Magnetic Resonance Spectroscopy as applied to epilepsy

Epilepsy is the most common serious disease of the brain. Magnetic Resonance Spectroscopy (MRS) is a novel imaging technique that offers the opportunity for co-localising biochemical information relating to metabolites specific to the study of epilepsy with high resolution MRI. Aims: The work included in this thesis was undertaken with two fundamental aims. The first was to apply a standardised MRS methodology in order to gain reproducible semi-quantitative information about the variation of relevant neuro-metabolites such as gamma amino butyric acid (GABA), glutamate (as glutamate plus glutamine [GLX]), N acetyl aspartate (NAA), myo-inositol (Ins) and creatine plus phosphocreatine (Cr) within epilepsy syndromes or pathological groups. The second main aim was to test a series of hypotheses relating to the regulation of the concentrations of these metabolites in the region of epileptic seizures, immediately following seizures and associated with particular medical and surgical treatment interventions. Methods: Seven experiments were performed in this thesis. In all seven studies the findings in the patient groups were compared against results from an acquired control group made up of healthy volunteers. In the first experiment [3.1] twenty patients with temporal lobe epilepsy, with (10), and without hippocampal sclerosis were studied using multi voxel magnetic resonance spectroscopic imaging (MRSI) sequences in order to examine for differences in the obtained metabolites N acetyl aspartate (NAA), creatine plus phosphocreatine (Cr), choline containing compounds (Cho), GLX and myo-inositol (Ins) across the pathological groups and against a control population. In experiments [3.2], [3.3], [3.4] and [3.6] an MRS protocol that incorporated a double quantum filter acquisition sequence was applied in order to allow measurement of GABA+ (a combined measure of GABA plus homocarnosine) in addition to measurement of the metabolites examined in [3.1]. Studies were performed in the occipital lobes in patients with idiopathic generalised epilepsy (IGE) (n =10) or occipital lobe epilepsy (n = 10) [3.2], in the frontal lobes in patients with IGE (n = 21) and within regions of the MRI visible pathology in patients with large focal malformations of cortical development (MCD, n =10) [3.4]. In the last experiment using this technique patients with hippocampal sclerosis and temporal lobe epilepsy (n = 16) were studied in the ipsilateral and also in the contralateral temporal lobes and following temporal lobe surgery (n = 10) [3.6]. In experiment [3.5] ten patients were examined whilst taking and when not taking sodium valproate in order to further examine for an effect of this medication on the measured metabolite concentrations. In experiment [3.7] ten patients were studied immediately after an epileptic seizure and then again during a subsequent inter-ictal period in order to examine for an influence of the recent seizure on the measured concentrations of the main metabolites. Results: MRSI in the temporal lobes in patients with temporal lobe epilepsy identified low NAA in the anterior hippocampus that was most severe in those patients with hippocampal sclerosis. GLX elevation was a feature in the patients without hippocampal sclerosis. Metabolic abnormality was most marked in the anterior compared to the posterior hippocampal regions. GABA+ levels were elevated in patients with MCD and in the ipsilateral temporal lobe in temporal lobe epilepsy associated with hippocampal sclerosis but levels were not altered in patients with IGE or OLE. GLX was also elevated in MCD in the region of MRI visible abnormality and in IGE patients when measured in the frontal lobes. Low NAA was a feature of TLE and MCD. Patients with IGE showed normal NAA levels in the occipital lobes but reduced frontal lobe concentrations. Cr concentrations were abnormal in the immediate post ictal period but normalised within 120 minutes. NAA was not altered and no significant change in lactate concentrations was observed. Finally sodium valproate treatment was associated with a reduction in the levels of Ins and with unchanged NAA and GLX levels. Main Conclusions: MRS techniques demonstrate metabolite abnormalities in epileptic patients. NAA is the most sensitive metabolite marker of chronic pathology but levels are insensitive to recent seizure history. These findings repeat earlier observations of the usefulness of NAA measurement in the assessment of chronic epilepsy whilst illustrating ongoing uncertainty as to the correct patho-physiological interpretation of reduced NAA levels. Measurable changes in the combined Cr signal are detectable whilst elevated lactate is not reliably observed following brief epileptic seizures at 1.5T. This finding indicates a potential role for MRS in functional activation studies. Malformations of cortical development have abnormal levels of both GABA+ and GLX and MCD sub-types may well demonstrate different metabolite profiles. This finding suggests that MRS could be a useful tool in the MRI classification of MCD and in the pre-surgical assessment of patients with focal malformations. Following successful temporal lobe surgery levels of NAA remain unchanged but NAA/Cr levels appear to normalise in the contralateral temporal lobe. NAA and GLX/NAA levels were altered in the frontal lobes but not in the occipital lobes in Idiopathic Generalised Epilepsy. This finding provides imaging support for frontal lobe dysfunction as a cause or consequence of IGE. Metabolite levels are affected by administered antiepileptic drugs. Sodium valproate reduces the levels of MRS visible Ins levels whilst topiramate and gabapentin appear to be associated with higher GABA+ levels. These findings may be of major importance in the assessment of treatment effect or in the investigation of patients with possible drug resistance. The effect of valproate on Ins levels may become particularly interesting in the light of a growing understanding of the role of astrocyte dysfunction in a range of neurological conditions which include migraine, epilepsy, Alzheimer’s disease, motor neurone disease and in ischaemic lesions

The impact of the UK COVID-19 pandemic on patient-reported health outcomes after stroke: a retrospective sequential comparison

BACKGROUND AND PURPOSE: The COVID-19 pandemic and related social isolation measures are likely to have adverse consequences on community healthcare provision and outcome after acute illnesses treated in hospital, including stroke. We aimed to evaluate the impact of the COVID-19 pandemic on patient-reported health outcomes after hospital admission for acute stroke. METHODS: This retrospective study included adults with acute stroke admitted to the University College Hospital NHS Foundation Trust Hyperacute Stroke Unit. We included two separate cohorts of consecutively enrolled patients from the same geographical population at two time points: 16th March-16th May 2018 (pre-COVID-19 pandemic); and 16th March-16th May 2020 (during the COVID-19 pandemic). Patients in both cohorts completed the validated Patient Reported Outcomes Measurement Information System-29 (PROMIS-29 version 2.0) at 30 days after stroke. RESULTS: We included 205 patients who were alive at 30 days (106 admitted before and 99 admitted during the COVID-19 pandemic), of whom 201/205 (98%) provided patient-reported health outcomes. After adjustment for confounding factors, admission with acute stroke during the COVID-19 pandemic was independently associated with increased anxiety (β = 28.0, p < 0.001), fatigue (β = 9.3, p < 0.001), depression (β = 4.5, p = 0.002), sleep disturbance (β = 2.3, p = 0.018), pain interference (β = 10.8, p < 0.001); and reduced physical function (β = 5.2, p < 0.001) and participation in social roles and activities (β = 6.9, p < 0.001). CONCLUSION: Compared with the pre-pandemic cohort, patients admitted with acute stroke during the first wave of the COVID-19 pandemic reported poorer health outcomes at 30 day follow-up in all domains. Stroke service planning for any future pandemic should include measures to mitigate this major adverse impact on patient health

Characteristics of ischaemic stroke associated with COVID-19

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is associated with coagulopathy causing venous and arterial thrombosis.1 2 Recent data from the pandemic epicentre in Wuhan, China, reported neurological complications in 36% of 214 patients with COVID-19; acute cerebrovascular disease (mainly ischaemic stroke) was more common among 88 patients with severe COVID-19 than those with non-severe disease (5.7% vs 0.8%).3 However, the mechanisms, phenotype and optimal management of ischaemic stroke associated with COVID-19 remain uncertain. We describe the demographic, clinical, radiological and laboratory characteristics of six consecutive patients assessed between 1st and 16th April 2020 at the National Hospital for Neurology and Neurosurgery, Queen Square, London, UK, with acute ischaemic stroke and COVID-19 (confirmed by reverse-transcriptase PCR (RT-PCR)) (table 1). All six patients had large vessel occlusion with markedly elevated D-dimer levels (≥1000μg/L). Three patients had multiterritory infarcts, two had concurrent venous thrombosis, and, in two, ischaemic strokes occurred despite therapeutic anticoagulation

Widespread extrahippocampal NAA/(Cr+Cho) abnormalities in TLE with and without mesial temporal sclerosis

MR spectroscopy has demonstrated extrahippocampal NAA/(Cr+Cho) reductions in medial temporal lobe epilepsy with (TLE-MTS) and without (TLE-no) mesial temporal sclerosis. Because of the limited brain coverage of those previous studies, it was, however, not possible to assess differences in the distribution and extent of these abnormalities between TLE-MTS and TLE-no. This study used a 3D whole brain echoplanar spectroscopic imaging (EPSI) sequence to address the following questions: (1) Do TLE-MTS and TLE-no differ regarding severity and distribution of extrahippocampal NAA/(Cr+Cho) reductions? (2) Do extrahippocampal NAA/(Cr+Cho) reductions provide additional information for focus lateralization? Forty-three subjects (12 TLE-MTS, 13 TLE-no, 18 controls) were studied with 3D EPSI. Statistical parametric mapping (SPM2) was used to identify regions of significantly decreased NAA/(Cr+Cho) in TLE groups and in individual patients. TLE-MTS and TLE-no had widespread extrahippocampal NAA/(Cr+Cho) reductions. NAA/(Cr+Cho) reductions had a bilateral fronto-temporal distribution in TLE-MTS and a more diffuse, less well defined distribution in TLE-no. Extrahippocampal NAA/(Cr+Cho) decreases in the single subject analysis showed a large inter-individual variability and did not provide additional focus lateralizing information. Extrahippocampal NAA/(Cr+Cho) reductions in TLE-MTS and TLE-no are neither focal nor homogeneous. This reduces their value for focus lateralization and suggests a heterogeneous etiology of extrahippocampal spectroscopic metabolic abnormalities in TLE

FcRn-mediated antibody transport across epithelial cells revealed by electron tomography

The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers, thereby providing the fetus or newborn with humoral immunity before its immune system is fully functional. In newborn rats, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. The pH difference between the apical (pH 6.0–6.5) and basolateral (pH 7.4) sides of intestinal epithelial cells facilitates the efficient unidirectional transport of IgG, because FcRn binds IgG at pH 6.0–6.5 but not at pH 7 or more. As milk passes through the neonatal intestine, maternal IgG is removed by FcRn-expressing cells in the proximal small intestine (duodenum and jejunum); remaining proteins are absorbed and degraded by FcRn-negative cells in the distal small intestine (ileum). Here we use electron tomography to make jejunal transcytosis visible directly in space and time, developing new labelling and detection methods to map individual nanogold-labelled Fc within transport vesicles and simultaneously to characterize these vesicles by immunolabelling. Combining electron tomography with a nonperturbing endocytic label allowed us to conclusively identify receptor-bound ligands, resolve interconnecting vesicles, determine whether a vesicle was microtubule-associated, and accurately trace FcRn-mediated transport of IgG. Our results present a complex picture in which Fc moves through networks of entangled tubular and irregular vesicles, only some of which are microtubule-associated, as it migrates to the basolateral surface. New features of transcytosis are elucidated, including transport involving multivesicular body inner vesicles/tubules and exocytosis through clathrin-coated pits. Markers for early, late and recycling endosomes each labelled vesicles in different and overlapping morphological classes, revealing spatial complexity in endo-lysosomal trafficking

Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential

Human IgG3 displays the strongest effector functions of all IgG subclasses but has a short half-life for unresolved reasons. Here we show that IgG3 binds to IgG-salvage receptor (FcRn), but that FcRn-mediated transport and rescue of IgG3 is inhibited in the presence of IgG1 due to intracellular competition between IgG1 and IgG3. We reveal that this occurs because of a single amino acid difference at position 435, where IgG3 has an arginine instead of the histidine found in all other IgG subclasses. While the presence of R435 in IgG increases binding to FcRn at neutral pH, it decreases binding at acidic pH, affecting the rescue efficiency—but only in the presence of H435–IgG. Importantly, we show that in humans the half-life of the H435-containing IgG3 allotype is comparable to IgG1. H435–IgG3 also gave enhanced protection against a pneumococcal challenge in mice, demonstrating H435–IgG3 to be a candidate for monoclonal antibody therapies

Maternal Immunization with Pneumococcal Surface Protein A Protects against Pneumococcal Infections among Derived Offspring

Pathogen-specific antibody plays an important role in protection against pneumococcal carriage and infections. However, neonates and infants exhibit impaired innate and adaptive immune responses, which result in their high susceptibility to pneumococci. To protect neonates and infants against pneumococcal infection it is important to elicit specific protective immune responses at very young ages. In this study, we investigated the protective immunity against pneumococcal carriage, pneumonia, and sepsis induced by maternal immunization with pneumococcal surface protein A (PspA). Mother mice were intranasally immunized with recombinant PspA (rPspA) and cholera toxin B subunit (CTB) prior to being mated. Anti-PspA specific IgG, predominantly IgG1, was present at a high level in the serum and milk of immunized mothers and in the sera of their pups. The pneumococcal densities in washed nasal tissues and in lung homogenate were significantly reduced in pups delivered from and/or breast-fed by PspA-immunized mothers. Survival after fatal systemic infections with various types of pneumococci was significantly extended in the pups, which had received anti-PspA antibody via the placenta or through their milk. The current findings strongly suggest that maternal immunization with PspA is an attractive strategy against pneumococcal infections during early childhood. (191 words

FcRn Overexpression in Transgenic Mice Results in Augmented APC Activity and Robust Immune Response with Increased Diversity of Induced Antibodies

Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance