The potential role of ocular and otolaryngological mucus proteins in myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating illness associated with a constellation of other symptoms. While the most common symptom is unrelenting fatigue, many individuals also report suffering from rhinitis, dry eyes and a sore throat. Mucin proteins are responsible for contributing to the formation of mucosal membranes throughout the body. These mucosal pathways contribute to the body's defense mechanisms involving pathogenic onset. When compromised by pathogens the epithelium releases numerous cytokines and enters a prolonged state of inflammation to eradicate any particular infection. Based on genetic analysis, and computational theory and modeling we hypothesize that mucin protein dysfunction may contribute to ME/CFS symptoms due to the inability to form adequate mucosal layers throughout the body, especially in the ocular and otolaryngological pathways leading to low grade chronic inflammation and the exacerbation of symptoms.

Dry eye symptoms and signs in United States Gulf War era veterans with myalgic encephalomyelitis/chronic fatigue syndrome.

BACKGROUND: To examine ocular symptoms and signs of veterans with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) diagnosis, ME/CFS symptoms, and controls. METHODS: This was a prospective, cross-sectional study of 124 South Florida veterans in active duty during the Gulf War era. Participants were recruited at an ophthalmology clinic at the Miami Veterans Affairs Hospital and evaluated for a diagnosis of ME/CFS, or symptoms of ME/CFS (intermediate fatigue, IF) using the Canadian Consensus criteria. Ocular symptoms were assessed via standardised questionnaires and signs via comprehensive slit lamp examination. Inflammatory blood markers were analysed and compared across groups. RESULTS: Mean age was 55.1 ± 4.7 years, 88.7% identified as male, 58.1% as White, and 39.5% as Hispanic. Ocular symptoms were more severe in the ME/CFS (n = 32) and IF (n = 48) groups compared to controls (n = 44) across dry eye (DE; Ocular Surface Disease Index [OSDI]: 48.9 ± 22.3 vs. 38.8 ± 23.3 vs. 19.1 ± 17.8, p < 0.001; 5 item Dry Eye Questionnaire [DEQ-5]: 10.8 ± 3.9 vs. 10.0 ± 4.6 vs. 6.6 ± 4.2, p < 0.001) and pain-specific questionnaires (Numerical Rating Scale 1-10 [NRS] right now: 2.4 ± 2.8 vs. 2.4 ± 2.9 vs 0.9 ± 1.5; p = 0.007; Neuropathic Pain Symptom Inventory modified for the Eye [NPSI-E]: 23.0 ± 18.6 vs. 19.8 ± 19.1 vs. 6.5 ± 9.0, p < 0.001). Ocular surface parameters and blood markers of inflammation were generally similar across groups. CONCLUSION: Individuals with ME/CFS report increased ocular pain but similar DE signs, suggesting that mechanisms beyond the ocular surface contribute to symptoms.

Sex-specific plasma lipid profiles of ME/CFS patients and their association with pain, fatigue, and cognitive symptoms.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex illness which disproportionally affects females. This illness is associated with immune and metabolic perturbations that may be influenced by lipid metabolism. We therefore hypothesized that plasma lipids from ME/CFS patients will provide a unique biomarker signature of disturbances in immune, inflammation and metabolic processes associated with ME/CFS. METHODS: Lipidomic analyses were performed on plasma from a cohort of 50 ME/CFS patients and 50 controls (50% males and similar age and ethnicity per group). Analyses were conducted with nano-flow liquid chromatography (nLC) and high-performance liquid chromatography (HPLC) systems coupled with a high mass accuracy ORBITRAP mass spectrometer, allowing detection of plasma lipid concentration ranges over three orders of magnitude. We examined plasma phospholipids (PL), neutral lipids (NL) and bioactive lipids in ME/CFS patients and controls and examined the influence of sex on the relationship between lipids and ME/CFS diagnosis. RESULTS: Among females, levels of total phosphatidylethanolamine (PE), omega-6 arachidonic acid-containing PE, and total hexosylceramides (HexCer) were significantly decreased in ME/CFS compared to controls. In males, levels of total HexCer, monounsaturated PE, phosphatidylinositol (PI), and saturated triglycerides (TG) were increased in ME/CFS patients compared to controls. Additionally, omega-6 linoleic acid-derived oxylipins were significantly increased in male ME/CFS patients versus male controls. Principal component analysis (PCA) identified three major components containing mostly PC and a few PE, PI and SM species-all of which were negatively associated with headache and fatigue severity, irrespective of sex. Correlations of oxylipins, ethanolamides and ME/CFS symptom severity showed that lower concentrations of these lipids corresponded with an increase in the severity of headaches, fatigue and cognitive difficulties and that this association was influenced by sex. CONCLUSION: The observed sex-specific pattern of dysregulated PL, NL, HexCer and oxylipins in ME/CFS patients suggests a possible role of these lipids in promoting immune dysfunction and inflammation which may be among the underlying factors driving the clinical presentation of fatigue, chronic pain, and cognitive difficulties in ill patients. Further evaluation of lipid metabolism pathways is warranted to better understand ME/CFS pathogenesis.

Modeling Neuroimmune Interactions in Human Subjects and Animal Models to Predict Subtype-Specific Multidrug Treatments for Gulf War Illness.

Gulf War Illness (GWI) is a persistent chronic neuroinflammatory illness exacerbated by external stressors and characterized by fatigue, musculoskeletal pain, cognitive, and neurological problems linked to underlying immunological dysfunction for which there is no known treatment. As the immune system and the brain communicate through several signaling pathways, including the hypothalamic-pituitary-adrenal (HPA) axis, it underlies many of the behavioral and physiological responses to stressors via blood-borne mediators, such as cytokines, chemokines, and hormones. Signaling by these molecules is mediated by the semipermeable blood-brain barrier (BBB) made up of a monocellular layer forming an integral part of the neuroimmune axis. BBB permeability can be altered and even diminished by both external factors (e.g., chemical agents) and internal conditions (e.g., acute or chronic stress, or cross-signaling from the hypothalamic-pituitary-gonadal (HPG) axis). Such a complex network of regulatory interactions that possess feed-forward and feedback connections can have multiple response dynamics that may include several stable homeostatic states beyond normal health. Here we compare immune and hormone measures in the blood of human clinical samples and mouse models of Gulf War Illness (GWI) subtyped by exposure to traumatic stress for subtyping this complex illness. We do this via constructing a detailed logic model of HPA-HPG-Immune regulatory behavior that also considers signaling pathways across the BBB to neuronal-glial interactions within the brain. We apply conditional interactions to model the effects of changes in BBB permeability. Several stable states are identified in the system beyond typical health. Following alignment of the human and mouse blood profiles in the context of the model, mouse brain sample measures were used to infer the neuroinflammatory state in human GWI and perform treatment simulations using a genetic algorithm to optimize the Monte Carlo simulations of the putative treatment strategies aimed at returning the ill system back to health. We identify several ideal multi-intervention strategies and potential drug candidates that may be used to treat chronic neuroinflammation in GWI.

Alterations in high-order diffusion imaging in veterans with Gulf War Illness is associated with chemical weapons exposure and mild traumatic brain injury.

The complex etiology behind Gulf War Illness (GWI) has been attributed to the combined exposure to neurotoxicant chemicals, brain injuries, and some combat experiences. Chronic GWI symptoms have been shown to be associated with intensified neuroinflammatory responses in animal and human studies. To investigate the neuroinflammatory responses and potential causes in Gulf War (GW) veterans, we focused on the effects of chemical/biological weapons (CBW) exposure and mild traumatic brain injury (mTBI) during the war. We applied a novel MRI diffusion processing method, Neurite density imaging (NDI), on high-order diffusion imaging to estimate microstructural alterations of brain imaging in Gulf War veterans with and without GWI, and collected plasma proinflammatory cytokine samples as well as self-reported health symptom scores. Our study identified microstructural changes specific to GWI in the frontal and limbic regions due to CBW and mTBI, and further showed distinctive microstructural patterns such that widespread changes were associated with CBW and more focal changes on diffusion imaging were observed in GW veterans with an mTBI during the war. In addition, microstructural alterations on brain imaging correlated with upregulated blood proinflammatory cytokine markers TNFRI and TNFRII and with worse outcomes on self-reported symptom measures for fatigue and sleep functioning. Taken together, these results suggest TNF signaling mediated inflammation affects frontal and limbic regions of the brain, which may contribute to the fatigue and sleep symptoms of the disease and suggest a strong neuroinflammatory component to GWI. These results also suggest exposures to chemical weapons and mTBI during the war are associated with different patterns of peripheral and central inflammation and highlight the brain regions vulnerable to further subtle microscale morphological changes and chronic signaling to nearby glia.

Role of estrogen receptor α and ß in preserving hippocampal function during aging.

The expression of the ERα and ERß estrogen receptors in the hippocampus may be important in the etiology of age-related cognitive decline. To examine the role of ERα and ERß in regulating transcription and learning, ovariectomized wild-type (WT) and ERα and ERß knockout (KO) mice were used. Hippocampal gene transcription in young ERαKO mice was similar to WT mice 6 h after a single estradiol treatment. In middle-age ERαKO mice, hormone deprivation was associated with a decrease in the expression of select genes associated with the blood-brain barrier; cyclic estradiol treatment increased transcription of these select genes and improved learning in these mice. In contrast to ERαKO mice, ERßKO mice exhibited a basal hippocampal gene profile similar to WT mice treated with estradiol and, in the absence of estradiol treatment, young and middle-age ERßKO mice exhibited preserved learning on the water maze. The preserved memory performance of middle-age ERßKO mice could be reversed by lentiviral delivery of ERß to the hippocampus. These results suggest that one function of ERß is to regulate ERα-mediated transcription in the hippocampus. This model is supported by our observations that knockout of ERß under conditions of low estradiol allowed ERα-mediated transcription. As estradiol levels increased in the absence of ERα, we observed that other mechanisms, likely including ERß, regulated transcription and maintained hippocampal-dependent memory. Thus, our results indicate that ERα and ERß interact with hormone levels to regulate transcription involved in maintaining hippocampal function during aging.

Disentangling the effects of PTSD from Gulf War Illness in male veterans via a systems-wide analysis of immune cell, cytokine, and symptom measures.

BACKGROUND: One-third of veterans returning from the 1990-1991 Gulf War reported a myriad of symptoms including cognitive dysfunction, skin rashes, musculoskeletal discomfort, and fatigue. This symptom cluster is now referred to as Gulf War Illness (GWI). As the underlying mechanisms of GWI have yet to be fully elucidated, diagnosis and treatment are based on symptomatic presentation. One confounding factor tied to the illness is the high presence of post-traumatic stress disorder (PTSD). Previous research efforts have demonstrated that both GWI and PTSD are associated with immunological dysfunction. As such, this research endeavor aimed to provide insight into the complex relationship between GWI symptoms, cytokine presence, and immune cell populations to pinpoint the impact of PTSD on these measures in GWI. METHODS: Symptom measures were gathered through the Multidimensional fatigue inventory (MFI) and 36-item short form health survey (SF-36) scales and biological measures were obtained through cytokine & cytometry analysis. Subgrouping was conducted using Davidson Trauma Scale scores and the Structured Clinical Interview for Diagnostic and statistical manual of mental disorders (DSM)-5, into GWI with high probability of PTSD symptoms (GWIH) and GWI with low probability of PTSD symptoms (GWIL). Data was analyzed using Analysis of variance (ANOVA) statistical analysis along with correlation graph analysis. We mapped correlations between immune cells and cytokine signaling measures, hormones and GWI symptom measures to identify patterns in regulation between the GWIH, GWIL, and healthy control groups. RESULTS: GWI with comorbid PTSD symptoms resulted in poorer health outcomes compared with both Healthy control (HC) and the GWIL subgroup. Significant differences were found in basophil levels of GWI compared with HC at peak exercise regardless of PTSD symptom comorbidity (ANOVA F = 4.7, P = 0.01,) indicating its potential usage as a biomarker for general GWI from control. While the unique identification of GWI with PTSD symptoms was less clear, the GWIL subgroup was found to be delineated from both GWIH and HC on measures of IL-15 across an exercise challenge (ANOVA F > 3.75, P < 0.03). Additional differences in natural killer (NK) cell numbers and function highlight IL-15 as a potential biomarker of GWI in the absence of PTSD symptoms. CONCLUSION: We conclude that disentangling GWI and PTSD by defining trauma-based subgroups may aid in the identification of unique GWI biosignatures that can help to improve diagnosis and target treatment of GWI more effectively.

Recent Research Trends in Neuroinflammatory and Neurodegenerative Disorders.

Neuroinflammatory and neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), traumatic brain injury (TBI) and Amyotrophic lateral sclerosis (ALS) are chronic major health disorders. The exact mechanism of the neuroimmune dysfunctions of these disease pathogeneses is currently not clearly understood. These disorders show dysregulated neuroimmune and inflammatory responses, including activation of neurons, glial cells, and neurovascular unit damage associated with excessive release of proinflammatory cytokines, chemokines, neurotoxic mediators, and infiltration of peripheral immune cells into the brain, as well as entry of inflammatory mediators through damaged neurovascular endothelial cells, blood-brain barrier and tight junction proteins. Activation of glial cells and immune cells leads to the release of many inflammatory and neurotoxic molecules that cause neuroinflammation and neurodegeneration. Gulf War Illness (GWI) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) are chronic disorders that are also associated with neuroimmune dysfunctions. Currently, there are no effective disease-modifying therapeutic options available for these diseases. Human induced pluripotent stem cell (iPSC)-derived neurons, astrocytes, microglia, endothelial cells and pericytes are currently used for many disease models for drug discovery. This review highlights certain recent trends in neuroinflammatory responses and iPSC-derived brain cell applications in neuroinflammatory disorders.

Ocular and inflammatory markers associated with Gulf War illness symptoms.

To examine the utility of ocular coherence tomography (OCT) metrics, in conjunction with systemic markers of inflammation, in identifying individuals with Gulf War Illness (GWI) symptoms. Prospective case-control study of 108 Gulf War Era veterans, split into 2 groups based on the presence of GWI symptoms, defined by the Kansas criteria. Information on demographics, deployment history, and co-morbidities were captured. 101 individuals underwent OCT imaging and 105 individuals provided a blood sample which was analyzed for inflammatory cytokines using an enzyme-linked immunosorbent assay-based chemiluminescent assay. The main outcome measure was predictors of GWI symptoms, examined with multivariable forward stepwise logistic regression analysis followed by receiver operating characteristic (ROC) analysis. The mean age of the population was 55 ± 4, 90.7% self-identified as male, 53.3% as White, and 54.3% as Hispanic. A multivariable model that considered demographics and co-morbidities found that a lower inferior temporal ganglion cell layer-inner plexiform layer (GCL‒IPL) thickness, higher temporal nerve fiber layer (NFL) thickness, lower interleukin (IL)-1ß levels, higher IL-1α levels, and lower tumor necrosis factor-receptor I levels correlated with GWI symptoms. ROC analysis demonstrated an area under the curve of 0.78 with the best cut-off value for the prediction model having a sensitivity of 83% and specificity of 58%. RNFL and GCL‒IPL measures, namely increased temporal thickness and decreased inferior temporal thickness, respectively, in conjunction with a number of inflammatory cytokines, had a reasonable sensitivity for the diagnosis of GWI symptoms in our population.

The Potential Role of Ocular and Otolaryngological Mucus Proteins in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating illness associated with a constellation of other symptoms. While the most common symptom is unrelenting fatigue, many individuals also report suffering from rhinitis, dry eyes and a sore throat. Mucin proteins are responsible for contributing to the formation of mucosal membranes throughout the body. These mucosal pathways contribute to the body's defense mechanisms involving pathogenic onset. When compromised by pathogens the epithelium releases numerous cytokines and enters a prolonged state of inflammation to eradicate any particular infection. Based on genetic analysis, and computational theory and modeling we hypothesize that mucin protein dysfunction may contribute to ME/CFS symptoms due to the inability to form adequate mucosal layers throughout the body, especially in the ocular and otolaryngological pathways leading to low grade chronic inflammation and the exacerbation of symptoms.

COVID-19 and Long COVID: Disruption of the Neurovascular Unit, Blood-Brain Barrier, and Tight Junctions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), could affect brain structure and function. SARS-CoV-2 can enter the brain through different routes, including the olfactory, trigeminal, and vagus nerves, and through blood and immunocytes. SARS-CoV-2 may also enter the brain from the peripheral blood through a disrupted blood-brain barrier (BBB). The neurovascular unit in the brain, composed of neurons, astrocytes, endothelial cells, and pericytes, protects brain parenchyma by regulating the entry of substances from the blood. The endothelial cells, pericytes, and astrocytes highly express angiotensin converting enzyme 2 (ACE2), indicating that the BBB can be disturbed by SARS-CoV-2 and lead to derangements of tight junction and adherens junction proteins. This leads to increased BBB permeability, leakage of blood components, and movement of immune cells into the brain parenchyma. SARS-CoV-2 may also cross microvascular endothelial cells through an ACE2 receptor-associated pathway. The exact mechanism of BBB dysregulation in COVID-19/neuro-COVID is not clearly known, nor is the development of long COVID. Various blood biomarkers could indicate disease severity and neurologic complications in COVID-19 and help objectively diagnose those developing long COVID. This review highlights the importance of neurovascular and BBB disruption, as well as some potentially useful biomarkers in COVID-19, and long COVID/neuro-COVID.

A common language for Gulf War Illness (GWI) research studies: GWI common data elements.

AIMS: The Gulf War Illness programs (GWI) of the United States Department of Veteran Affairs and the Department of Defense Congressionally Directed Medical Research Program collaborated with experts to develop Common Data Elements (CDEs) to standardize and systematically collect, analyze, and share data across the (GWI) research community. MAIN METHODS: A collective working group of GWI advocates, Veterans, clinicians, and researchers convened to provide consensus on instruments, case report forms, and guidelines for GWI research. A similar initiative, supported by the National Institute of Neurologic Disorders and Stroke (NINDS) was completed for a comparative illness, Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), and provided the foundation for this undertaking. The GWI working group divided into two sub-groups (symptoms and systems assessment). Both groups reviewed the applicability of instruments and forms recommended by the NINDS ME/CFS CDE to GWI research within specific domains and selected assessments of deployment exposures. The GWI CDE recommendations were finalized in March 2018 after soliciting public comments. KEY FINDINGS: GWI CDE recommendations are organized in 12 domains that include instruments, case report forms, and guidelines. Recommendations were categorized as core (essential), supplemental-highly recommended (essential for specified conditions, study types, or designs), supplemental (commonly collected, but not required), and exploratory (reasonable to use, but require further validation). Recommendations will continually be updated as GWI research progresses. SIGNIFICANCE: The GWI CDEs reflect the consensus recommendations of GWI research community stakeholders and will allow studies to standardize data collection, enhance data quality, and facilitate data sharing.

Associations of Immune Genetic Variability with Gulf War Illness in 1990-1991 Gulf War Veterans from the Gulf War Illness Consortium (GWIC) Multisite Case-Control Study.

Gulf War illness (GWI) encompasses a constellation of persistent debilitating symptoms associated with significant changes in central nervous system (CNS) and immune functioning. Currently, there is no validated biomarker for GWI risk susceptibility. Given the impact of immune responses linked to GWI symptomology, genetic variability that causes persistent inflammatory/immune alterations may be key. This Boston University-based Gulf War Illness Consortium (GWIC) study investigated the impact of single nucleotide polymorphisms (SNPs) in variants of immune and pain genetic markers IL1B, IL2, IL6, IL6R, IL10, TNF, TGF, TLR2, TLR4, MD2, MYD88, BDNF, CRP, ICE, COMT and OPRM1 on GWI occurrence in a Caucasian subset of Gulf War (GW) veterans with (cases, n = 170) and without (controls, n = 34) GWI. Logistic regression modeling created a prediction model of GWI risk that associated genetic variability in TGF (rs1800469, p = 0.009), IL6R (rs8192284, p = 0.004) and TLR4 (rs4986791, p = 0.013) with GWI occurrence. This prediction model was specific and sensitive, with a receiver operator characteristic area under the curve of 71.4%. This is the first report of immune genetic variability being predictive of GWI and warrants validation in larger independent cohorts. Future reports will present interactions of these genetic risk factors with other characteristics of GW service.

Brain-Immune Interactions as the Basis of Gulf War Illness: Clinical Assessment and Deployment Profile of 1990-1991 Gulf War Veterans in the Gulf War Illness Consortium (GWIC) Multisite Case-Control Study.

The Boston University-based Gulf War Illness Consortium (GWIC) is a multidisciplinary initiative developed to provide detailed understanding of brain and immune alterations that underlie Gulf War illness (GWI), the persistent multisymptom disorder associated with military service in the 1990-1991 Gulf War. The core GWIC case-control clinical study conducted in-depth brain and immune evaluation of 269 Gulf War veterans (223 GWI cases, 46 controls) at three U.S. sites that included clinical assessments, brain imaging, neuropsychological testing, and analyses of a broad range of immune and immunogenetic parameters. GWI cases were similar to controls on most demographic, military, and deployment characteristics although on average were two years younger, with a higher proportion of enlisted personnel vs. officers. Results of physical evaluation and routine clinical lab tests were largely normal, with few differences between GWI cases and healthy controls. However, veterans with GWI scored significantly worse than controls on standardized assessments of general health, pain, fatigue, and sleep quality and had higher rates of diagnosed conditions that included hypertension, respiratory and sinus conditions, gastrointestinal conditions, and current or lifetime depression and post-traumatic stress disorder. Among multiple deployment experiences/exposures reported by veterans, multivariable logistic regression identified just two significant GWI risk factors: extended use of skin pesticides in theater (adjusted OR = 3.25, p = 0.005) and experiencing mild traumatic brain injury during deployment (OR = 7.39, p = 0.009). Gulf War experiences associated with intense stress or trauma (e.g., participation in ground combat) were not associated with GWI. Data and samples from the GWIC project are now stored in a repository for use by GWI researchers. Future reports will present detailed findings on brain structure and function, immune function, and association of neuroimmune measures with characteristics of GWI and Gulf War service.

Gulf War Illness Clinical Trials and Interventions Consortium (GWICTIC): A collaborative research infrastructure for intervention and implementation.

AIMS: There is an inadequate portfolio of treatments for Gulf War Illness (GWI), a complex disease involving multiple organ systems, and early-phase clinical trials are hampered by many logistical problems. To address these challenges, the Gulf War Illness Clinical Trials and Interventions Consortium (GWICTIC) was formed with the aims of (i) creating a collaborative consortium of clinical and scientific researchers that will rapidly implement rigorous and innovative phase I and II clinical trials for GWI, (ii) perform at least four phase I or II clinical trials, (iii) provide a foundation of scalable infrastructure and management in support of the efficient and successful operation of the GWICTIC, and (iv) partner with the Boston Biorepository, Recruitment & Integrated Network for GWI and other GWI investigators to develop a common data element platform for core assessments and outcomes. MAIN METHODS: The GWICTIC brings together a multidisciplinary team of researchers at several institutions to provide scientific innovation, statistical and computational rigor, and logistical efficiency in the development and implementation of early-phase low-risk clinical trials for GWI. The GWICTIC core trials adhere to a Veteran-centered philosophy and focus on interventions with multiple mechanistic targets to maximize the likelihood of efficacy. To support rapid and efficient study startup and implementation across the GWI research community, the GWICTIC will share infrastructure with investigator-initiated research studies funded under separate mechanisms. SIGNIFICANCE: The GWICTIC will leverage the efficiencies of centralized research support and innovative trial designs to address several longstanding needs in the GWI interventions research community.

Aging alters the expression of genes for neuroprotection and synaptic function following acute estradiol treatment.

This study used microarray analysis to examine age-related changes in gene expression 6 and 12 h following a single estradiol injection in ovariectomized mice. Estradiol-responsive gene expression at the 6 h time point was reduced in aged (18 months) animals compared with young (4 months) and middle-aged (MA, 12 months) mice. Examination of gene clustering within biological and functional pathways indicated that young and MA mice exhibited increased expression of genes for cellular components of the synapse and decreased expression of genes related to oxidative phosphorylation and mitochondrial dysfunction. At the 12 h time point, estradiol-responsive gene expression increased in aged animals and decreased in young and MA mice compared with the 6 h time point. Gene clustering analysis indicated that aged mice exhibited increased expression of genes for signaling pathways that are rapidly influenced by estradiol. The age differences in gene expression for rapid signaling pathways may relate to disparity in basal pathway activity and estradiol mediated activation of rapid signaling cascades.

Treatment Avenues in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Split-gender Pharmacogenomic Study of Gene-expression Modules.

PURPOSE: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating multisymptom illness impacting up to 1 million people in the United States. As the pathogenesis and etiology of this complex condition are unclear, prospective treatments are limited. Identifying US Food and Drug Administration-approved drugs that may be repositioned as treatments for ME/CFS may offer a rapid and cost-effective solution. METHODS: Here we used gene-expression data from 33 patients with Fukuda-defined ME/CFS (23 females, 10 males) and 21 healthy demographically comparable controls (15 females, 6 males) to identify differential expression of predefined gene-module sets based on nonparametric statistics. Differentially expressed gene modules were then annotated via over-representation analysis using the Consensus Pathway database. Differentially expressed modules were then regressed onto measures of fatigue and cross-referenced with drug atlas and pharmacogenomics databases to identify putative treatment agents. FINDINGS: The top 1% of modules identified in males indicated small effect sizes in modules associated with immune regulation and mitochondrial dysfunction. In females, modules identified included those related to immune factors and cardiac/blood factors, returning effect sizes ranging from very small to intermediate (0.147 < Cohen δ < 0.532). Regression analysis indicated that B-cell receptors, T-cell receptors, tumor necrosis factor α, transforming growth factor ß, and metabolic and cardiac modules were strongly correlated with multiple composite measures of fatigue. Cross-referencing identified genes with pharmacogenomics data indicated immunosuppressants as potential treatments of ME/CFS symptoms. IMPLICATIONS: The findings from our analysis suggest that ME/CFS symptoms are perpetuated by immune dysregulation that may be approached via immune modulation-based treatment strategies.

Hepatocyte growth factor and alternative splice variants - expression, regulation and implications in osteogenesis and bone health and repair.

INTRODUCTION: Bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes. These pluripotent cells secrete hepatocyte growth factor (HGF), which regulates cell growth, survival, motility, migration, mitogenesis and is important for tissue development/regeneration. HGF has four splice variants, NK1, NK2, NK3, and NK4 which have varying functions and affinities for the HGF receptor, cMET. HGF promotes osteoblastic differentiation of MSCs into bone forming cells, playing a role in bone development, health and repair. AREAS COVERED: This review will focus on the effects of HGF in osteogenesis, bone repair and bone health, including structural and functional insights into the role of HGF in the body. EXPERT OPINION: Approximately 6.2 million Americans experience a fracture annually, with 5-10% being mal- or non-union fractures. HGF is important in priming MSCs for osteogenic differentiation in vitro and is currently being studied to assess its role during bone repair in vivo. Due to the high turnover rate of systemic HGF, non-classic modes of HGF-treatment, including naked-plasmid HGF delivery and the use of HGF splice variants (NK1 & NK2) are being studied to find safe and efficacious treatments for bone disorders, such as mal- or non-union fractures.

TAp63γ and ΔNp63ß promote osteoblastic differentiation of human mesenchymal stem cells: regulation by vitamin D3 Metabolites.

The transcription factor p63 is required for skeletal formation, and is important for the regulation of 1α,25(OH)2D3 receptor (VDR) in human mesenchymal stem cells (hMSC). Herein we report that TAp63γ and ΔNp63ß appear to be an integral part of the osteoblastic differentiation of hMSC and are differentially regulated by the vitamin D3 metabolites 1α,25(OH)2D3 and 24R,25(OH)2D3. We compared the endogenous expression of p63 isoforms (TA- and ΔNp63) and splice variants (p63α, -ß, -γ), in naive hMSC and during osteoblastic differentiation of hMSC. TAp63α and -ß were the predominant p63 variants in naive, proliferating hMSC. In contrast, under osteoblastic differentiation conditions, expression of p63 changed from the TAp63α and -ß to the TAp63γ and ΔNp63ß variants. Transient overexpression of the p63 variants demonstrated that TAp63ß, ΔNp63ß, and ΔNp63γ increased alkaline phosphatase activity and ΔNp63α and -γ increased the expression of mRNA for osteocalcin and osterix. Our results support the hypothesis that TAp63α and -ß promote a naive state in hMSC. Moreover, TAp63γ is increased during and promotes early osteoblastic differentiation through the expression of pro-osteogenic genes; VDR, Osterix, Runx2 and Osteopontin. ΔNp63ß also appears to support osteogenic maturation through increased alkaline phosphatase activity. Treatment with 1α,25(OH)2D3 increased the expression of mRNA for ΔNp63, while addition of 24R,25(OH)2D3 increased the expression of TA- and ΔNp63γ variants. These novel findings demonstrate for the first time that p63 variants are differentially expressed in naive hMSC (TAp63α,ß), are important during the osteoblastic differentiation of hMSC (TAp63γ and ΔNp63ß), and are differentially regulated by the vitamin D3 metabolites, 1α,25(OH)2D3 and 24R,25(OH)2D3. The molecular nuances and mechanisms of osteoblastic differentiation presented here will hopefully improve our understanding of bone development, complications in bone repair (mal- and non-union fractures), osteoporosis and possibly lead to new modalities of treatment.

24R,25-dihydroxyvitamin D3 promotes the osteoblastic differentiation of human mesenchymal stem cells.

Although 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is considered the most biologically active vitamin D3 metabolite, the vitamin D3 prohormone, 25-hydroxyvitamin D3 [25(OH)D3], is metabolized into other forms, including 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]. Herein we show that 24R,25(OH)2D3 is fundamental for osteoblastic differentiation of human mesenchymal stem cells (hMSCs). Our approach involved analyses of cell proliferation, alkaline phosphatase activity, and pro-osteogenic genes (collagen 1A1, osteocalcin, vitamin D receptor [VDR], vitamin D3-hydroxylating enzymes [cytochrome P450 hydroxylases: CYP2R1, CYP27A1, CYP27B1 and CYP24A1]) and assessment of Ca(2+) mineralization of extracellular matrix. 24R,25(OH)2D3 inhibited hMSC proliferation, decreased 1α-hydroxylase (CYP27B) expression, thereby reducing the ability of hMSCs to convert 25(OH)D3 to 1α,25(OH)2D3, and promoted osteoblastic differentiation through increased alkaline phosphatase activity and Ca(2+) mineralization. 24R,25(OH)2D3 decreased expression of the 1α,25(OH)2D3 receptor, VDR. 24R,25(OH)2D3 but not 1α,25(OH)2D3 induced Ca(2+) mineralization dependent on the absence of the glucocorticoid analog, dexamethasone. To elucidate the mechanism(s) for dexamethasone-independent 1α,25(OH)2D3 inhibition/24R,25(OH)2D3 induction of Ca(2+) mineralization, we demonstrated that 1α,25(OH)2D3 increased whereas 24R,25(OH)2D3 decreased reactive oxygen species (ROS) production. 25(OH)D3 also decreased ROS production, potentially by conversion to 24R,25(OH)2D3. Upon inhibition of the vitamin D3-metabolizing enzymes (cytochrome P450s), 25(OH)D3 increased ROS production, potentially due to its known (low) affinity for VDR. We hypothesize that vitamin D3 actions on osteoblastic differentiation involve a regulatory relationship between 24R,25(OH)2D3 and 1α,25(OH)2D3. These results implicate 24R,25(OH)2D3 as a key player during hMSC maturation and bone development and support the concept that 24R,25(OH)2D3 has a bioactive role in the vitamin D3 endocrine system.