Culturing Adult Stem Cells for Cell-Based Therapeutics: Neuroimmune Applications

Pluripotent stem cells can be successfully isolated from a variety of tissues from adult organisms. This fact opens the exciting possibility of cell-based therapies for a large number of clinical treatments. However, the development of optimized protocols to obtain, grow, and cryopreserve cells, as well as that of effective clinical treatment procedures, is no easy task. The therapeutic potential of cells expanded in vitro depends on a multitude of factors including isolation procedures, donor and tissue types, expansion and preservation methods, etc. Researchers are investing great efforts to determine which of these many variables significantly impact downstream performance of in vitro expanded stem cells by studying associated changes in molecular profiles and their effect on the host immune system. This chapter reviews the current status of stem cell production and its derivatives, which are paving the way to different treatments in the clinic. Due to the research interests of our labs, particular emphasis is placed on the potential benefits of stem cell-based therapeutics for the treatment of spinal cord injuries and the neuroimmune disease myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) not only derived from differentiation and cell engraftment mechanisms but also due to the anti-inflammatory and immunoregulatory capacities of these cells

Complement Component C1q as a Potential Diagnostic Tool for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Subtyping

Myalgic encephalomyelitis; Blood analytics; DiagnosisEncefalomielitis miálgica; Análisis de sangre; DiagnósticoEncefalomielitis miàlgica; Anàlisi de sang; DiagnòsticBackground: Routine blood analytics are systematically used in the clinic to diagnose disease or confirm individuals’ healthy status. For myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a disease relying exclusively on clinical symptoms for its diagnosis, blood analytics only serve to rule out underlying conditions leading to exerting fatigue. However, studies evaluating complete and large blood datasets by combinatorial approaches to evidence ME/CFS condition or detect/identify case subgroups are still scarce. Methods: This study used unbiased hierarchical cluster analysis of a large cohort of 250 carefully phenotyped female ME/CFS cases toward exploring this possibility. Results: The results show three symptom-based clusters, classified as severe, moderate, and mild, presenting significant differences (p < 0.05) in five blood parameters. Unexpectedly the study also revealed high levels of circulating complement factor C1q in 107/250 (43%) of the participants, placing C1q as a key molecule to identify an ME/CFS subtype/subgroup with more apparent pain symptoms. Conclusions: The results obtained have important implications for the research of ME/CFS etiology and, most likely, for the implementation of future diagnosis methods and treatments of ME/CFS in the clinic.This research was partially funded by the Association of Patients with ME/CFS and Fibromyalgia in Catalonia, Spain (ACAF; www.fibromyalgia.cat (accessed on 23 March 2019) to J.C.-M. and J.A.-M., and by a UCV 2020-270-001 grant to E.O

Pressure Point Threshold and ME/CFS comorbidity as Indicators of Physiotherapy Response in Fibromyalgia

Current pharmacological treatments of Fibromyalgia (FM) are merely symptom palliative, as clinical trials have so far failed to provide overall benefits without associated harms. Polypharmacy often leads to patient´s health deterioration and chronic drug use to an eventual lack of patient´s response. Emerging evidence support that physiotherapy treatments based on mechanical triggers improve FM symptoms and therefore could be used for therapeutic purposes by themselves, or in combination with current pharmacological treatments, as part of integrative medicine programs. However, a paucity of studies rigorously and systematically evaluating this possibility exists. This study uses scores from validated standardized questionnaires, algometer pressure point threshold (PPT) readings and responses from a custom self-developed questionnaire to determine the impact of a pressure-controlled manual protocol on FM hyperalgesia/allodynia, fatigue and patient´s quality of life. The results show that patient´s baseline sensitivity to pain inversely correlates with treatment response in FM. Moreover, patients presenting comorbid ME/CFS do not seem to respond to the applied therapy as those presenting FM only. Thus, pretreatment PPTs and ME/CFS comorbidity may serve as indicators to predict patient´s response to physiotherapy programs based on mechanical triggers, as the one evaluated here. These unexpected findings grant further explorations including the study of gene expression profiles associating to patient´s treatment response in the blood collection of samples generated by this study.Medicin

Lack of evidence for retroviral infections formerly related to chronic fatigue in Spanish Fibromyalgia patients

The etiology of fibromyalgia and chronic fatigue syndrome (FM/CFS) is currently unknown. A recurrent viral infection is an attractive hypothesis repeatedly found in the literature since it would explain the persistent pain and tiredness these patients suffer from. The initial striking link of two distinct orphan retroviruses: the gamma retroviruses murine leukemia virus (MLV)-related virus and the delta retrovirus T-lymphotropic virus type 2 (HTLV-2) to chronic fatigue have not been confirmed to date. Results: Genomic DNA (gDNA) from 75 fibromyalgia patients suffering from chronic fatigue and 79 age-matched local healthy controls were screened for the presence of MLV-related and HTLV-2 related proviral sequences. The XMRV env gene was amplified in 20% of samples tested (24% patients/15% healthy controls). Unexpectedly, no PCR amplifications from independent gDNA preparations of the same individuals were obtained. None of the positive samples showed presence of contaminating murine sequences previously reported by other investigators, neither contained additional regions of the virus making us conclude that the initial env amplification came from spurious air-driven amplicon contaminants. No specific HTLV-2 sequences were obtained at any time from any of the 154 quality-controlled gDNA preparations screened. Conclusions: Previous associations between MLV-related or HTLV-2 retrovirus infection with chronic fatigue must be discarded. Thus, studies showing positive amplification of HTLV-2 sequences from chronic fatigue participants should be revised for possible undetected technical problems. To avoid false positives of viral infection, not only extreme precautions should be taken when nested-PCR reactions are prepared and exhaustive foreign DNA contamination controls performed, but also consistent amplification of diverse regions of the virus in independent preparations from the same individual must be demanded. The fact that our cohort of patients did not present evidence of any of the two types of retroviral infection formerly associated to chronic fatigue does not rule out the possibility that other viruses are involved in inciting or maintaining fibromyalgia and/or chronic fatigue conditions.Medicin

Pressure Point Thresholds and ME/CFS Comorbidity as Indicators of Patient’s Response to Manual Physiotherapy in Fibromyalgia

Current pharmacological treatments of Fibromyalgia (FM) are merely symptom palliative, as clinical trials have so far failed to provide overall benefits without associated harms. Polypharmacy often leads to patient’s health deterioration and chronic drug use to an eventual lack of patient’s response. Emerging evidence supports that physiotherapy treatments based on mechanical triggers improve FM symptoms and therefore could be used for therapeutic purposes by themselves or in combination with current pharmacological treatments, as part of integrative medicine programs. However, a paucity of studies rigorously and systematically evaluating this possibility exists. This study uses scores from validated standardized questionnaires, algometer pressure point threshold (PPT) readings and responses from a custom self-developed questionnaire to determine the impact of a pressure-controlled custom manual protocol on FM hyperalgesia/allodynia, fatigue and patient’s quality of life. The results show that patient’s baseline sensitivity to pain inversely correlates with treatment response in FM. Moreover, post-stratification analysis unexpectedly reveals that patients presenting comorbid ME/CFS do not seem to respond to the applied therapy as those presenting FM only. Therefore, pre-treatment PPTs and ME/CFS comorbidity may serve as indicators to predict patient’s response to physiotherapy programs based on mechanical triggers. Further exploration of these findings is granted. In addition, the study of gene expression profiles in the blood collection generated by this study should help unveil the molecular mechanisms behind patient’s differential response to manual therapy.Medicin

La infección crónica por el gamma retrovirus xenotrópico relacionado con el virus de la leucemia murina (XMRV) en patología humana: ¿Realidad o artefacto de laboratorio?

El nuevo virus xenotrópico relacionado con el virus de la leucemia murina (XMRV), único gamma retrovirus identificado en humanos hasta la fecha, se aisló de especímenes de cáncer de próstata en el año 2006, y en el 2009 se asoció impactantemente con el síndrome de fatiga crónica (SFC), enfermedad debilitante de sintomatología compleja que cursa, frecuentemente, con alteraciones de la enzima antiviral ribonucleasa L (RNasa L), al igual que el cáncer de próstata hereditario. La imposibilidad de replicar estos hallazgos y la presencia de secuencias virales similares halladas en kits comerciales y preparaciones de polimerasas “hotstart” que contienen anticuerpos derivados de hibridomas plantean la posibilidad de que las secuencias amplificadas en los estudios iniciales del XMRV puedan corresponder a contaminaciones con ADN murino y que el nuevo XMRV no sea más que un artefacto creado por procesos de recombinación en el seno de un xenoinjerto. Sin embargo, una contaminación casual no permitiría explicar la desigual distribución de secuencias virales halladas entre pacientes y controles sanos, ni cómo fue posible hallar secuencias genómicas específicas humanas flanqueando las secuencias terminales repetidas largas (LTRs) virales del XMRV en tejidos de pacientes de cáncer de próstata. ¿Podría ser que además de un contaminante habitual de reactivos de laboratorio el XMRV fuese un gamma retrovirus humano genuino? El desarrollo de ensayos serológicos específicos y sensibles del XMRV combinados con rigurosos controles resultará indispensable para dar respuesta definitiva a esta importante pregunta.The new and only human gammaretrovirus identified to date: the xenotropic murine leukemia virus-related virus (XMRV) was isolated back in 2006 from prostate cancer specimens and strikingly associated to Chronic Fatigue Syndrome (CFS) in the year 2009. CFS is a multisymptom debilitating disease often showing an altered ribonuclease L (RNase L) enzyme activity, as it occurs in hereditary prostate cancer. The irreproducibility of these findings and the identification of related viral sequences in commercial kits and “hotstart” polymerase preparations containing hybridoma-derived antibodies suggest that the XMRV sequences found in the initial studies may correspond to murine DNA contaminations and that the XMRV might well be an artefact generated by recombination processes that took place within a xenograft. However, a random contaminating event cannot explain the unequal distribution of viral sequences found between patients and healthy controls, nor the presence of human genomic sequences flanking the XMRV long terminal repeats (LTRs) in prostate cancer specimens. Would it be possible that the XRMV is a frequent reagent contaminant but a human genuine gammaretrovirus as well? Highly specific and sensitive serologic assays combined with proper strict controls will be required to answer this important question.Medicin

Impact of Long-Term Cryopreservation on Blood Immune Cell Markers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome : Implications for Biomarker Discovery

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmune disorder characterized by numerous symptoms of unknown etiology. The ME/CFS immune markers reported so far have failed to generate a clinical consensus, perhaps partly due to the limitations of biospecimen biobanking. To address this issue, we performed a comparative analysis of the impact of long-term biobanking on previously identified immune markers and also explored additional potential immune markers linked to infection in ME/CFS. A correlation analysis of marker cryostability across immune cell subsets based on flow cytometry immunophenotyping of fresh blood and frozen PBMC samples collected from individuals with ME/CFS (n = 18) and matched healthy controls (n = 18) was performed. The functionality of biobanked samples was assessed on the basis of cytokine production assay after stimulation of frozen PBMCs. T cell markers defining Treg subsets and the expression of surface glycoprotein CD56 in T cells and the frequency of the effector CD8 T cells, together with CD57 expression in NK cells, appeared unaltered by biobanking. By contrast, NK cell markers CD25 and CD69 were notably increased, and NKp46 expression markedly reduced, by long-term cryopreservation and thawing. Further exploration of Treg and NK cell subsets failed to identify significant differences between ME/CFS patients and healthy controls in terms of biobanked PBMCs. Our findings show that some of the previously identified immune markers in T and NK cell subsets become unstable after cell biobanking, thus limiting their use in further immunophenotyping studies for ME/CFS. These data are potentially relevant for future multisite intervention studies and cooperative projects for biomarker discovery using ME/CFS biobanked samples. Further studies are needed to develop novel tools for the assessment of biomarker stability in cryopreserved immune cells from people with ME/CFS

The European ME/CFS Biomarker Landscape project: an initiative of the European network EUROMENE.

Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) is a common and severe disease with a considerable social and economic impact. So far, the etiology is not known, and neither a diagnostic marker nor licensed treatments are available yet. The EUROMENE network of European researchers and clinicians aims to promote cooperation and advance research on ME/CFS. To improve diagnosis and facilitate the analysis of clinical trials surrogate markers are urgently needed. As a first step for developing such biomarkers for clinical use a database of active biomarker research in Europe was established called the ME/CFS EUROMENE Biomarker Landscape project and the results are presented in this review. Further we suggest strategies to improve biomarker development and encourage researchers to take these into consideration for designing and reporting biomarker studies

Unravelling myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) : Gender-specific changes in the microRNA expression profiling in ME/CFS

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystem illness characterized by medically unexplained debilitating fatigue with suggested altered immunological state. Our study aimed to explore peripheral blood mononuclear cells (PBMCs) for microRNAs (miRNAs) expression in ME/CFS subjects under an exercise challenge. The findings highlight the immune response and inflammation links to differential miRNA expression in ME/CFS. The present study is particularly important in being the first to uncover the differences that exist in miRNA expression patterns in males and females with ME/CFS in response to exercise. This provides new evidence for the understanding of differential miRNA expression patterns and post-exertional malaise in ME/CFS. We also report miRNA expression pattern differences associating with the nutritional status in individuals with ME/CFS, highlighting the effect of subjects' metabolic state on molecular changes to be considered in clinical research within the NINDS/CDC ME/CFS Common Data Elements. The identification of gender-based miRNAs importantly provides new insights into gender-specific ME/CFS susceptibility and demands exploration of sex-suited ME/CFS therapeutics

Relationships between Problematic Cannabis Use and Risky Behaviors in Spanish Adolescents

This study examined the relations between problematic cannabis use, physical assault, and getting involved in a motor vehicle accident under the influence of cannabis in a sample of adolescents randomly selected from 25 public and semiprivate high schools in Alicante (Spain). Participants (n = 648) completed The Spanish National Standardized Survey about drug use in high school adolescents (ESTUDES, 2017), which includes the cannabis abuse screening test (CAST). Prevalence of cannabis use across the life-span and within the past 30 days was 37.5% and 17.4%, respectively. CAST scores were associated with an increased risk of driving under the effects of cannabis, riding shotgun, and physical assault, but not with an increased risk of having a motor vehicle accident. There were no differences between boys and girls in the association of problematic cannabis use with risky behaviors. This result highlights the importance of comprehensive prevention and education strategies for adolescents at high risk of cannabis use.This study has been funded by Ayuntamiento de Alicante. Plan Municipal de Drogodependencia (AYTOALICANTE3-18I). Delegación del Gobierno para el Plan Nacional sobre Drogas