| 1. |
- Vaziri Sani, Fariba, et al.
(författare)
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A novel triple mix radiobinding assay for the three ZnT8 (ZnT8-RWQ) autoantibody variants in children with newly diagnosed diabetes
- 2011
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier. - 0022-1759 .- 1872-7905. ; 371:1-2, s. 25-37
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Tidskriftsartikel (refereegranskat)abstract
- Background and aims: Autoantibodies against the zinc transporter 8 (ZnT8A) are common in type 1 diabetes (T1D). ZnT8A analyses are complicated by the fact that there are three variants of the autoantigen at amino acid position 325 representing ZnT8-R (Arginine), ZnT8-W (Tryptophan) and ZnT8-Q (Glutamin). The aims of the study were: 1) to develop an autoantigen triple mix Radio-Binding Assay (RBA) for ZnT8A: 2) to identify the individual ZnT8-R,-W,-QA reactivity and 3) to validate the triple mix ZnT8A RBA in children with newly diagnosed T1D. less thanbrgreater than less thanbrgreater thanMethods: Serum samples were obtained from 2664 (56% males, n = 1436) patients in the Swedish nationwide Better Diabetes Diagnosis (BDD) study representing patients with T1D (97%, n = 2582), T2D (1.7%, n = 46), MODY (1.0%, n = 28) and secondary diabetes (0.3%, n = 8). cDNA coding for the C-terminal end of each variant was prepared by site-directed mutagenesis and subcloned into a high efficiency in vitro transcription translation vector. The ZnT8 variants were labeled with 35S-methionine and used in a standard RBA separating free from autoantibody-bound autoantigen with Protein A-Sepharose. less thanbrgreater than less thanbrgreater thanResults: ZnT8-TripleA was detected in 1678 (65%) patients with T1D, 4 (9%) T2D, 3 (11%) MODY and in none (0%) of the patients with secondary diabetes. Among the T1D patients ZnT8-RA was detected in 1351 (52%) patients, ZnT8-WA in 1209(47%) and ZnT8-QA in 790(31%) demonstrating that 1661 (64%) had one or several ZnT8A. The ZnT8-TripleA assay showed a false positive rate of 1.9% (n = 49). Only 1.2% (n = 32) of the T1D patients were false negative for ZnT8-TripleA compared to 0/46(0%) of the T2D patients. The precision (intra assay CV) and reproducibility (inter assay CV) of the ZnT8-TripleA assay did not differ from the RBA of the individual ZnT8 variants. less thanbrgreater than less thanbrgreater thanConclusion: We conclude that the ZnT8-TripleA assay had low false positive and false negative rates. The ZnT8-TripleA assay would therefore be highly suitable not only to analyze patient with newly diagnosed diabetes but also for screening the general population since this assay demonstrated high sensitivity and very high specificity.
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| 2. |
- Andersen, Marie Louise M., et al.
(författare)
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Association between autoantibodies to the Arginine variant of the Zinc transporter 8 (ZnT8) and stimulated C-peptide levels in Danish children and adolescents with newly diagnosed type 1 diabetes
- 2012
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Ingår i: Pediatric Diabetes. - : Hindawi Limited. - 1399-543X .- 1399-5448. ; 13:6, s. 454-462
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Tidskriftsartikel (refereegranskat)abstract
- Background The zinc transporter 8 (ZnT8) was recently identified as a common autoantigen in type 1 diabetes (T1D) and inclusion of ZnT8 autoantibodies (ZnT8Ab) was found to increase the diagnostic specificity of T1D. Objectives The main aims were to determine whether ZnT8Ab vary during follow-up 1 year after diagnosis, and to relate the reactivity of three types of ZnT8Ab to the residual stimulated C-peptide levels during the first year after diagnosis. Subjects A total of 129 newly diagnosed T1D patients <15 years was followed prospectively 1, 3, 6, and 12 months after diagnosis. Methods Hemoglobin A1c, meal-stimulated C-peptide, ZnT8Ab, and other pancreatic autoantibodies were measured at each visit. Patients were genotyped for the rs13266634 variant at the SLC30A8 gene and HLA-DQ alleles. Results The levels of all ZnT8Ab [ZnT8Arg (arginine), ZnT8Trp (tryptophan), ZnT8Gln (glutamine)] tended to decrease during disease progression. A twofold higher level of ZnT8Arg and ZnT8Gln was associated with 4.6%/5.2% (p = 0.02), 5.3%/8.2% (p = 0.02) and 8.9%/9.7% (p = 0.004) higher concentrations of stimulated C-peptide 3, 6, and 12 months after diagnosis. The TT genotype carriers of the SLC30A8 gene had 45.8% (p = 0.01) and 60.1% (p = 0.002) lower stimulated C-peptide 6 and 12 months after diagnosis compared to the CC and the CT genotype carriers in a recessive model. Conclusions The levels of the Arg variant of the ZnT8 autoantibodies are associated with higher levels of stimulated C-peptide after diagnosis of T1D and during follow-up. Carriers of the TT genotype of the SLC30A8 gene predict lower stimulated C-peptide levels 12 months after diagnosis.
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| 3. |
- Nilsson, Anna-Lena, et al.
(författare)
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Relationship Between Ljungan Virus Antibodies, HLA-DQ8, and Insulin Autoantibodies in Newly Diagnosed Type 1 Diabetes Children
- 2013
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Ingår i: Viral Immunology. - : Mary Ann Liebert Inc. - 0882-8245 .- 1557-8976. ; 26:3, s. 207-215
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Tidskriftsartikel (refereegranskat)abstract
- Environmental factors, including viral infections, may explain an increasing and fluctuating incidence of childhood type 1 diabetes (T1D). Ljungan virus (LV) isolated from bank voles have been implicated, but it is unclear whether LV contributes to islet autoimmunity, progression to clinical onset, or both, of T1D. The aim was to test whether LV antibodies (LVAb) were related to HLA-DQ and islet autoantibodies in newly diagnosed T1D patients (n = 676) and controls (n = 309). Patients, 0-18 years of age, diagnosed with T1D in 1996-2005 were analyzed for LVAb, HLA-DQ genotypes, and all seven known islet autoantibodies (GADA, IA-2A, IAA, ICA, ZnT8RA, ZnT8WA, and ZnT8QA). LVAb at 75th percentile, defined as cut off, was 90 (range 6-3936) U/mL and 4th quartile LVAb were found in 25% (170/676) of which 64% were < 10 (n = 108, p < 0.0001), and 27% were < 5 (n = 45; p < 0.0001) years old. The 4th quartile LVAb in children < 10 years of age correlated to HLA DQ2/8, 8/8, and 8/X (p < 0.0001). Furthermore, in the group with 4th quartile LVAb, 55% were IAA positive (p = 0.01) and correlation was found between 4th quartile LVAb and IAA in children < 10 years of age (p = 0.035). It is concluded that 1) LVAb were common among the young T1D patients and LVAb levels were higher in the younger age groups; 2) 4th quartile LVAb correlated with IAA; and 3) there was a correlation between 4th quartile LVAb and HLA-DQ8, particularly in the young patients. The presence of LVAb supports the notion that prior exposure to LV may be associated with T1D.
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| 4. |
- Nilsson, Sara, et al.
(författare)
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A mutation in factor I that is associated with atypical hemolytic uremic syndrome does not affect the function of factor I in complement regulation.
- 2007
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 44:8, s. 1835-1844
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Tidskriftsartikel (refereegranskat)abstract
- Factor I (FI) is the major complement inhibitor that degrades Ob and C4b in the presence of cofactors such as factor H (FH) and membrane cofactor protein (MCP). Recently, mutations and polymorphisms in complement regulator molecules FH and MCP but also in FI have been associated with atypical hemolytic uremic syndrome (aHUS). HUS is a disorder characterized by hemolytic anemia, thrombocytopenia and acute renal failure. In this study, we report three unrelated patients with an identical heterozygous mutation, G261D, in the FI heavy chain who developed severe aHUS at different time points in their lives. Two of the patients also have polymorphisms in FH previously associated with risk of developing aHUS. Testing in particular one patient and control serum samples we did not observe major differences in complement hemolytic activity, FI plasma levels or the capability to degrade C4b or Ob. A recombinant protein was produced in order to analyze the functional consequences of the mutation. Mutant FI had a slightly different migration pattern during electrophoresis under reducing conditions. An alteration due to alternative splicing or glycosylation was ruled out, thus the altered migration may be due to proximity of the mutation to a cysteine residue. The recombinant mutant FI degraded Ob and C4b in a manner comparable to wild-type protein. In conclusion, despite the association between the heterozygous mutation in FI and aHUS we did not observe any abnormalities in the function of FI regarding complement regulation.
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| 5. |
- Skärstrand, Hanna, et al.
(författare)
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Antigenicity and epitope specificity of ZnT8 autoantibodies in type 1 diabetes.
- 2012
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Ingår i: Scandinavian Journal of Immunology. - : Wiley. - 1365-3083 .- 0300-9475.
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Tidskriftsartikel (refereegranskat)abstract
- The SNP rs13266634 encodes either an Arginine (R) or a Tryptophan (W) (R325W) at the amino acid position 325 in the Zinc Transporter 8 (ZnT8) protein. Autoantibodies (Ab) that recognize ZnT8R, ZnT8W or both at the polymorphic site are common in newly diagnosed type 1 diabetes (T1D) patients. The epitope specificity and affinity of ZnT8Ab are poorly understood, but may be of importance for the prediction and clinical classification of T1D. Therefore, the aims were to 1) determine the immunogenicity of short (318-331) ZnT8 peptides in mice, and 2) test the affinity of short and long (268-369) ZnT8 proteins in T1D patients positive for either ZnT8WAb or ZnT8RAb. Sera from BALB/cByJ mice immunized with short R, W or Q (Glutamine) ZnT8 peptides were tested for ZnT8-peptide antibodies in ELISA and radiobinding assay (RBA). Using reciprocal permutation experiment, short synthetic ZnT8R and ZnT8W (318-331) and long in vitro transcription translation ZnT8R and ZnT8W (268-369) proteins, were tested in competitive RBA with R- and W- monospecific T1D sera samples. All mouse sera developed non-epitope specific peptide-antibodies in ELISA and only 6/12 mice had ZnT8-RWQ antibodies in RBA. Both long ZnT8R and ZnT8W (268-369) but not any short, proteins displaced labeled ZnT8 (268-369) proteins in binding to T1D ZnT8Ab-specific sera. The reciprocal cross-over tests showed that half maximal displacement varied 2-11-fold indicating variable affinity of patient ZnT8Ab, signifying crucial autoantibody epitope spreading. The present approach should make it possible to dissect the importance of the R325W ZnT8 autoantigen epitope in the T1D pathogenesis.
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| 6. |
- Skärstrand, Hanna, et al.
(författare)
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ZnT8 autoantibody epitope specificity and affinity examined with recombinant ZnT8 variant proteins in specific ZnT8R and ZnT8W autoantibody positive type 1 diabetes patients.
- 2015
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Ingår i: Clinical and Experimental Immunology. - : Oxford University Press (OUP). - 0009-9104 .- 1365-2249. ; 179:2, s. 220-229
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Tidskriftsartikel (refereegranskat)abstract
- Variant specific zinc transporter 8 autoantibodies (ZnT8A) against either arginine (R) or tryptophan (W) at amino acid (aa) position 325 of the zinc transporter 8 (ZnT8) has been identified in T1D patients. Reciprocal cross-over tests revealed differences in half-maximal binding to indicate variable affinity of patient ZnT8 autoantibodies. Insufficient recombinant ZnT8 variant proteins have precluded detailed analyses of ZnT8 autoantibody affinity. The aims in the present study were to 1) generate recombinant ZnT8R- and ZnT8W-aa275-369 proteins 2) test the ZnT8R- and ZnT8W-aa275-369 proteins in reciprocal competitive radiobinding assays (RBA) against ZnT8R- and ZnT8W-aa268-369 labeled with (35) S-methionine, and 3) determine the specificity and affinity of sera specific for either ZnT8 Arginine (R) or ZnT8 Tryptophan (W) autoantibodies in newly diagnosed type 1 diabetes (T1D) patients. The results demonstrate first that it was possible to produce recombinant human MBP-ZnT8aa275-369 protein purified to homogeneity for RBA reciprocal competition experiments. Second, high titer ZnT8WA sera diluted to half maximal binding showed significant specificity for respective variants of either ZnT8R or ZnT8W. Third, ZnT8WA positive sera showed high affinity for ZnT8W compared to ZnT8RA for ZnT8R. These data demonstrate that T1D patients may have single amino acid specific autoantibodies directed against either ZnT8R or ZnT8W and that the autoantibody affinity to the respective variant may be different. Further studies are needed to assess the mechanisms by which variant specific ZnT8A of variable affinity develop and their possible role in the pathogenic process leading to the clinical onset of T1D.
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