1,721,122 research outputs found
The Application of the International Consensus Criteria to Assess Prevalence of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis in Australia
No full textChronic Fatigue Syndrome, also referred to as Myalgic Encephalomyelitis (CFS/ME) is receiving greater public health attention as a complex condition that results in substantial functional impairment. It is largely characterised by severe, medically unexplained fatigue and is associated with a broad range of further symptoms. Diagnosis remains a particular challenge and case definitions remain the primary tool in the absence of reliable and consistent clinical and biological markers. Several case definitions have been proposed and these differ significantly in symptom criteria, comorbid considerations and exclusion of other conditions, thus representing contrasting clinical profiles. This contributes to large differences in reported population, clinical, and laboratory findings reported for CFS/ME.
A systematic review of worldwide prevalence studies was performed and demonstrated that the Fukuda et al. definition has been adopted as a standard. Furthermore, no studies had adopted the more recently proposed International definition. Despite the numerous studies demonstrating the public health impact of CFS/ME abroad, only one report was found estimating CFS/ME prevalence in the Australian population but this report predated the Fukuda and more recent case definitions. In the absence of a prevalence estimate for Australia, a meta-analysis was performed to pool worldwide prevalence figures of Fukuda defined cases. The pooled prevalence for self-reporting assessment was 3.28% (95% CI: 2.24–4.33) and 0.76% (95% CI: 0.23–1.29) for clinical assessment.Thesis (PhD Doctorate)Doctor of Philosophy (PhD)School of Medical ScienceGriffith HealthFull Tex
Investigation of Cellular and Genetic Mechanisms Required for Natural Killer Cell Cytotoxic Activity in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis
No full textChronic Fatigue Syndrome/ Myalgic Encephalomyelitis (CFS/ME) is a complex disorder associated with disabling and unremitting fatigue, flu-like symptoms, muscle and joint pain, post-exertional malaise and cognitive impairments. CFS/ME is a heterogeneous illness and the cause of CFS/ME is currently unknown. Consequently, there is no biological marker to diagnose the illness, and case definitions are used to identify CFS/ME patients. CFS/ME is a multi-system disorder that has been associated with impairments in the functions of the endocrine, immune, central and autonomic nervous systems. Evidence for chronic immune dysfunction in CFS/ME has been reported by several groups, where significantly reduced Natural Killer (NK) cell cytotoxic activity has been the most predominant and consistent finding. NK cells consist of CD56brightCD16dim/- NK cells which produce immunoregulatory cytokines, and CD56dimCD16+ NK cells which induce cytotoxic lysis of target cells infected by viruses, bacteria and parasites, or cells that have been malignantly transformed. The granule secretory pathway mediates NK cell cytotoxic activity and includes contact, adhesion, activation, granule polarisation and degranulation. Cytotoxic lysis of target cells is predominantly induced by the granule secretory pathway and, as such, has been associated with the cytotoxic dysfunction reported in CFS/ME patients. The aim of this thesis was to investigate cellular and genetic components required for NK cell cytotoxic activity to identify a potential mechanism of reduced cytotoxic activity in CFS/ME patients.Thesis (PhD Doctorate)Doctor of Philosophy (PhD)School of Medical ScienceGriffith HealthFull Tex
Epidemiology of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis: An Assessment of Case Definitions, Health Related Quality of Life, and Therapeutic Behaviours
No full textChronic Fatigue Syndrome or Myalgic Encephalomyelitis (CFS/ME) is chronic debilitating disorder of no known cause. It is characterised by chronic fatigue that is not due to ongoing exertion or due to another medical condition, and becomes markedly worse following minimal activity. The fatigue is further accompanied by a multitude of other symptoms affecting many body systems [1].
Patients suffer a substantial loss in quality of life, with severe disruption to daily activities. This disorder is heterogeneous in nature, with patients experiencing varied symptoms, at different severities, frequencies and duration. Patients also experience periods of relapse and remission [2].
The unknown aetiology and patho-mechanism has hindered the development of laboratory based diagnostic tools, thus diagnosis is solely based on symptom criteria. However, with the inconsistent presentation, no standardised diagnostic criteria have been established, and several criteria exist varying widely in the description of the illness [3, 4]. The lack of standardisation has resulted in CFS/ME becoming an umbrella term [5]. Patients who suffer from general chronic fatigue but do not meet the other criteria, or have fatigue as a result of an underlying condition, are often misdiagnosed with CFS/ME. Alternatively, those that may have CFS/ME are often misdiagnosed as another illness due to lack of recognition [6].Thesis (Masters)Master of Medical Research (MMedRes)School of Medical ScienceGriffith HealthFull Tex
Examination of Innate and Adaptive Immune Cells in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis Patients with Varying Degrees of Symptom Severity
No full textThe immune system has a critical influence on the maintenance of physiological homeostasis. To date, immunological dysfunction, particularly reduced natural killer (NK) cell cytotoxic activity in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) patients has been consistently observed. CFS/ME is a severely debilitating illness, with no known pathomechanism and diagnosis is made according to symptom specific criteria. CFS/ME is characterised by persistent and unexplained fatigue, alongside a range of symptoms, including: post-exertional neuroimmune exhaustion, neurological, immune, gastrointestinal, genitourinary and energy metabolism impairments. However, a symptom specific criterion provides complications for diagnosis, particularly as symptoms may be qualitative. CFS/ME is also a heterogeneous illness, with patients experiencing moderate to severe symptoms. CFS/ME patients with moderate symptoms are those who have reduced mobility and ability to perform their routine daily activities. CFS/ME patients with severe symptoms are usually homebound and/or restricted to a wheelchair. The debilitating nature of CFS/ME creates an economic burden and contributes largely to health resources, affecting CFS/ME patients as well as the wider community. In Australia, the annual cost to the community per CFS/ME patient, with a prevalence rate of 0.2% is $729.3 million (based on 2012 estimates and earlier prevalence studies).Thesis (PhD Doctorate)Doctor of Philosophy (PhD)School of Medical ScienceGriffith HealthFull Tex
Laboratory Analysis, Imaging, and Treatment Outcomes of Central Nervous System Demyelinating Disease
No full textNatural killer (NK) cell phenotypes have been reported to be implicated in the pathogenesis of multiple sclerosis (MS). Several studies have observed reduced peripheral numbers, reduced cytotoxic activity, and altered CD56Dim and CD56Bright NK cell phenotypes. In this thesis I have investigated NK cell cytotoxicity, calcium mobilisation and transient receptor potential melastatin 3 (TRPM3) surface expression. NK cell cytotoxic activity and calcium signalling were examined in CD56Dim and CD56Bright NK cells before and after stimulation with ionomycin, pregnenolone sulphate, 2-aminoethoxydiphenyl borate and thapsigargin. Purified NK cells were labelled with antibodies to determine TRPM3, CD69 and CD107a surface expression using flow cytometry. Twenty-two people with MS and 22 healthy controls were recruited for this project. Twelve of the 22 MS cases previously received alemtuzumab and the remaining 10 were on no medication. TRPM3 was significantly increased in untreated MS compared with healthy controls and treated MS (p = 0.034). There was a significant decrease in CD69 surface expression on CD56Dim phenotype NK cells for untreated MS (p = 0.031) and treated MS (p = 0.036) compared to controls. Calcium mobilisation in CD56Bright NK cells and to a lesser extent CD56Dim NK cells between healthy controls, treated and untreated MS was also altered. In conclusion, these investigations suggest variations in TRPM3 expression and calcium mobilisation of NK cells may be implicated in the pathogenesis of MS. Further investigation is required to determine the mechanism by which alemtuzumab alters calcium signalling in NK cells.
Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory disease of the central nervous system associated with antibodies to aquaporin-4 (AQP4) which has distinct clinical, radiological and pathological features, but also has some overlap with MS and myelin oligodendrocyte glycoprotein (MOG) antibody associated disease. Early recognition of NMOSD is important because of differing responses to both acute and preventive therapy. Magnetic resonance imaging (MRI) has proven helpful in this process. Key MRI clues to the diagnosis of NMOSD are longitudinally extensive lesions of the optic nerve (more than half the length) and spinal cord (3 or more vertebral segments), bilateral optic nerve lesions and lesions of the optic chiasm, area postrema, floor of the IV ventricle, periaqueductal grey matter, hypothalamus and walls of the III ventricle. Other NMOSD specific lesions are denoted by their unique morphology: heterogeneous lesions of the corpus callosum, "cloud-like" Gd-enhancing white matter lesions and "bright spotty" lesions of the spinal cord. The specificity of other lesions described in NMOSD, including linear periventricular periependymal lesions and 'patch' subcortical white matter lesions, are less certain. The use of advanced MR imaging techniques is yielding further useful information about focal degeneration of the thalamus and optic radiation in NMOSD and suggests that paramagnetic rim patterns and changes in normal appearing white matter are specific to MS. MR imaging is crucial in the early recognition of NMOSD and in directing testing for AQP4 antibodies and guiding immediate acute treatment decisions. Increasingly, MR imaging is playing a role in diagnosing seronegative cases of NMOSD.
NMOSD and MS are inflammatory diseases of the CNS. Overlap in the clinical and MRI features of NMOSD and MS means that distinguishing these conditions can be difficult. With the aim of evaluating the diagnostic utility of MRI features in distinguishing NMOSD from MS, I have conducted a cross-sectional analysis of imaging data and developed predictive models to distinguish the two conditions. NMOSD and MS MRI lesions were identified and defined through a literature search. AQP4 antibody seropositive NMOSD cases were collected together with age- and sex-matched MS cases. MRI of orbits, brain and spine were reported by at least two blinded reviewers. MRI brain or spine was available for 166/168 (99%) of cases. Longitudinally extensive (odds ratio [OR]=203), "bright spotty" (OR 93.8), whole (axial; OR=57.8) or gadolinium (Gd) enhancing (OR=28.6) spinal cord lesions, bilateral (OR=31.3) or Gd-enhancing (OR=15.4) optic nerve lesions, and nucleus tractus solitarius (OR=19.2), periaqueductal (OR=16.8) or hypothalamic (OR=7.2) brain lesions were associated with NMOSD. Ovoid (OR=0.029), Dawson's finger (OR 0.031), pyramidal corpus callosum (OR=0.058), periventricular (OR=0.136), temporal lobe (OR=0.137) and T1 black hole (OR=0.154) brain lesions were associated with MS. A score-based algorithm and a decision tree determined by machine learning accurately predicted more than 85% of both diagnoses using first available imaging alone. I have confirmed NMOSD and MS specific MRI features and combined these in predictive models that can accurately identify more than 85% of cases as either AQP4 seropositive NMOSD or MS.
NMOSD is associated with significant morbidity and mortality. Several therapies have been recommended for NMOSD and more recently clinical trials have demonstrated efficacy for three monoclonal antibody therapies. I present an observational study of treatment response in NMOSD. This was a retrospective, unblinded, observational study of treatment efficacy of rituximab, traditional immunosuppressive therapy and MS therapies (used inadvertently) in people with AQP4 antibody positive NMOSD. Treatment efficacy was assessed using annualised relapse rate (ARR), time to first relapse and Expanded Disability Status Scale scores. Complete relapse and treatment data were available for 43/68 (63%) of AQP4 antibody positive NMOSD cases covering 74 episodes of treatment. In a time to first relapse analysis rituximab showed a risk ratio of 0.23 (95% CI 0.08 - 0.65) when compared with no treatment and there was a nonsignificant reduction in ARR of 35% compared to pre-treatment. The MS therapy, β-interferon (p = 0.0002), and cyclophosphamide (p = 0.0034) were associated with an increased ARR compared to pre-treatment. A lower final EDDS was seen for Rituximab (median 4.0 [range 0.0 - 7.0]; p = 0.042) and traditional immunosuppressive therapy (median 4.0 [range 0.0 - 8.0]; p = 0.016) compared to β-interferon (median 6.0 [range 4.0 - 7.5]). These data provide additional support for the use of rituximab in preference to traditional immunosuppressive agents and MS disease modifying therapies as first line treatment of NMOSD.Thesis (PhD Doctorate)Doctor of PhilosophySchool of Medicine & DentistryGriffith HealthFull Tex
T helper cell cytokine profiles after endurance exercise
Get PDFEndurance exercise can cause immunosuppression and increase the risk of upper respiratory illness. The present study examined changes in the secretion of T helper (Th) cell cytokines after endurance exercise. Ten highly trained road cyclists [mean+/-SEM: age 24.2+/-1.7 years; height 1.82+/-0.02 m; body mass 73.8+/-2.0 kg; peak oxygen uptake 65.9+/-2.3 mL/(kg.min)] performed 2 h of cycling exercise at 90% of the second ventilatory threshold. Peripheral blood mononuclear cells were isolated and stimulated with phytohemagglutinin. Plasma cortisol concentrations and the concentration of Th1/Th2/Th17 cell cytokines were examined. Data were analyzed using both traditional statistics and magnitude-based inferences. Results revealed a significant decrease in plasma cortisol at 4-24 h postexercise compared with pre-exercise values. Qualitative analysis revealed postexercise changes in concentrations of plasma cortisol, IL-2, TNF, IL-4, IL-6, IL-10, and IL-17A compared with pre-exercise values. A Th1/Th2 shift was evident immediately postexercise. Furthermore, for multiple cytokines, including IL-2 and TNF (Th1), IL-6 and IL-10 (Th2), and IL-17 (Th17), no meaningful change in concentration occurred until more than 4 h postexercise, highlighting the duration of exercise-induced changes in immune function. These results demonstrate the importance of considering "clinically'' significant versus statistically significant changes in immune cell function after exercise
Impaired surface expression of Transient Receptor Potential Melastatin 2 and 3 ion channels lowers Natural Killer Cell Cytotoxic Activity in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome
No full textMyalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) is a disabling condition characterised by unexplained chronic fatigue that is associated with immune, neurological (including autonomic), musculoskeletal, cardiovascular and gastrointestinal symptoms [1, 2]. Currently, accurate diagnosis remains challenging in the absence of a clinical or laboratory test. Although the aetiology of ME/CFS remains undefined, a significant reduction in natural killer (NK) cell cytotoxicity is consistently reported in ME/CFS patients compared with healthy controls (HC) [3-6]. NK cells are effector lymphocytes of the innate immune system principally responsible for recognising and responding to pathogen invasion [7]. Approximately 90% of peripheral NK cells are CD56DimCD16+ which are highly cytotoxic and kill infected, tumour or ‘missing self’ cells through cytotoxic processes [8]. Conversely, the CD56BrightCD16Dim/- subset is responsible for immunosurveillance and cytokine production [9]. Importantly, NK cells require calcium (Ca2+) to regulate various cellular functions, such as cell differentiation, cell division, apoptosis, transcription, and cytotoxicity [10].
Transient Receptor Potential (TRP) channels are a group of unique ion channels whereby majority are highly selective to Ca2+ [11]. Functionally, TRP ion channels regulate “threat” stimuli, such as pain, thermosensation, mechanosensation, pathogens, and chemicals, via sensory transduction pathways. TRPM2 and TRPM3 are cation channels ubiquitously expressed throughout the human body and expressed in almost all cell types, such as NK cells. Both TRPM members are highly permeable to Ca2+, therefore are critical regulators of Ca2+-dependent pathways, such as NK cell cytotoxicity. TRPM2 is activated by adenine dinucleotides (ADPR, cADPR, NAADP, ꞵ-NAD), reactive oxygen species (hydrogen peroxide and OH-), and intracellular ([Ca2+]i). Conversely, TRPM3 is potently activated by neural steroids, such as pregnenolone sulphate (PregS) and nifedipine. Upon stimulation, TRPM2 and TRPM3 ion channels trigger a rapid influx of Ca2+ influx and rise in [Ca2+]i, which subsequently facilitates intracellular pathways for Ca2+ release from cellular organelles.
Recently, TRPM2 has emerged as a key receptor in mediating Ca2+-induced anti-tumour activity in mouse NK cells via synergistic activation with CD38. CD38 generates Ca2+ mobilizing secondary messengers, such as ADPR, to activate TRPM2. Rah et al., determined inhibition of sustained tumour-induced Ca2+ signals and degranulation in mouse NK cells following 8-bromoadenosine diphosphoribose (8-Br-ADPR) treatment [12]. Moreover, N6-Benzoyladenosine-3′,5′-cyclic monophosphate (N6-Bnz-cAMP) significantly increased intracellular ADPR, however was inhibited in Ca2+-free conditions [12]. Currently, no in vitro studies have examined the phenotype and function of TRPM2 ion channels on human NK cell subsets, notably in ME/CFS research. Conversely, five single nucleotide polymorphisms in NK cells associated with TRPM3 have been previously identified in ME/CFS patients [13]. Reduced TRPM3 surface expression and impaired Ca2+ influx has furthermore been identified on NK cells in ME/CFS patients [14].
Given TRPM2 and TRPM3 are both critical regulators for Ca2+ signalling in NK cells, the overall aim of this thesis was to investigate the role of TRPM2 and TRPM3 in mediating NK cell cytotoxicity to identify a potential mechanism of reduced NK cell cytotoxic activity in ME/CFS patients. Study one aimed to develop an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cell subsets using an antibody that has not been previously used with flow cytometry. Applying this optimised methodology, study two aimed to quantify TRPM2 and CD38 surface expression on NK cell subsets at baseline and post in vitro drug treatments (N6-Bnz-cAMP and 8-Br-ADPR) in ME/CFS patients and HC. NK cell cytotoxicity was furthermore measured at baseline and post in vitro drug treatments (N6-Bnz-cAMP and 8-Br-ADPR) between groups. Lastly, study three aimed to examine the clinical presentation in a moderate-severe ME/CFS group, as well as measure NK cell cytotoxicity post in vitro drug treatment with TRPM3 agonists, PregS, nifedipine and ononetin, in ME/CFS patients and HC.
Age and sex matched HC were included in study one. Age and sex-matched ME/CFS patients meeting the Canadian Consensus Criteria (CCC) and HC were included in studies two and three. All participants donated 85ml of whole blood and peripheral NK cells were isolated. TRPM2 and CD38 surface expression was measured on CD56DimCD16+ and CD56BrightCD16Dim/- subsets, as well as NK cell cytotoxicity at baseline and post in vitro drug treatments by flow cytometry. Drug treatments included: interleukin-2, N6-Bnz-cAMP, 8-Br-ADPR, PregS, nifedipine and ononetin.
Study one determined 1:50 as the optimal primary TRPM2 antibody dilution following a two-hour incubation period. TRPM2 surface expression with and without CD38 co-expression significantly increased between 1:300 and 1:50 primary TRPM2 antibody dilutions following a two-hour incubation period on both CD56DimCD16+ and CD56BrightCD16Dim/- NK cell subsets. On the CD56DimCD16+ subset only, TRPM2 and CD38 surface expression also significantly increased at 1:50 compared with 1:100. Moreover, TRPM2 surface expression significantly decreased between 1:50 and 1:5 TRPM2 antibody dilution following a two-hour incubation period. This significant decrease highlights the high-dose hook effect, whereby the highly concentrated 1:5 antibody dilution saturated both capture and detection TRPM2 antibodies. Study two identified a significant overexpression of the TRPM2 ion channel on NK cell subsets in ME/CFS patients compared with HC. No significant differences in NK cell cytotoxicity were observed between or within groups post N6-Bnz-cAMP and 8-Br-ADPR drug treatments.
Lastly, study three revealed no signficiant differences in NK cell cytotoxicity post PregS and nifedipine drug treatments, as well as subsequent blocking with ononentin in both groups. In both Study 2 and Study 3, viral infections and various clinical ME/CFS symptoms were significantly associated with reduced NK cell cytotoxicity in ME/CFS patients. Associations included: pain, cognitive difficulties, sleep disturbances, sensory impairments, thermostatic instability and gastrointestinal disturbances, possibly involving TRPM2 and TRPM3.
In conclusion, the results of this thesis are the first to develop a novel and optimal in vitro methodology to measure TRPM2 and CD38 surface expression on human NK cell subsets using flow cytometry. This thesis is also the first to report overexpressed TRPM2 ion channels on NK cell subsets in ME/CFS patients. Oxidative stress induced by viral infections is hypothesised to cause this overexpression in TRPM2 ion channels in ME/CFS patients as previously reported. Overexpressed TRPM2 ion channels may cause mitochondrial dysfunction, cellular death, DNA damage, and disruption to MAPK pathways following uncontrolled increases in [Ca2+]i. Collectively, these processes interfere with downstream Ca2+-dependent pathways, such as NK cell cytotoxicity, which was found to be significantly reduced at baseline in ME/CFS patients compared to HC.
The drug-treated NK cell cytotoxicity results may reflect the sensitivity of the cytotoxic assay to capture the TRPM2 and TRPM3 drug-modulatory effects on NK cell cytotoxicity. Given the drugs were incubated in the media and cells for more than 24 hours, the TRPM2 and TRPM3 ion channels may have undergone repetitive activation and inhibition cycles. Consequently, this may have activated and caused sustained long-term Ca2+-dependent pathways which may have potentially resulted in disrupted gene expression, irreversible cellular death and the development of NK cell hyporesponsiveness.
Furthermore, a primary limitation with TRPM2 is the lack of potent and specific pharmacological tools. Consequently, the TRPM2 signals may have been reduced or lost, subsequently modulating activation of downstream NK cell cytotoxic processes, such as the extracellular signal-regulated protein kinase 1/2 and mitogen-activated protein kinase pathways. Taken together, this thesis warrants the identification of additional experiments with a more appropriate time-sensitivity to capture the pharmacological effects of specific cellular mechanisms of interest, as well as the identification and development of more potent, specific, and non-toxic pharmacological tools targeting TRPM2. Additional rationales include co-localisation of between TRPM2 and CD38 and the involvement of TRPM2 and TRPM3 spliced isoforms.
A common feature shared amongst the significant associations between reduced NK cell cytotoxicity clinical ME/CFS symptoms is the high expression and functional activity of TRPM2 and TRPM3 in the CNS, which functions as the control centre for these physiological systems. However, additional quantitative tests examining these clinical functions, such as nociceptive pain and thermoregulation, are required to definitvely associate the possible roles of TRPM2 and TRPM3 activity and the unique clinical presentation of ME/CFS. Interestingly, positive correlations were determined between reduced NK cell cytotoxicity and overexpressed TRPM2 ion channels on both NK cell subsets within the ME/CFS group. ROC analyses also revealed diagnostic potential for reduced NK cell cytotoxicity and overexpressed TRPM2 ion channels in ME/CFS patients. Collectively, these results highlight a relationship between TRPM2 and reduced NK cell cytotoxicity in ME/CFS. Therefore, furthur investigations in this vital area of ME/CFS research will assist in the validation of TRPM2 and TRPM3 as potential biological markers to further understand the unique pathomechanism of ME/CFS and facilitate the development of targeted therapeutic interventions to improve the quality of life of ME/CFS patients.Thesis (PhD Doctorate)Doctor of Philosophy (PhD)School of Medical ScienceGriffith HealthFull Tex
Implications of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome on quality of life and the role of transient receptor potential melastatin 3 ion channels on natural killer cells in the pathomechanism of illness
No full textMyalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a serious disorder of unknown aetiology with an abstruse pathomechanism. Diagnosis is often delayed due to the lack of laboratory tests and specific diagnostic criteria as well as the lack of effective treatments that heightens the burden of illness. ME/CFS is characterised by post-exertional malaise and neuroimmune exhaustion, accompanied by symptoms that are neurological, immunological, endocrinological, gastrointestinal, and genitourinary in nature. To date, the cause and origin remain ambiguous, hence patients often try different treatment options to improve health, but without much success. The burden of ME/CFS on quality of life (QoL) is a key component of this thesis, therefore, the first aim was to determine the impact of ME/CFS on QoL.
For the first phase of this thesis, using an online survey, self-reported data was collected from 480 individuals diagnosed with ME/CFS (46.0 ± 12.3 years). A multivariate linear regression was used to determine the correlation between ME/CFS symptom presentation with QoL. Health-related QoL (HRQoL) was significantly impaired in ME/CFS patients across all eight SF-36 domains when compared with Australian general population norms obtained from the Australian Bureau of Statistics (p<0.001). ME/CFS patients reported the lowest SF-36 scores for physical role of illness on limitations (4.1 ± 15.1). Lower QoL scores in limitations due to physical functioning were associated with gender (p<0.001, adjusted R2=0.236), high body mass index (BMI) (p=0.015, adjusted R2=0.236), unemployment (p<0.001, adjusted R2=0.236), cognitive difficulties (p=0.014, adjusted R2=0.236), sensory disturbances (p<0.001, adjusted R2=0.236) and cardiovascular symptoms (p<0.001, adjusted R2=0.236). Low scores for limitations due to pain were associated with high BMI (p=0.008, adjusted R2=0.497), flu-like symptoms (p=0.037, adjusted R2=0.497) and body temperature intolerances (p=0.035, adjusted R2=0.497), as well as frequent visits to their general practitioner (p=0.035, adjusted R2=0.497). Reduced well-being QoL scores were associated with smoking status (p=0.013, adjusted R2=0.155), psychiatric comorbidity (p<0.001, adjusted R2=0.155), sleep disturbances (p<0.001, adjusted R2=0.155) and gastrointestinal upset (p=0.038, adjusted R2=0.155).
Immunological dysfunction is a key aspect of the presentation of ME/CFS, the significantly reduced natural killer (NK) cell cytotoxicity has been consistently reported. However, this the consistence of impaired NK cell cytotoxicity was yet to be validated through systematic review. In phase II, a systematic review was completed to investigate NK cell cytotoxicity in ME/CFS patients compared with HC. Impaired NK cell cytotoxicity was a consistent immunological feature in ME/CFS patients, and this systematic review confirmed the validity of NK cell cytotoxicity as a reliable vector for research in ME/CFS. Therefore, NK cells were used as a cell model for ME/CFS research in phases III and IV of this thesis.
NK cell cytotoxicity heavily relies on long-term sustained influx of calcium (Ca2+) to successfully activate function through intracellular signalling proteins, polarisation of cytolytic granules and release of cytolytic proteins. Therefore, addition focus of this thesis was to investigation Ca2+ mobilisation and characterise transient receptor potential melastatin 3 (TRPM3) channels. TRPM3 ion channels are highly permeable to Ca2+, and have critical roles in pain perception, thermosensation and inflammation. In the laboratory, TRPM3 can be activated using pregnenolone sulfate (PregS) and inhibited using ononetin. Recently, treatment of NK cells in vitro using naltrexone hydrochloride (NTX) has restored TRPM3 function in ME/CFS patients. It is hypothesised that some ME/CFS subsets may be the result of ion channelopathy, and that this research will contribute to the understanding of TRPM3 and Ca2+ mobilisation to the mechanism of ME/CFS.
NK cells were isolated from whole blood of participants using negative immunomagnetic selection. In phase III, NK cells were isolated from n=15 ME/CFS patients (43.93±2.94 years, 60% female) and n=15 healthy controls (HC) (44.2±3.14 years, 60% female). The co-localisation of TRPM3 with Ca2+ signalling protein, phosphatidylinositol 4,5-bisphosphate (PIP2), after regulation with TRPM3 specific drugs including PregS and ononetin, was investigated using immunofluorescence. Finally, in phase IV, NK cells were isolated from n=10 ME/CFS patients (43.9±10.71 years, 90% female) and n=10 HC (44.1±10.39, 90% female) to investigate TRPM3-dependent Ca2+ influx using live-cell immunofluorescent imaging.
Inferential analysis of co-localisation between HC and ME/CFS patients revealed a significant reduction in co-localisation of TRPM3 with PIP2 in ME/CFS patients under control conditions (p=0.0021). Interestingly, a significant increase in co-localisation of TRPM3 with PIP2 was reported in ME/CFS patients compared with HC following ononetin (p=0.0408). NK cell cytotoxicity was significantly reduced in ME/CFS patients compared with HC at baseline (p=0.0362). Overnight interleukin-2 (IL-2) stimulation significantly enhanced NK cell cytotoxicity in both HC and ME/CFS patients (p<0.0001). However, no significant difference was reported in NK cell cytotoxicity within groups following pharmacological treatment supplemented with IL-2.
TRPM3-dependent Ca2+ influx was determined following stimulation with PregS using three measurements: slope, half-time response (T1/2) and amplitude. Baseline Ca2+ influx was determined. The amplitude and T1/2 response were significantly reduced in ME/CFS patients compared with HC (p<0.0001). Ca2+ influx measurements for slope (p<0.0001), amplitude (p<0.0001) and T1/2 (p=0.0123) were significantly lower in ME/CFS patients compared with HC following overnight stimulation with IL-2. Interestingly, overnight treatment of isolated NK cells with NTX restored TRPM3-dependent Ca2+ influx in ME/CFS patients. There was no longer a significant difference between groups for slope and T1/2 response. A significant increase in amplitude was reported in ME/CFS patients compared with HC (p<0.0001).
In conclusion, ME/CFS is a serious condition and associated with reduced HRQoL emphasising the significant burden of illness, the importance of renewed public health policy, and increased research funding for ME/CFS patients. This thesis was the first to report on the significant association of low QoL scores with symptom presentation and severity in a large cohort of Australian ME/CFS patients. An example was the significant association found between reduced QoL and flu-like symptoms. ME/CFS is a multifactorial condition, and a key feature is immunological dysfunction, specifically reduced NK cell cytotoxicity. However, as ME/CFS is an ambiguous and multifactorial condition, the use of an appropriate model is vital for research. This thesis reports novel findings to support and confirm NK cell cytotoxicity is a consistent finding and model for research into the pathomechanism of ME/CFS. While TRPM3 ion channel dysfunction is well documented using whole-cell patch clamp, the intracellular signalling pathways involved in TRPM3 dysfunction is elusive. This research reveals, for the first time, the potential role of PIP2 in TRPM3 ion channel dysfunction. The potential crosstalk of signalling proteins involved in TRPM3, PIP2 and IL-2 signalling pathways in lymphocytes provides a foundation for future research in ME/CFS. The characterisation of TRPM3 ion channels provides an important target for not only elucidating the pathomechanism of ME/CFS, but also provides a potential therapeutic target. By targeting TRPM3 ion channels through pharmacotherapeutic modalities, namely NTX, this thesis reported novel findings for the restoration of intracellular Ca2+ influx in NK cells of ME/CFS patients using immunofluorescent imaging. TRPM3 is widely expressed in many cell and tissue types throughout the human body and by targeting TRPM3 ion channels with NTX, this may improve the multisystemic clinical presentation of ME/CFS patients, which may increase the QoL of these patients. Importantly, the use of a reliable and accessible cellular model for undertaking research into the pathomechanism of ME/CFS may advance research across disciplines and allow for the discovery of pharmacotherapeutic interventions to improve HRQoL.Thesis (PhD Doctorate)Doctor of Philosophy (PhD)School of Pharmacy & Med SciGriffith HealthFull Tex
Vasoactive neuropeptides in clinical ophthalmology: An association with autoimmune retinopathy?
No full textThe mammalian eye is protected against pathogens and inflammation in a relatively immune-privileged environment. Stringent mechanisms are activated that regulate external injury, infection, and autoimmunity. The eye contains a variety of cells expressing vasoactive neuropeptides (VNs), and their receptors, located in the sclera, cornea, iris, ciliary body, ciliary process, and the retina. VNs are important activators of adenylate cyclase, deriving cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). Impairment of VN function would arguably impede cAMP production and impede utilization of ATP. Thus VN autoimmunity may be an etiological factor in retinopathy involving perturbations of purinergic signaling. A sound blood supply is necessary for the existence and functional properties of the retina. This paper postulates that impairments in the endothelial barriers and the blood-retinal barrier, as well as certain inflammatory responses, may arise from disruption to VN function. Phosphodiesterase inhibitors and purinergic modulators may have a role in the treatment of postulated VN autoimmune retinopathy.Full Tex
Understanding myalgic encephalomyelitis
No full textMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a severe condition characterized by post-exertional neuroimmune exhaustion (PENE) accompanied by neurological, immunological, gastrointestinal (GI), and mitochondrial disturbances (1). The global prevalence of ME/CFS is ∼1%, affecting 17 million to 24 million people (2). ME/CFS is heterogeneous not only in symptom presentation but also illness trajectories, which can be worsening, plateauing, improving, or relapsing-remitting. Approximately 25% of patients with ME/CFS are considered severe and are bound to their homes. Although the etiology of ME/CFS is elusive, a large proportion of patients (∼60%) report post-infectious onset, such as after Epstein-Barr virus infection (3). The recent emergence of a chronic post-infectious condition, called Long Covid, overlaps considerably with ME/CFS in immunological, mitochondrial, and neurological dysfunctions (4). These similarities have resulted in increased interest and acceptance of ME/CFS as a disease and may stimulate research, the development of a diagnostic test, and pharmacotherapeutic interventions in ME/CFS that may be applied to Long Covid.Full Tex
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