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

Niegowska, Magdalena; Rapini, Novella; Piccinini, Simona; Mameli, Giuseppe; Caggiu, Elisa; Bitti, Maria Luisa Manca; Sechi, Leonardo Antonio

doi:10.1038/srep22266

Cited by 36 publications

(34 citation statements)

References 32 publications

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“…finding is consistent with the detection of high levels of serum antibodies directed against homologous peptide epitopes derived from ZnT8 TMD and Mycobacterium avium subspecies paratuberculosis, a putative environmental agent triggering or accelerating T1D in Sardinian and Italian populations (34).…”

Section: Znt8 Cell Surface Antigen and Autoimmunitysupporting

confidence: 89%

A subclass of serum anti-ZnT8 antibodies directed to the surface of live pancreatic β-cells

Merriman¹,

Huang²,

et al. 2018

Journal of Biological Chemistry

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The islet-specific zinc transporter ZnT8 is a major self-antigen found in insulin granules of pancreatic β-cells. Frequent insulin secretion exposes ZnT8 to the cell surface, but the humoral antigenicity of the surface-displayed ZnT8 remains unknown. Here we show that a membrane-embedded human ZnT8 antigen triggered a vigorous immune response in knock-out mice. Approximately 50% of serum immunoreactivities toward ZnT8 were mapped to its transmembrane domain that is accessible to extracellular ZnT8 antibody (ZnT8A). ZnT8A binding was detected on live rat insulinoma INS-1E cells, and the binding specificity was validated by a CRISPR/Cas9 mediated knock-out. Applying established ZnT8A assays to purified serum antibodies from patients with type 1 diabetes, we detected human ZnT8A bound to live INS-1E cells, whereas a knock-out specifically reduced the surface binding. Our results demonstrate that ZnT8 is a cell surface self-antigen, raising the possibility of a direct involvement in antibody-mediated β-cell dysfunction and cytotoxicity.

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Section: Znt8 Cell Surface Antigen and Autoimmunitysupporting

confidence: 89%

A subclass of serum anti-ZnT8 antibodies directed to the surface of live pancreatic β-cells

Merriman¹,

Huang²,

et al. 2018

Journal of Biological Chemistry

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“…32 subjects at risk for T1D (n = 19 males and n = 13 females, mean age 8.90±3.52 years) and age-matched healthy volunteers (HC; n = 42, mean age 6.90±3.55 years) were selected in blind from a dataset of children and youth recruited for a previous study [ 16 ] upon periodical visits at the Tor Vergata University Hospital of Rome, Italy. Additionally, further time-point samples of 11 T1D at-risk children were included, giving in total 50 samples.…”

Section: Methodsmentioning

confidence: 99%

“…Each assay included a highly positive control serum for data normalization with Abs reactivity established at 1.0 arbitrary units (U/ml). Optimal positivity thresholds for the homologs were set as previously reported [ 16 ]. Cut-off value discriminating between samples positive and negative for L5P was calculated based on ROC curve and accounted for 0.83 U/ml with specificity of 83.33%.…”

Section: Methodsmentioning

confidence: 99%

“…Our earlier reports demonstrated that MAP epitopes making part of glucan branching protein (MAP1,4-αgbp), putative regulator for proline utilization (MAP2404c) and two portions of cation efflux membrane protein (MAP3865c) induce high seroreactivity in children at risk for T1D [ 16 ]. The selected peptides present sequence homology to human proinsulin (PI) or zinc transporter 8 (ZnT8); Abs against both proteins circulate in blood of individuals with T1D even before manifestation of clinical symptoms and are used to diagnose autoimmune diabetes [ 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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Aims/HypothesisAlthough 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.MethodsPlasma 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.ResultsResponses 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%.ConclusionsMAP-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.

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“…The technical difficulty of growing MAP in culture lead us to search for an alternative method to grow MAP more rapidly to determine the relationship of MAP and CD and to multiple other potential conditions such as multiple sclerosis, and type I diabetes mellitus (3)(4)(5). In the natural environment where MAP is present, dependence of MAP on mycobactin for growth is likely to be provided by another Mycobacterium to produce mycobactin if the dependence is absolute, and experimentally it has been shown that the mycobactin dependence of MAP is not absolute (1,6,7).…”

Section: Introductionmentioning

confidence: 99%

Alternative Growth Behavior ofMycobacterium AviumSubspecies and Staphylococci with Implications for Clinical Microbiology and Blood Culture

P¹,

Lm²,

Jt³

et al. 2016

Preprint

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Rapid culture of Mycobacterium avium subspecies paratuberculosis (MAP) from patients remains a challenge. During the process of developing a rapid culture method for MAP, we found that there is an alternative growth behavior present in MAP, MAH (Mycobacterium avium subspecies hominissuis) and other bacteria such as Staphylococcus aureus, and Staphylococcus pseudintermedius. The bacterial DNA, RNA and proteins are present in the supernatants of the liquid culture media after routine microcentrifugation. When cultured in the solid media plate, there are a limited number of colonies developed for MAP and MAH disproportionate to the growth. We believe there is an alternative growth behavior for MAP, MAH and other bacteria similar to "phenoptosis". Based on the alternative bacterial growth behavior, we tested 62 blood culture specimens that have been reported negative by routine automated blood culture method after 5 days of incubation. We used alternative culture media and molecular diagnostic techniques to test these negative culture bottles, and we found a large percentage of bacterial growth by alternative culture media (32%) and by molecular PCR amplification using 16s rDNA primer set and DNA sequencing (69%). The sensitivity of detection by the molecular PCR/sequencing method is significantly higher than by routine automated blood culture. Given the challenge of early diagnosis of sepsis in the hospital setting, it is necessary to develop more sensitive and faster diagnostic tools to guide clinical practice and improve the outcome of sepsis management.

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

Cited by 36 publications

References 32 publications

A subclass of serum anti-ZnT8 antibodies directed to the surface of live pancreatic β-cells

A subclass of serum anti-ZnT8 antibodies directed to the surface of live pancreatic β-cells

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

Alternative Growth Behavior ofMycobacterium AviumSubspecies and Staphylococci with Implications for Clinical Microbiology and Blood Culture

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