Magdalena Niegowska

Natalizumab Therapy Modulates miR-155, miR-26a and Proinflammatory Cytokine Expression in MS Patients

MicroRNAs fine-tune the regulation of Th1/Th17 lymphocyte subsets in multiple sclerosis. We investigated the expression of miRNAs (previously associated with mycobacterial and viral infections) in MS patients and healthy donors (HD) following 6 months natalizumab therapy. In addition, Th1/Th17 cytokines and the presence of anti-EBNA1/VCA IgG in MS patients with different pattern of miRNA expression have been evaluated. MiR-155, miR-26a, miR-132, miR-146a and Th1/Th17 cytokines expression was detected by RT-real time PCR; moreover anti-EBNA1 and VCA IgG titres were measured by ELISA. We observed an up-regulation of miR-155 (p value = 0.009) and miR-132 (p value = 0.04) in MS patients compared to HD. In MS patients, IL-17a (p = 0.037), IFN γ (p = 0.012) and TNFα (p = 0.015) but not IL-6 were over-expressed compared to HD. Two different miRNAs patterns associated to the expression of different cytokines were observed in the MS cohort. Moreover, a down-regulation of miR-155 and miR-26a was seen in MS patients during and after natalizumab therapy. MS patients that over-expressed miR-155 showed a higher EBNA1 IgG titer than MS patients with high levels of miR-26a. In conclusions the expression of particular miRNAs modulates the pro-inflammatory cytokine expression and the humoral response against EBV and this expression is natalizumab regulated.

Type 1 Diabetes at-risk children highly recognize Mycobacterium avium subspecies paratuberculosis epitopes homologous to human Znt8 and Proinsulin

Mycobacterium avium subspecies paratuberculosis (MAP) has been previously associated to T1D as a putative environmental agent triggering or accelerating the disease in Sardinian and Italian populations. Our aim was to investigate the role of MAP in T1D development by evaluating levels of antibodies directed against MAP epitopes and their human homologs corresponding to ZnT8 and proinsulin (PI) in 54 T1D at-risk children from mainland Italy and 42 healthy controls (HCs). A higher prevalence was detected for MAP/ZnT8 pairs (62,96% T1D vs. 7,14% HCs; p < 0.0001) compared to MAP/PI epitopes (22,22% T1D vs. 9,52% HCs) and decreasing trends were observed upon time-point analyses for most peptides. Similarly, classical ZnT8 Abs and GADA decreased in a time-dependent manner, whereas IAA titers increased by 12%. Responses in 0–9 year-old children were stronger than in 10–18 age group (75% vs. 69,1%; p < 0.04). Younger age, female sex and concomitant autoimmune disorders contributed to a stronger seroreactivity suggesting a possible implication of MAP in multiple autoimmune syndrome. Cross-reactivity of the homologous epitopes was reflected by a high correlation coefficient (r2 > 0.8) and a pairwise overlap of positivity (>83% for MAP/ZnT8).

Recognition of ZnT8, Proinsulin, and Homologous MAP Peptides in Sardinian Children at Risk of T1D Precedes Detection of Classical Islet Antibodies

As numerous studies put in evidence the increasing incidence of type 1 diabetes (T1D) in children, an early diagnosis is of great importance to define correct treatment and diet. Currently, the identification of classical islet autoantibodies is the primary biomarker for diagnosis in subjects at risk, especially in pediatric patients. Recent studies suggest that detection of antibodies against ZnT8 protein in preclinical phase can predict the development of T1D. We previously demonstrated a significant association of Mycobacterium avium subspecies paratuberculosis (MAP) with T1D in adult Sardinian patients. To enforce this finding, we investigated the presence of antibodies against ZnT8 and proinsulin (PI) with respective homologous epitopes: MAP3865c133–141/ZnT8186–194, MAP3865c125–133/ZnT8178–186, MAP2404c70–85/PI46–61, and MAP1,4αgbp157–173/PI64–80, in 23 children at risk for T1D, formerly involved in the TRIGR study, and 22 healthy controls (HCs). Positivity to anti-MAP and homologous human peptides was detected in 48% of at-risk subjects compared to 5,85% HCs, preceding appearance of islet autoantibodies. Being MAP easily transmitted to humans with infected cows milk and detected in retail infant formulas, MAP epitopes could be present in extensively hydrolyzed formula and act as antigens stimulating β-cell autoimmunity.

Antibodies against Proinsulin and Homologous MAP Epitopes Are Detectable in Hashimoto's Thyroiditis Sardinian Patients, an Additional Link of Association.

Hashimoto’s thyroiditis (HT) is the prevailing organ-specific autoimmune disease in Sardinia, often complicated with other autoimmune disorders, most commonly type 1 diabetes (T1D). While numerous studies describe levels of anty-thyroid antibodies (Abs) in T1D patients, few papers evaluate the status of anti-islet autoimmunity in subjects affected by HT. Previously, we portrayed Mycobacterium avium subspecies paratuberculosis (MAP) as an environmental factor strongly associated with both diseases. In this study, we analyzed plasma of Sardinian HT patients (n=177) and healthy controls (HCs; n=175) for the presence of Abs against proinsulin and MAP-derived homologous epitopes: MAP1,4αgbp157-173/PI64-80 were recognized by 5,08% and 18,64% of HT vs 0,57% and 7,43% of HCs (AUC=0,6 for both; p<0,0003 and 0,002, respectively), whereas the prevalence of Abs against MAP2404c70-85/PI46-61 peptides was higher but not significant in patients when compared to HCs. In women (n=152), Abs against MAP1,4αgbp157-173 were detected in 12,50% of HT vs 2,75% of HCs (AUC=0,63; p<0,0002), while positivity to its human homolog PI64-80 was observed in 16,42% of HT vs 6,42% of HCs (AUC=0,61; p<0,001). In men (n=25), a significant anti-PI46-61 Abs levels were detected in 4% of HT vs none of the HCs (AUC=0,7; p<0,003). Age-related analyses revealed the highest prevalence between 31-40 years old (45,83%) in the total study population and among males (33,33%); in contrast, women had a higher seroreactivity between 51-60 years (42,11%). A further follow-up and determination of anti-islet Abs levels is needed to evaluate the association of immune responses directed against the MAP/PI homologous peptides with progression to overt diabetes in HT subjects.

Mycobacterium avium subspecies paratuberculosis and myelin basic protein specific epitopes are highly recognized by sera from patients with Neuromyelitis optica spectrum disorder

Epstein-Barr virus (EBV) is the main environmental agent associated to neuromyelitis optica spectrum disorder (NMOSD). Following to studies reporting an increased prevalence of antibodies against peptides derived from Mycobacterium avium subsp. paratuberculosis (MAP) homologous to EBV and human epitopes (MBP85-98, IRF5424-434) in multiple sclerosis (MS), we investigated whether seroreactivity to these antigens display a NMOSD-specific pattern. The sera of 34 NMOSD patients showed elevated levels of antibodies against MAP and MBP compared to healthy controls (44% vs. 5%, p < 0.0002 and 50% vs. 2%, p < 0.0001, respectively), while, unlike in MS, responsiveness to EBV was similar.

Increased seroreactivity to proinsulin and homologous mycobacterial peptides in latent autoimmune diabetes in adults

Latent Autoimmune Diabetes in Adults (LADA) is a slowly progressing form of immune-mediated diabetes that combines phenotypical features of type 2 diabetes (T2D) with the presence of islet cell antigens detected in type 1 diabetes (T1D). Heterogeneous clinical picture have led to the classification of patients based on the levels of antibodies against glutamic acid decarboxylase 65 (GADA) that correlate with clinical phenotypes closer to T1D or T2D when GADA titers are high or low, respectively. To date, LADA etiology remains elusive despite numerous studies investigating on genetic predisposition and environmental risk factors. To our knowledge, this is the first study aimed at evaluation of a putative role played by Mycobacterium avium subsp. paratuberculosis (MAP) as an infective agent in LADA pathogenesis. MAP is known to cause chronic enteritis in ruminants and has been associated with autoimmune disorders in humans. We analyzed seroreactivity of 223 Sardinian LADA subjects and 182 healthy volunteers against MAP-derived peptides and their human homologs of proinsulin and zinc transporter 8 protein. A significantly elevated positivity for MAP/proinsulin was detected among patients, with the highest prevalence in the 32-41-year-old T1D-like LADA subgroup, supporting our hypothesis of a possible MAP contribution in the development of autoimmunity.

Epstein–Barr virus infection is associated to patients with multiple myeloma and monoclonal gammopathy of undetermined significance

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant disorder associated with a progression to multiple myeloma (MM). MM is a plasma cell malignancy with a chronic lymphoproliferative disorder. Epstein–Barr virus (EBV) has been associated with different types of lymphoma. The aim of this study was to evaluate whether there is an association between MM, MGUS, and EBV in patients by serological and molecular methods. The results showed only a serological IgG response against EBNA-1 in MGUS and MM patients, not against viral capsid antigen (VCA) EBV antigen. Molecular analyses display a significant EBV DNA in premalignant and malignant plasma cell disorders; in particularly we observed an overexpression of latent EBV LMP2A gene in MM and MGUS patients. These data display a possible contribute of EBV, during viral latent cell cycle, to MM cell survival through a possible deregulation of RAS pathway induced by a latent membrane protein 2A (LMP2A) of EBV. MM is a plasma cell malignancy which develops in the bone marrow and, when symptomatic, is clinically characterized by renal failure, immunosuppression with repeated infections, cytopenia, and bone lesions. The pathogenesis of this disorder is dominated by a variety of genomic abnormalities often related to genetic instability but also the complex interactions between malignant plasma cells and their microenvironment play a fundamental role.[1] MGUS is a premalignant plasma cell disorder associated with a 1% per year risk of progression to MM. It is defined by a serum monoclonal protein <30 g/L, <10% clonal plasma cells in the bone marrow and the absence of endorgan damage that can be attributed to the plasma cell proliferative disorder.[2] The potential role for EBV in lymphomagenesis was first advocated in the ‘African’ form of Burkitt lymphoma. Since then EBV has been also associated with the development of other lymphomas, including classical Hodgkin lymphoma, diffuse large B-cell lymphoma and natural killer/T-cell lymphoma.[3] Much less is known about the possible role of EBV in the disease history of patients with MM. In fact, apart from few case reports describing the onset of possibly EBV-related plasmacytomas in strongly immunodeficient patients,[4–9] a single study has tried to systematically address this scenario. In paraffin-embedded bone marrow biopsies DNA of EBV was detected slightly more frequently in MM patients than in age-matched controls.[10] In the present study, the possible role of EBV in the pathogenesis of MM was extensively evaluated by serological and molecular analysis in patients with MM and MGUS. A case–control study was performed on samples from 34 MM patients (16 males and 18 females, mean age 65.5 ± 7.11), 25 subjects with MGUS (12 were males and 13 females; mean age 67.0 ± 8.17) and 25 age and sex matched healthy controls (HCs; 14 males and 11 females; mean age 64.6 ± 7.5). The diagnosis of MM and MGUS was performed according to international guidelines.[2] Among MM patients according to Durie and Salmon classification 9%, 15%, and 76% were in Stage I, II, and III respectively while 74% and 26% were in Stage A and B. According to the International Scoring System 15%, 47%, and 38% were in Group I, II, and III, respectively. 60%, 32%, 4%, and 4% of the subjects were characterized by IgG-kappa, IgG-lambda, IgM-kappa, and IgA-lambda, respectively. All patients and donors had given informed consent. The study had been approved by the Local Ethics Committee. Presence and levels of IgG antibodies against EBV antigen-specific EBNA-1 and VCA were evaluated in sera using commercially available kit anti-EBV EBNA and VCA IgG ELISA (Bio-Rad, ref. 807017 and 807019). Detection of EBV genome in peripheral blood mononuclear cells (PBMCs) derived from samples previously analyzed for EBNA-1 and VCA antibodies response was attained by selective amplification of the qualitative and quantitative EBV DNA

Seroreactivity against Specific L5P Antigen from Mycobacterium avium subsp. paratuberculosis in Children at Risk for T1D

Aims/Hypothesis Although numerous environmental agents have been investigated over the years as possible triggers of type 1 diabetes (T1D), its causes remain unclear. We have already demonstrated an increased prevalence of antibodies against peptides derived from Mycobacterium avuim subsp. paratuberculosis (MAP) homologous to human zinc transporter 8 protein (ZnT8) and proinsulin in Italian subjects at risk for or affected by T1D. In this study, we compared titers of the previously detected antibodies with seroreactivity to MAP lipopentapetide (L5P) that recently emerged as a strong immunogenic component able to specifically distinguish MAP from other mycobacteria. Methods Plasma of 32 children and youth at risk for T1D including follow-up samples and 42 age-matched healthy controls (HC) recruited at the Tor Vergata University Hospital in Rome was analyzed by indirect ELISA for the presence of antibodies against MAP-derived epitopes MAP3865c133–141, MAP3865c125-133, MAP2404c70-85 and MAP1,4αgbp157-173 along with their ZnT8 and proinsulin homologs. The data were analyzed through two-tailed Mann-Whitney U test and relation between variables was determined by principal component analysis. Results Responses to L5P were not detectable in subjects whose initial seroreactivity to MAP peptides and their human homologs was lost in follow-up samples, whereas anti-L5P antibodies appeared constantly in individuals with a stable immunity against MAP antigens. The overall coincidence in positivity to L5P and the four MAP epitopes both in children at risk for T1D and HC exceeded 90%. Conclusions MAP-derived homologs may cross-react with ZnT8 and proinsulin peptides inducing immune responses at a young age in subjects predisposed for T1D. Thus, L5P may have a diagnostic value to immediately indicate the presence of anti-MAP seroreactivity when evaluation of a more complex antibody status is not required. Almost complete coincidence in responses to both types of antigens lends support to the involvement of MAP in T1D.

Rheumatoid arthritis patient antibodies highly recognize IL-2 in the immune response pathway involving IRF5 and EBV antigens

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by a progressive joint damage due to largely unknown environmental factors acting in concert with risk alleles conferring genetic susceptibility. A major role has been attributed to viral infections that include past contacts with Epstein-Barr virus (EBV) and, more recently, to non-protein coding sequences of human endogenous retrovirus K (HERV-K) integrated in the human genome. Molecular mimicry between viral and self proteins is supposed to cause the loss of immune tolerance in predisposed hosts. There are evidences that anti-IL-2 antibodies (Abs) are present in subjects affected by autoimmune diseases and may be responsible for alterations in regulatory T cell responses. In this study, we evaluated the levels of Abs against IL-2, viral epitopes and interferon regulatory factor 5 (IRF5) in 140 RA patients and 137 healthy controls (HCs). Ab reactivity reached the highest levels for IRF5, EBV and IL-2 (56%, 44% and 39%, respectively) in RA with significantly lower values among HCs (7–9%, p < 0.0001), which suggests a possible cross-reaction between IRF5/EBV homologous antigens and shifts in T cell balance disrupted by anti-IL-2 Abs.

Soluble BAFF Level Is Not Correlated to Mycobacterium avium Subspecies Paratuberculosis Antibodies and Increases After Interferon-β Therapy in Multiple Sclerosis Patients

B cells are being recognized as one of the major players in the pathogenesis of multiple sclerosis (MS). The B cell activating factor (BAFF) system plays an essential role in B cell homeostasis and function in the periphery. Mycobacterium avium subspecies paratuberculosis (MAP) has been previously associated to MS in Sardinia. Antibodies against a MAP surface protein, MAP_2694, have been found significantly associated to MS patients, and this response was modified by interferon-β therapy. Increased BAFF levels following IFN-β therapy have been also described in MS patients. In this study, we evaluated whether soluble BAFF levels are comparable in men and women affected by MS and performed a correlation of the reported BAFF increase in MS patients under IFN-β therapy with changes of humoral response against MAP_2694. For these reasons, we investigated 44 MS patients before and after IFN-β therapy. A significant difference of BAFF levels was found between men and women with MS; moreover, we confirmed that IFN-β therapy strongly induces BAFF serum levels, but this was not related to the modification of immunological response against MAP_2694. In conclusion, our study highlights that IFN-β therapy induces the potent B cell survival factor BAFF without alterations of the humoral immune response against MAP.