell counts and can be considered as sensitive and reliable markers of mastitis. Advanced proteomic methods were developed and used to further characterise the changes taking place in the proteins expressed in normal and mastitic milk. The methodology for analysis of the bovine milk proteome was successfully established. This part of the work concluded that the patterns of protein expression of clinically mastitic milk showed clear differences from that of normal milk. Further analysis of milk samples from a mild subclinical model of Staphylococcus aureus mastitis showed that alterations to the milk p:oteome were only minimal. However this study was useful in identifying areas worthy of future research. Recent publications on APP research speculate upon the local production ofM-SAA3 in the bovine mammary gland. The final objective of this study was therefore to investigate the expression of MSAA3 in bovine mammary tissue. Our studies confirm the local production of M-SAA3 in the bovine mammary gland and its up-regulation during bovine mastitis. This finding may provide further information on a possible role for this protein. Overall the findings detailed in this thesis indicate that APPs in milk are valuable markers of mastitis and additionally that the ease of collection of milk by non-invasive methods suggest that milk has potential for the discovery of disease biomarkers

Differential protein profiling as a potential multi-marker approach for TSE diagnosis

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177This "proof of concept" study, examines the use of differential protein expression profiling using surface enhanced laser desorption and ionisationtime of flight mass spectrometry (SELDI-TOF) for the diagnosis of TSE disease. Spectral output from all proteins selectively captured from individual murine brain homogenate samples, are compared as "profiles" in groups of infected and non-infected animals. Differential protein expression between groups is thus highlighted and statistically significant protein "peaks" used to construct a panel of disease specific markers. Studies at both terminal stages of disease and throughout the time course of disease have shown a disease specific protein profile or "disease fingerprint" which could be used to distinguish between groups of TSE infected and uninfected animals at an early time point of disease. Results Our results show many differentially expressed proteins in diseased and control animals, some at early stages of disease. Three proteins identified by SELDI-TOF analysis were verified by immunohistochemistry in brain tissue sections. We demonstrate that by combining the most statistically significant changes in expression, a panel of markers can be constructed that can distinguish between TSE diseased and normal animals. Conclusion Differential protein expression profiling has the potential to be used for the detection of disease in TSE infected animals. Having established that a "training set" of potential markers can be constructed, more work would be required to further test the specificity and sensitivity of the assay in a "testing set". Based on these promising results, further studies are being performed using blood samples from infected sheep to assess the potential use of SELDI-TOF as a pre-mortem blood based diagnostic.https://doi.org/10.1186/1471-2334-9-1889pubpub

Differential protein profiling as a potential multi-marker approach for TSE diagnosis

Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177This "proof of concept" study, examines the use of differential protein expression profiling using surface enhanced laser desorption and ionisationtime of flight mass spectrometry (SELDI-TOF) for the diagnosis of TSE disease. Spectral output from all proteins selectively captured from individual murine brain homogenate samples, are compared as "profiles" in groups of infected and non-infected animals. Differential protein expression between groups is thus highlighted and statistically significant protein "peaks" used to construct a panel of disease specific markers. Studies at both terminal stages of disease and throughout the time course of disease have shown a disease specific protein profile or "disease fingerprint" which could be used to distinguish between groups of TSE infected and uninfected animals at an early time point of disease. Results Our results show many differentially expressed proteins in diseased and control animals, some at early stages of disease. Three proteins identified by SELDI-TOF analysis were verified by immunohistochemistry in brain tissue sections. We demonstrate that by combining the most statistically significant changes in expression, a panel of markers can be constructed that can distinguish between TSE diseased and normal animals. Conclusion Differential protein expression profiling has the potential to be used for the detection of disease in TSE infected animals. Having established that a "training set" of potential markers can be constructed, more work would be required to further test the specificity and sensitivity of the assay in a "testing set". Based on these promising results, further studies are being performed using blood samples from infected sheep to assess the potential use of SELDI-TOF as a pre-mortem blood based diagnostic.https://doi.org/10.1186/1471-2334-9-1889pubpub

Cognitive behaviour therapy plus aerobic exercise training to increase activity in patients with myotonic dystrophy type 1 (DM1) compared to usual care (OPTIMISTIC):Study protocol for randomised controlled trial

Peer reviewedPublisher PD

A pessimistic view of optimistic belief updating

Received academic wisdom holds that human judgment is characterized by unrealistic optimism, the tendency to underestimate the likelihood of negative events and overestimate the likelihood of positive events. With recent questions being raised over the degree to which the majority of this research genuinely demonstrates optimism, attention to possible mechanisms generating such a bias becomes ever more important. New studies have now claimed that unrealistic optimism emerges as a result of biased belief updating with distinctive neural correlates in the brain. On a behavioral level, these studies suggest that, for negative events, desirable information is incorporated into personal risk estimates to a greater degree than undesirable information (resulting in a more optimistic outlook). However, using task analyses, simulations and experiments we demonstrate that this pattern of results is a statistical artifact. In contrast with previous work, we examined participants’ use of new information with reference to the normative, Bayesian standard. Simulations reveal the fundamental difficulties that would need to be overcome by any robust test of optimistic updating. No such test presently exists, so that the best one can presently do is perform analyses with a number of techniques, all of which have important weaknesses. Applying these analyses to five experiments shows no evidence of optimistic updating. These results clarify the difficulties involved in studying human ‘bias’ and cast additional doubt over the status of optimism as a fundamental characteristic of healthy cognition

A novel formulation of inhaled sodium cromoglicate (PA101) in idiopathic pulmonary fibrosis and chronic cough: a randomised, double-blind, proof-of-concept, phase 2 trial

Background Cough can be a debilitating symptom of idiopathic pulmonary fibrosis (IPF) and is difficult to treat. PA101 is a novel formulation of sodium cromoglicate delivered via a high-efficiency eFlow nebuliser that achieves significantly higher drug deposition in the lung compared with the existing formulations. We aimed to test the efficacy and safety of inhaled PA101 in patients with IPF and chronic cough and, to explore the antitussive mechanism of PA101, patients with chronic idiopathic cough (CIC) were also studied. Methods This pilot, proof-of-concept study consisted of a randomised, double-blind, placebo-controlled trial in patients with IPF and chronic cough and a parallel study of similar design in patients with CIC. Participants with IPF and chronic cough recruited from seven centres in the UK and the Netherlands were randomly assigned (1:1, using a computer-generated randomisation schedule) by site staff to receive PA101 (40 mg) or matching placebo three times a day via oral inhalation for 2 weeks, followed by a 2 week washout, and then crossed over to the other arm. Study participants, investigators, study staff, and the sponsor were masked to group assignment until all participants had completed the study. The primary efficacy endpoint was change from baseline in objective daytime cough frequency (from 24 h acoustic recording, Leicester Cough Monitor). The primary efficacy analysis included all participants who received at least one dose of study drug and had at least one post-baseline efficacy measurement. Safety analysis included all those who took at least one dose of study drug. In the second cohort, participants with CIC were randomly assigned in a study across four centres with similar design and endpoints. The study was registered with ClinicalTrials.gov (NCT02412020) and the EU Clinical Trials Register (EudraCT Number 2014-004025-40) and both cohorts are closed to new participants. Findings Between Feb 13, 2015, and Feb 2, 2016, 24 participants with IPF were randomly assigned to treatment groups. 28 participants with CIC were enrolled during the same period and 27 received study treatment. In patients with IPF, PA101 reduced daytime cough frequency by 31·1% at day 14 compared with placebo; daytime cough frequency decreased from a mean 55 (SD 55) coughs per h at baseline to 39 (29) coughs per h at day 14 following treatment with PA101, versus 51 (37) coughs per h at baseline to 52 (40) cough per h following placebo treatment (ratio of least-squares [LS] means 0·67, 95% CI 0·48–0·94, p=0·0241). By contrast, no treatment benefit for PA101 was observed in the CIC cohort; mean reduction of daytime cough frequency at day 14 for PA101 adjusted for placebo was 6·2% (ratio of LS means 1·27, 0·78–2·06, p=0·31). PA101 was well tolerated in both cohorts. The incidence of adverse events was similar between PA101 and placebo treatments, most adverse events were mild in severity, and no severe adverse events or serious adverse events were reported. Interpretation This study suggests that the mechanism of cough in IPF might be disease specific. Inhaled PA101 could be a treatment option for chronic cough in patients with IPF and warrants further investigation

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Physical, cognitive, and mental health impacts of COVID-19 after hospitalisation (PHOSP-COVID): a UK multicentre, prospective cohort study

Background The impact of COVID-19 on physical and mental health and employment after hospitalisation with acute disease is not well understood. The aim of this study was to determine the effects of COVID-19-related hospitalisation on health and employment, to identify factors associated with recovery, and to describe recovery phenotypes. Methods The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a multicentre, long-term follow-up study of adults (aged ≥18 years) discharged from hospital in the UK with a clinical diagnosis of COVID-19, involving an assessment between 2 and 7 months after discharge, including detailed recording of symptoms, and physiological and biochemical testing. Multivariable logistic regression was done for the primary outcome of patient-perceived recovery, with age, sex, ethnicity, body-mass index, comorbidities, and severity of acute illness as covariates. A post-hoc cluster analysis of outcomes for breathlessness, fatigue, mental health, cognitive impairment, and physical performance was done using the clustering large applications k-medoids approach. The study is registered on the ISRCTN Registry (ISRCTN10980107). Findings We report findings for 1077 patients discharged from hospital between March 5 and Nov 30, 2020, who underwent assessment at a median of 5·9 months (IQR 4·9–6·5) after discharge. Participants had a mean age of 58 years (SD 13); 384 (36%) were female, 710 (69%) were of white ethnicity, 288 (27%) had received mechanical ventilation, and 540 (50%) had at least two comorbidities. At follow-up, only 239 (29%) of 830 participants felt fully recovered, 158 (20%) of 806 had a new disability (assessed by the Washington Group Short Set on Functioning), and 124 (19%) of 641 experienced a health-related change in occupation. Factors associated with not recovering were female sex, middle age (40–59 years), two or more comorbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial but only weakly associated with the severity of acute illness. Four clusters were identified with different severities of mental and physical health impairment (n=767): very severe (131 patients, 17%), severe (159, 21%), moderate along with cognitive impairment (127, 17%), and mild (350, 46%). Of the outcomes used in the cluster analysis, all were closely related except for cognitive impairment. Three (3%) of 113 patients in the very severe cluster, nine (7%) of 129 in the severe cluster, 36 (36%) of 99 in the moderate cluster, and 114 (43%) of 267 in the mild cluster reported feeling fully recovered. Persistently elevated serum C-reactive protein was positively associated with cluster severity. Interpretation We identified factors related to not recovering after hospital admission with COVID-19 at 6 months after discharge (eg, female sex, middle age, two or more comorbidities, and more acute severe illness), and four different recovery phenotypes. The severity of physical and mental health impairments were closely related, whereas cognitive health impairments were independent. In clinical care, a proactive approach is needed across the acute severity spectrum, with interdisciplinary working, wide access to COVID-19 holistic clinical services, and the potential to stratify care. Funding UK Research and Innovation and National Institute for Health Research

Epitope Mapping of Fc gamma RIIa Monoclonal Antibodies

FcγRIIa (CD32) is an IgG receptor which has been shown to be important in autoimmune disease pathology. IV.3, 8.7, and 7.30 are anti-FcγRIIa monoclonal antibodies (mAbs), which block the interaction between FcγRIIa and complex IgG. In this study, the three mAbs were demonstrated to inhibit FcγRIIa function. The determination of the precise epitopes of the IV.3, 8.7, and 7.30 mAbs may become a potential approach for designing inhibitors for FcγRIIa. The epitope of IV.3, 8.7, and 7.30 were determined using chimeric receptors based on the extracellular domains of FcγRIIa and the FcεRI a chain. The epitopes for IV.3 was found to be mapped on amino acid residues 132-137, while 8.7 and 7.30 were on amino acid residues 112-119 and 157-162. Based on the crystal 3D model of FcγRIIa molecule, these amino acid sequences are clustered together forming a contiguous region within the ligand binding site of the receptor