Transfection of Vitamin D3-Induced Tolerogenic Dendritic Cells for the Silencing of Potential Tolerogenic Genes. Identification of CSF1R-CSF1 Signaling as a Glycolytic Regulator.

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising strategy to re-establish immune tolerance in autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (VitD3-tolDC) has been widely tested because of their immune regulatory properties. To identify molecules and pathways involved in the generation of VitD3-tolDC, we established an easy and fast gene silencing method based on the use of Viromer blue to introduce siRNA into monocytes on day 1 of culture differentiation. The analysis of the effect of CD209 (DC-SIGN) and CD115 (CSF1R) down-modulation on the phenotype and functionality of transfected VitD3-tolDC revealed a partial role of CD115 in their tolerogenicity. Further investigations showed that CSF1R-CSF1 signaling is involved in the induction of cell metabolic reprogramming, triggering glycolysis to produce high amounts of lactate, a novel suppressive mechanism of T cell proliferation, recently found in autologous tolerogenic dendritic cells (ATDCs).

Immunological Pattern in IgA Nephropathy.

The current gold-standard diagnostic technique for IgA nephropathy (IgAN), the leading form of primary glomerulonephritis, is renal biopsy. CD89 (the main IgA receptor) is expressed on the surface of monocytes and plays a role in disease pathogenesis. Immunocomplexes formed by sCD89 (soluble form) and Gd-IgA1 are related to disease prognosis. We hypothesize that reduced CD89 surface expression on monocytes may be a marker of disease severity. We aimed to analyze leukocyte subpopulations in peripheral blood and CD89 surface expression on monocytes in a prospective study of 22 patients and 12 healthy subjects (HS). Leukocyte subpopulations and CD89 expression were analyzed by flow cytometry. IgAN patients had a higher percentage of activated and effector memory CD4+ and CD8+ T lymphocytes, a lower percentage of transitional B lymphocytes and plasmablasts, and a higher percentage of CD56dimCD16+ NK cells and myeloid dendritic cells compared with HS. Correlations between reduced CD89 expression levels on nonclassical monocytes, histological findings of a poor prognosis on renal biopsy and baseline renal function were observed. IgAN patients show a characteristic immunological pattern in peripheral blood. A reduced expression level of CD89 on nonclassical monocytes identifies patients with a worse renal prognosis.

Th1Th17CM Lymphocyte Subpopulation as a Predictive Biomarker of Disease Activity in Multiple Sclerosis Patients under Dimethyl Fumarate or Fingolimod Treatment.

Peripheral blood biomarkers able to predict disease activity in multiple sclerosis (MS) patients have not been identified yet. Here, we analyzed the immune phenotype of T lymphocyte subpopulations in peripheral blood samples from 66 RRMS patients under DMF (n = 22) or fingolimod (n = 44) treatment, by flow cytometry. A correlation study between the percentage and absolute cell number of each lymphocyte subpopulation with the presence of relapses or new MRI lesions during 12-month follow-up was performed. Patients who had undergone relapses showed at baseline higher percentage of Th1CM cells (relapsed: 11.60 ± 4.17%vs. nonrelapsed: 9.25 ± 3.17%, p < 0.05) and Th1Th17CM cells (relapsed: 15.65 ± 6.15%vs. nonrelapsed: 10.14 ± 4.05%, p < 0.01) before initiating DMF or fingolimod treatment. Kaplan-Meier analysis revealed that patients with Th1Th17CM (CD4+CCR7+CD45RA-CCR6+CXCR3+) cells > 11.48% had a 50% relapse-free survival compared to patients with Th1Th17CMcells < 11.48% whose relapse-free survival was 88% (p = 0.013, log-rank test). Additionally, a high percentage of Th1Th17CM cells was also found in patients with MRI activity (MRI activity: 14.02 ± 5.87%vs. no MRI activity: 9.82 ± 4.06%, p < 0.01). Our results suggest that the percentage of Th1Th17CM lymphocytes at baseline is a predictive biomarker of activity during the first 12 months of treatment, regardless of the treatment.

HLA-DR Expression on Monocytes and Sepsis Index Are Useful in Predicting Sepsis.

The reduction of mortality in patients with sepsis depends on the early identification and treatment of at-risk patients. The aim was to evaluate the HLA-DR expression on the surface of monocytes (MHLA-DR ratio), the sepsis index (CD64 expression on neutrophils/MHLA-DR ratio), and C-reactive protein (CRP) with the development of sepsis. We prospectively enrolled 77 critically ill patients, 59 with stroke and 18 with traumatic brain injuries. The biomarkers were tested at the baseline and 3, 6, 9, 12, and 15 days later. Most patients (71%) developed sepsis (4.2 ± 1.3 days after admission). On day 3, those subsequently developing sepsis had lower levels of MHLA-DR+ (81.7 ± 16.2% vs. 88.5 ± 12.1%, p < 0.05) and higher sepsis indexes (0.19 ± 0.19 vs. 0.08 ± 0.08, p < 0.01) than those not developing sepsis. The MHLA-DR ratio slowly recovered before day 6, while the sepsis index remained raised in septic patients up to day 9 (p < 0.05). To predict the development of sepsis, optimal cut-offs were CRP levels > 106.90 mg/mL (74.19% sensitivity, 69.49 specificity) and MHLA-DR expression rate < 72.80% (45.31% sensitivity, 89.47% specificity). The periodic monitoring of the MHLA-DR expression together with CRP and sepsis index may help to identify patients in the ICU at increased risk of developing sepsis.

Seroprevalence of SARS-CoV-2 in Patients with Multiple Sclerosis under Disease-Modifying Therapies: A Multi-Centre Study.

BACKGROUND: The EMCOVID project conducted a multi-centre cohort study to investigate the impact of COVID-19 on patients with Multiple Sclerosis (pwMS) receiving disease-modifying therapies (DMTs). The study aimed to evaluate the seroprevalence and persistence of SARS-CoV-2 antibodies in MS patients enrolled in the EMCOVID database. The DMTs were used to manage MS by reducing relapses, lesion accumulation, and disability progression. However, concerns arose regarding the susceptibility of pwMS to COVID-19 due to potential interactions between SARS-CoV-2 and the immune system, as well as the immunomodulatory effects of DMTs. METHODS: This prospective observational study utilized data from a Multiple Sclerosis and COVID-19 (EMCOVID-19) study. Demographic characteristics, MS history, laboratory data, SARS-CoV-2 serology, and symptoms of COVID-19 were extracted for pwMS receiving any type of DMT. The relationship between demographics, MS phenotype, DMTs, and COVID-19 was evaluated. The evolution of SARS-CoV-2 antibodies over a 6-month period was also assessed. RESULTS: The study included 709 pwMS, with 376 patients providing samples at the 6-month follow-up visit. The seroprevalence of SARS-CoV-2 antibodies was higher among pwMS than the general population, with Interferon treatment being significantly associated with greater seroprevalence (16.9% vs. 8.4%; p 0.003). However, no other specific DMT showed a significant association with antibody presence. A total of 32 patients (8.5%) tested positive for IgG, IgM, or IgA antibodies against SARS-CoV-2 at baseline, but then tested negative at 6 months. Most of the pwMS in the cohort were asymptomatic for COVID-19 and, even among symptomatic cases, the prognosis was generally favourable. CONCLUSION: pwMS undergoing DMTs exhibited a higher seroprevalence of COVID-19 than the general population. Interferon treatment was associated with a higher seroprevalence, suggesting a more robust humoral response. This study provides valuable insights into the seroprevalence and persistence of SARS-CoV-2 antibodies in pwMS and contributes to our understanding of the impact of COVID-19 amongst this population.

Increased Natural Killer Cells Are Associated with Alcohol Liver Fibrosis and with T Cell and Cytotoxic Subpopulations Change.

Natural killer (NK) cells play a therapeutic role in liver fibrosis (LF). We aimed to analyze NK cells in heavy drinkers without cirrhosis or decompensated liver disease and establish correlations with other related subpopulations. Data on sociodemographic characteristics, alcohol consumption, laboratory parameters, and immunophenotyping of NK (CD16+/CD56+), T (CD3+), B (CD19+), NKT (CD16+/CD56+/CD3+), and cytotoxic (CD3-CD8+) cells were collected. Fibrosis-4 (FIB-4) scores were used to compare patients without (FIB-4 < 1.45) and with (FIB-4 > 3.25) advanced LF (ALF). We included 136 patients (76% male) with a mean age of 49 years who had a 15-year alcohol use disorder (AUD) and alcohol consumption of 164 g/day. Patients with ALF (n = 25) presented significantly lower absolute total lymphocyte, T cell, B cell, and NKT cell numbers than patients without LF (n = 50; p < 0.01). However, the NK cells count was similar (208 ± 109 cells/µL vs. 170 ± 105 cells/µL) in both groups. The T cells percentage was lower (80.3 ± 5.6% vs. 77 ± 7%; p = 0.03) and the NK cells percentage was higher (9.7 ± 5% vs. 13 ± 6%; p = 0.02) in patients with ALF than in those without LF. The percentages of NK cells and T cells were inversely correlated in patients without (r = -0.65, p < 0.01) and with ALF (r = -0.64; p < 0.01). Additionally, the NK cells and CD3-CD8+ cell percentages were positively correlated in patients without (r = 0.87, p < 0.01) and with (r = 0.92; p < 0.01) ALF. Conclusions: Heavy drinkers without decompensated liver disease showed an increase in NK cells related to T cells lymphopenia and an increase in cytotoxic populations. The interaction of NK cells with other subpopulations may modify alcohol-related liver disease progression.

Follow up of the Humoral Response in Healthcare Workers after the Administration of Two Dose of the Anti SARS-CoV-2 Vaccines-Effectiveness in Delta Variant Breakthrough Infections.

The implementation of vaccination among healthcare workers (HCWs) allowed the management of the pandemic in a manner that differed from that in the first waves. It has been demonstrated that the mRNA vaccines elicit good humoral responses but that there are still breakthrough infections. In summer 2021, a fifth wave emerged, despite the good coverage of HCWs in Spain. We aimed to study the SARS-CoV-2 IgG antibody levels as a marker to predict the possibility of Delta variant infections after vaccination after a seroepidemiological campaign. Of the 5000 participants, a total of 4902 (98.04%) showed a positive result in the serological anti-S test and only 98 (1.96%) were negative. Among the 4368 fully vaccinated participants, only in five cases was the serology negative. Of the total number of participants that received antibody results during the study, 162 were PCR positive in the subsequent two months. Among these, 151 were fully vaccinated (two doses). Significant differences between antibody BAU/mL levels were found between PCR positive and non-PCR positive participants (p < 0.01). The median of BAU/mL was higher in those vaccinated patients with no infection (1260 BAU/mL; 465−2080) versus infected patients (661 BAU/mL; 361−2080). These data support the idea that vaccines play an important role in the control of the pandemic, especially among HCWs at the time of the Delta variant circulation. More studies with other variants of concern must be performed in order to establish a correlation between the levels of IgG and the new infections.

Skewed Cellular Distribution and Low Activation of Functional T-Cell Responses in SARS-CoV-2 Non-Seroconvertors.

The role of T cells in the control of SARS-CoV-2 infection has been underestimated in favor of neutralizing antibodies. However, cellular immunity is essential for long-term viral control and protection from disease severity. To understand T-cell immunity in the absence of antibody generation we focused on a group of SARS-CoV-2 Non-Seroconvertors (NSC) recovered from infection. We performed an immune comparative analysis of SARS-CoV-2 infected individuals stratified by the absence or presence of seroconversion and disease severity. We report high levels of total naïve and low effector CD8+ T cells in NSC. Moreover, reduced levels of T-cell activation monitored by PD-1 and activation-induced markers were observed in the context of functional SARS-CoV-2 T-cell responses. Longitudinal data indicate the stability of the NSC phenotype over three months of follow-up after infection. Together, these data characterized distinctive immunological traits in NSC including skewed cellular distribution, low activation and functional SARS-CoV-2 T-cell responses. This data highlights the value of T-cell immune monitoring in populations with low seroconversion rates in response to SARS-CoV-2 infection and vaccination.

Seroprevalence of SARS-CoV-2 in a Cohort of Patients with Multiple Sclerosis under Disease-Modifying Therapies.

Background: Disease-modifying therapies (DMTs) used to treat multiple sclerosis (MS) alter the immune system and therefore increase the risk of infection. There is growing concern about the impact of COVID-19 on patients with MS (pwMS), especially those treated with DMTs. Methods: This is a single-center prospective observational study based on data from the Esclerosis Múltiple y COVID-19 (EMCOVID-19) study. Demographic characteristics, MS history, laboratory data and SARS-CoV-2 serology, and symptoms of COVID-19 in pwMS treated with any DTM were extracted. The relationship among demographics, MS status, DMT, and COVID-19 was evaluated. Results: A total of 259 pwMS were included. The administration of interferon was significantly associated with the presence of SARS-CoV-2 antibodies (26.4% vs. 10.7%, p = 0.006). Although patients taking interferon were significantly older (49.1 vs. 43.5, p = 0.003), the association of interferon with the presence of SARS-CoV-2 antibodies was still significant in the multivariate analysis (OR 2.99 (1.38; 6.36), p = 0.006). Conclusions: According to our data, pwMS present a higher risk of COVID-19 infection compared with results obtained from the general population. There is no evidence of a worse COVID-19 outcome in pwMS. DMTs did not significantly change the frequency of COVID-19, except for interferon; however, these findings must be interpreted with caution given the small sample of pwMS taking each DMT.

Identifying Changes in Peripheral Lymphocyte Subpopulations in Adult Onset Type 1 Diabetes.

T- and B-lymphocytes play an important role in the pathogenesis of type 1 diabetes (T1D), a chronic disease caused by the autoimmune destruction of the insulin-producing cells in the pancreatic islets. Flow cytometry allows their characterization in peripheral blood, letting to investigate changes in cellular subpopulations that can provide insights in T1D pathophysiology. With this purpose, CD4+ and CD8+ T cells (including naïve, central memory, effector memory and terminally differentiated effector (TEMRA), Th17 and Tregs) and B cells subsets (naïve, unswitched memory, switched memory and transitional B cells) were analysed in peripheral blood of adult T1D patients at disease onset and after ≥2 years using multiparametric flow cytometry. Here we report changes in the percentage of early and late effector memory CD4+ and CD8+ T cells as well as of naïve subsets, regulatory T cells and transitional B cells in peripheral blood of adult patients at onset of T1D when compared with HD. After 2 years follow-up these changes were maintained. Also, we found a decrease in percentage of Th17 and numbers of T cells with baseline. In order to identify potential biomarkers of disease, ROC curves were performed being late EM CD4 T cell subset the most promising candidate. In conclusion, the observed changes in the percentage and/or absolute number of lymphocyte subpopulations of adult T1D patients support the hypothesis that effector cells migrate to the pancreas and this autoimmune process perseveres along the disease. Moreover, multiparametric flow allows to identify those subsets with potential to be considered biomarkers of disease.

Combined Therapy of Vitamin D3-Tolerogenic Dendritic Cells and Interferon-ß in a Preclinical Model of Multiple Sclerosis.

Autologous antigen-specific therapies based on tolerogenic dendritic cells (tolDC) offer the possibility to treat autoimmune diseases by restoring homeostasis and targeting specifically autoreactive responses. Here, we explore the hypothesis that systemic inflammation occurring in autoimmune diseases, such as multiple sclerosis (MS), can generate a disease-specific environment able to alter the functionality of tolDC. In this context in fact, a combined therapy of tolDC with an immunomodulatory treatment could potentiate the beneficial effect of this antigen-specific cell therapy. For this purpose, we analyzed the efficacy of a combined therapy based on the use of vitamin D3 (VitD3)-tolDC plus interferon beta (IFN-beta) in MS. VitD3-tolDC were generated from healthy donors and MS patients and co-cultured with allogeneic peripheral blood mononuclear cells, in the presence or absence of IFN-beta. In vitro, VitD3-tolDC treatment reduced the percentage of activated T cells and allogeneic proliferation, whereas VitD3-tolDC+IFN-beta treatment enhanced the suppressive ability of VitD3-tolDC and, additionally, induced a shift towards a Th2 profile. To determine the clinical benefit of the combined therapy, C57BL/6-experimental autoimmune encephalomyelitis (EAE)-induced mice were treated with antigen-specific VitD3-tolDC and/or IFN-beta. Treatment of EAE mice with combined therapy ameliorated the disease course compared to each monotherapy. These results suggest that a combined therapy based on antigen-specific VitD3-tolDC and IFN-beta may represent a promising strategy for MS patients.

Detection of Autoantibodies in Bronchoalveolar Lavage in Patients with Diffuse Interstitial Lung Disease. / Detección de autoanticuerpos en el lavado broncoalveolar en pacientes con enfermedad pulmonar intersticial difusa.

INTRODUCTION: Serum autoantibodies support the diagnosis of interstitial lung disease (ILD) related to systemic autoimmune diseases (SAD-ILD). Nevertheless, their presence in the bronchoalveolar lavage (BAL) has not been explored. OBJECTIVES: To demonstrate the presence of autoantibodies in the BAL of ILD patients at onset of clinical evaluation, its relation with serum autoantibodies and to analyze clinical features of patients with autoantibodies in BAL. METHODS: Autoantibodies against extractable nuclear antigens (ENAs) were analyzed by immunoblot in the BAL of 155 patient with suspected diagnosis of ILD and 10 controls. RESULTS: Seven ENAs were detected in the BAL of 19 patients (Anti-Ro52, Anti-Ro60, CENP-B, Anti-La, Jo-1, Sm/RNP and Anti-SL70). The most frequent ENA was anti-Ro52 (13 patients; 68,4% of positives ones). Seven patients presented more than one ENAs. Fourteen were diagnosed of SAD-ILD, 3 of interstitial pneumonia with autoimmune features, one of non-specific idiopathic pneumonia and other of silicosis. In 10 cases (52%) IgA autoantibodies were also detected. The autoantibodies observed in BAL were also detected in the serum of 17 patients (90%). There were no significant clinical differences with the patients with SAD-ILD or interstitial pneumonia with autoimmune features with patients with negative BAL. CONCLUSION: The study of ENAs in BAL is feasible and can be a useful tool in the ILD initial algorithm, specifically sustaining the suspected diagnosis of SAD-ILD.

Vitamin D3-Induced Tolerogenic Dendritic Cells Modulate the Transcriptomic Profile of T CD4+ Cells Towards a Functional Hyporesponsiveness.

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most robust approaches due to their immune regulatory properties, which are currently being tested in clinical trials. However, the mechanisms that vitD3-tolDC trigger for the induction of tolerance remain elusive. For this reason, we performed a full phenotypical, functional, and transcriptomic characterization of T cells upon their interaction with autologous, antigen-specific vitD3-tolDC. We observed a strong antigen-specific reduction of T cell proliferation, combined with a decrease in the relative prevalence of TH1 subpopulations and IFN-γ production. The analysis of the transcriptomic profile of T CD4+ cells evidenced a significant down-modulation of genes involved in cell cycle and cell response to mainly pro-inflammatory immune-related stimuli, highlighting the role of JUNB gene as a potential biomarker of these processes. Consequently, our results show the induction of a strong antigen-specific hyporesponsiveness combined with a reduction on the TH1 immune profile of T cells upon their interaction with vitD3-tolDC, which manifests the regulatory properties of these cells and, therefore, their therapeutic potential in the clinic.

Loss of naive T lymphocytes is associated with advanced liver fibrosis in alcohol use disorder.

BACKGROUND: Alcohol use disorder (AUD) is associated with changes in cellular immunity. The objective of the present study was to analyze the contribution of AUD to the differentiation of T cells and associations with advanced liver fibrosis (ALF). METHODS: This cross-sectional study included patients admitted for treatment of AUD between 2013 and 2016. T cell immune-phenotyping defined four profiles of cellular differentiation according to the expression of CCR7 and CD45RA: naive T cells, central memory (TCM) cells, effector memory (TEM) cells, and terminal effector (TEMRA) cells. CD4+ memory cells were subdivided into Th1, Th2, and Th17 according to the expression of CXCR3 and CCR6. The stages of cellular differentiation were compared to healthy controls. ALF was defined as FIB-4 > 3.25. RESULTS: Seventy-nine patients (81% men) with a median age of 50 years (IQR: 45-56 years) and median ethanol consumption of 150 g/day (IQR: 100-200 g/day) were included in the study. Compared to healthy controls, patients with AUD had fewer CD4+ naive cells (p < 0.001), more TCM and TEM cells (p = 0.003 and p = 0.050, respectively), and larger Th2 populations (p = 0.03). Among CD8+ cells, the percentage of TCM, TEM, and TEMRA were higher in patients with AUD than in the healthy controls (p < 0.05). Patients with ALF had fewer CD4+ and CD8+ naive cells (p < 0.05) and more CD4+ memory cells than patients without ALF. CONCLUSIONS: Altered lymphocyte differentiation in AUD patients suggests immunosenescence. An increase in memory cells and decrease in naive cells is associated with ALF.

Seroprevalence of SARS-CoV-2 IgG specific antibodies among healthcare workers in the Northern Metropolitan Area of Barcelona, Spain, after the first pandemic wave.

BACKGROUND: The rapid spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) around the world has caused a global pandemic, infecting millions of individuals, with an unprecedented impact in health care systems worldwide. Healthcare workers are one of the risk groups that need to be well protected, due to their strategic role in patient management, presently and in prevention of healthcare needs for future outbreaks. Here, we present the results of the first SARS-CoV-2 seroprevalence study in the Northern Metropolitan Area of Barcelona, Spain. METHODS: IgG SARS-CoV-2 antibodies were analyzed in serum samples from 7563 healthcare workers of the Northern Metropolitan Area of Barcelona. Samples were collected after the first pandemic wave (from May 4th to May 22nd, 2020) and were analyzed by automated chemiluminescence assays. All samples were tested for IgG anti-S1/S2. Participant samples with negative or equivocal results but with analytical signals above the limit of detection and/or previously confirmed COVID-19 diagnosis were also tested for IgG anti-Nucleocapsid. RESULTS: A total of 779 of 7563 (10.3%) healthcare workers were positive for anti-SARS-CoV-2 IgG (specific for either S1/S2 or N antigens). No significant differences were observed between those working at primary care or at the reference hospital. Interestingly, among 341 participants with a confirmed COVID-19 diagnosis, 36 (10.55%) tested negative for SARS-CoV-2 IgG (both S1/S2 and recombinant N antigen). CONCLUSION: Seroprevalence of anti-SARS-CoV-2 IgG in the healthcare workers of the North Metropolitan Area of Barcelona was higher than in the general population in the same geographical area. Safety measures have to be stressed in order to protect these essential workers from future pandemic waves.

MAP7 and MUCL1 Are Biomarkers of Vitamin D3-Induced Tolerogenic Dendritic Cells in Multiple Sclerosis Patients.

The administration of autologous tolerogenic dendritic cells (tolDC) has become a promising alternative for the treatment of autoimmune diseases, such as multiple sclerosis (MS). Specifically, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of the most widely studied approaches, as it has evidenced significant immune regulatory properties, both in vitro and in vivo. In this article, we generated human vitD3-tolDC from monocytes from healthy donors and MS patients, characterized in both cases by a semi-mature phenotype, secretion of IL-10 and inhibition of allogeneic lymphocyte proliferation. Additionally, we studied their transcriptomic profile and selected a number of differentially expressed genes compared to control mature and immature dendritic cells for their analysis. Among them, qPCR results validated CYP24A1, MAP7 and MUCL1 genes as biomarkers of vitD3-tolDC in both healthy donors and MS patients. Furthermore, we constructed a network of protein interactions based on the literature, which manifested that MAP7 and MUCL1 genes are both closely connected between them and involved in immune-related functions. In conclusion, this study evidences that MAP7 and MUCL1 constitute robust and potentially functional biomarkers of the generation of vitD3-tolDC, opening the window for their use as quality controls in clinical trials for MS.

Distinct pattern of peripheral lymphocyte subsets in Graves' disease with persistency of anti-TSHR autoantibodies.

Background: Graves' disease (GD) is characterized by the production of autoantibodies against the TSHR (TRAbs). With long-term treatment, serum concentrations of TRAbs decline but in some patients, despite being clinically stable, TRAbs persist for many years.Objective: To investigate whether GD patients with persistence of TRAbs constitute a subset of patients that could be identified by phenotypic analysis of circulating lymphocytes, suggesting disease heterogeneity.Materials and methods: Peripheral blood lymphocytes (including naïve, memory and effector T and B cells, Th17, regulatory T cells (Treg), recent thymic emigrants (RTEs) and double positive CD4+CD8+ (DP) cells) were analysed by flow cytometry in a cross-sectional study in 25 clinically stable GD patients, five patients at onset of GD disease and 40 healthy donors (HDs).Results: GD patients with persistence of TRAbs showed a lower percentage of Treg and lower absolute numbers of central and effector memory CD8+ T cells than HD. No differences in RTEs were found in peripheral blood from GD patients compared to HD. Stable GD patients had higher percentage of DP cells of effector phenotype than HD.Conclusions: Using extensive phenotypic analysis of lymphocyte subpopulations, it is possible to detect changes that help to identify patients with persistent TSHR antibodies and may contribute to understand why the autoimmune response is maintained.

Partial remission and early stages of pediatric type 1 diabetes display immunoregulatory changes. A pilot study.

Type 1 diabetes (T1D) is a chronic metabolic disease of unknown etiology that results from ß-cell destruction. The onset of the disease, which arises after a long asymptomatic period of autoimmune attack, may be followed by a relapsing and remitting progression, a phenomenon that is most evident during the partial remission phase (PR). This stage lasts for a few months, shows minor requirements of exogenous insulin and could be explained by a recovery of immunological tolerance. This study aims to identify new biomarkers at early stages of pediatric T1D that reflect immunoregulatory changes. To that end, pediatric patients with T1D (n = 52) and age-related control subjects (n = 30) were recruited. Immune response-related molecules and lymphocyte subsets were determined starting at T1D onset and until the second year of progression. Results showed that circulating TGF-ß levels decreased during PR, and that betatrophin concentration was increased in all the considered stages without differing among studied checkpoints. Moreover, an increase of regulatory T, B and NK subsets was found during T1D progression, probably reflecting an attempt to restore self-tolerance. By contrast, a reduction in monocyte levels was observed at the early stages of diabetes. The results reveal significant changes in immunological parameters during the different early stages of T1D in children, which could ultimately serve as potential biomarkers to characterize the progression of T1D.

Optimal response to dimethyl fumarate is mediated by a reduction of Th1-like Th17 cells after 3 months of treatment.

AIM: Dimethyl fumarate (DMF) is one of the most promising therapies for relapsing-remitting multiple sclerosis (RRMS) patients since it has shown immunomodulatory and neuroprotective effects. However, a percentage of RRMS patients do not exhibit an optimal response to DMF. The objective of this study was to identify early biomarkers of treatment response by analyzing changes in peripheral leukocyte subpopulations directly in whole blood samples. METHODS: A longitudinal and prospective study analyzing peripheral blood leukocyte subpopulations in 22 RRMS patients before initiating DMF treatment (baseline) and at 1, 3, 6, and 12 months of follow-up was performed. Differences between no evidence of disease activity (NEDA) and ongoing disease activity (ODA) patients were analyzed. RESULTS: The beneficial effect of DMF was associated with a specific depletion of memory CD4+ and CD8+ T lymphocytes and B cells. Importantly, only NEDA patients showed (a) a shift from a pro- to an antiinflammatory profile, with an increase of Th2 cells and a decrease of Th1-like Th17 lymphocytes; and (b) an increase of regulatory CD56bright NK cells. CONCLUSION: The optimal response to DMF is mediated by a shift to antiinflammatory and immunoregulatory profile, which puts forward Th1-like Th17 lymphocytes as a potential early biomarker of treatment response.