Maria Inmaculada Dominguez-Mozo

Type 1 Diabetes (T1D) is thought to result from the interaction of genetic and environmental factors, with different studies highlighting a potential role for the gut microbiota and its metabolites in modulating immune responses and disease development. We hypothesized that patients with T1D exhibited altered levels of circulating bacterial metabolites compared with healthy controls (HC), and that these metabolite profiles were associated with key demographic, clinical, and analytical features of the disease. A total of 91 T1D patients and 58 HC were recruited. Plasma samples were collected and analyzed with gas chromatography coupled to mass spectrometry for the detection of the metabolites: short-chain fatty acids (SCFAs: acetate [AA], propionate [PA], isobutyrate [IBA], butyrate [BA], isovalerate [IVA], valerate [VA], and methyl valerate [MVA]), medium-chain fatty acids (MCFAs: hexanoate [HxA] and heptanoate [HpA]) and para-cresol (p-cresol). We also calculated the ratios between the different SCFAs with AA. T1D patients showed significantly higher circulating AA levels than HC, along with reduced PA/AA and IBA/AA ratios, indicating an altered SCFA profile. SCFA diversity was lower in T1D patients, with reduced detection of BA, and total SCFA levels were increased mainly due to elevated AA. AA levels were higher and SCFA ratios lower in women with T1D compared with healthy women, while p-cresol levels were higher in men with T1D than in healthy men. In T1D patients, AA levels positively correlated with HbA1c, whereas PA/AA, IBA/AA, and BA/AA ratios showed negative correlations, particularly in women. MV/AA and non-AA/AA ratios were inversely associated with glucose levels, again, mainly in women. p-cresol levels correlated positively with age and ferritin and were higher in T1D patients with liver dysfunction or hypertension. Therefore, we can conclude that T1D is associated with a marked alteration in circulating gut-derived metabolites, characterized by increased AA levels, particularly in women, and an imbalance in SCFA ratios that correlates with glycemic control. These findings, together with the associations observed for p-cresol and metabolic comorbidities, support a role for the gut microbiota–host metabolic axis in T1D.

INTRODUCTION: While the exact cause of Type 1 Diabetes (T1D) remains unclear, it is widely believed that both genetic and environmental factors contribute to the development of the disease. Recent research has explored the potential role of gut microbiota and its metabolites in modulating immune responses and influencing the development of autoimmune diseases like T1D. With this purpose, we designed a study: 1. to compare the levels of different bacterial metabolites in plasma samples of T1D patients and healthy controls (HC). 2. to correlate the levels of these metabolites with different demographic, clinical and analytical variables collected from the T1D patients. METHODS: A total of 91 T1D patients were recruited. Plasma samples were collected and analyzed with gas chromatography coupled to mass spectrometry for the detection of the metabolites: short-chain fatty acids (SCFA: acetate [AA], propionate [PA], isobutyrate [IBA], butyrate [BA], isovalerate [IVA], valerate [VA] and methyl valerate [MVA]), medium-chain fatty acids (MCFA: hexanoate [HxA] and heptanoate [HpA]) and para-cresol (p-cresol). We also calculated the ratios between the different SCFA with AA. RESULTS: 1. AA levels were significantly higher in T1D patients than in HC (p=0.0009). PA/AA and IBA/AA ratios were significantly higher in HC (p=0.0004 and p=0.0001, respectively). 2. Glycated haemoglobin (HbA1c) was positively correlated with AA levels (p=0.0001; r=0.406) and a significant negative correlation with a rSpearman< -0.3 was found for PA/AA, IBA/AA and BA/AA ratios. 3. p-cresol correlated with Ferritin levels (p=0.04; r=0.362); besides, p-cresol levels were lower in T1D patients with a normal liver profile (p=0.002) and in T1D patients without hypertension (p=0.005). CONCLUSIONS: Serum levels of bacterial metabolites were significantly different in T1D patients. AA levels were significantly increased in T1D patients and p-cresol was higher in T1D patients with liver disturbances and hypertension. To develop strategies to restore gut microbiota health and immune balance could be essential for the control of T1D.

Background: MicroRNAs are small non-coding RNA that regulate gene expression at a post-transcriptional level affecting several cellular processes including inflammation, neurodegeneration and remyelination. Different patterns of miRNAs expression have been demonstrated in multiple sclerosis compared to controls, as well as in different courses of the disease. For these reason they have been postulated as promising biomarkers candidates in multiple sclerosis.Objective: to correlate serum microRNAs profile expression with disability, cognitive functioning and brain volume in patients with remitting-relapsing multiple sclerosis.Methods: cross-sectional study in relapsing-remitting multiple sclerosis patients treated with glatiramer acetate. Disability was measured with Expanded Disability Status Scale (EDSS) and cognitive function was studied with Symbol Digit Modalities Test (SDMT). Brain volume was analyzed with automatic software NeuroQuant^®.Results: We found an association between miR.146a.5p (r_s:0.434, p=0.03) and miR.9.5p (r_s:0.516, p=0.028) with EDSS; and miR-146a.5p (r_s:-0.476, p=0.016) and miR-126.3p (r_s:-0.528, p=0.007) with SDMT. Regarding to the brain volume, miR.9.5p correlated with thalamus (r_s:-0.545, p=0.036); miR.200c.3p with pallidum (r_s:-0.68, p=0.002) and cerebellum (r_s:-0.472, p=0.048); miR-138.5p with amygdala (r_s:0.73, p=0.016) and pallidum (r_s:0.64, p=0.048); and miR-223.3p with caudate (r_s:0.46, p=0.04).Conclusions: These data support the hypothesis of microRNA as potential biomarkers in this disease. More studies are needed to validate these results and to better understand the role of microRNAs in the pathogenesis, monitoring and therapeutic response of multiple sclerosis.

PHERV-W ENV and syncytin-1, the envelope proteins of the human endogenous retrovirus W family (HERV-W), have been proposed as etiological factors for MS development. In addition, herpesviruses, such as the Epstein-Barr virus (EBV) and the human herpesvirus 6A/B (HHV-6A/B), have been also strongly associated with the disease. This work aims to study the possible link between viral loads and antibody titers against EBV and HHV-6A/B and the pHERV-W ENV/syncytin-1 protein/gene expression. For this purpose, we conducted a 12-month longitudinal study involving 98 RRMS patients. Peripheral blood samples were obtained from each patient. Serum antibody titers against EBV and HHV-6A/B were determined by ELISA, while viral loads were analyzed using qPCR. HLA MS-related alleles were also genotyped. pHERV-W ENV/syncytin-1 protein and gene expression levels in immune cells were assessed by flow cytometry and qPCR, respectively. We found that the 12-month variation of the pHERV-W ENV gene expression levels positively correlated with the variation of the EBV viral load, especially in those patients with high baseline EBV loads. Therefore, these results could support previous studies pointing to the transactivation of pHERV-W ENV by EBV. However, further studies are needed to better understand this possible relationship.

Background and purpose: Short‐chain fatty acids (SCFAs) can have pro‐ or anti‐inflammatory properties, but their relationship with multiple sclerosis (MS) relapses during pregnancy remains unknown. This study aimed to explore SCFA profiles in MS patients during pregnancy and to assess their association with the appearance of relapses during pregnancy and postpartum.Methods: We prospectively included 53 pregnant MS patients and 21 healthy control women. Patients were evaluated during pregnancy and puerperium. SCFAs were measured by liquid chromatography–mass spectrometry.Results: Sixteen patients (32%) had relapses during pregnancy or puerperium, and 37 (68%) did not. All MS patients showed significant increases in acetate levels during pregnancy and the postpartum period compared to non‐MS women. However, propionate and butyrate values were associated with disease activity. Their values were higher in nonrelapsing patients and remained similar to the control group in relapsing patients. The variable that best identified active patients was the propionate/acetate ratio. Ratios of p < 0.01). Most nonrelapsing patients showed values of >0.36, which were similar to those in healthy pregnant women.Conclusions: Low propionate/acetate ratio values during the first trimester of gestation identified MS patients at risk of relapses during pregnancy and the postpartum period.

Although the etiology of multiple sclerosis (MS) is still unknown, it is commonly accepted that environmental factors could contribute to the disease. The objective of this study was to analyze the humoral response to Epstein-Barr virus, human herpesvirus 6A/B and cytomegalovirus, and the levels of 25-hydroxyvitamin D (25(OH)D) and the three main short-chain fatty acids (SCFA), propionate (PA), butyrate (BA) and acetate (AA), in MS patients and healthy controls (HC) to understand how they could contribute to the pathogenesis of the disease. With this purpose, we analyzed the correlations among them and with different clinical variables and a wide panel of cell subsets. We found statistically significant differences for most of the environmental factors analyzed when we compared MS patients and HC, supporting their possible involvement in the disease. The strongest correlations with the clinical variables and the cell subsets analyzed were found for 25(OH)D and SCFAs levels. A correlation was also found between 25(OH)D and PA/AA ratio, and the interaction between these factors negatively correlated with interleukin 17 (IL-17)-producing CD4+ and CD8+ T cells in untreated MS patients. Therapies that simultaneously increase vitamin D levels and modify the proportion of SCFA could be evaluated in the future.

Human herpesvirus-6A (HHV-6A) and −6B (HHV-6B) might be involved in the etiopathogenesis of multiple sclerosis (MS), especially the HHV-6A. We aim at assessing, for the first time in the scientific literature, the HHV-6A/B microRNAs in MS patients. We analyzed the miRNAs of HHV-6A: miR-U86, and −6B: hhv6b-miR-Ro6-1, −2, −3-3p, −3-5p, and −4 in paired samples of serum and CSF of 42 untreated MS patients and 23 patients with other neurological diseases (OND), using Taqman MicroRNA Assays. Intrathecal HHV-6A/B antibody production and anti-HHV-6A/B IgG/IgM levels in serum were measured. MS clinical data were available. We detected the following miRNAs: hhv6b-miR-Ro6-2 (serum: MS:97.7%, OND:95.7%; CSF: MS:81%, OND:86.4%), 3-3p (serum: MS:4.8%, OND:0%; CSF: MS:2.4%, OND:4.5%), −3-5p (serum: MS:95.2%, OND:91.3%; CSF: MS:50%, OND:54.5%), and miR-U86 (serum: MS:54.8%, OND:47.8%; CSF: MS:11.9%, OND:9.1%). In the serum of the whole population (MS and OND patients) we found a significant correlation between the levels of hhv6b-miR-Ro6-2 and −3-5p (Spearman r = 0.839, pcorr = 3E-13), −2 and miR-U86 (Spearman r = 0.578, pcorr = 0.001) and −3-5p and miR-U86 (Spearman r = 0.698, pcorr = 1.34E-5); also in the CSF, between hhv6b-miR-Ro6-2 and −3-5p (Spearman r = 0.626, pcorr = 8.52E-4). These correlations remained statistically significant when both populations were considered separately. The anti-HHV-6A/B IgG levels in CSF and the intrathecal antibody production in positive MS patients for hhv6b-miR-Ro6-3-5p were statistically significant higher than in the negative ones (pcorr = 0.006 and pcorr = 0.036). The prevalence of miR-U86 (30.8%) in the CSF of individuals without gadolinium-enhancing lesions was higher (p = 0.035) than in the ones with these lesions (0%); however, the difference did not withstand Bonferroni correction (pcorr = 0.105). We propose a role of HHV-6A/B miRNAs in the maintenance of the viral latency state. Further investigations are warranted to validate these results and clarify the function of these viral miRNAs.

Response to interferon-beta (IFN-beta) treatment is heterogeneous in multiple sclerosis (MS). We aimed to search for biomarkers predicting no evidence of disease activity (NEDA) status upon IFN-beta treatment in MS. 119 patients with relapsing–remitting MS (RRMS) initiating IFN-beta treatment were included in the study, and followed prospectively for 2 years. Neutralizing antibodies (NAb) were explored in serum samples obtained after 6 and 12 months of IFN-beta treatment. Soluble cytokines and blood lymphocytes were studied in basal samples by ELISA and flow cytometry, respectively. 9% of patients developed NAb. These antibodies were more frequent in patients receiving IFN-beta 1b than in those treated subcutaneous (p = 0.008) or intramuscular (p < 0.0001) IFN-beta 1a. No patient showing NAb remained NEDA during follow-up. Basal immunological variables are also associated with patient response. Percentages below 3% of CD19 + CD5 + cells (AUC 0.74, CI 0.63–0.84; OR 10.68, CI 3.55–32.15, p < 0.0001; Likelihood ratio 4.28) or above 2.6% of CD8 + perforin + T cells (AUC 0.79, CI 0.63–0.96; OR 6.11, CI 2.0–18.6, p = 0.0009; Likelihood ratio 5.47) increased the probability of achieving NEDA status during treatment. Basal blood immune cell subsets contribute to identify MS patients with a high probability of showing an optimal response to IFN-beta.

One of the risk factor to develop progressive multifocal leukoencephalopathy (PML) among natalizumab-treated patients is the presence and high levels of anti-JCV antibodies. Our purpose was to test the association of different clinical and demographic variables with the presence and levels of anti-JCV antibodies in a Spanish cohort of patients with multiple sclerosis (MS) during natalizumab treatment. All patients with MS from two hospitals with at least one measure of the anti-JCV antibodies levels (2011–2014) were recruited, among them were two PML cases. Anti-JCV antibody levels were assessed using two-step ELISA. A total of 1061 patients (16·3% natalizumab-treated) participated in this study. The seropositivity rate of anti-JCV antibodies was 58·2%. It increased with age (P_corrected = 0·00005) and was lower among HLA-DRB1*15:01 carriers (P_corrected = 0·049). The two patients with PML were HLA-DRB1*15:01 carriers. We had at least three quarterly anti-JCV antibody measurements (index value) from 137 patients, whose levels did not increase during natalizumab treatment. However, 5·8% of these patients had an increase of the index value higher of one point in a maximum of 6 months, something that was more frequently observed (P = 0·054) among patients treated with immunosuppressant prior to natalizumab onset. Old age and HLA-DRB1*15:01 were the factors that influence positively and negatively, respectively, our anti-JCV antibody prevalence, although our both PML cases were HLA-DRB1*15:01carriers. Most of our patients showed a stable anti-JCV antibody index values during natalizumab treatment.

Objective To assess the effectiveness and safety of fingolimod use in a Spanish clinical practice setting. Methods Retrospective study with multiple sclerosis patients who received at least 1 fingolimod dose between January 2004 and January 2015. Effectiveness and safety data were collected during the entire treatment of each patient. Analysis was performed for the total population and stratified according to prior treatment, sex, and age at treatment initiation. Results A total of 167 patients were included, 50.9% had prior immunomodulator use, 33.5% natalizumab use, and 15.6% were naive patients. The annual relapse rate (ARR) decreased for the total population at month 12 (62%) and month 24 (84%) (P < 0.0001, in both cases); for naive patients (P < 0.05) and patients with prior immunomodulator use (P < 0.0001); for patients with prior natalizumab use, the ARR kept low after treatment initiation (0.23). After 24 months, the proportion of relapse-free patients was 70% or greater and disability progression-free patients was 80% or greater. No significant differences were observed when the results were compared by prior treatment, sex, or age. Thirty-two patients (19.2%) reported adverse drug reactions and 9.6% discontinued: 4.8% due to adverse drug reactions and 4.8% for lack of effectiveness. Conclusions The results support fingolimod use due to clinical effectiveness, tolerability, and ease of administration.