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Epstein–Barr virus and Mycobacterium avium subsp. paratuberculosis (MAP) have been associated to multiple sclerosis (MS). We searched for antibodies against the homologous peptides Epstein–Barr virus nuclear antigen 1 (EBNA1)400–413, MAP_0106c protein (MAP)121–132, and myelin basic protein (MBP)85–98 on a MS Sardinian cohort, showing that these antibodies are highly prevalent among MS patients compared to healthy controls. Competitive assay demonstrated that antibodies recognizing EBNA1400–413 and MAP121–132 cross-react with MBP85–98, possibly through a molecular mimicry mechanism. Indeed, the fact that peptides from different pathogens can be cross-recognized by antibodies targeting self-epitopes supports the hypothesis that EBV and MAP might trigger autoimmunity through a common target.

Introduction: Recent findings propose that Mycobacterium avium subsp. paratuberculosis (MAP) infection could act as risk factor in favoring multiple sclerosis (MS) progression. SLC11A1 is a gene associated with mycobacterial survival in the host and it may be involved in the induction and maintenance of autoimmune disease. Methodology: In this preliminary study, 100 MS patients and 100 healthy controls (HCs) from Sardinia were enrolled. Eight single nucleotide polymorphisms (SNPs) in the SLC11A gene were searched by PCR RFLP - genotyping. IS900 specie specific PCR was undertaken to search for MAP presence. Indirect ELISA was performed to asses if MS patients displayed a stronger humoral response against MAP2694 protein compared to the HCs. Results: Only rs2276631 SNP was associated with MS. MAP DNA was detected in 23 out of 100 MS patients (23%) and in 7 out of 100 HCs (7%). A strong humoral response against MAP2694 protein was detected in 36% of MS patients and only in 3% of HCs. A correlation between ELISA sero-positivity and the rs2276631 SNP was also found. Conclusion: Our preliminary results suggest that the Sardinian population might be prone to develop autoimmune disease due to polymorphisms in immunomodulating the SLC11A1 gene, which is important in the immune response against intracellular bacteria such as MAP.

Sardinia acts as an ideal setting for multiple sclerosis (MS) studies because its prevalence of MS is one of the highest worldwide. Several pathogens have been investigated amongst 119 Sardinian MS patients and 117 healthy controls to determine whether they might have a role in triggering MS in genetically predisposed individuals. Mycobacterium avium subsp. paratuberculosis (MAP) and Epstein Barr virus DNA were detected in 27.5% and 17.3%, respectively, of the MS patients. Moreover an extremely high humoral immune response against MAP recombinant protein MAP FprB (homologous to human myelin P0) was observed, whereas no significant results were found against Mycobacterium tuberculosis FprA and Helicobacter pylori HP986 protein.

Mycobacterium avium ss. paratuberculosis (MAP) is an intracellular pathogen recently associated with multiple sclerosis (MS). Aiming to identify immunodominant epitopes belonging to MS related protein MAP2694 (UniProt accession no. Q73WG6), we investigated the binding activity of selected peptides against MS Sardinian sera. An overlapping 9-mers peptide library was synthesized spanning the entire aminoacidic sequence of the protein. Peripheral blood was collected from 47 MS patients and 42 sex and age matched healthy volunteers and subjected to enzyme-linked immunosorbent assay (ELISA) in order to investigate the reaction against the linear peptides generated. Two out of 58 synthetic 9-mers were strongly recognized by MS patients' antibodies compared to controls. A competitive inhibition assay demonstrated that these two epitopes are immunodominant antibody targets within MAP2694 protein, as sera pre-adsorbed with these peptides were able to significantly block the antibody reaction to the MAP2694 protein, even if at a lesser extent than MAP2694 protein itself.

Over recent years, there has been an increasing acknowledgment of the diversity that exists among Mycobacterium tuberculosis clinical isolates. To facilitate comparative studies aimed at deciphering the relevance of this diversity to human disease, an unambiguous and easily interpretable method of strain classification is required. Presently, the most effective means of assigning isolates into a series of unambiguous lineages is the method of Gagneux et al. (S. Gagneux et al., Proc. Natl. Acad. Sci. USA 103:2869-2873, 2006) that involves the PCR-based detection of large sequence polymorphisms (LSPs). In this manner, isolates are classified into six major lineages, the majority of which display a high degree of geographic restriction. Here we describe an independent replicate of the Gagneux study carried out on 798 isolates collected over a 6-year period from mostly foreign-born patients resident on the island of Montreal, Canada. The original trends in terms of bacterial genotype and patient ethnicity are remarkably conserved within this Montreal cohort, even though the patient distributions between the two populations are quite distinct. In parallel with the LSP analysis, we also demonstrate that “clustered” tuberculosis (TB) cases defined through restriction fragment length polymorphism (RFLP) analysis (for isolates with ≥6 IS 6110 copies) or RFLP in combination with spoligotyping (for isolates with 6110 copies) do not stray across the LSP-defined lineage boundaries. However, our data also demonstrate the poor discriminatory power of either RFLP or spoligotyping alone for these low-IS 6110 -copy-number isolates. We believe that this independent validation of the LSP method should encourage researchers to adopt this system in investigations aimed at elucidating the role of strain variation in TB.

Multiple sclerosis (MS) is thought to be triggered by environmental factors acting on a genetic predisposition. Sardinians share a homogeneous genetic background and boast one of the highest MS prevalence worldwide. We investigated Epstein–Barr virus (EBV) antibody prevalence in Sardinian population by ELISA. Our results show a higher serum prevalence of EBNA-1 IgG in MS patients compared to healthy controls. Moreover, analyzing a subset of patients treated for 6 months with IFN-β, we observed a decrease in their EBNA-1 specific IgG titers. These results confirm previous findings and strengthen the association between EBV and MS in Sardinia.

Introduction: The Helicobacter pylori (HP) reinfection rate seems to be higher in developing countries than in developed ones. An increased seroprevalence of HP has also been reported in patients with type 1 diabetes (T1D) and Hashimoto’s thyroiditis (HT). Mycobacterium avium subsp. paratuberculosis (MAP) has been linked to both T1D and HT. Quite a few lines of evidence indicate that autoantibodies against several epitopes belonging to human zinc transporter 8 (ZnT8) cross-recognize the homologous MAP3865c epitopes in both T1D and HT patients. HP may play a role in HT disease, most likely acting through a molecular mimicry mechanism that targets ZnT8 as reported for MAP and the two autoimmune diseases. Methodology: An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of antibodies against several epitopes deriving from HP proteins, which are highly homologous to the immunodominant ZnT8 peptides previously identified: ZnT8178–186 and ZnT8186–194. Results: None of the HP peptides tested were significantly recognized when the humoral responses of 92 HT patients and 91 healthy volunteers were analyzed. Conclusions: These findings do not support a triggering role for HP (through ZnT8 mimicking) in HT. If a molecular mimicry phenomenon is taking place, it involves a different self-antigen. Moreover, the negative outcome of the experiments performed stresses the fact that sharing stretches of sequence homology is relevant, but not enough to trigger an antibody-mediated cross-recognition.

Mycobacterium avium subspecies paratuberculosis (MAP) asymptomatic infection is speculated to play a role in type 1 diabetes (T1D) among Sardinian subjects. Data obtained analyzing a pediatric population from mainland Italy lends support to the hypothesis, which envisions MAP as an environmental factor at play in T1D pathogenesis. Aiming to investigate the likelihood of cross-recognition between linear determinants shared by self (proinsulin) and non-self (MAP) proteins, 59 children with new onset T1D and 60 healthy controls (HCs) from continental Italy were enrolled in the study. Serum samples were subjected to indirect enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies (Abs) toward four homologues MAP/proinsulin epitopes. The rate of MAP infection (42.4% in T1D children and 5% in HCs; p0.0001) was estimated searching for Abs against MAP specific protein MptD. The homologous MAP2404c70-85 and proinsulin (PI)46-61 peptides were recognized by 42.4 and 39% of new-onset T1D children and only in 5% of HCs (AUC = 0.76, AUC = 0.7, p0.0001); whereas the prevalence of Abs against MAP 1,4-α-gbp157-173 and PI64-80 peptides was 45.7 and 49.1% in new-onset T1D children, respectively, compared with 3.3% of HCs (AUC = 0.74 and p0.0001 in both). Pre-incubation of MAP Ab-positive sera with proinsulin peptides was able to block the binding to the correspondent MAP epitopes, thus showing that Abs against these homologous peptides are cross-reactive. MAP/Proinsulin Ab mediated cross-recognition, most likely via molecular mimicry, maybe a factor in accelerating and/or initiating T1D in MAP-infected children. Indeed, it is known that anti-proinsulin and anti-Insulin autoantibodies are the earliest to appear.

There are several pieces of evidence indicating that Mycobacterium avium subspecies paratuberculosis (MAP) infection is linked to type 1 diabetes (T1D) in Sardinian patients. An association between MAP and T1D was recently observed in an Italian cohort of pediatric T1D individuals, characterized by a different genetic background. It is interesting to confirm the prevalence of anti-MAP antibodies (Abs) in another pediatric population from continental Italy, looking at several markers of MAP presence. New-onset T1D children, compared to age-matched healthy controls (HCs), were tested by indirect enzyme-linked immunosorbent assay for the presence of Abs toward the immunodominant MAP3865c/ZnT8 homologues epitopes, the recently identified C-terminal MAP3865c281-287 epitope and MAP-specific protein MptD. Abs against MAP and ZnT8 epitopes were more prevalent in the sera of new-onset T1D children compared to HCs. These findings support the view that MAP3865c/ZnT8 cross-reactivity is involved in the pathogenesis of T1D, and addition of Abs against these peptides to the panel of existing T1D biomarkers should be considered. It is important now to investigate the timing of MAP infection during prospective follow-up in at-risk children to elucidate whether Ab-titers against these MAP/ZnT8 epitopes are present before T1D onset and if so if they wane after diagnosis.

Bacillus Calmette-Guérin (BCG) and Mycobacterium avium subspecies paratuberculosis (MAP) have been associated with multiple sclerosis (MS). Clinical data indicates that BCG vaccination exerts anti-inflammatory effects in MS; conversely, MAP is thought to be one of the possible infectious factors responsible of MS through a molecular mimicry mechanism. A peptide-based indirect ELISA was used to detect antibodies against the encephalitogenic myelin oligodendrocyte glycoprotein (MOG)35-55 epitope, and two mycobacterial peptides sharing sequence homology with the latter: MAP_2619c352-361/BCG_1224355-364 and BCG_3329c64-74. Among 40 MS patients and 39 healthy volunteers included in the study, only MOG35-55 was capable of inducing a significantly higher humoral response in MS subjects compared to controls. Indeed, 11 out of 40 MS subjects (27.5%) and only 2 out of 39 controls (5%) were antibody-positive for MOG35-55 (p=0.01, AUC=0.65). These findings strengthen the importance of MOG35-55 in MS pathogenesis. The MAP and BCG MOG-homologues epitopes investigated were not recognized in MS patients. Overall, the results allow us concluding that sharing homology of linear epitopes is necessary but not sufficient to induce antibody-mediated cross-reactivity.

Background: A large number of reports indicate the association of Epstein-Barr virus (EBV), and Mycobacterium avium subsp. paratuberculosis (MAP) with multiple sclerosis (MS). Objective: To gain a better understanding of the role of these two pathogens, we investigated the host response induced by selected antigenic peptides. Methods: We examined both humoral and cell-mediated responses against peptides deriving from EBV tegument protein BOLF1, the MAP_4027 and the human interferon regulatory factor 5 (IRF5424–434) homolog in several MS patients and healthy controls (HCs). Results: Antibodies against these peptides were highly prevalent in MS patients compared to HCs. Concerning MS patients, BOLF1305–320, MAP_402718–32 and IRF5424–434 peptides were able to induce mainly Th1-related cytokines secretion, whereas Th2-related cytokines were down-regulated. Flow cytometry analyses performed on a subset of MS patients highlighted that these peptides were capable of inducing the release of pro-inflammatory cytokines: IFN-γ and TNF-α by CD4+ and CD8+ T lymphocytes, and IL-6 and TNF-α by CD14+ monocyte cells. Conclusion: Our data demonstrated that both EBV and MAP epitopes elicit a consistent humoral response in MS patients compared to HCs, and that the aforementioned peptides are able to induce a T-cell-mediated response that is MS correlated.

Purpose: MAP3865c, a Mycobacterium avium subspecies paratuberculosis (MAP) cell membrane protein, has a relevant sequence homology with zinc transporter 8 (ZnT8), a beta-cell membrane protein involved in Zn++ transportation. Recently, antibodies recognizing MAP3865c epitopes have been shown to cross-react with ZnT8 in type 1 diabetes patients. The purpose of this study was to detect antibodies against MAP3865c peptides in patients with high-risk proliferative diabetic retinopathy and speculate on whether they may somehow be involved in the pathogenesis of this severe retinal disorder. Methods: Blood samples were obtained from 62 type 1 and 80 type 2 diabetes patients with high-risk proliferative diabetic retinopathy and 81 healthy controls. Antibodies against 6 highly immunogenic MAP3865c peptides were detected by indirect ELISA. Results: Type 1 diabetes patients had significantly higher rates of positive antibodies than controls. Conversely, no statistically significant differences were found between type 2 diabetes patients and controls. After categorization of type 1 diabetes patients into two groups, one with positive, the other with negative antibodies, we found that they had similar mean visual acuity (∼0.6) and identical rates of vitreous hemorrhage (28.6%). Conversely, Hashimoto's thyroiditis prevalence was 4/13 (30.7%) in the positive antibody group and 1/49 (2%) in the negative antibody group, a statistically significant difference (P = 0.016). Conclusions: This study confirmed that type 1 diabetes patients have significantly higher rates of positive antibodies against MAP/ZnT8 peptides, but failed to find a correlation between the presence of these antibodies and the severity degree of high-risk proliferative diabetic retinopathy. The significantly higher prevalence of Hashimoto's disease among type 1 diabetes patients with positive antibodies might suggest a possible common environmental trigger for these conditions.

Our group has recently demonstrated that Mycobacterium avium subspecies paratuberculosis (MAP) infection significantly associates with T1D in Sardinian adult patients. Due to the potential role played by MAP in T1D pathogenesis, it is relevant to better characterize the prevalence of anti-MAP antibodies (Abs) in the Sardinian population, studying newly diagnosed T1D children. Therefore, we investigated the seroreactivity against epitopes derived from the ZnT8 autoantigen involved in children at T1D onset and their homologous sequences of the MAP3865c protein. Moreover, sera from all individuals were tested for the presence of Abs against: the corresponding ZnT8 C-terminal region, the MAP specific protein MptD, the T1D autoantigen GAD65 and the T1D unrelated Acetylcholine Receptor. The novel MAP3865c281–287 epitope emerges here as the major C-terminal epitope recognized. Intriguingly ZnT8186–194 immunodominant peptide was cross-reactive with the homologous sequences MAP3865c133–141, strengthening the hypothesis that MAP could be an environmental trigger of T1D through a molecular mimicry mechanism. All eight epitopes were recognized by circulating Abs in T1D children in comparison to healthy controls, suggesting that these Abs could be biomarkers of T1D. It would be relevant to investigate larger cohorts of children, followed over time, to elucidate whether Ab titers against these MAP/Znt8 epitopes wane after diagnosis.

Background and aims: A demographic analysis in the Mediterranean island of Sardinia revealed marked differences in extreme longevity across the 377 municipalities and particularly identified a mountain inner area where the proportion of oldest subjects among male population has one of the highest validated value worldwide. The cause(s) of this unequal distribution of male longevity may be attributed to a concurrence of environmental, lifestyle and genetic factors. Methods and results: In this study we focussed on some lifestyle and nutrition variables recorded in the island's population in early decades of 20th century, when agricultural and pastoral economy was still prevalent, and try to verify through ecological spatial models if they may account for the variability in male longevity. By computing the Extreme Longevity Index (the proportion of newborns in a given municipality who reach age 100) the island's territory was divided in two areas with relatively higher and lower level of population longevity. Most nutritional variables do not show any significant difference between these two areas whereas a significant difference was found with respect to pastoralism (P = 0.0001), physical activity estimated by the average slope of the territory in each municipality (P = 0.0001), and average daily distance required by the active population to reach the usual workplace (P = 0.0001). Conclusion: Overall, these findings suggest that factors affecting the average energy expenditure of male population such as occupational activity and geographic characteristics of the area where the population mainly resides, are important in explaining the spatial variation of Sardinian extreme longevity. © 2011 Elsevier B.V.

Mycobacterium avium subsp. paratuberculosis (MAP) infection is highly spread in the ruminant herds of Sardinia, in the Western Mediterranean. The objective of this study was to investigate prevalence of MAP infection in association with Multiple Sclerosis (MS) using clinical specimen from patients and controls. We analyzed samples for the presence of MAP specific DNA and to demonstrate humoral response to a MAP protein (MAP2694), a predicted homologue of the T-cell receptor gamma-chain/complement component 1 of the host. We found presence of MAP DNA in 42% of the MS patients and an extremely significant humoral immune response revealed by the MS patients against the MAP protein. In our opinion, this is the first report that significantly associates MAP infection with MS. Further studies will be required to confirm if MAP could be one of the triggers of MS, according to the molecular mimicry theory, in susceptible (and genetically at risk) individuals.

BackgroundThe etiology of type 1 diabetes mellitus (T1DM) is still unknown; numerous studies are performed to unravel the environmental factors involved in triggering the disease. SLC11A1 is a membrane transporter that is expressed in late endosomes of antigen presenting cells involved in the immunopathogenic events leading to T1DM. Mycobacterium avium subsp. paratuberculosis (MAP) has been reported to be a possible trigger in the development of T1DM.Methodology/principal findingsFifty nine T1DM patients and 79 healthy controls were genotyped for 9 polymorphisms of SLC11A1 gene, and screened for the presence of MAP by PCR. Differences in genotype frequency were evaluated for both T1DM patients and controls. We found a polymorphism in the SLC11A1 gene (274C/T) associated to type 1 diabetic patients and not to controls. The presence of MAP DNA was also significantly associated with T1DM patients and not with controls.Conclusions/significanceThe 274C/T SCL11A1 polymorphism was found to be associated with T1DM as well as the presence of MAP DNA in blood. Since MAP persists within macrophages and it is also processed by dendritic cells, further studies are necessary to evaluate if mutant forms of SLC11A1 alter the processing or presentation of MAP antigens triggering thereby an autoimmune response in T1DM patients.