Mucosal Immunity and the Gut-Microbiota-Brain-Axis in Neuroimmune Disease.

Recent advances in next-generation sequencing (NGS) technologies have opened the door to a wellspring of information regarding the composition of the gut microbiota. Leveraging NGS technology, early metagenomic studies revealed that several diseases, such as Alzheimer's disease, Parkinson's disease, autism, and myalgic encephalomyelitis, are characterized by alterations in the diversity of gut-associated microbes. More recently, interest has shifted toward understanding how these microbes impact their host, with a special emphasis on their interactions with the brain. Such interactions typically occur either systemically, through the production of small molecules in the gut that are released into circulation, or through signaling via the vagus nerves which directly connect the enteric nervous system to the central nervous system. Collectively, this system of communication is now commonly referred to as the gut-microbiota-brain axis. While equally important, little attention has focused on the causes of the alterations in the composition of gut microbiota. Although several factors can contribute, mucosal immunity plays a significant role in shaping the microbiota in both healthy individuals and in association with several diseases. The purpose of this review is to provide a brief overview of the components of mucosal immunity that impact the gut microbiota and then discuss how altered immunological conditions may shape the gut microbiota and consequently affect neuroimmune diseases, using a select group of common neuroimmune diseases as examples.

Single-nucleotide polymorphisms in a cohort of significantly obese women without cardiometabolic diseases.

BACKGROUND/OBJECTIVES: Obesity is an important risk factor for the development of diseases such as diabetes mellitus, hypertension, and dyslipidemia; however, a small number of individuals with long-standing obesity do not present with these cardiometabolic diseases. Such individuals are referred to as metabolically healthy obese (MHO) and potentially represent a subgroup of the general population with a protective genetic predisposition to obesity-related diseases. We hypothesized that individuals who were metabolically healthy, but significantly obese (BMI ≥ 35 kg/m2) would represent a highly homogenous subgroup, with which to investigate potential genetic associations to obesity. We further hypothesized that such a cohort may lend itself well to investigate potential genotypes that are protective with respect to the development of cardiometabolic disease. SUBJECTS/METHODS: In the present study, we implemented this novel selection strategy by screening 892 individuals diagnosed as Class 2 or Class 3 obese and identified 38 who presented no manifestations of cardiometabolic disease. We then assessed these subjects for single-nucleotide polymorphisms (SNPs) that associated with this phenotype. RESULTS: Our analysis identified 89 SNPs that reach statistical significance (p < 1 × 10-5), some of which are associated with genes of biological pathways that influences dietary behavior; others are associated with genes previously linked to obesity and cardiometabolic disease as well as neuroimmune disease. This study, to the best of our knowledge, represents the first genetic screening of a cardiometabolically healthy, but significantly obese population.

Evaluation of four clinical laboratory parameters for the diagnosis of myalgic encephalomyelitis.

BACKGROUND: Myalgic encephalomyelitis (ME) is a complex and debilitating disease that often initially presents with flu-like symptoms, accompanied by incapacitating fatigue. Currently, there are no objective biomarkers or laboratory tests that can be used to unequivocally diagnosis ME; therefore, a diagnosis is made when a patient meets series of a costly and subjective inclusion and exclusion criteria. The purpose of the present study was to evaluate the utility of four clinical parameters in diagnosing ME. METHODS: In the present study, we utilized logistic regression and classification and regression tree analysis to conduct a retrospective investigation of four clinical laboratory in 140 ME cases and 140 healthy controls. RESULTS: Correlations between the covariates ranged between [- 0.26, 0.61]. The best model included the serum levels of the soluble form of CD14 (sCD14), serum levels of prostaglandin E2 (PGE2), and serum levels of interleukin 8, with coefficients 0.002, 0.249, and 0.005, respectively, and p-values of 3 × 10-7, 1 × 10-5, and 3 × 10-3, respectively. CONCLUSIONS: Our findings show that these parameters may help physicians in their diagnosis of ME and may additionally shed light on the pathophysiology of this disease.

Nutritional modulation of the intestinal microbiota; future opportunities for the prevention and treatment of neuroimmune and neuroinflammatory disease.

The gut-brain axis refers to the bidirectional communication between the enteric nervous system and the central nervous system. Mounting evidence supports the premise that the intestinal microbiota plays a pivotal role in its function and has led to the more common and perhaps more accurate term gut-microbiota-brain axis. Numerous studies have identified associations between an altered microbiome and neuroimmune and neuroinflammatory diseases. In most cases, it is unknown if these associations are cause or effect; notwithstanding, maintaining or restoring homeostasis of the microbiota may represent future opportunities when treating or preventing these diseases. In recent years, several studies have identified the diet as a primary contributing factor in shaping the composition of the gut microbiota and, in turn, the mucosal and systemic immune systems. In this review, we will discuss the potential opportunities and challenges with respect to modifying and shaping the microbiota through diet and nutrition in order to treat or prevent neuroimmune and neuroinflammatory disease.

Humoral Immunity Profiling of Subjects with Myalgic Encephalomyelitis Using a Random Peptide Microarray Differentiates Cases from Controls with High Specificity and Sensitivity.

Myalgic encephalomyelitis (ME) is a complex, heterogeneous illness of unknown etiology. The search for biomarkers that can delineate cases from controls is one of the most active areas of ME research; however, little progress has been made in achieving this goal. In contrast to identifying biomarkers that are directly involved in the pathological process, an immunosignature identifies antibodies raised to proteins expressed during, and potentially involved in, the pathological process. Although these proteins might be unknown, it is possible to detect antibodies that react to these proteins using random peptide arrays. In the present study, we probe a custom 125,000 random 12-mer peptide microarray with sera from 21 ME cases and 21 controls from the USA and Europe and used these data to develop a diagnostic signature. We further used these peptide sequences to potentially uncover the naturally occurring candidate antigens to which these antibodies may specifically react with in vivo. Our analysis revealed a subset of 25 peptides that distinguished cases and controls with high specificity and sensitivity. Additionally, Basic Local Alignment Search Tool (BLAST) searches suggest that these peptides primarily represent human self-antigens and endogenous retroviral sequences and, to a minor extent, viral and bacterial pathogens.

Decreased Numbers of CD57+CD3- Cells Identify Potential Innate Immune Differences in Patients with Autism Spectrum Disorder.

BACKGROUND/AIM: Autism spectrum disorders (ASD) are complex, and severe heterogeneous neurodevelopmental pathologies with accepted but complex immune system abnormalities. Additional knowledge regarding potential immune dysfunctions may provide a greater understanding of this malady. The aim of this study was to evaluate the CD57(+)CD3(-) mature lymphocyte subpopulation of natural killer cells as a marker of immune dysfunction in ASD. MATERIALS AND METHODS: Three-color flow cytometry-based analysis of fresh peripheral blood samples from children with autism was utilized to measure CD57(+)CD3(-) lymphocytes. RESULTS: A reduction of CD57(+)CD3(-) lymphocyte count was recorded in a significant number of patients with autism. DISCUSSION AND CONCLUSION: We demonstrated that the number of peripheral CD57(+)CD3(-) cells in children with autism often falls below the clinically accepted normal range. This implies that a defect in the counter-regulatory functions necessary for balancing pro-inflammatory cytokines exists, thus opening the way to chronic inflammatory conditions associated with ASD.

Human dendritic cells transfected with a human papilloma virus-18 construct display decreased mobility and upregulated cytokine production.

The marked depletion of dendritic cells (DCs) in skin cancers, as well as preneoplastic and neoplastic cervical epithelium, suggests a central role for DCs in productive human papillomavirus (HPV) infection and cancer promotion. It has been suggested that HPV may facilitate tumor development by reducing DC density, contributing to a decrease in local immune surveillance. In this study, we have examined the response of human DCs transfected with a construct containing the HPV18 genome and their subsequent expression of papilloma virus proteins. Transfected cells expressed the L1 major capsid protein and upregulated E6 and E7 oncoprotein transcripts as detected by RT-PCR. Transfection of DCs also resulted in a significant increase in cytokine production. Finally, we observed that HPV18 transfection decreased the migratory activity of DCs. Our data indicate that HPV transfection of DCs leads to changes in migratory activity and cytokine production, which potentially can suppress or delay immune responses to viral antigens. Additionally, changes in cytokine production by HPV-transformed human fibroblasts and human cervical epithelial cells revealed that the migratory and antigen-presenting functions of DCs may be impaired by the suppressive effects of cytokines produced by HPV-infected epithelial and stromal cells.

High-throughput 16S rRNA gene sequencing reveals alterations of intestinal microbiota in myalgic encephalomyelitis/chronic fatigue syndrome patients.

Human intestinal microbiota plays an important role in the maintenance of host health by providing energy, nutrients, and immunological protection. Intestinal dysfunction is a frequent complaint in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients, and previous reports suggest that dysbiosis, i.e. the overgrowth of abnormal populations of bacteria in the gut, is linked to the pathogenesis of the disease. We used high-throughput 16S rRNA gene sequencing to investigate the presence of specific alterations in the gut microbiota of ME/CFS patients from Belgium and Norway. 43 ME/CFS patients and 36 healthy controls were included in the study. Bacterial DNA was extracted from stool samples, PCR amplification was performed on 16S rRNA gene regions, and PCR amplicons were sequenced using Roche FLX 454 sequencer. The composition of the gut microbiota was found to differ between Belgian controls and Norwegian controls: Norwegians showed higher percentages of specific Firmicutes populations (Roseburia, Holdemania) and lower proportions of most Bacteroidetes genera. A highly significant separation could be achieved between Norwegian controls and Norwegian patients: patients presented increased proportions of Lactonifactor and Alistipes, as well as a decrease in several Firmicutes populations. In Belgian subjects the patient/control separation was less pronounced, however some abnormalities observed in Norwegian patients were also found in Belgian patients. These results show that intestinal microbiota is altered in ME/CFS. High-throughput sequencing is a useful tool to diagnose dysbiosis in patients and could help designing treatments based on gut microbiota modulation (antibiotics, pre and probiotics supplementation).

Plasmacytoid dendritic cells in the duodenum of individuals diagnosed with myalgic encephalomyelitis are uniquely immunoreactive to antibodies to human endogenous retroviral proteins.

Myalgic encephalomyelitis (ME) is a debilitating illness of unknown etiology characterized by neurocognitive dysfunction, inflammation, immune abnormalities and gastrointestinal distress. An increasing body of evidence suggests that disruptions in the gut may contribute to the induction of neuroinflammation. Therefore, reports of human endogenous retroviral (HERV) expression in association with neuroinflammatory diseases prompted us to investigate the gut of individuals with ME for the presence of HERV proteins. In eight out of 12 individuals with ME, immunoreactivity to HERV proteins was observed in duodenal biopsies. In contrast, no immunoreactivity was detected in any of the eight controls. Immunoreactivity to HERV Gag and Env proteins was uniquely co-localized in hematopoietic cells expressing the C-type lectin receptor CLEC4C (CD303/BDCA2), the co-stimulatory marker CD86 and the class II major histocompatibility complex HLA-DR, consistent with plasmacytoid dendritic cells (pDCs). Although the significance of HERVs present in the pDCs of individuals with ME has yet to be determined, these data raise the possibility of an involvment of pDCs and HERVs in ME pathology. To our knowledge, this report describes the first direct association between pDCs and HERVs in human disease.

Role of psychological aspects in both chronic pain and in daily functioning in chronic fatigue syndrome: a prospective longitudinal study.

In addition to fatigue, many patients with chronic fatigue syndrome (CFS) experience chronic musculoskeletal pain. We aimed at examining the role of catastrophizing, coping, kinesiophobia, and depression in the chronic pain complaints and in the daily functioning of CFS patients. A consecutive sample of 103 CFS patients experiencing chronic widespread musculoskeletal pain completed a battery of questionnaires evaluating pain, daily functioning, and psychological characteristics (depression, kinesiophobia, pain coping, and catastrophizing). Thirty-nine patients participated in the 6-12-month follow-up, consisting of questionnaires evaluating pain and pressure pain algometry. Correlation and linear regression analyses were performed to identify predictors. The strongest correlations with pain intensity were found for catastrophizing (r = -.462, p < .001) and depression (r = -.439, p < .001). The stepwise multiple regression analysis revealed that catastrophizing was both the immediate main predictor for pain (20.2%) and the main predictor on the longer term (20.1%). The degree of depression was responsible for 10% in the observed variance of the VAS pain after 6-12 months. No significant correlation with pain thresholds could be revealed. The strongest correlations with daily functioning at baseline were found for catastrophizing (r = .435, p < .001) and depression (r = .481, p < .001). Depression was the main predictor for restrictions in daily functioning (23.1%) at baseline. Pain catastrophizing and depression were immediate and long-term main predictors for pain in patients with CFS having chronic widespread musculoskeletal pain. They were also correlated to daily functioning, with depression as the main predictor for restrictions in daily functioning at baseline.

The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro.

Previous research has suggested that human herpesvirus-6 (HHV-6) may integrate into host cell chromosomes and be vertically transmitted in the germ line, but the evidence--primarily fluorescence in situ hybridization (FISH)--is indirect. We sought, first, to definitively test these two hypotheses. Peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per milliliter of blood. FISH confirmed that HHV-6 DNA colocalized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, and that the integration site was identical among members of the same family. Integration of the HHV-6 genome into TTAGGG telomere repeats was confirmed by additional methods and sequencing of the integration site. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome. We next asked whether HHV-6A infection of naïve cell lines could lead to integration. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6, the virus integrated into telomeres. Reactivation of integrated HHV-6A virus from individuals' PBMCs as well as cell lines was successfully accomplished by compounds known to induce latent herpesvirus replication. Finally, no circular episomal forms were detected even by PCR. Taken together, the data suggest that HHV-6 is unique among human herpesviruses: it specifically and efficiently integrates into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.

Increased d-lactic Acid intestinal bacteria in patients with chronic fatigue syndrome.

Patients with chronic fatigue syndrome (CFS) are affected by symptoms of cognitive dysfunction and neurological impairment, the cause of which has yet to be elucidated. However, these symptoms are strikingly similar to those of patients presented with D-lactic acidosis. A significant increase of Gram positive facultative anaerobic faecal microorganisms in 108 CFS patients as compared to 177 control subjects (p<0.01) is presented in this report. The viable count of D-lactic acid producing Enterococcus and Streptococcus spp. in the faecal samples from the CFS group (3.5 x 10(7) cfu/L and 9.8 x 10(7) cfu/L respectively) were significantly higher than those for the control group (5.0 x 10(6) cfu/L and 8.9 x 10(4) cfu/L respectively). Analysis of exometabolic profiles of Enterococcus faecalis and Streptococcus sanguinis, representatives of Enterococcus and Streptococcus spp. respectively, by NMR and HPLC showed that these organisms produced significantly more lactic acid (p<0.01) from (13)C-labeled glucose, than the Gram negative Escherichia coli. Further, both E. faecalis and S. sanguinis secrete more D-lactic acid than E. coli. This study suggests a probable link between intestinal colonization of Gram positive facultative anaerobic D-lactic acid bacteria and symptom expressions in a subgroup of patients with CFS. Given the fact that this might explain not only neurocognitive dysfunction in CFS patients but also mitochondrial dysfunction, these findings may have important clinical implications.

Severe versus Moderate criteria for the new pediatric case definition for ME/CFS.

The new diagnostic criteria for pediatric ME/CFS are structurally based on the Canadian Clinical Adult case definition, and have more required specific symptoms than the (Fukuda et al. Ann Intern Med 121:953-959, 1994) adult case definition. Physicians specializing in pediatric ME/CFS referred thirty-three pediatric patients with ME/CFS and 21 youth without the illness. Those who met ME/CFS criteria were separated into Severe and Moderate categories. Significant differences were found for symptoms within each of the six major categories: fatigue, post-exertional malaise, sleep, pain, neurocognitive difficulties, and autonomic/neuroendocrine/immune manifestations. In general, the results showed participants who met the Severe ME/CFS criteria reported the highest scores, the Moderate ME/CFS group show scores that were a little lower, and the control group evidenced the lowest scores. Findings indicate that the Pediatric Case Definition for ME/CFS can distinguish between those with this illness and controls, and between those with Severe versus Moderate manifestations of the illness.

Detection of herpesviruses and parvovirus B19 in gastric and intestinal mucosa of chronic fatigue syndrome patients.

BACKGROUND: Human herpesvirus-6 (HHV-6), Epstein-Barr virus and parvovirus B19 have been suggested as etiological agents of chronic fatigue syndrome but none of these viruses is consistently detected in all patients. However, active viral infections may be localized in specific tissues, and, therefore, are not easily detectable. The aim of this study was to investigate the presence of HHV-6, HHV-7, EBV and parvovirus B19 in the gastro-intestinal tract of CFS patients. PATIENTS AND METHODS: Using real-time PCR, viral DNA loads were quantified in gastro-intestinal biopsies of 48 CFS patients and 35 controls. RESULTS: High loads of HHV-7 DNA were detected in most CFS and control biopsies. EBV and HHV-6 were detected in 15-30% of all biopsies. Parvovirus B19 DNA was detected in 40% of the patients versus less than 15% of the controls. CONCLUSION: Parvovirus B19 may be involved in the pathogenesis of CFS, at least for a subset of patients. The gastro-intestinal tract appears as an important reservoir of infection for several potentially pathogenic viruses.

A comparison of patients with chronic fatigue syndrome in two "ideologically" contrasting clinics.

Aim of the present study was to compare chronic fatigue syndrome (CFS) patients, attending 2 "ideologically" contrasting clinics for CFS, on various patient and illness characteristics. Fifty-nine CFS patients of each clinic, located in Leuven and Brussels (Belgium), participated. Patients did not differ with regard to age, levels of fatigue, psychopathology, and self-efficacy. However, patients from the psychosocially-oriented clinic had a lower level of education, reported more progressive illness onset, and attributed their illness more to psychological causes. Patients in the biologically-oriented clinic reported more pain, and showed higher levels of social functioning, motivation and vitality, as well as fewer limitations related to emotional problems. It is concluded that CFS patients attending the 2 clinics could not be distinguished along dualistic biological/psychosocial lines, but those reporting sudden illness onset and making somatic attributions were more likely to be represented in the biologically-oriented clinic.

Exercise performance and chronic pain in chronic fatigue syndrome: the role of pain catastrophizing.

OBJECTIVES: This study aimed to examine the associations between bodily pain, pain catastrophizing, depression, activity limitations/participation restrictions, employment status, and exercise performance in female patients with chronic fatigue syndrome (CFS) who experience widespread pain. DESIGN: Cross-sectional observational study. SETTING: A university-based clinic. PATIENTS: Thirty-six female CFS patients who experienced widespread pain. OUTCOME MEASURES: Patients filled in the Medical Outcomes Short-Form 36 Health Status Survey, the Chronic Fatigue Syndrome Activities and Participation Questionnaire, the Beck Depression Inventory, and the Pain Catastrophizing Scale, and underwent a maximal exercise stress test with continuous monitoring of electrocardiographic and ventilatory parameters. RESULTS: Pain catastrophizing was related to bodily pain (r = -0.70), depression (r = 0.55), activity limitations/participation restrictions (r = 0.68), various aspects of quality of life ( r varied between -0.51 and -0.64), and exercise capacity (r varied between -0.41 and -0.61). Based on hierarchical multiple regression analysis, pain catastrophizing accounted for 41% of the variance in bodily pain in female CFS patients who experience chronic widespread musculoskeletal pain. Among the three subscale scores of the Pain Catastrophizing Scale, helplessness and rumination rather than magnification were strongly related to bodily pain. Neither pain catastrophizing nor depression was related to employment status. CONCLUSIONS: These data provide evidence favoring a significant association between pain catastrophizing, bodily pain, exercise performance, and self-reported disability in female patients with CFS who experience widespread pain. Further prospective longitudinal studying of these variables is required.

Lower frequency of IL-17F sequence variant (His161Arg) in chronic fatigue syndrome patients.

Chronic fatigue syndrome (CFS) is characterized by immune dysfunctions including chronic immune activation, inflammation, and alteration of cytokine profiles. T helper 17 (Th17) cells belong to a recently identified subset of T helper cells, with crucial regulatory function in inflammatory and autoimmune processes. Th17 cells are implicated in allergic inflammation, intestinal diseases, central nervous system inflammation, disorders that may all contribute to the pathophysiology of CFS. IL-17F is one of the pro-inflammatory cytokines secreted by Th17 cells. We investigated the association between CFS and the frequency of rs763780, a C/T genetic polymorphism leading to His161Arg substitution in the IL-17F protein. The His161Arg variant (C allele) antagonizes the pro-inflammatory effects of the wild-type IL-17F. A significantly lower frequency of the C allele was observed in the CFS population, suggesting that the His161Arg variant may confer protection against the disease. These results suggest a role of Th17 cells in the pathogenesis of CFS.

Unravelling intracellular immune dysfunctions in chronic fatigue syndrome: interactions between protein kinase R activity, RNase L cleavage and elastase activity, and their clinical relevance.

This study examined possible interactions between immunological abnormalities and symptoms in CFS. Sixteen CFS patients filled in a battery of questionnaires, evaluating daily functioning, and underwent venous blood sampling, in order to analyse immunological abnormalities. Ribonuclease (RNase) L cleavage was associated with RNase L activity (rs=0.570; p=0.021), protein kinase R (PKR) (rs=0.716; p=0.002) and elastase activity (rs=0.500; p=0.049). RNase L activity was related to elastase (rs=0.547; p=0.028) and PKR activity (rs=0.625; p=0.010). RNase L activity (rs=0.535; p=0.033), elastase activity (rs=0.585; p=0.017) and RNase L cleavage (rs=0.521; p=0.038) correlated with daily functioning. This study suggests that in CFS patients an increase in elastase activity and subsequent RNase L cleavage is accompanied by increased activity of both the PKR and RNase L enzymes. RNase L and elastase activity are related to daily functioning, thus evidence supporting the clinical importance of these immune dysfunctions in CFS patients was provided.

Defining the occurrence and influence of alpha-delta sleep in chronic fatigue syndrome.

BACKGROUND: Patients with chronic fatigue syndrome (CFS) present a disordered sleep pattern and frequently undergo polysomnography to exclude a primary sleep disorder. Such studies have shown reduced sleep efficiency, a reduction of deep sleep, prolonged sleep initiation, and alpha-wave intrusion during deep sleep. Deregulation of the 2-5A synthetase/RNase L antiviral pathway and a potential acquired channelopathy are also found in a subset of CFS patients and could lead to sleep disturbances. This article compiles a large sleep study database on CFS patients and correlates these data with a limited number of immune parameters as it has been thought that RNase L could be associated with these sleep disturbances. METHODS: Forty-eight patients who fulfilled 1994 Centers for Disease Control and Prevention criteria for CFS underwent extensive medical evaluation, routine laboratory testing, and a structured psychiatric interview. Subjects then completed a complaint checklist and a two-night polysomnographic investigation. RNase L analysis was performed by gel electrophoresis using a radiolabeled 2',5'-oligoadenylate trimer. Basic descriptive statistical parameters were calculated. RESULTS: Patients experienced a prolonged sleep latency, showed a low sleep efficiency index, and had a low percentage of slow wave sleep. The present alpha-delta intrusion correlated with anxiety; no correlations appeared, however, between alpha-delta sleep and immunologic parameters, including RNase L. CONCLUSIONS: The main findings are 1) validation of sleep latency problems and other sleep disturbances as already suggested by several authors; 2) alpha-delta intrusion seems associated with anxiety; and 3) elevated RNase L did not correlate with alpha-delta sleep.

Comparison of two exercise testing protocols in patients with chronic fatigue syndrome.

This study examined whether a linear exercise stress-testing protocol generated different peak exercise performance variables than a stepwise exercise testing protocol in patients with chronic fatigue syndrome (CFS). We conducted a comparative study with patients randomly allocated to one of two exercise testing protocols. Twenty-eight women with CFS completed two self-reported measures (the CFS Symptom List and the CFS Activities and Participation Questionnaire) and then performed until exhaustion either the linear or the stepwise exercise testing protocol with continuous monitoring of physiological variables (heart rate and oxygen uptake). At baseline, we found no significant differences in demographic features and health status between groups (p > 0.05). Based on ratio peak workload/peak oxygen uptake, mechanical efficiency was lower among the subjects performing the stepwise protocol (p = 0.002). When we analyzed the mean linear regression slope values between oxygen uptake and workload from each subject's minute-by-minute exercise data points, we found that mechanical efficiency was lower among the subjects performing the stepwise protocol (p = 0.039). Apart from mechanical efficiency, we found no differences in exercise performance data between groups (p > 0.05). Our results suggest that the difference between linear and stepwise exercise protocols cannot account for all discrepancies of previous studies on exercise performance data in women with CFS, but they do suggest that the nature of the exercise testing protocol influences mechanical efficiency in these patients. Further study is warranted.