Sandra Bahia Ramos

Role of adaptive and innate immune cells in chronic fatigue syndrome/myalgic encephalomyelitis

Perturbations in immune processes are a hallmark of a number of autoimmune and inflammatory disorders. Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is an inflammatory disorder with possible autoimmune correlates, characterized by reduced NK cell activity, elevations in regulatory T cells (Tregs) and dysregulation in cytokine levels. The purpose of this article is to examine innate and adaptive immune cell phenotypes and functional characteristics that have not been previously examined in CFS/ME patients. Thirty patients with CFS/ME and 25 non-fatigued controls were recruited for this study. Whole blood samples were collected from all participants for the assessment of cell phenotypes, functional properties, receptors, adhesion molecules, antigens and intracellular proteins using flow cytometric protocols. The cells investigated included NK cells, dendritic cells, neutrophils, B cells, T cells, γδT cells and Tregs. Significant changes were observed in B-cell subsets, Tregs, CD4(+)CD73(+)CD39(+) T cells, cytotoxic activity, granzyme B, neutrophil antigens, TNF-α and IFN-γ in the CFS/ME patients in comparison with the non-fatigued controls. Alterations in B cells, Tregs, NK cells and neutrophils suggest significant impairments in immune regulation in CFS/ME and these may have similarities to a number of autoimmune disorders.

Cytokines in the Cerebrospinal Fluids of Patients with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Objectives. Previous research has provided evidence for dysregulation in peripheral cytokines in patients with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). To date only one study has examined cytokines in cerebrospinal fluid (CSF) samples of CFS/ME patients. The purpose of this pilot study was to examine the role of cytokines in CSF of CFS/ME patients. Methods. CSF was collected from 18 CFS/ME patients and 5 healthy controls. The CSF samples were examined for the expression of 27 cytokines (interleukin- (IL-) 1β, IL-1ra, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, basic FGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, and VEGF) using the Bio-Plex Human Cytokine 27-plex Assay. Results. Of the 27 cytokines examined, only IL-10 was significantly reduced in the CFS/ME patients in comparison to the controls. Conclusions. This preliminary investigation suggests that perturbations in inflammatory cytokines in the CSF of CFS/ME patients may contribute to the neurological discrepancies observed in CFS/ME.

A Preliminary Comparative Assessment of the Role of CD8+ T Cells in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis and Multiple Sclerosis

Background. CD8+ T cells have putative roles in the regulation of adaptive immune responses during infection. The purpose of this paper is to compare the status of CD8+ T cells in Multiple Sclerosis (MS) and Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). Methods. This preliminary investigation comprised 23 CFS/ME patients, 11 untreated MS patients, and 30 nonfatigued controls. Whole blood samples were collected from participants, stained with monoclonal antibodies, and analysed on the flow cytometer. Using the following CD markers, CD27 and CD45RA (CD45 exon isoform 4), CD8+ T cells were divided into naïve, central memory (CM), effector memory CD45RA− (EM), and effector memory CD45RA+ (EMRA) cells. Results. Surface expressions of BTLA, CD127, and CD49/CD29 were increased on subsets of CD8+ T cells from MS patients. In the CFS/ME patients CD127 was significantly decreased on all subsets of CD8+ T cells in comparison to the nonfatigued controls. PSGL-1 was significantly reduced in the CFS/ME patients in comparison to the nonfatigued controls. Conclusions. The results suggest significant deficits in the expression of receptors and adhesion molecules on subsets of CD8+ T cells in both MS and CFS/ME patients. These deficits reported may contribute to the pathogenesis of these diseases. However, larger sample size is warranted to confirm and support these encouraging preliminary findings.

Characterisation of cell functions and receptors in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME)

BackgroundAbnormal immune function is often an underlying component of illness pathophysiology and symptom presentation. Functional and phenotypic immune-related alterations may play a role in the obscure pathomechanism of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). The objective of this study was to investigate the functional ability of innate and adaptive immune cells in moderate and severe CFS/ME patients. The 1994 Fukuda criteria for CFS/ME were used to define CFS/ME patients. CFS/ME participants were grouped based on illness severity with 15 moderately affected (moderate) and 12 severely affected (severe) CFS/ME patients who were age and sex matched with 18 healthy controls. Flow cytometric protocols were used for immunological analysis of dendritic cells, monocytes and neutrophil function as well as measures of lytic proteins and T, natural killer (NK) and B cell receptors.ResultsCFS/ME patients exhibited alterations in NK receptors and adhesion markers and receptors on CD4+T and CD8+T cells. Moderate CFS/ME patients had increased CD8+ CD45RA effector memory T cells, SLAM expression on NK cells, KIR2DL5+ on CD4+T cells and BTLA4+ on CD4+T central memory cells. Moderate CFS/ME patients also had reduced CD8+T central memory LFA-1, total CD8+T KLRG1, naïve CD4+T KLRG1 and CD56dimCD16− NK cell CD2+ and CD18+CD2+. Severe CFS/ME patients had increased CD18+CD11c− in the CD56dimCD16− NK cell phenotype and reduced NKp46 in CD56brightCD16dim NK cells.ConclusionsThis research accentuated the presence of immunological abnormalities in CFS/ME and highlighted the importance of assessing functional parameters of both innate and adaptive immune systems in the illness.

Analysis of the Relationship between Immune Dysfunction and Symptom Severity in Patients with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME)

Objective: Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is a disabling illness, characterised by persistent, debilitating fatigue and a multitude of symptoms. Immunological alterations are prominent in CFS/ME cases, however little is known about the relationship between CFS/ME severity and the extent of immunological dysfunction. The purpose of this study was to assess innate and adaptive immune cell phenotypes and function of two groups of CFS/ME patients, bedridden (severe) and mobile (moderate). Methods: CFS/ME participants were defined using the Centres for Disease Prevention and Control (1994 CDC) Criteria for CFS/ME. Participants were grouped into healthy controls (n=22, age=40.14 ± 2.38), moderate/ mobile (n=23; age=42.52 ± 2.63) and severe/bedridden (n=18; age=39.56 ± 1.51) CFS/ME patients. Flow cytometric protocols were used to examine neutrophil, monocyte, dendritic cells (DCs), iNKT, Treg, B, γδ and CD8+ T cell phenotypes, NK cytotoxic activity and receptors. Results: The present data found that CFS/ME patients demonstrated significant decreases in NK cytotoxic activity, transitional and regulatory B cells, γδ1T cells, KIR2DL1/DS1, CD94+ and KIR2DL2/L3. Significant increases in CD56-CD16+NKs, CD56dimCD16- and CD56brightCD16-/dim NKs, DCs, iNKT phenotypes, memory and naive B cells were also shown in CFS/ME participants. Severe CFS/ME patients demonstrated increased CD14-CD16+ DCs, memory and naive B cells, total iNKT, iNKT cell and NK phenotypes compared to moderate CFS/ME patients. Conclusion: This study is the first to determine alterations in NK, iNKT, B, DC and γδ T cell phenotypes in both moderate and severe CFS/ME patients. Immunological alterations are present in innate and adaptive immune cells and sometimes, immune deregulation appears worse in CFS/ME patients with more severe symptoms. It may be appropriate for CFS/ME patient severity subgroups to be distinguished in both clinical and research settings to extricate further immunological pathologies that may not have been previously reported.

Immune Abnormalities in Patients Meeting New Diagnostic Criteria for Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Background: Immunological abnormalities have been identified in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients fulfilling the 1994 Centers for Disease Control diagnostic criteria. Significant developments have been made to diagnostic criteria, but potential immunological markers have not been assessed in patients fulfilling these latest clinical requirements. Therefore, this study evaluated immunological parameters in patients that also fulfill the latest diagnostic criteria available known as the International Consensus Criteria. Methods: The Immunological investigations including Natural Killer cell activity and phenotyping studies for dendritic cells, neutrophils, B cells and regulatory T cells were performed on whole blood samples collected from all participants using flow cytometric protocols. The physical functioning of all participants was also evaluated using scores from the Short Form Health Survey, and the World Health Organization Disability Adjustment Schedule. Results were compared according 1994 Centers of Disease Control and Prevention defined patients, and International Consensus Criteria defined patients, and healthy controls. Results: Natural killer cell activity was consistently and significantly decreased, and regulatory T cells were significantly increased in both patient groups compared to healthy controls. Differences were found in human neutraphil antigens and expression of natural killer cell receptors between patient groups. Highly significant correlations were also found between physical status and some immune parameters in International Consensus Criteria defined patients. Conclusion: This preliminary investigation on different diagnostic criteria suggests that the International Consensus Criteria may be more effective a detecting salient differences in the immune system.

Serum Immune Proteins in Moderate and Severe Chronic Fatigue Syndrome/Myalgic Encephalomyelitis Patients

Immunological dysregulation is present in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME), with recent studies also highlighting the importance of examining symptom severity. This research addressed this relationship between CFS/ME severity subgroups, assessing serum immunoglobulins and serum cytokines in severe and moderate CFS/ME patients. Participants included healthy controls (n= 22), moderately (n = 22) and severely (n=19) affected CFS/ME patients. The 1994 Fukuda Criteria defined CFS/ME and severity scales confirmed mobile and housebound CFS/ME patients as moderate and severe respectively. IL-1β was significantly reduced in severe compared with moderate CFS/ME patients. IL-6 was significantly decreased in moderate CFS/ME patients compared with healthy controls and severe CFS/ME patients. RANTES was significantly increased in moderate CFS/ME patients compared to severe CFS/ME patients. Serum IL-7 and IL-8 were significantly higher in the severe CFS/ME group compared with healthy controls and moderate CFS/ME patients. IFN-γ was significantly increased in severe CFS/ME patients compared with moderately affected patients. This was the first study to show cytokine variation in moderate and severe CFS/ME patients, with significant differences shown between CFS/ME symptom severity groups. This research suggests that distinguishing severity subgroups in CFS/ME research settings may allow for a more stringent analysis of the heterogeneous and otherwise inconsistent illness.

Examination of Single Nucleotide Polymorphisms (SNPs) in Transient Receptor Potential (TRP) Ion Channels in Chronic Fatigue Syndrome Patients

Background The transient receptor potential (TRP) superfamily in humans comprises 27 cation channels with permeability to monovalent and divalent cations. These channels are widely expressed within humans on cells and tissues and have significant sensory and regulatory roles on most physiological functions. Chronic fatigue syndrome (CFS) is an unexplained disorder with multiple physiological impairments. OBJECTIVES The purpose of this study was to determine the role of TRPs in CFS. Methods The study comprised 115 CFS patients (age = 48.68 ± 1.06 years) and 90 nonfatigued controls (age = 46.48 ± 1.22 years). CFS patients were defined according to the 1994 Center for Disease Prevention and Control criteria for CFS. A total of 240 single nucleotide polymorphisms (SNPs) for 21 mammalian TRP ion channel genes (TRPA1, TRPC1, TRPC2, TRPC3, TRPC4, TRPC6, TRPC7, TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8, TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and TRPV6) were examined via the Agena Biosciences iPLEX Gold assay. Statistical analysis was performed using the PLINK analysis software. Results Thirteen SNPs were significantly associated with CFS patients compared with the controls. Nine of these SNPs were associated with TRPM3 (rs12682832; P < 0.003, rs11142508; P < 0.004, rs1160742; P < 0.08, rs4454352; P < 0.013, rs1328153; P < 0.013, rs3763619; P < 0.014, rs7865858; P ≤ 0.021, rs1504401; P ≤ 0041, rs10115622; P ≤ 0.050), while the remainder were associated with TRPA1 (rs2383844; P ≤ 0.040, rs4738202; P ≤ 0.018) and TRPC4 (rs6650469; P ≤ 0.016, rs655207; P ≤ 0.018). Conclusion The data from this pilot study suggest an association between TRP ion channels, predominantly TRPM3 and CFS. This and other TRPs identified may contribute to the etiology and pathomechanism of CFS.

Pilot Study of Natural Killer Cells in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis and Multiple Sclerosis.

Patients with chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) and multiple sclerosis (MS) suffer from debilitating fatigue which is not alleviated by rest. In addition to the fatigue‐related symptoms suffered by patients with CFS/ME and MS, dysfunction of the immune system and, in particular, reduced natural killer (NK) cell cytotoxic activity has also been reported in CFS/ME and MS. The purpose of this pilot study was to compare NK cellular mechanisms in patients with CFS/ME and MS to investigate potential dysfunctions in the NK cell activity pathway. Flow cytometry protocols assessed CD56dimCD16+ and CD56brightCD16+/− NK cell expression of adhesion molecules, NK activating and inhibiting receptors, NK cell maturation and lytic proteins. All participants in this study were female and included 14 patients with CFS/ME, nine patients with MS and 19 non‐fatigued controls. The patient groups and the non‐fatigued controls were not taking any immunosuppressive or immune‐enhancing medications. In the MS cohort, KIR2DL5 was significantly increased on CD56brightCD16+/− NK cells and expression of CD94 was significantly increased on CD56dimCD16+ NK cells in comparison with the controls. Co‐expression of CD57 and perforin was significantly increased on CD56dimCD16+ NK cells from patients with CFS/ME compared to the MS and non‐fatigued control participants. The results from this pilot study suggest that NK cells from patients with CFS/ME and MS may have undergone increased differentiation in response to external stimuli which may affect different mechanisms in the NK cell cytotoxic activity pathway.

Characterisation of B cell Subsets and Receptors in Chronic Fatigue Syndrome Patients

Limited immunological changes have been previously reported in B cell phenotype in Chronic Fatigue Syndrome (CFS) patients, so there is no clear established role of B cells in the pathophysiology of CFS patients. The aim of this study was to evaluate B cells subsets including naive, memory naive, memory switched, memory non-switched, double negative, transitional, plasmablasts, HLA-DR + , plasma and regulatory B cells (Breg) in CFS patients compared with non-fatigued controls. B cell activation markers (CD81, CD21) and surface receptors (CD79a/b, IgM, IgD, IgA, IgE) were also examined in CFS patients compared with non-fatigued controls. 46 CFS patients (age=50.00 ± 2.00 years) and 34 non-fatigued controls (age=49.00 ± 2.16 years) participated in the study. The percentage of BCR IgM + B cells was significantly increased in the CFS group compared with non-fatigued controls (p=0.037). Similarly, there was a significant decrease in the CD1d + B cells in the CFS group compared with nonfatigued controls (p=0.046). No additional differences in B cell phenotypes, activation markers and surface receptors were found in the CFS patients compared with the non-fatigued control group. The differences observed in the B cell phenotype of CFS patients compared with non-fatigued controls may explain some of the disturbances in the immune homeostasis, however whether this is causal or the consequence of immunological imbalances previously reported in CFS patients requires further investigation.