Australian CFS Science Symposium Overview by Rosamund Vallings
This is just a fantastic review by Dr. Vallings of a really exciting Conference Down Under. Lots of new research much of it directed at the immune system and the central nervous system. There are studies on the brain stem, autopsies, CNS inflammation, NPY, neurodegeneration….. Notice that the basal ganglia in the brain show up several times (this is not the first time)… Well done Australia! (Headings and photos added)
International Science Symposium on ME/CFS, 3-4 December 2010, Bond University, Queensland, Australia.
Nancy Klimas and Homeostatic imbalance – The first paper was by Nancy Klimas (Miami,USA), and she presented a systems biology approach to ME/CFS. She described CFS is a disorder of homeostatic imbalance. She briefly outlined her 25 year history of involvement with this illness, when initially she worked on the theory of a chronic immune activation syndrome, with an immunological focus. It was next recognised as a neuro-inflammatory disorder, and now genomics have become involved. She listed and described some of her current research work.
One study involved an exercise challenge to induce relapse, looking at the gene expression and immune changes before, immediately after and 4 hours later. 3 matched groups were studied: Gulf War illness, CFS and controls. The exercise challenge was 8 minutes on an exercycle with measurement of VO2 max. The gene expression showed significant differences in those with GWI and CFS. (By case definition GWI and CFS meet the same criteria). Immunological pathways were similarly affected – these were mainly inflammatory, and the immune cascade led to many symptoms 4 hours later. Symptoms involved the endocrine, immune, autonomic and neurological systems. The genes regulating NK function which included abnormal perforin and granzyme levels were affected.
She then went on to describes Broderick’s 3 basic elements of analysis of immune signals, and related this to the states after the 8 minute challenge:
1. Those that looked different
2. Those that hang out with a different crowd
3. Those that behave differently (altered response dynamics)
In this study there was persistent inflammation, a surge in immune interaction and an IL-1 “splash” effect. There was a huge cascade effect in 8 minutes and persisting 4 hours later. Homeostasis is “messed up” and needs to remodel.
There is a need to focus on autonomic and immune therapies which do interface with each other. This study confirms that graded exercise is not good for those with CFS, and patients must stop exercise well short of the aerobic threshold. Breaks between exercise need to be twice as long as the duration of the exercise.
Perry on Infection and Sickness Behavior– Hugh Perry (Southampton, UK) discussed the adaptive and maladaptive components of what he describes as “sickness behaviour”. He then focussed on the language of “sickness” in relation to the way the systems behave during inflammation, for example “feeling ill” with pain and fever. He described sickness behaviour as an organised strategy which is not “bad”.
Infection leads to an inflammatory response with release of cytokines, which then communicate with the brain and cause symptoms such as malaise, fever and depression. Systemic inflammation activates selective brain regions, with different challenges activating different regions. This mechanism works through the macrophages in the brain via the blood-brain-barrier. Endothelial cells communicate with the macrophages via the microglia. This is an important part of homeostasis, and is usually transient.
He then went on to talk about chronic neurological disease when microglia increase in number and activation and become “primed”. Exaggerated sickness behaviour then occurs in those with chronic brain disease, in response to infection. The microglia release cytokines very readily if already primed. A maladaptive pathway develops.
One study involved the follow up of 300 Alzheimer’s disease (AD) patients 2 monthly for 6 months. Those who had an infection had a more rapid mental decline, while those who had suffered no infection showed no change. Other “behaviours” also changed greatly as a result of infection. He described obesity, smoking, age and grey hair as all contributing to earlier AD as all these have inflammatory effects.
He concluded by saying that systemic infections lead to distortion and maladaption exhibited by sickness behaviour, because of the primed microglia. This in turn leads to accelerated progression of brain disease. He said that a vaccination can be used as a challenge to demonstrate changes. Functional MRI has more use at detecting these changes.
Fletcher on NPY, a Potential Biomarker for ME/CFS – Mary Ann Fletcher (Miami, Florida) presented her work on biomarkers for CFS. The goal in CFS research has been to find a biomarker or combination of biomarkers. This will enhance the ability to diagnose and demonstrate severity of the illness, define subsets and help to manage trials.
Natural killer (NK) cells were studied initially looking at function and the diminution of perforin and granzyme. 176 CFS patients showed significantly lower function in NK cells compared to controls. She then went on to describe how neuropeptide-Y (NPY) is involved in the stress reaction with increase in norepinephrine and NPY from the sympathetic nerve endings. In a controlled study, NPY was considerably higher in CFS compared to controls.
Use of receiver operating curve (ROC) analysis was described, and this showed discrimination between CFS patients and controls. Using ROC, NPY was found to be 80% sensitive in CFS, (which is better than the PSA test we use to help diagnose cancer of the prostate). NPY also correlates with markers of disease severity. Other potential biomarkers using this technique included 10 of 16 cytokines measured, NK cell cell function and dipepdyl peptidase/CD26 which is indicative of immune activation. This is all part of a complex integrated system.
In the future exercise challenge will be included in testing this paradigm, and computer analysis will be developed to stimulate research in further clinical trials. These abnormalities may have applications in other diseases.
Donovan on ME/CFS Autopsies – Dominic O’Donovan, (Cambridge,UK) a neuropathologist presented the results of autopsy on 4 patients who had a specialist diagnosis of CFS:
1. A 32 year old male with a 20 year history of CFS, who died of a suicide overdose of medication. Spinal cord and brain at autopsy showed excess corpora amylacea, which was in excess of normal ageing. There were intermediate filaments closely related to glial cells, and maybe within the glia rather than the axons. No evidence of ganglionitis. (EBV negative)
2. A female of 32 with a 5 year history. She had refused any medical help and been bedridden and refused food and water. She finally died of renal failure. The pathology showed a focal chronic inflammatory infiltrate (T8 lymphocyes) in the dorsal root ganglia. (EBV negative).
3. A female of 43 – an assisted suicide in Switzerland with a barbiturate overdose. The brain showed global ischaemia, but this was likely due to the drugs used. Dorsal root ganglia showed mild excess of lymphocytic nodules of nageotte but with no obvious inflammation, but this could represent a subtle chronic inflammatory state.
4. A female of 31 whose death may have been due to opiate ingestion. There was some toxic demyelination with spinal subarachnoid haemorrgae, but she was on warfarin. There was some mild possible chronic ganglionitis.
Differential diagnosis here was discussed and would have included AIDs, Sjorgren’s syndrome, varicella zoster and paraneoplastic disease.
These results have raised the possibility that some cases of CFS may have sensory or autonomic ganglionitis. A specific brain and tissue bank in the UK is proposed.
Sukocheva on ME/CFS Autopsy – Olga Sukocheva (Adelaide, Australia) presented the immunohistochemical and microbiological post mortem findings in a 20 year old patient with fatal idiopathic encephalopathy. This patient had been diagnosed with CFS following a severe encephalitic like illness aged 10. There was evidence of inflammatory damage with suppression of microglial cells. Down regulation of ankyrin B was detected in the white matter of the hippocampus.
There was no significant difference in ankyrin G. Tests for Coxiella burnetii and Legionella were instituted. C.burnetii antigens were present in astrocytes, and in the microglial cells in the grey matter of the hippocampus. C.burnetii antigen was also found in spleen and liver macrophages,lymphoid tissue, bone marrow, lung and heart tissues. Legionella antigen was not found.
Dan Peterson on Viral Infections Dan Peterson (Nevada, USA) started his talk with a brief overview of the incidence and effects of CFS in the USA. He then went on to describe research problems, such as the varied definition, heterogeneity of patients, lack of biomarkers, patient self-selection, researcher bias and lack of funding. He described a number of “scientific journeys” undertaken in CFS research. He stressed the importance of the bringing together of the patient, biotechnology, database informantics, genomics and clinical medical guidelines. Diseases can now be defined from a molecular perspective. Networking and collaboration are keys to successful research. There needs to be large-scale clinical data gathering, with international biospecimen collection.
He then went on to discuss the importance of looking at viral infections in CFS. Leukotropic herpes viruses particularly HHV6, HCMV and EBV are among a number of major candidates in CFS. He reported on large studies in which active HHV6 was detected in 28%, HCMV in 29% and EBV in 51%. 10% were co-infected. Active EBV infection significantly correlates with the presence of auto-antibodies, with antibodies directed at thyroid peroxidase and parietal cells.
Up to 30% of patients may respond to antiviral medication.
Brenu on Immune Dysregulation – Ekua Brenu (Queensland, Australia) had looked at innate and adaptive immunity in CFS. It was postulated that her study could assist in developing biomarkers. The study involved 253 patients and 100 controls. Studies were undertaken at zero and 6 months. Cytotoxic activity of NK cells and CD8+T cells was significantly reduced. Perforin and granzyme activity was reduced. When looking at NK cell phenotypes, CD56 bright cells were significantly diminished. Cytokine secretion from CD4+T cells showed significant elevation of IL-10, IFN-Á and TNF-·. FOXP3 expression was also heightened in the CFS group. Vaso-active intestinal peptide (VIP) receptors were also investigated and found to be significantly elevated.
De Meirleir on XMRV Kenny de Meirlier (Brussels, Belgium): Because chronic activation of the immune system is present in progressive HIV and is a better predictor of disease outcome than viral load, it is important to test the hypothesis that a similar pattern may be observed in XMRV positive CFS patients. 16 XMRV positive patients (using culture assay) had a large number of tests performed.
These patients were found to have reduced lymphocyte numbers and CD-57+lymphocytes reduced, as observed in HIV. There was evidence of an activated innate immune system (increased elastase activity and C4a). sCD14 was significantly higher than expected, and this correlated with plasma lipopolysaccharide (LPS) a proinflammatory component of the gram-negative bacterial envelope. Low stool IgA indicated dysfunctional mucosa-associated lymphomal tissue in XMRV positive patients. Serum IL-8,IL-10,MCP-1 and MIP-1‚ are increased and might constitute a biological signature for viral infection.
This all provides supportive evidence for microbial translocation being part of the pathology of XMRV +ve patients.
He described a Norwegian study of severely disabled CFS patients in which the plasma LPS was elevated in those with a low Karnofsky score. This suggests a leaky gut syndrome. Stool analysis in CFS patients has indicated overgrowth of enterococci, streptococci and fungi with diminished E.Coli count. This can lead to overproduction of hydrogen sulphide which is toxic to mitochondria and affects ATP.
Kwiatek on Brainstem Dysfunction – Richard Kwiatek (Adelaide, Australia) is a rheumatologist with a particular interest in neuro-imaging. MRI was performed to look for brainstem dysfunction in CFS. Whole-brain optimised voxel-based volumetry and novel quantification of T1-weighted and T-2 weighted signal levels in structural MRI were used. Voxels build a 3-D map of the brain. In the CFS patients seated pulse pressure was reduced, and seated heart rate and asleep heart rate were increased, compared to controls. This was then correlated with brain change, other symptoms and fatigue.
Prefrontal white matter volume reduced with increasing sleeping heart rate in CFS with the opposite in controls. Midbrain white matter volume reduced with increasing fatigue. There was a strong correlation between total brainstem grey matter volume and seated pulse pressure in the CFS patients.
Brainstem grey matter changes suggest a failure of cerebrovascular auto-regulation, potentially mediated by astrocytes. Astrocyte dysfunction may therefore be central to CFS pathogenesis. There seems to be disrupted autonomic nervous system homeostasis. He does not feel it is reduced blood volume that will be causing this.
Marmion on Q-fever – Barrie Marmion (Adelaide, Australia) has studied Q-fever and its aftermath for many years. There were 11 suffering from post Q-fever fatigue syndrome out of 39 who had had the acute illness in one study cited. The C.burnetii antigen persists, and causes immune modulation with gene expression and symptoms. Usually it is continuous from the initial onset, but episodic relapses may occur due to re-infection or inadvertent Q-fever vaccination. IL-6 is elevated and IL-2 is down. The symptoms fit the criteria for a diagnosis of CFS.
3 Q-fever groups were studied and there were differences in the frequency of carriage of HLA-DR B1*11 and of IFN-Á. 35% were positive in the post-Q-fever syndrome group, and the levels were low in the controls and Q-fever recovered group and the Q-fever endocarditis group.. These differences support the concept of different immune states in chronic Q fever, determined by genetic variations in host immune responses, rather than by the properties of C.burnetii.
Boullerne on CFS and MS Anne Boullerne (Illinois, USA) discussed the issue of chronic fatigue in relation to CFS and MS. She described MS as a characteristic auto-immune disorder. She outlined the differences in incidence, symptoms, duration of illness etc. She emphasised that while MS is a neuro-immune disease, CFS is an acquired severe complex system dysfunction. In MS there is oligoclonal IgG in the CSF in 95% of cases, and brain lesions with T and B cells are seen on MRI.
She asks the question “Is gliosis present in CFS?” In CFS MRI abnormalities maybe found such as small punctate subcortical white matter intensities in the frontal lobes, small ventricular volume, slow blood flow and some atrophy.
She had looked at functional MRI in relation to control imagery and visual imagery. Both were found to be slower in CFS compared to controls. Changes associated with finger tapping and auditory monitoring correlated with subjective fatigue and brain response during challenge involving memory.
Using M.R.Spectroscopy, there was an increase incholine in the basal ganglia, no significant difference in glutathione, and ventricular lactate was elevated. There was no alteration in levels of GABA and glutamate.
In a rat model for Gulf War Syndrome, using pyridostigmine, there was no gliosis and no increased permeability of the blood brain barrier.
A possible auto-immunity including vasoactive neuropeptides is hypothesised.
Tate on Molecular Biomarkers and RNase L – Warren Tate (Dunedin, NZ) and his team have just initiated a study to develop tools that can accurately detect molecular changes within cells in response to double-stranded RNA (dsRNA) relevent to CFS. He explained how recent XMRV findings had stimulated research and a need for a bank of genetic material. Biomarkers need to be established as well as less specific markers to reflect changes in global homeostasis.There needs to be targetting of a vulnerable point in the biology of XMRV viral RNA that determines the ratio of its structural and enzyme proteins.
He went on to describe types of biomarkers:
1. Specific such as in a cell undergoing apoptosis: RNaseL, PKR, phosphorylation of PKR etc
2. Specific biomarkers of disturbed homeostasis
3. General biomarkers – marking global disturbed homeostasis of various organs
He explained the RNaseL activation pathway. RNaseL cleavage may be specific to CFS. He is currently studying the ratio of the RNaseL terminal fragment to uncleaved protein. He will also be looking at abnormal PKR activation. This is cleaved by caspase to form the 37D fragment. This undergoes phosphorylation which can be measured – the protein-synthesis factor e1F2·. These 2 phosphorylation events will be detected by specific antibodies against the phosphopeptides of the 2 proteins.
Feinstein on Droxidopa and Combatting Neuro-degeneration Douglas Feinstein (Illinois,Chicago) presented study of noradrenergic treatments for neuro-degenerative diseases. Glial cells are activated producing neurotoxins, which induce neuronal damage and leukocyte infiltration into the CNS. Noradrenaline regulates glial inflammatory responses, exerts neuroprotective effects and helps maintain the integrity of the blood brain barrier (BBB). Dysregulation of noradrenaline signalling could exacerbate disease. The supposed reductions of noradrenaline increase inflammatory responses, the amyloid burden and neurotropic factors.
Noradrenaline is mainly produced in the locus coeruleus (LC). This part of the brain is damaged in Alzheimer’s and Parkinsonism. LC loss correlates with plaque and tangle numbers. The question was asked “does increasing noradrenaline in the CNS improve things?” The drug Droxidopa is a precursor of noradrenaline. This drug is in phase 3 trials for neurogenic orthostatic hyopotension. In mice the drug leads to improvement in plaques and learning. This drug used in MS and experimental auto-immune encephalmyelitis (EAE) showed stabilisation compared to controls. This trial indicates that the LC is significantly damaged in MS and EAE.
Noradrenaline directed therapies need to be considered if there is perhaps also LC disturbance in CFS.
Ganea on VIP – Doina Ganea (Philadelphia,USA) spoke about Vasoactive Intestinal Peptide (VIP) – an endogenous and exogenous immunomodlator. VIP downregulates the innate immune response by inhibiting the release of pro-inflammatory cytokines, chemokines and nitric oxide by activated macrophages, microglia and dendritic cells. It also affects the adaptive immune response by reducing the co-stimulatory capacity of antigen-presenting cells, and by inducing Th2 type responses. She had looked at several diseases, such as collagen-induced arthritis and autoimmune encephalmyelitis. She had used dendritic cells generated in the presence of VIP/PACAP as immunomodulatory agents, with positive results.
Carson on CCL21 and Immune related CNS Pathology – Monica Carson (California,USA) had studied the CNS expression of the classic chemokine CCL21. This is a predisposing factor for auto-immunity due to the proliferation induced pre-activation. It thus contributes to chronic inflammatory disease and auto-immunity. Experimental work was done using mice. Resulting data indicated that CCL21 expression within the CNS has the potential to contribute to T-cell mediated CNS pathology. This could occur via homeostatic priming of CD4+T cell lymphocytes outside the CNS, and CD4+T cell migration into parenchymal site after infection with organisms such as toxoplasma.
Staines on Vaso-active Neuropeptides – Donald Staines (Gold Coast, Australia) rounded off the formal papers with a presentation looking at novel treatments in CFS. He considered whether auto-immunity affecting vaso-active neuropeptides suggest a pathomechanism. ME/CFS may be associated with auto-immunity affecting the function of vaso-active neuropeptides, such as VIP and PACAP (pituitary adenylate cyclase activating peptide). Upsets in adenylate cyclase (AC) signalling and cAMP functioning possibly involving ATP toxicity may be a feature of VN auto-immunity.
Purinergic receptors such as ATP negatively regulate AC. He outlined some basic biochemical principles to clarify things; AC amplifies incoming intracellular signals; PACAP is an acetylcholine co-transmitter; AC is involved in long term potentiation and enhanced maintenance of neuronal activity. VIP/PACAP synergism is involved with potentiation of cardiac firing, anti-apoptosis function, cAMP and insulin control, hypoxia regulation and glutamate metabolism. Purinergic signalling is involved in centrally mediated pain (neuropathic pain).
He then went on to describe some likely treatment possibilities based on these principles. These included purigenic signalling modulators, VIP/PACAP mimics/analogues, phosphodiesterase inhibitors: eg Rolipram (toxic), Ibudilast, Roflumilast; B cell depletors (Rituximab); chondroitinase; VIP liposomes and lentivirus agents. Some of these could be considered for clinical trials.