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Scooped by
Gilbert C FAURE
November 20, 2023 4:13 AM
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Despite the prevalence of autoimmune conditions, like the thyroid disease Hashimoto's, finding help can prove frustrating and expensive. Patients may rack up big bills as they search for a diagnosis.
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Scooped by
Gilbert C FAURE
November 17, 2021 7:58 AM
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BackgroundAutoimmune thyroid disease (AITD) is characterized by thyroid dysfunction and deficits in the autoimmune system. Growing attention has been paid toward the field of gut microbiota over the last few decades.
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Suggested by
Société Francaise d'Immunologie
June 17, 2019 6:56 AM
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Summary There is currently scarce knowledge of the immunological profile of patients with latent autoimmune diabetes mellitus in the adult (LADA) when compared with healthy controls (HC) and patients with classical type 1 diabetes (T1D) and type 2 diabetes (T2D). The objective of this study was to investigate the cellular immunological profile of LADA patients and compare to HC and patients with T1D and T2D. All patients and age‐matched HC were recruited from Uppsala County. Peripheral blood mononuclear cells were isolated from freshly collected blood to determine the proportions of immune cells by flow cytometry. Plasma concentrations of the cytokine interleukin (IL)‐35 were measured by enzyme‐linked immunosorbent assay (ELISA). The proportion of CD11c+CD123– antigen‐presenting cells (APCs) was lower, while the proportions of CD11c+CD123+ APCs and IL‐35+ tolerogenic APCs were higher in LADA patients than in T1D patients. The proportion of CD3–CD56highCD16+ natural killer (NK) cells was higher in LADA patients than in both HC and T2D patients. The frequency of IL‐35+ regulatory T cells and plasma IL‐35 concentrations in LADA patients were similar to those in T1D and T2D patients, but lower than in HC. The proportion of regulatory B cells in LADA patients was higher than in healthy controls, T1D and T2D patients, and the frequency of IL‐35+ regulatory B cells was higher than in T1D patients. LADA presents a mixed cellular immunological pattern with features overlapping with both T1D and T2D. Introduction Latent autoimmune diabetes mellitus in the adult (LADA) 1 accounts for 9% of all adult onset diabetes 1. Phenotypically, LADA patients share characteristics of both type 1 diabetes (T1D) (antibodies against the insulin‐producing β cells) and type 2 diabetes (T2D) patients (aged >35 years and non‐insulin‐dependent at onset) 2, 3. LADA also shares genetic features of both T1D and T2D 4. Despite the presence of autoantibodies, predominantly against glutamic acid decarboxylase (GAD), the disease progress is much slower than in T1D. At present, the focus on the immunological characterization of LADA has concerned the presence of autoantibodies, with only a few studies of other components of the immune system, such as low‐grade proinflammatory markers or cellular immunology 4-6. So far there are no published reports describing the cellular immunological profile that characterizes LADA patients in comparison to healthy individuals, T1D as well as T2D patients, simultaneously. Innate immune cells, such as dendritic cells (DCs), are the main players during early development of autoimmune diabetes 7-10. Allen et al. have reported that plasmacytoid dendritic cells (pDCs) are expanded during the early development of T1D and enhance the islet autoantigen presentation to T cells through an immune complex capture 11. In line with this, it has been shown that interferon (IFN)‐α‐producing pDCs augment the T helper type 1 (Th1) response in T1D and the number of IFN‐α‐producing pDCs are increased in T1D patients compared to healthy controls 12. Therefore, it is of interest to investigate CD11c+CD123–human leucocyte antigen (HLA)‐DR+Lin– (CD11c+CD123–) antigen‐presenting cells (APCs), CD11c+CD123+HLA‐DR+Lin– (CD11c+CD123+) APCs and CD11c–CD123+HLA‐DR+Lin– (CD11c–CD123+) pDCs in LADA patients. Herein, these subsets of DCs were analysed using flow cytometry, as described previously 13. Neutrophils have been found to be reduced in recent‐onset T1D 14, 15, and CD15low neutrophils (low‐density granular neutrophils) have also been shown to have a pathogenic role in systemic autoimmunity 16. In accordance with this, Neuwrith et al. have reported a role of CD15low cells in T1D development 17. Alterations in the frequency of CD16 monocytes has been associated with diabetes complications 18. Natural killer (NK) cells participate in innate immune responses and in animal models of T1D, it has been reported that depletion of NK cells prevents the development of T1D 19, 20. An altered frequency of total number of NK cells has been found in LADA, but not in T2D, patients 21, 22. Furthermore, it has been reported in children with T1D that NK cells have defects in signalling and function that might contribute to the pathogenesis of T1D 23. NK cell subsets could be defined based on the relative expression of CD16 and CD56, i.e. CD56dim (CD56lowCD16+ and CD56lowCD16–) and CD56bright (CD56highCD16+ and CD56highCD16–), as reviewed by Poli et al. 24. CD56dim NK cells are considered to be mature NK cells and CD56bright cells are immature cells. CD56dim NK cells have cytotoxic properties while CD56bright NK cells are proinflammatory, as they produce IFN‐γ 25. In the present study we determined the proportion of both CD56dim and CD56bright NK cell subpopulations using flow cytometry. Adaptive immune cells such as T (CD4+ and CD8+) and B (CD19+) cells also play a major role in autoimmunity and metabolic disorders 26-30. An altered phenotype of CD4+, CD8+ and CD19+ cells has been reported in LADA patients 5, 31-33. Therefore, in this study we determined the proportions of these cells in LADA patients when compared to T1D and T2D patients. Immune cells that produce anti‐inflammatory cytokines, e.g. interleukin (IL)‐10, transforming growth factor (TGF)‐β and IL‐35, are known as regulatory immune cells, i.e. regulatory T (Treg) cells, regulatory B (Breg) cells and tolerogenic APCs 34-36. Treg cells have received a great deal of attention, as these cells play a major role in maintaining the homeostasis of the immune system and preventing the development of autoimmune diseases 37, 38. A reduced number of Treg cells has been reported in different autoimmune diseases, including T1D 39. Treg cells can be identified by using the cell surface markers CD4, CD25 and CD127 and the intracellular forkhead box (Fox) transcription factor FoxP3 40. Breg cells can be identified by using the cell surface markers CD19, CD24, CD38 and CD40 41. Treg cells, Breg cells and APCs produce IL‐35 34-36, 42-44. This cytokine is a novel anti‐inflammatory cytokine, which belongs to the IL‐12 cytokine family, and has two subunits, Ebi3 and IL‐12p35 42, 43. It has previously been reported that IL‐35 suppresses autoimmunity and plays a putative role in tumour biology 35, 36, 42, 45-47. We have found that the systemic administration of IL‐35 prevented the development of autoimmune diabetes in the multiple low‐dose streptozotocin (MLDSTZ) mouse model, and effectively reversed established disease in both the MLDSTZ and non‐obese diabetic mouse models of T1D by maintaining the normal suppressive phenotype of Treg cells 46. We also found that IL‐35 levels were higher in T1D patients with residual C‐peptide compared to T1D patients with no measurable residual C‐peptide 48. Thus, our previous findings indicate that IL‐35 may prevent the loss of β cell mass. The possible alteration in the frequencies of IL‐35‐producing cells in LADA patients has not yet been investigated. In this study we aimed to determine the proportions of different innate, adaptive and regulatory immune cells in order to elucidate the immunological profile that characterizes LADA in comparison to healthy controls, T1D and T2D patients. Materials and methods Informed consent The Uppsala County regional ethics board approved this study, and the reported investigations were carried out in accordance with the principles of the Declaration of Helsinki, as revised in 2000. All participants were provided with oral and written information about the study, and signed a written consent. Inclusion The All New Diabetes in Uppsala (ANDiU) study (www.andiu.se) is an ongoing study with the aim of describing the incidence of diabetes subgroups in Uppsala County, Sweden (population approximately 300 000). Patients newly diagnosed with diabetes (ADA criteria) are included. Upon registration patients are classified into diabetes subtypes according to criteria shared with the All New Diabetics in Scania (ANDIS) study (http://andis.ludc.med.lu.se; Principle Investigator: Leif Groop) in southern Sweden. Criteria for LADA diagnosis are age ≥35 years, GAD antibody (GADA) positivity (>20 IE/ml) and fasting (f)C‐peptide levels ≥0·3 nmol/l. T1D criteria are GADA positivity and fC‐peptide <0·3 nmol/l, whereas for T2D age ≥35 years, GADA negativity and fC‐peptide >0·72 nmol/l are used as diagnostic criteria. For all three diagnoses there should be no history of pancreatitis, pancreatic cancer or other causes of secondary diabetes. There are no unified criteria for LADA diagnosis and classification, but the criteria used in ANDiU and ANDIS are in line with previous literature 3. The exception is fC‐peptide, which is used in ANDiU and ANDIS as an indicator of remaining insulin production to separate LADA from T1D 49, 50. It replaces the insulin therapy criterion (i.e. no insulin therapy within 6–12 months of diagnosis) that is often used, which is open to subjectivity as it reflects the assessment made by the treating physician. From ANDiU we recruited patients classified as LADA and T2D. T1D patients and healthy controls were recruited at Uppsala University Hospital and Eriksberg Primary Health Care Clinic, Uppsala, respectively. T1D and T2D patients, as well as healthy controls, were age‐, sex‐ and body mass index (BMI)‐matched to the LADA patients. Exclusion criteria were ongoing hormonal treatment (ATC code H01‐03), use of non‐steroid anti‐inflammatory drugs (NSAID, ATC code M01), ongoing chemotherapy treatment (ATC code L01‐04) and other autoimmune disease (e.g. hypo/hyperthyroidism, rheumatic disease, vitiligo, psoriasis, coeliac disease, Addison’s disease and inflammatory bowel disease). Table 1 provides characteristics of the included patients and Supporting information, Fig. S1 shows a flow‐chart of the inclusion process of the study. Controls (n = 13) T1D (n = 16) LADA (n = 14) T2D (n = 16) Age (years) 61·6 ± 2·9 66·0 ± 1·4 65·7 ± 2·4 64·0 ± 2·0 Sex (M%/F%) 62%/38% 73%/27% 57%/43% 70%/30% BMI (kg/m2) 28·9 ± 4·0 26·5 ± 0·7 27·9 ± 0·5 29·1 ± 1·0 Age at onset n.a. 19·7 ± 1·8 63·6 ± 1·5b 60·0 ± 2·0b Disease duration n.a. 46·1 ± 2·2 4·9 ± 0·3b 5·3 ± 0·3a, b HbA1c (mmol/mol) 35·8 ± 0·7 58·9 ± 3·2a 45·8 ± 2·3b 52·27 ± 4·0a fC‐peptide (nmol/l) 1·10 ± 0·11 0·22 ± 0·13a 0·95 ± 0·14b 1·30 ± 0·12b, c fp‐glucose (mmol/l) 5·9 ± 0·1 11·7 ± 1·0a 8·0 ± 0·5b 9·1 ± 0·6a, b GAD antibody IgG (IE/ml) 0 ± 0 508 ± 205 337 ± 190 1 ± 0 IA‐2 antibody IgG (IE/ml) 1 ± 0 25 ± 7 387 ± 295 1 ± 0 Hb (g/l) 145 ± 2 142 ± 3 145 ± 3 141 ± 4 Lkc (109/l) 6·0 ± 0·4 5·8 ± 0·4 6·6 ± 0·8 5·4 ± 0·2 CRP (mg/l) 1·2 ± 0·4 1·6 ± 0·3 1·3 ± 0·2 2·4 ± 0·8 All values are given as mean ± standard error of the mean (s.e.m.). Multiple comparison of means by analysis of variance (anova), Tukey’s post‐hoc test. a P‐value versus controls < 0·05; b P‐value versus T1D < 0·05; c P‐value versus LADA < 0·05. GADA = glutamate decarboxylase 65 antibody; eGFR = creatinine estimated glomerular filtration rate; TSH = thyroid‐stimulating hormone; LADA = latent autoimmune diabetes mellitus in the adult (LADA); Ig = immunoglobulin; BMI = body mass index; n.a. = not available; HbAlc = haemoglobinA1c; CRP = C‐reactive peptide; fC‐peptide = fasting C‐peptide; fp‐glucose = fasting plasma glucose; Lkc = leucocyte count. Routine laboratory parameters Blood samples were collected after overnight fasting. All routine laboratory parameters were analysed at the Central Clinical Chemistry Laboratory at Uppsala University Hospital, which is certified by the External Quality Assessment Service (EQUALIS) (equalis.se/en). On inclusion, blood haemoglobin 51, blood leucocyte count (Lkc), plasma C‐reactive protein (CRP), serum transglutaminase [immunoglobulin (Ig)A] antibodies, serum thyroxine (T4), serum triiodothyronine (T3), serum thyroid‐stimulating hormone (TSH), serum TSH‐receptor 18 antibody, serum thyroid peroxidase antibody, plasma IgA antibody, serum cortisol, 21‐hydroxylase 18 antibody, serum GADA, fasting serum C‐peptide, fasting plasma glucose and blood haemoglobinA1c (HbA1c) were analysed. Glomerular filtration rate (GFR) was estimated based on creatinine‐levels calculated with the MDRD formula 52. Flow cytometry Freshly isolated peripheral blood mononuclear cells (PBMCs) were prepared using Histopaque‐1077 (Sigma, St Louis, MO, USA), as described earlier 48. PBMCs were stained with antibodies for surface markers (Supporting information, Table S1). The cells were thereafter fixed and permeabilized with fixation permeabilization buffer (eBioscience, San Diego, CA, USA) for intracellular markers (Supporting information, Table S1). The samples were run on LSR II Fortesa (Becton Dickinson, Franklin Lakes, NJ, USA) using DivaDacker software 53 and 1 million events were counted for analysis. Fluorescence minus one (FMO), isotype and single‐stained controls were used for gating strategies. The flow cytometry standard (FCS) files were analysed on FlowLogic software (Inivai Technologies, Mentone, Australia). Representative gating strategies are shown in Supporting information, Fig. S2–S5. IL‐35 measurements in plasma Plasma IL‐35 concentrations were determined by using an enzyme‐linked immunosorbent assay (ELISA) kit (Cloud‐Clone Corporation, Wuhan, China). Statistics Differences in means between healthy controls, T1D and T2D patients compared to LADA patients were calculated by one‐way analysis of variance (anova) with Dunnett’s post‐hoc test for immunological profiles. Comparisons between all groups were performed by anova with Tukey’s post‐hoc test for research subjects’ characteristics (Table 1). A P‐value < 0·05 was considered as a significant difference between groups. Statistical analyses were carried out in GraphPad Prism version 6.0TM. Results Innate immune cell responses We observed a lower proportion of CD11c+CD123– APCs in LADA patients than in healthy controls and T1D patients (Fig. 1a). Furthermore, the proportion of CD11c+CD123– APCs among HLA‐DR+Lin– cells was lower in LADA patients than in T1D patients (Fig. 1b), whereas the proportions of pDCs and pDCs among HLA‐DR+Lin– were not altered in LADA patients (Fig. 1c,d). We did not observe any difference in the proportion of CD11c+CD123+ APCs (Fig. 1e), but the proportion of CD11c+CD123+ cells among HLA‐DR+Lin– was higher in patients with LADA than in patients with T1D (Fig. 1f). We did not find any alteration in the proportions of CD15low neutrophils and CD16+ monocytes in LADA patients (Supporting information, Fig. S6a,b). We also examined different subsets of NK cells and did not find any alteration in LADA patients compared to the other groups regarding the proportions of CD3–CD56lowCD16+, CD3–CD56lowCD16– and CD3–CD56highCD16– NK cells (Fig. 2a–c). However, the proportion of CD3–CD56highCD16+ NK cells was higher in LADA patients compared to both healthy controls and T2D patients (Fig. 2d). We did not observe any alteration in the proportions of CD3+CD56+ NK T cells between the groups (Supporting information, Fig. S6c). Adaptive immune cell responses The proportions of CD3+, CD4+CD25– and CD8+ T cells were not altered in LADA patients when compared to the other groups (Supporting information, Fig. S7a–c). Similarly, the proportion of CD19+ cells was not altered (Supporting information, Fig. S7d). Regulatory immune cell responses The proportions of CD4+CD25+CD127–FoxP3+ cells (Treg cells) and CD4+CD25+CD127–FoxP3+Helios+thymic‐derived T cells (tTreg) were similar in all four groups (Supporting information, Fig. S8a,b). Similarly, the proportions of Treg cells as well as the tTreg cells among CD4+ T cells were similar in all four groups (Supporting information, Fig. S8c,d). However, the proportions of IL‐35+ cells among Treg and tTreg cells in patients with LADA were similar in patients with T1D and T2D, but lower when compared to healthy controls (Fig. 3a,b). We also analysed the proportion of CD19+CD24+CD40+CD38+ cells (Breg cells), and observed a higher proportion of these cells in LADA patients when compared to healthy controls and patients with T1D and T2D (Fig. 3c). The proportion of IL‐35+ cells among Breg cells was higher in LADA patients when compared to T1D patients (Fig. 3d). Subsequently, we analysed the proportion of IL‐35+ tolerogenic APCs. Interestingly, the proportion of IL‐35+ cells among APCs was higher in patients with LADA compared to patients with T1D (Fig. 3e). Patients with LADA had IL‐35 concentrations in blood plasma that were similar to those in patients with T1D and T2D, but lower compared to those in healthy controls (Fig. 3f). Discussion We determined the proportions of different immune cells to elucidate the immunological patterns that distinguish patients with LADA compared not only to healthy individuals, but also to patients with T1D and T2D. With regard to the innate and adaptive immune cells, we found that LADA shares common features with both T1D and T2D. The proportion of CD11c+CD123– APCs in LADA patients was between that observed in T1D and T2D patients. Similarly, the proportion of CD11c+CD123+ cells among the HLA‐DR+Lin– was highest in T2D patients and lowest in T1D patients, whereas the proportion in LADA patients was between that of T1D and T2D patients. Neutrophils have previously been claimed to play a role in the early development of both T1D 14, 15, 54 and T2D 55. In the present study, we did not find any alteration in the proportion of CD15low neutrophils in LADA patients when compared to the other study groups. It cannot be excluded that early transient alterations during disease development as described for T1D 14, 15 had occurred, as our study subjects with LADA had a mean disease duration of almost 5 years. It has been reported previously that the proportion of CD3–CD56+CD16+ NK cells is decreased in LADA patients 22. Instead, we observed an increased proportion of CD3–CD56highCD16+ NK cells in LADA patients compared to both healthy controls and T2D patients. The reason for the discrepancy between the studies is unknown, but it could be because we used freshly prepared PBMCs instead of cryopreserved cell preparations. CD56high or CD56bright NK cells produce IFN‐γ 25. The proportions of these NK cells were similar in LADA and T1D patients. This result illustrates that, like T1D, LADA is also a Th1 response disorder. The frequency of FoxP3+ Treg cells was similar in all groups. However, the proportion of IL‐35+ cells among Treg cells in patients with LADA was similar to those in patients with T1D and T2D, but lower when compared to healthy controls. This may suggest a common functional defect of Treg cells in LADA, T1D and T2D patients. This idea was further supported because the circulating concentrations of IL‐35 in patients with LADA were similar to patients with T1D and T2D, but lower than in than healthy controls. We have previously shown that decreased IL‐35 production by Treg cells is associated with a functional defect 46. Also, previous findings of a decreased frequency of FoxP3+ cells among CD4+CD25intermediate cells in LADA patients when compared to healthy controls 5, and a decreased Foxp3 gene expression in CD4+ T cells in LADA patients, are considered with defective Treg cell functionality 56. In patients with T1D, it has been reported that Treg cells lose their FoxP3 expression due to DNA methylation in the Foxp3 gene, which may cause a defective function in Treg cells and result in a decreased number of Treg cells 57. Interestingly, Li et al. also reported an abnormal DNA methylation in peripheral CD4+ T cells of LADA patients 33, which indicates a dysfunction of Treg cells 58. Breg cells play an important role in maintaining the autoimmune and/or inflammatory response 35, 36. We found that the frequency of Breg cells was higher in LADA patients compared to all other groups. These results are in agreement with a previous report, where Deng et al. found the lowest frequency of IL‐10‐producing B cells in T1D patients when compared to LADA and T2D patients 32. Our observed lower proportion of IL‐35+ cells among Breg cells in T1D than LADA patients suggests that the response of IL‐35+ Breg cells in LADA patients is similar to that of T2D patients, which might protect against β cell destruction by immunological insults in LADA patients. Although the proportions of IL‐35+ Breg cells and IL‐35+ tolerogenic APCs were higher in LADA than in T1D patients, the circulating concentrations of IL‐35 were similar in LADA and T1D patients. This could be due to other IL‐35‐producing immune cells, such as iTr35 59 and macrophages 60. These particular populations have not been investigated in the present study. APCs (CD123– APCs) and pDCs (CD123+ APCs) prime T cells to react against the insulin‐producing β cells. In the present study, we found that the numbers of APCs were lower in LADA than in T1D patients. This indicates that the initial immune response is lower in LADA patients, which might cause a delay in the development of an autoimmune response. Tolerogenic APCs protect β cells in animal models of T1D 44, and Dixon et al. reported that tolerogenic APCs produce IL‐35 in human peripheral blood 34. We analysed the frequency of IL‐35+ cells among APCs in our study and found that IL‐35+ cells were more frequent in LADA than in T1D patients, which may contribute to a milder disease development. In a previous study, we found that systemic treatment with IL‐35 in two different animal models of T1D prevented destruction of β cells 46. However, as this is an association study it is impossible to determine whether or not the immunological differences are causal. We observed changes in the proportions of different immune cells in LADA patients compared to healthy controls and other diabetes patients. Our conclusion is that the changes in APC and Breg cell numbers in LADA patients are more similar to those also observed in T2D patients, whereas the changes in NK cell numbers are more similar to those found in T1D patients. There are no differences in IL‐35 concentrations and IL‐35+ Treg cell numbers in between LADA, T1D and T2D patients, but in all three conditions these are lower than the healthy individuals. Thus, our analysis of several subsets of immune cells reveals that LADA patients present a mixture of cellular immunological changes with features overlapping with both T1D and T2D. Acknowledgements The technical assistance from research nurses Violeta Armijo Del Valle, Rebecka Hilmius and Karin Kjellström is gratefully acknowledged. The present study was supported financially by the Swedish Research Council (2017‐01343, 921‐2014‐7054), EXODIAB, the Swedish Diabetes Foundation, Diabetes Wellness Sverige, the Swedish Child Diabetes Fund, SEB Diabetesfonden, O.E. och Edla Johanssons vetenskapliga stiftelse and the Novo Nordisk Foundation. Disclosures The authors declare that there are no conflicts of interest associated with this paper. Author contributions M. M. and K. S. contributed to the design of the study, conducted experiments, acquired and analysed data, and participated in writing the manuscript. Z. L., D. E., J. S. and S. S. contributed to the design of the study, analysed data and participated in the writing of the manuscript. P. O. C. designed the study, analysed data and wrote the manuscript. All authors read and approved the final version. K. S. is the guarantor of this work and, as such, had access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Supporting Information References
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Scooped by
Gilbert C FAURE
January 10, 2019 4:22 AM
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Abstract Between 50% and 86% of patients with autoimmune hepatitis (AIH) relapse after immunosuppression withdrawal; long‐term immunosuppression is associated with increased risk of neoplasias and infections. Chloroquine diphosphate (CQ) is an immunomodulatory drug that reduces the risk of flares in rheumatologic diseases. Our aims were to investigate the efficacy and safety of CQ for maintenance of biochemical remission of AIH in a double‐blind randomized trial and to define a subgroup that obtained a greater benefit from its use. A total of 61 patients with AIH in histologic remission (90.1% AIH type 1 [AIH‐1]) were randomized to receive CQ 250 mg/day or placebo for 36 months. Of the 61 patients, 31 received CQ and 30 placebo. At baseline, clinical, laboratory, histologic findings, and human leukocyte antigen (HLA) profile were similar between the two groups. Relapse‐free survival was significantly higher in the CQ group compared to the placebo group (59.3% and 19.9%, respectively P = 0.039). For those patients completing 3‐year treatment, relapse rates were 41.6% and 0% after CQ and placebo withdrawal, respectively. Factors associated with a higher risk of relapse in multiple Cox regression were placebo use (hazard ratio, 2.4; 95% confidence interval [CI], 1.055.5; P = 0.039) and anti‐soluble liver antigen/liver‐pancreas (anti‐SLA/LP) seropositivity (hazard ratio, 5.4; 95% CI, 1.91‐15.3; P = 0.002). Although it was not possible to define a subgroup that obtained a greater benefit from CQ according to anti‐SLA/LP reactivity or HLA profile, 100% of patients who were anti‐SLA/LP‐positive (+) relapsed with placebo compared to 50% with CQ (P = 0.055). In the CQ group, 54.8% had side effects and 19.3% interrupted the drug regimen. Conclusion: CQ safely reduced the risk of relapse of AIH, but it was not possible to define a subgroup that obtained a greater benefit with CQ use, probably because of sample size. Abbreviations – negative + positive AIH autoimmune hepatitis AIH‐1/2 AIH type 1/2 ALT alanine aminotransferase anti‐LKM1 anti‐liver/kidney microsomal antibody type 1 anti‐SLA/LP anti‐soluble liver antigen/liver‐pancreas CI confidence interval CQ chloroquine diphosphate HLA human leukocyte antigen HLA‐DQ human leukocyte antigen isotype DQ HLA‐DR human leukocyte antigen isotype DR IAIHG International Autoimmune Hepatitis Group IgG immunoglobulin G The goal of treatment in autoimmune hepatitis (AIH) is to achieve normalization of aminotransferase and immunoglobulin G (IgG) levels with absent or minimal histologic activity to prevent disease progression. A sustained remission of treatment after its withdrawal is the most desirable endpoint. However, most patients relapse within the first year and need reintroduction of treatment, and only approximately 20% achieve this goal.1-3 Patients who present multiple relapses evolve more frequently for liver cirrhosis, death due to liver disease, and the need for liver transplantation than those who remain in sustained remission.4 A significant advance in the treatment of AIH would be the identification of drugs with a good safety profile and few side effects that would allow the maintenance of AIH remission after withdrawal of the immunosuppressive treatment. Antimalarial drugs have been prescribed for almost 400 years and are used in the treatment of systemic lupus erythematosus with benefits in symptomatic control, general and disease‐free survival, and reduction of severity of disease exacerbations.5, 6 Chloroquine diphosphate (CQ) has multiple mechanisms of action in immune response; this could justify its use in the treatment of AIH. CQ 250 mg/day was used for maintenance of disease remission after immunosuppression withdrawal in a preliminary pilot study with 14 patients in histologic remission; in that study, the CQ‐treated group had a 6.49‐fold lower risk of disease relapse.7 In 2 patients who were anti‐soluble liver antigen/liver‐pancreas (anti‐SLA/LP)(+) and who relapsed after CQ withdrawal, its reintroduction as monotherapy (on request) resulted in normalization of aminotransferases and, in one case, histologic remission. The aims of this study were to evaluate whether CQ is safe and effective in preventing relapse of AIH in patients with histologic remission of the disease after immunosuppression withdrawal and whether there is a specific patient profile in which it could be more beneficial. Participants and Methods Trial Design and Participants This was an interventional, single‐center, randomized, double‐blind, controlled, parallel‐group trial of 36 months of CQ versus placebo in patients with AIH in histologic remission. The authors designed the protocol, monitored patients, acquired and maintained the data, and performed statistical analysis. The protocol was approved by the local ethics committees and registered in the ClinicalTrials.gov database (NCT01980745). All subjects signed a written informed consent before enrollment, and the study was performed according to good clinical practice and the Declaration of Helsinki. Patients 18 to 70 years of age with a probable/definite diagnosis of AIH according to International Autoimmune Hepatitis Group (IAIHG) criteria8 who had biochemical and histologic remission after immunosuppressive therapy were eligible for enrollment in this study. Exclusion criteria were overlapping syndromes with primary biliary cholangitis and primary sclerosing cholangitis, decompensated liver cirrhosis (presence of ascites, hepatic encephalopathy, spontaneous bacterial peritonitis, acute variceal bleeding, or Child‐Turcotte‐Pugh B and C classification), enrollment in a previous study with antimalarial drugs, and pregnancy. Interventions All patients received initial treatment with azathioprine and prednisone, most of them in doses of 50 mg/day and 30 mg/day, respectively. There were monthly reductions in prednisone dose and increases in dose of azathioprine if necessary (up to 2 mg/kg/day if tolerated) in order to maintain the levels of aminotransferases within the normal reference range with the lowest dose possible. Doses of prednisone ranged from 5 to 15 mg/day and azathioprine from 50 to 150 mg/day during maintenance treatment. After normalization of hepatic enzymes with stable doses of the drugs, there were no additional adjustments in the doses (except in the cases of adverse effects that justified some modification) until the liver biopsy was performed to evaluate histologic remission, which never occurred before 18 months of normal aminotransferases and 2 years of treatment. Histologic remission was defined as absence or presence of minimal interface hepatitis (hepatitis activity index <4) according to the IAIHG definition.2 The histologic evaluation was performed by experienced liver pathologists according to the Brazilian Society of Pathology.9 Following confirmation of histologic remission, patients were randomized to receive either a fixed dose of CQ (250 mg/day, drug A) or placebo (drug B). CQ was chosen because it was the only formulation available for dispensing by the public health system. In the first month after enrollment, patients received CQ or placebo in addition to the immunosuppressive regimen that induced histologic remission; this was azathioprine and prednisone in all cases. During the second month, azathioprine was discontinued immediately, with weekly weaning of 2.5 mg of the prednisone maintenance dose. Subsequently, the patients remained on drug A or drug B for up to 36 months, disease relapse, significant side effects, pregnancy, or willing withdrawal from the study. Follow‐up was for at least 1 year after the end of the protocol. Primary Outcomes The primary outcome was the maintenance of biochemical response following immunosuppression withdrawal, which was defined as aminotransferase levels within the normal reference range. Relapse of AIH was defined as elevation of aminotransferase levels above twice the normal upper reference value in at least two different measurements taken within a 15‐day interval during which there was no tendency for liver enzymes to fall or stabilize. Routine blood tests and clinical evaluation were performed monthly during the first 6 months of the study, every 2 months or earlier if necessary until the end of the first year, and every 3 months until the end of the study. Secondary Outcomes Secondary outcomes were to determine whether there was a specific group of patients who would benefit most from the ingestion of CQ and to assess the safety profile of the drug. Autoantibody Testing Anti‐nuclear, anti‐smooth muscle, anti‐liver/kidney microsomal antibody type 1 (anti‐LKM1), anti‐liver cytosol type 1, and anti‐mitochondrial antibodies were detected by indirect immunofluorescence; performed on frozen unfixed rodent substrate that included kidney, liver, and stomach; and were considered positive if the titers were greater than or equal to 1:40 according to IAIHG diagnosis criteria.10 The fluorescent patterns of anti‐nuclear and anti‐microfilament antibodies were detected by indirect immunofluorescence on human epithelial type 2 (HEp2) cells and human fibroblasts. Anti‐SLA/LP antibodies were detected by enzyme‐linked immunosorbent assay using the commercial kits Quanta Lite SLA (Inova Diagnostics Inc., San Diego, CA) and/or anti‐SLA/LP IgG (EUROIMMUN, Luebeck, Germany), according to the manufacturer’s instructions, which consider positive values as ≥25 units and ≥1, respectively. Human Leukocyte Antigen Typing Human leukocyte antigen (HLA) typing was performed with DNA samples previously extracted using the dodecyltrimethylammonium bromide (DTAB)/cetyltrimethylammonium bromide (CTAB) technique and stored at −20°C. For the amplification reaction of the HLA isotype DR (HLA‐DR) and HLA isotype DQ (HLA‐DQ) genes, specific primers were used to test the polymorphism in exon 2. The presence of the allele or allele group was based on the presence or absence of the amplified product. The determination of alleles or groups of class II alleles was performed by polymerase chain reaction (primer‐specific sequence) with Micro SSP HLA DNA typing trays (One Lambda). The reaction was performed following the manufacturer’s instructions. Treatment‐Related Side Effects The relationship between the drug and side effects was defined according to the Naranjo algorithm, which determines the probability that an adverse reaction to the drug is actually due to the drug and not the result of other factors.11 The severity of side effects was classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03.12 All patients underwent ophthalmologic evaluation before inclusion in the study and at least one annual evaluation according to protocol thereafter for the detection of retinal antimalarial deposition signals. Sample Size Determination Our main hypothesis to be tested was whether there is a difference in AIH relapse rate between patients who use CQ and patients who use placebo. The rates of AIH relapse were estimated in a pilot study, which resulted in relapse of 23.5% in the CQ group and 72.2% in a historical group without medication.7 Assuming an accrual rate of 7 patients per year, a sample size of 31 patients was defined to allow the study to estimate a minimum detectable difference of 37.2% using a 2‐sided Fisher’s exact test with 80% power at a 5% level of significance. Randomization Eligible patients were randomly assigned to receive CQ or placebo after withdrawal of immunosuppression by simple randomization and were allocated to each group in a 1:1 ratio. A medical assistant unaware of the study generated the random allocation sequence by a draw with 62 numbered papers: 31 named drug A and 31 named drug B. One of the trial investigators enrolled and assigned participants to the proposed interventions after randomization. After patients agreed to participate in the study, the principal investigator checked each patient’s list number and determined which drug was destined for that number. The drugs were formulated at the hospital pharmacy as tablets with an identical external appearance. The research assistant and the patients were all blind to the type of medication used. After the study was completed, the pharmacist revealed the composition of the tablets initially named drugs A and B to the researchers. The secrecy of the formulation was maintained until the end of the study. Statistical Analysis For statistical analysis, all patients undergoing randomization (except those who refused medication immediately after enrollment) were included regardless of the time of follow‐up or withdrawal due to side effects or pregnancy. Relapse‐free survival was defined as the time between a patient’s enrollment and the end of treatment (36 months) or the occurrence of side effects leading to discontinuation of the drug and the subject being censored from the study. Time to relapse was defined as the period between the immunosuppression withdrawal and the biochemical relapse of the disease. Relapse‐free survival curves were estimated using the Kaplan‐Meier method and compared by the log‐rank test. The hazard ratios with their respective 95% confidence intervals (CIs) were estimated using the simple Cox regression. A multivariate regression model was fitted using clinically relevant covariates for AIH relapse defined in previous studies. The interactions between the drug and autoantibody reactivity and HLA profile were evaluated by multivariate Cox regression to investigate whether there would be any subgroup of patients with additional benefits with the use of CQ. The proportional hazard assumption was verified using the Schoenfeld residues.13 Baseline characteristics were compared with Fisher’s exact test for categorical variables and t test or Mann‐Whitney test for quantitative variables. The normality and homoscedasticity hypotheses were evaluated by the Anderson‐Darling14 and Levene15 tests, respectively. All hypothesis tests were 2‐sided and considered P < 0.05 as statistically significant. R, version 3.0.2 (R Core Team, 2013), was used for all calculations. Results Patients were enrolled from May 2002 to October 2011, and the follow‐up ended in 2015. All patients except 1 had a definite diagnosis of AIH before treatment according to IAIHG criteria. One patient who could not undergo a liver biopsy due to the presence of ascites had “probable diagnosis” with a score of 13 points. Clinical, laboratory, and histologic data at diagnosis are shown in Table 1. A total of 62 patients were randomized to receive CQ 250 mg/day (n = 31) or placebo (n = 31) for 36 months. Except for 1 patient in the placebo group who refused treatment immediately after randomization, all patients received their assigned treatment (Fig. 1). The mean follow‐up time was 3,526 ± 1,233 days. The median time of drug intake was 329 days (22‐1,275 days).The median age at diagnosis of AIH was 26.86 years (2.7‐63.4 years), and mean treatment duration time before immunosuppression withdrawal was 5.9 ± 4.08 years (median time, 4.56 years). Approximately 90% had AIH‐1; 23.3% of all patients had anti‐SLA/LP seropositivity. Out of 61 patients, 56 had an initial liver biopsy (91.8%), and hepatic cirrhosis was detected in 52.4%. At baseline, demographic, clinical, laboratory, and histologic data were similar between CQ and placebo groups (Table 2). When the HLA profile was analyzed, HLA‐DR3 (26/61 patients) and HLA‐DR13 (26/61 patients) were the most frequent, followed by HLA‐DR4 (17/61 patients) and HLA‐DR7 (13/61 patients). In the CQ group, there was a lower frequency of HLA‐DR3 (35.5% versus 50%), HLA‐DR4 (19.3% versus 36.7%), and HLA‐DR7 (16.1% versus 26.7%) compared to the placebo group and a higher frequency of DR13 (51.6% versus 33.4%). However, these differences did not reach statistical significance (Table 3). There was no specific HLA‐DR profile significantly related to anti‐SLA/LP seropositivity, although 50% harbored DR3. Data at Diagnosis n (%) Presentation of disease at onset Asymptomatic* 6 (9.8) Acute exacerbation of chronic AIH † 44 (72.2) Insidious onset ‡ 11 (18) Extrahepatic autoimmune diseases 8 (13.1) § AST ‖ (mean ± SD) 26.04 ± 20.1 ALT ‖ (mean ± SD) 22.83 ± 21.2 Alkaline phosphatase ‖ (mean ± SD) 1.73 ± 1.05 Gamma‐glutamyltransferase ‖ (mean ± SD) 4.91 ± 4.1 Albumin g/dL (mean ± SD) 3.37 ± 0.68 Total bilirubin mg/dL (mean ± SD) 9.13 ± 9.08 INR (mean ± SD) 1.5 ± 0.41 Gamma globulin ‖ (mean ± SD) 1.97 ± 1.06 Histologic findings ¶ n (%) Rosettes of hepatocytes 33 (58.9) Plasma cells inflammatory infiltrate 23 (41) Interface hepatitis 52 (92.8) Submassive necrosis 4 (7.14) * Diagnosis due to elevation of liver enzymes or ultrasonographic alterations during follow‐up of extrahepatic diseases (rheumatologic and endocrinologic diseases). † Presentation similar to viral hepatitis, with jaundice, coluria, and fecal acolia with or without signs of portal hypertension. ‡ Nonspecific symptoms of fatigue, general ill health, right upper quadrant pain, lethargy, malaise, anorexia, weight loss, nauseas, pruritus, polyarthargia. § Thyroid disease (n = 2), ulcerative colitis (n = 2), systemic erythematosus lupus (n = 2), Sjögren syndrome (n = 1), idiopathic thrombocytopenic purpura (n = 1). ‖ Values are shown as number of times above the upper normal limits. ¶ Percentage calculated considering 56 patients. Of these patients, 5 patients did not have liver biopsy at diagnosis due to presence of ascites and/or coagulopathy. Liver fibrosis staging according to Brazilian Society of Pathology Classification of chronic hepatitis.9 Abbreviations: AST, aspartate aminotransferase; INR, international normalized ratio. CQ (n = 31) Placebo (n = 30) P Value Female sex (%) 26 (83.9) 26 (86.7) 1 Age at diagnosis of AIH (mean ± SD) 29.4 ± 17.9 30.5 ± 17.1 0.98 Age at study inclusion (mean ± SD) 37.7 ± 16.1 39.1 ± 16.9 0.8 AIH classification (%) 0.27 Type 1 29 (93.5) 26 (86.7) Type 2 1 (3.2) 0 (0) Type 3 (isolated anti‐SLAP/LP) 1 (3.2) 1 (3.3) No serologic markers 0 (0) 3 (10) Anti‐SLA/LP seropositivity (%)* 6 (20) 8 (26.7) 0.76 Antimicrofilament seropositivity (%)† 11 (37.9) 16 (55.2) 0.29 Liver cirrhosis at diagnosis (%)‡ 14 (50) 18 (64.3) 0.42 Advanced fibrosis (F3/F4) at study inclusion (%)§ 18 (58) 16 (53.3) 0.8 Arterial hypertension (%) 3 (9.7) 5 (16.7) 0.47 Diabetes mellitus (%) 4 (12.9) 5 (16.7) 0.73 Normal gamma globulin/IgG levels|| at inclusion (%) 20 (64.5) 23 (76.7) 0.4 Immunosuppression at histologic remission (mg/day) (mean ± SD) Azathioprine 90.7 ± 26.6 97.5 ± 22.1 0.47 Prednisone 8.5 ± 2.8 8.9 ± 2.1 0.8 Treatment time before inclusion at study (years, mean ± SD) 6 ± 4.55 5.8 ± 3.53 0.44 *In 1 patient of the CQ group, it was not possible to assess anti‐SLA/LP seropositivity in initial screening. Regarding differences between patients who were anti‐SLA(+) or (–), patients who were anti‐SLA(+) had lower levels of alkaline phosphatase, gamma‐glutamyltranspeptidase, and bilirubin. There were no differences in histologic findings, IgG levels, albumin, INR, and aminotransferases (data not shown). †Information available in 29 patients in each group. ‡Two patients in each group did not have liver biopsy at diagnosis due to presence of ascites and/or coagulopathy. Liver fibrosis staging according to Brazilian Society of Pathology Classification of chronic hepatitis.9 §Liver biopsy was insufficient to stage liver fibrosis in 1 patient without impairing the degree of inflammatory activity. Classification of liver fibrosis according to METAVIR fibrosis score. ||Gamma globulin and/or IgG levels within the normal range (gamma globulin <1.5 g/dL and IgG <1,538 mg/dL). Abbreviation: INR, international normalized ratio. HLA Profile Chloroquine (n = 31) n (%) Placebo (n = 30) n (%) DR1 4 (12.9) 2 (6.7) DR3 11 (35.5) 15 (50)* DR4 6 (19.3) 11 (36.7)† DR7 5 (16.1) 8 (26.7)‡ DR8 0 (0) 1 (3.3) DR9 2 (6.4) 0 (0) DR10 1 (3.2) 0 (0) DR11 4 (12.9) 2 (6.7) DR13 16 (51.6) 10 (33.4)§ DR14 4 (12.9) 1 (3.3) DR15 3 (9.7) 4 (13.3) DR16 3 (9.7) 2 (6.7) DQ02 14 (45.1) 14 (46.7) DQ04 2 (6.4) 5 (16.7) DQ05 10 (32.2) 5 (16.7) DQ06 16 (51.6) 12 (40)|| DQ07 4 (12.9) 3 (10) DQ08 6 (19.3) 5 (16.7) There were no significant statistical differences in HLA‐DR and HLA‐DQ profiles between the CQ and placebo groups (P > 0.05). *P = 0.3 †P = 0.16 ‡P = 0.36 §P = 0.2 ||P = 0.44 Primary Outcomes Patients in the CQ group completed the study more frequently (38.7% versus 13.3%) and took the medication for a longer period compared to those in the placebo group (616 ± 484.8 versus 386 ± 377.4 days, respectively). The mean alanine aminotransferase (ALT) level at relapse was 271.1 ± 301.5 U/L (reference value <31 U/L). Patients who relapsed restarted immunosuppressive therapy without clinical or laboratory signs of liver dysfunction. Factors associated with higher risk of AIH relapse by univariate Cox regression were anti‐SLA/LP seropositivity, placebo randomization, and HLA‐DR3 profile (Fig. 2; Table 4). In the multivariate analysis, factors associated with AIH relapse were anti‐SLA/LP seropositivity and placebo randomization. Relapse‐free survival was significantly lower in the placebo group compared to the CQ group (CQ group, 59.3% [43.1%‐81.6%] versus placebo group, 19.9% [7.9%‐50.2%]; hazard ratio, 2.4; 95% CI, 1.05‐5.5; P = 0.039) and in patients with anti‐SLA/LP seropositivity (hazard ratio, 5.4; 95% CI, 1.91‐15.3; P = 0.002) (Table 5). After CQ/placebo withdrawal at the end of the study at 3 years, 5 of 12 patients (41.6%) in the CQ group relapsed compared to none of 4 patients in the placebo group. Relapse‐free survival 6 months after the study ended was 60% in the CQ group and 100% in the placebo group (P = 0.03) because no patient in the placebo group who maintained biochemical remission during the 3‐year follow‐up relapsed after the study’s end. Variable Hazard Ratio (95% CI) P Value Droga B (placebo) 2.51 (1.19‐5.25) 0.015 Anti‐SLA/LP seropositivity 2.84 (1.34‐5.98) 0.006 HLA‐DR3 2.04 (1‐4.16) 0.049 HLA‐DQ6 0.53 (0.25‐1.1) 0.089 HLA‐DR13 0.63 (0.3‐1.31) 0.21 HLA‐DR7 0.77 (0.32‐1.89) 0.57 HLA‐DR4 2.02 (0.96‐4.27) 0.066 Antimicrofilament seropositivity 1.24 (0.61‐2.54) 0.55 Classification of AIH* AIH‐2 0 (0‐infinity) 0.99 AIH‐3 0.26 (0.04‐1.93) 0.19 Liver cirrhosis at diagnosis 0.71 (0.34‐1.49) 0.36 Advanced fibrosis at study randomization (F3/F4) 1.09 (0.54‐2.24) 0.8 Normal gamma globulin level at randomization 1.14 (0.51‐2.56) 0.74 Diabetes mellitus 0.38 (0.09‐1.58) 0.18 Age at diagnosis 1 (0.98‐1.02) 0.71 *Considering type 1 AIH as the reference group; (AIH‐3 ‐ anti‐SLA/LP as sole marker). Variable Hazard Ratio (95% CI) P Value Placebo intake 2.4 (1.05‐5.5) 0.039 Anti‐SLA/LP seropositivity 5.4 (1.91‐15.3) 0.002 Age at diagnosis of AIH 1 (0.98‐1.03) 0.99 Normal gamma globulin levels at inclusion 0.54 (0.2‐1.45) 0.22 HLA‐DR3 1.84 (0.73‐4.63) 0.19 HLA‐DR13 0.9 (0.29‐2.79) 0.86 Secondary Outcomes We investigated whether a subgroup showed greater benefit with antimalarial use. Of the 14 patients who had anti‐SLA/LP reactivity, 8 were randomized for placebo and 6 for CQ. Of the 6 patients randomized to receive CQ, 3 relapsed compared to all patients in the placebo group (50% versus 100%, respectively; P = 0.055). The anti‐SLA/LP(+) patients had significantly higher levels of aminotransferases at AIH relapse compared to the anti‐SLA/LP‐negative (–) group (460 ± 429 U/L versus 165 ± 120 U/L; P = 0.048; normal reference value up to 31 U/L). The aminotransferase levels in the relapse of patients with anti‐SLA/LP(+) were higher in the placebo group compared to the CQ group (541 ± 475 U/L versus 243.3 ± 183 U/L), but this difference was not statistically significant (P = 0.33). It was not possible to establish an interaction between the drug and anti‐SLA/LP seropositivity (hazard ratio, 2.17; 95% CI, 0.57‐8.23; P = 0.254) by multivariable Cox analysis. It was also not possible to identify a subgroup achieving the greatest benefit according to HLA profile, AIH type, and antimicrofilament seropositivity (Table 6). Variable Hazard Ratio (95% CI) P Value Antimicrofilament seropositivity 3.82 (0.75‐19.38) 0.10 Anti‐SLA/LP 1.41 (0.28‐7.03) 0.67 HLA‐DR3 0.87 (0.2‐3.86) 0.85 HLA‐DR4 0.98 (0.17‐5.76) 0.98 HLA‐DR7 0.49 (0.07‐3.24) 0.46 HLA‐DR13 0.89 (0.19‐4.13) 0.88 Side Effects In the CQ group, 54.8% of the patients had some side effects compared with 16.7% in the placebo group. The discontinuation due to side effects occurred in 19.3% of patients in the CQ group and 10% in the placebo group. The majority of adverse events in the CQ group were classified as possible according to the Naranjo algorithm; none were classified as definite (Table 7). Adverse Event Chloroquine (n = 31) n (%) Placebo (n = 30) n (%) Any AE 17 (54.8) 5 (16.7) Discontinuation due to AE 6 (19.3) 3 (10) Classification according to Naranjo algorithm Definite (0) Definite (0) Probable (4)* Probable (0) Possible (12)† Possible (3) Doubtful (1)‡ Doubtful (2) Grade 3/4 0 0 Grade 2 Neuropathy 2 (6.4) 0 Dermatologic 6 (19.3) 2 (6.6) Seizure 1 (3.2) 0 Arthralgia 0 1 (3.3) Grade 1 Headache 2 (6.4) 1 (3.3) Worsening renal function 1 (3.2) 0 Dyspepsia 3 (9.7) 0 Myalgia 1 (3.2) 0 Dermatologic 3 (9.7) 0 Retinopathy 1 (3.2) 1 (3.3) *Probable causality between drug and adverse reaction: exfoliative dermatitis (1), neuropathy (1), pruritus (1), impairment of renal function (1). †Possible causality between drug and adverse reaction: headache (2), convulsion (1), nausea (1), cutaneous hyperpigmentation (3), pruritus (4), retinopathy (1), cutaneous papules (1), neuropathy (1), inappetence (1). ‡Doubtful causality between drug and adverse reaction: myalgia (1). Abbreviation: AE, adverse event. There were no grade 3 or 4 side effects. Most side effects in the CQ group were dermatologic and included pruritus (5 patients), skin darkening (3 patients), sporadic pruritic papules (1 patient), and skin lesions similar to pellagra (1 patient). Withdrawal of medications was necessary in only one case; the other symptoms were controlled with antihistamines, skin hydration, and vitamin B3 replacement. The most serious side effects were seizures (1 patient), worsening renal function (1 patient), and peripheral neuropathy (2 patients). Despite drug withdrawal improvement, there was no security that these adverse events were drug related. According to the Naranjo algorithm, none of the side effects were classified as definite in relation to the drug. The seizure was classified as possible (although the patient reported irregular use of anticonvulsants), the impaired renal function as probable (the patient had a further diagnosis of Gitelman’s syndrome), and the two cases of neuropathy as likely and possible (1 patient had the diagnosis of carpal tunnel syndrome). Two patients, 1 in the CQ group (after 19 months) and 1 in the placebo group (after 24 months), had suspicion of CQ deposition in the macula during ophthalmologic evaluations. Although none of these patients had visual loss and the suspicion was not confirmed during follow‐up, the drug was discontinued in both cases. Discussion This was a double‐blind randomized study of 61 patients with AIH in biochemical and histologic remission that demonstrated CQ was safe and effective to significantly reduce the risk of relapse 2.5 times in a period up to 3 years after immunosuppression withdrawal. Antimalarial drugs have immunomodulatory effects and have already been used in other autoimmune diseases. In systemic lupus erythematosus, for which they were better studied, they have beneficial effects on general and disease‐free survival, with a reduction in the risk and delay of quiescent disease reactivation, improvement of the metabolic profile, and protection against occurrence of serious infections.5, 6, 16 Their possible mechanisms of action in the pathophysiology of AIH include inhibition of the processing and presentation of antigens, inhibition of cytokine release (interleukins 1, 2, and 6; tumor necrosis factor α; and interferon‐γ), inhibition of the T helper 17 response (inhibition of the release of interleukins 6, 17, and 22), decreased natural killer cell activity, inhibition of polymorphonuclear chemotaxis, and inhibition of the activity of cytotoxic T lymphocytes and clusters of differentiation (CD)4+ T lymphocytes with autoreactivity.17, 18 In the current study, possible biases that could have interfered with the results were avoided. Patients were blindly randomized, the immunosuppressive treatment was maintained along with CQ for 1 month before withdrawal, and all subjects had at least 18 months of aminotransferase normalization and more than 2 years of immunosuppressive treatment before randomization, as recommended by IAIHG in relation to attempted withdrawal of medication. It appears that duration of treatment may influence relapse rates because patients treated continuously for more than 4 years had more sustained remission rates than those treated for less than 4 years.19 In the current study, the mean duration of treatment before immunosuppressive withdrawal was approximately 6 years. Liver biopsies were performed to confirm histologic remission because approximately 55% of the patients with liver enzymes and normal IgG levels still present interface hepatitis in the biopsy and disease relapse in case of treatment discontinuation.20 However, even normal histology does not completely rule out the possibility of disease relapse because it may occur even in patients with histologic remission.21 The option for the maintenance of immunosuppressive treatment that induced histologic remission during the first month of the study was justified because there is a delay in achieving effective plasma concentrations and stable serum concentrations of CQ are only achieved after 4 to 6 weeks.18 Another reason for this choice was to reduce the risk of confusing possible enzymatic alterations related to immunosuppression withdrawal with hepatotoxicity by the antimalarial, at least in the first month of introduction of a new medication.22 The beneficial effect of CQ on protection against disease relapse may be evidenced by several findings in this study. Patients who used the medication were more apt to complete the study and used the medication for a longer period than those who used the placebo. In addition, after cessation of the drug at the end of the study, relapse in the CQ group was significantly higher than in the placebo group, showing that the antimalarial was actually protecting patients against relapse. The relapse‐free survival was 59.3% in the CQ group and 19.9% in the placebo group during the 3 years of treatment, and this difference was also statistically significant. It can be argued that 59.3% is still a low relapse‐free survival when compared to maintenance of lifelong immunosuppressive therapy, with or without corticosteroids.1-3 These options are associated with the countless side effects of corticosteroids and the increased risk of neoplasias and bacterial and fungal infections secondary to thiopurine drugs, which justifies the search for new therapies.23 The use of immunomodulatory drugs without immunosuppressive properties to maintain AIH remission could add options to this scenario. Another aspect to consider is the adopted definition of disease relapse as the elevation of aminotransferase levels above twice the upper normal limit as proposed by the 1999 IAIHG criteria8 rather than 3 times as proposed by other guidelines.1, 2 This concept certainly increased the rates of relapse diagnosis. Another controversial point is the discontinuation of treatment because of side effects. In several cases, the treatment was interrupted because of the occurrence of clinical manifestations that were not clearly related to CQ. The withdrawal rate due to adverse effects was 19.3% in the CQ group, which may have influenced the final results because these patients did not relapse while taking the medication but after antimalarial withdrawal. In addition, this relapse rate occurred over a 3‐year period during which the patients remained without the side effects of corticosteroids and with the possibility of secondary gains in metabolic profile and bone density. With respect to autoantibody reactivity, anti‐SLA/LP and anti‐LKM1 are associated with an increased risk of relapse, and a meta‐analysis suggested never stopping therapy when the former is present.24 In the current study, anti‐SLA/LP(+) patients had higher levels of aminotransferases at disease relapse compared to anti‐SLA/LP(–) patients, and the relapse rates in this subgroup were 100% in the placebo group and 50% in the CQ group. However, when attempting to establish the relationship between anti‐SLA/LP reactivity and the drug used, the difference was not statistically significant; this may be related to sample size and should be confirmed in further studies. A study reporting a 20‐year experience with AIH treatment in childhood25 showed that all anti‐LKM1(+) patients relapsed after treatment discontinuation. In our series, there was one case of AIH‐2 in the CQ group. This patient completed the study without relapse and had biochemical relapse (aspartate aminotransferase, 197 U/L; ALT, 531 U/L [normal value, <31 U/L]) after approximately 90 days of its withdrawal. The HLA profile determines different clinical presentations and different responses to treatment, resulting in an autoimmune response of greater or lesser severity. Caucasian patients with HLA‐DR3 present earlier and more aggressive disease onset with inferior response to corticosteroid treatment compared to patients with HLA‐DR4.2, 26, 27 In Brazil, the susceptibility to AIH‐1 is primarily linked to HLA‐DR13, and there is a secondary association with DR3.28 In the current study, patients with AIH‐1 with HLA‐DR3 or HLA‐DR13 were the most common profiles. A study of AIH‐1 in North America evaluated 26 patients with non‐HLA‐DR3/DR4 and showed that patients in whom the only marker of susceptibility was HLA‐DR13 had lower rates of relapse after treatment withdrawal than those whose sole marker of susceptibility was HLA‐DR3.29 These findings are similar to those found in our study in which the presence of HLA‐DR3 was associated with a higher risk of relapse. Regarding HLA‐DQ, the two serotypes most frequently found in our series were DQ2 and DQ6. This finding is similar to findings in Brazilian and Argentinean patients with AIH‐1 in which susceptibility to the disease with the DR13‐DQ6 haplotype was observed.28, 30 The presence of HLA‐DQ6 resulted in a trend of lower risk of relapse (P = 0.089) with a risk ratio of 0.53, which should be evaluated in studies with a larger sample size. In a Brazilian study of HLA typing, a significant increase in DQ6 was observed in strong linkage disequilibrium with DR13.28 In the present study, 22 of the 28 patients with HLA‐DQ6 had at least one DR13 allele, which was associated with a lower risk of disease relapse compared to HLA‐DR3.29 Therefore, it remains an open question whether this protective effect is actually related to HLA‐DQ6 or to HLA‐DR13. Concerning the safety of antimalarial use, although hydroxychloroquine causes fewer side effects than CQ in patients with rheumatic diseases, CQ is 2‐3 times more potent than hydroxychloroquine.31 This probably influenced the rates of side effects. To minimize the risk of side effects, only patients with cirrhosis with preserved liver function were included because CQ binds moderately (60%) to plasma proteins and undergoes primarily hepatic metabolism by cytochrome P450 enzymes; in addition, impairments in liver function could affect drug metabolism.18, 32 Treatment suspension rates due to side effects were probably overestimated because of the inflexibility adopted in attributing a causal relationship between the symptoms presented during treatment and drug use. In addition, no higher incidence of side effects was observed when compared to published data, and there were no third‐ or fourth‐degree side effects in the current study. The most commonly reported side effects related to the use of CQ, occurring in up to 20% of patients, are dyspeptic and dermatologic symptoms, which were the most frequent in this series. Most of the time, dermatologic findings were reverted in this study with the use of antihistamines and hydration, without impairment of quality of life. The dyspeptic symptoms, which are more common in the first few weeks of treatment, usually improve over time and are minimized by dose reduction or fractionation and postprandial administration18, 33; this was not necessary in any of our patients. Neuromyopathy and seizures are rare events with few reported cases,18, 33, 34 which was also the case in the present study. One of the most feared side effects is retina damage by the antimalarial, which is usually irreversible and dose dependent and time dependent. It is present in less than 1% of patients after 5 years of use but reaches 20% after 20 years. In this series, retinopathy was detected in one case in the CQ group and one in the placebo group, without visual losses. Some risk factors associated with its occurrence are a cumulative total dose of 460 g or daily dose above 250 mg (or >3 mg/kg/day for individuals with low weight), age over 60 years, liver or kidney dysfunction, maculopathy, or retinal preexisting disease.35 In the case of a 50‐year‐old patient in the CQ group, injury suspicion occurred 19 months after starting the medication, the dose by weight was 2.84 mg/kg/day, and there was no renal or hepatic dysfunction. After drug discontinuation, the suspected lesion was completely reversed in less than 6 months of ophthalmologic follow‐up. It is worth remembering that, like antimalarials, long‐term use of corticosteroids is also associated with visual risk, such as glaucoma and cataracts, and a periodic ophthalmologic assessment is also recommended in patients who use long‐term medication. Some criticisms of this study could be made, such as the reduced sample size, the use of CQ instead of hydroxychloroquine, the early withdrawal of antimalarial drugs following the occurrence of some side effects even without a well‐documented causal relationship with the drug, and suspension in pregnancy. Despite the fact that hydroxychloroquine has a better safety profile in rheumatologic diseases, particularly with regard to retinopathy occurrence, a study with 940 patients with several rheumatic disorders revealed that 28% of patients had side effects with CQ compared to 15% with hydroxychloroquine. However, the interruption rate due to side effects was higher with CQ but due to clinical inefficacy was higher with hydroxychloroquine.36 Therefore, in the current study, the use of CQ was probably related to a higher rate of adverse events but maybe with a lower rate of disease relapse. CQ was the only formulation available for dispensing in our Public Health System, but the side effects most frequently reported in our patients were cutaneous and gastrointestinal; these were usually temporary and easy to alleviate. The placebo group also had a high rate of treatment discontinuation, probably reflecting the rigor of investigators in relation to the side effects due to the off‐label use of the drug in the treatment of AIH. On the other hand, the high rates of discontinuation were minimized by the evaluation of relapse‐free survival by the Kaplan‐Meier method because the patient could be observed until its censorship. Although the drug is considered relatively safe during pregnancy and already used in patients with antiphospholipid syndrome, there are no adequate and well‐controlled studies evaluating the safety and efficacy of CQ in pregnancy, justifying its withdrawal in the current study with more decreases in the sample size. The long enrollment period may have added some biases in this study, such as the changes in the AIH criteria for disease remission. The most recent AIH guidelines suggest that biochemical remission should include the normalization of aminotransferases and IgG levels. When the present study was planned and patient recruitment was started, the 1999 IAIHG recommendations, which are still valid, were the reference, and their considerations on complete remission, including histologic remission, were strictly followed. Liver biopsies at the end of the 3‐year CQ administration were not performed because the criteria for AIH relapse after full complete response were based only on biochemical results. However, this long period of time is justified by the low incidence/prevalence of the disease and the fact that the study is unicentric. This in turn also allowed better control of other confounding factors that could influence the results, such as inflammatory activity in liver biopsies before inclusion, standardization of the exams collected, criteria for suspension of the medication, and poststudy follow‐up. In conclusion, CQ safely and significantly reduces the risk of relapsing AIH in remission following discontinuation of immunosuppressive therapy; however, a subgroup with greater benefit from its use has yet to be defined. The side effects presented were mild and more frequently controlled with the use of symptomatic medication without affecting quality of life. The high rates of adverse effects and discontinuation of the drug were a concern in the study but should be interpreted with caution because of the stringent criteria for discontinuation of treatment in the case of side effects resulting from the “off‐label” treatment of AIH with CQ. Possibly the use of hydroxychloroquine instead of CQ could be a safer and better tolerated option to maintain AIH remission, but this should be investigated in further studies. The most outstanding finding of our study is the possibility of using a drug without immunosuppressive effect as part of the treatment of AIH. This creates the expectation that in the long term other drugs with immunomodulatory action can be formulated, allowing an increase of the scarce therapeutic options available for the treatment of the disease. In order to definitely include the drug as a possible therapeutic option, we should consider carrying out multicenter studies, including different countries and populations, to draw more solid conclusions regarding the role of antimalarial drugs in the treatment of AIH. Potential conflict of interest Nothing to report. References
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Rescooped by
Gilbert C FAURE
from Laboratory Medicine
April 21, 2018 2:16 AM
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There are at least 100 different autoimmune diseases that can affect any organ of the body. In the USA, it is estimated that autoimmune diseases affect between 5% and 8% of the population (14.7–23.5 million people), but individual autoimmune diseases like Addison’s disease, autoimmune neuropathies and ACNA vasculitis can certainly be classified as rare (affecting less than 200,000 people [USA] or fewer than 1 in 2000 of the population [Europe]). Nevertheless, autoimmune diseases often require lifelong treatment, are considerably more prevalent among women and are among the 10 leading causes of mortalilty in women under 65 years. It is also interesting to compare the prevalence of autoimmune diseases to, for example, heart disease (22%) and cancer (9%), which seem to have a much higher public profile [1] and research support with funding quoted in billions of dollars rather than the millions for autoimmune diseases. The incidence of autoimmune diseases is increasing. For example, the International Diabetes Atlas reported that in 2017 there were 10 million more adults worldwide with type I diabetes that in 2015, and if this trend continues, by 2045, 67 million Europeans will be living with type I diabetes [2]. Over the last 20 years, our ability to detect and quantify the antibodies involved in autoimmune diseases has improved, and gradually such tests are becoming incorporated onto high throughput platforms often in Clinical Blood Sciences. However, autoantibody measurements can lack clinical specificity and sensitivity and show high variation between platforms and tests, and certified reference materials are only recently available for only a small number of autoantibodies. The number of requests is increasing far beyond what would be expected for a tests for rare conditions and with low pretest probabilities. Numerical reports give no impression of the “uncertainty” of the result, and the enthusiasm for automated analysis and reporting puts the responsibility on the requesting clinician to interpret the result, often without understanding of the limitations of the test. This issue of Clinical Chemistry and Laboratory Medicine highlights the diversity of autoimmune disease from organ-specific conditions to systemic multiorgan disease. It reminds us that there is significant overlap between Clinical Chemistry and Immunology and that we have an important role the diagnosis and management of a variety of autoimmune disease. We can clearly see how much work is being done to investigate, understand and improve autoantibody testing, but there is still much to be done to optimise the requesting, the analytical process and the interpretation of these complex assays. More than 90% of cases of Addison’s disease have an autoimmune aetiology with antibodies to adrenal tissue and more specifically antibodies to 21-hydroxylase being markers of the disease. The symptoms of adrenal insufficiency can be subtle, but patients may also present as an emergency in Addisonian crisis. It is also well known that Addison’s is associated with other autoimmune endocrinopathies, e.g. Grave’s disease, hypothyroidism or type I diabetes. Manso et al. [3] describe a patient with autoimmune hypothyroidism where the patient’s family history led to the finding of positive anti-adrenal antibodies. Further investigation confirmed the diagnosis of subclinical Addison’s and treatment was started. Their literature review showed that only 1% of patients with autoimmune thyroid disease had anti-adrenal-cortex antibodies. Use of a more sensitive ELISA-based assay increased the detection of antibodies to 21-hydroxylase in children with autoimmune thyroid disease to approximately 4 percent, although even at this frequency, it would only be worth checking for anti-adrenal antibodies in patient with clinical features or family history. Surprisingly, anti-adrenal-cortex antibodies are found in a much greater proportion of patients with premature ovarian failure and in chronic hypoparathyroidism, so screening for antibodies to 21-hydroxylase in such patients may be useful. Classically, adrenal antibodies have been detected by indirect immunofluorescence. This can be technically demanding, and the interpretation of the staining needs considerable training and experience. del Pilar Larosa et al. [4] have reported the development of an ELISA for 21-hydroxylase, the enzyme, which is the important antigen within the adrenal cortex. They report good specificity and sensitivity for Addison’s disease so this may be a better method for supporting a diagnosis of Addison’s than the qualitative immunofluorescence techniques. Type I diabetes also has an autoimmune aetiology, and in patients where the clinical and biochemical presentation is complex, antibodies to insulin, glutamic acid decarboxylase (GAD), insulinoma antigen (IA2) and zinc transporter 8 (ZnT8) may be measured to improve the diagnostic power. Censi et al. [5] report the case of a patient with high blood insulin concentrations, hypoglycaemia and a high serum concentration of antibodies to insulin. It is important that we are aware of these antibodies because they loosely bind to insulin, which is gradually released independent of the normal feedback mechanism generating hypoglycaemia. These circulating anti-insulin autoantibodies also have the capacity to interfere in analyses that may be used in the investigation of unexplained hypoglycaemia, e.g. insulin, C-peptide and proinsulin immunoassays. There has been a dramatic improvement in the understanding of autoimmune mediated neuropathies over the last 20 years with autoantibody responses to gangliosides being implicated in their pathology. These conditions can be acute or chronic; they can affect the motor, sensory or autonomic nerves; and the presenting features can be variable. Improved diagnosis with prompt treatment could prevent irreversible nerve damage and consequent disability. Klehmet et al. [6] describe a multiplex approach to detecting antibodies IgG and IgM antibodies to 11 ganglioside antigens in patients with autoimmune neuropathies, other neurological conditions and normal subjects. The IgM anti-sulphatyl, IgM anti-GM1 and IgM anti-GD1b antibodies showed a higher frequency in the inflammatory neuropathy patients compared to patients with other neurological conditions. This is early data, and the authors suggest that further investigation is warranted, but it does show the power of multiplex-type analysis in identifying markers for further directed investigation. The term “phospholipid” antibodies like the term “neurological” antibodies describes antibodies to a number of distinct antigenic targets. In both situations, the tissues where these antigens are expressed are throughout the body generating widespread symptoms. Phospholipids, including cardiolipin and β2 glycoprotein 1, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol, are an integral part of all cell membranes. Antibodies to these components are associated with arterial or venous thrombosis. There are well-defined diagnostic criteria for antiphospholipid syndrome, but only measurements of IgG and IgM antibodies to cardiolipin and/or β2 glycoprotein 1 are included alongside lupus anticoagulant (an adaptation of a clotting test). Antibodies to the other phospholipids have generally been considered to have limited diagnostic value; however, Zhang et al. [7] have evaluated the clinical relevance of antibodies to a phosphatidylserine-prothrombin complex. They found increased concentrations of IgG and IgM antibodies to phosphatidylserine-prothrombin complex in their patients with antiphospholipid syndrome, although in their study, cardiolipin antibodies showed the best clinical performance. Interestingly, antibodies to the phosphatidylserine-prothrombin complex were detected in patients with clinical features of antiphospholipid syndrome, but negative antibodies to the classical antigens, suggesting that although the majority of patients show cardiolipin antibodies, other antigenic specificities may be important in individual patients. Antinuclear antibodies (ANAs) is a commonly requested autoantibody with importance in the diagnosis of systemic autoimmune rheumatic diseases such as systemic lupus erythematosus, Sjogren syndrome, etc. ANAs have classically been detected by a manual indirect immunofluorescence method, which needs good laboratory skills and the ability to recognise the diverse and often complex patterns seen at the microscope reading of the slides. Samples with positive staining patterns have reflex testing by ELISA-based assay for antibodies to specific antigens. The deskilling of laboratories and the simple volume of requests are driving automation for ANA testing. Willems et al. [8] report a review of almost 10,000 patients who were tested for ANA by indirect immunofluorescence and an automated ANA “screen”. They report reasonable concordance and comparable receiver operator curves between the two methods. However, simply looking at the comparison does not tell the whole story. The data were also analysed by a requestor, who showed a significantly higher PPV for systemic autoimmune rheumatic diseases when testing was requested by clinicians specialised in systemic rheumatic disease. They concluded that the performance of immunoassay and indirect immunofluorescence depends on the specific disease being considered and that for optimum value both assay types are best used in combination and results interpreted in the context of the clinical picture. Antibodies to the extractable nuclear antigens Ro/SSA and La/SSB are found in a number of systemic connective tissue diseases, e.g. Sjogren syndrome, systemic lupus erythematosus, etc. These antibodies, if present, cross the placenta from mum to baby as part of the active IgG transport. The Ro and La antigens are expressed in foetal cardiac tissue from the 18th to the 24th week, particularly on the surface of cardiac myocytes, and in a proportion of patients, the antibodies cause inflammation, fibrosis and calcification of foetal cardiac conduction tissues leading to atrioventricular block. Antibodies to the p200 epitope of the Ro52/SSA antigen appear to have a critical pathogenic role the development of foetal heart block. Mattia et al. [9] have evaluated the performance of two “in-house” assays for anti-p200 antibodies in pregnant women positive for anti- SSA/Ro52 antibodies. They did find differences in assay performance, but further investigation may identify an analytical process for anti-p-200 antibodies that may help identify patients who are at a significantly increased risk future pregnancy being affected by foetal heart block. Antineutrophil cytoplasmic antibodies (ANCAs) are autoantibodies that are mainly directed against the cytoplasmic antigens proteinase 3 and myeloperoxidase of neutrophils and the monocytes. Their presence is associated with systemic vasculitides and in particular granulomatosis with polyangiitis, microscopic polyangiitis and Churg-Straus syndrome. Like ANAs, indirect immunofluorescence and ELISA-based assay are used together to detect and quantify these antibodies with a similar drive to replace the qualitative tests with automated quantitative analyses. Certified reference preparations for IgG anti-proteinase 3 and IgG anti-myeloperoxidase are only recently available and are not yet embedded in the analytical process, making comparison of results over time and between methods difficult. Borderline positive results, irrespective of the method used, present the greatest interpretative challenge with limited data on their significance. Watad et al. [10] present a retrospective study of ANCA investigations and conclude that patients with borderline-positive IgG anti-myeloperoxidase antibodies and positive ANCA staining by indirect immunofluorescence have a poorer outcome in terms of their renal function than patients with negative ANCA staining. Autoantibodies are complex analytes; the antigens that we use in the analysis vary subtly between companies, and even within a company between different lots, the antibodies that we are trying to detect can vary between patients and even within a patient during the disease course. The demand for autoantibody testing, particularly for the rare autoimmune diseases, is increasing beyond logic, but instead of educating users and optimising demand, we are increasing the capacity for measurement with high throughput automated systems. The analytical speed and detection systems introduce another source of variation into a group of tests where robust standardisation does not exist. The papers in this issue of Clinical Chemistry and Laboratory Medicine clearly show the relevance of autoantibody testing in a diverse range of disease. However, none of the analytes described in these eight papers were measured with respect to certified reference materials; as a scientific community, we need to embrace standardisation (or harmonisation) for autoantibody testing. Some of these papers also review large amounts of data, e.g. 14,555 ANCA results over a 10-year period [10] and 9856 ANA results over a 2-year period [8]. We consider the approximate incidence and prevalence of ANCA vasculitis (incidence 2/100,000 and prevalence 26/100,000) and SLE (incidence 5/100,000 and prevalence 52/100,000), but we may question why so many of these tests were requested when the pretest probability is so low. We have two enormous challenges – to ensure that test tests are requested appropriately and to improve the analysis and reduce the variability of autoantibody testing. References 1. Directorate General for Internal Policies, Policy Department A: Economic and Scientific policy. Workshop Autoimmune Diseases – Modern Diseases, Brussels 25th Sep 2017 Proceedings. Available at: http://www.europarl.europa.eu/cmsdata/133620/ENVI%202017-09%20WS%20Autoimmune%20diseases%20%20PE%20614.174%20(Publication).pdf. ; 2. International Diabetes Federation IDF Diabetes Atlas, 8th ed. Available at: http://www.diabetesatlas.org/resources/2017-atlas.html. ; 3. Manso J, Pezzani R, Scarpa E, Gallo N, Betterle C. The natural history of autoimmune addison’s disease with a non-classical presentation: a case report and review of literature. Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-1108. [Epub ahead of print]. PubMedGoogle Scholar 4. del Pilar Larosa M, Chen S, Steinmaus N, Macrae H, Guo L, Masiero S, et al. A new ELISA for autoantibodies to steroid 21-hydroxylase. Clin Chem Lab Med 2017. doi: 10.1515/cclm-2017-0456. [Epub ahead of print]. PubMedGoogle Scholar 5. Censi S, Albergoni MP, Gallo N, Plebani M, Boscaro M, Betterle C. Insulin autoimmune syndrome (Hirata’s disease) in an Italian patient: a case report and review of the literature. Clin Chem Lab Med 2017. doi: 10.1515/cclm-2017-0392. [Epub ahead of print]. Google Scholar 6. Klehmet J, Märschenz S, Ruprecht K, Wunderlich B, Büttner T, Hiemann R, et al. Analysis of anti-ganglioside antibodies by a line immunoassay in patients with chronic-inflammatory demyelinating polyneuropathies (CIDP). Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-0792. [Epub ahead of print]. PubMedGoogle Scholar 7. Zhang S, Wu Z, Zhang W, Zhao J, Norman GL, Zeng X, et al. Antibodies to phosphatidylserine/prothrombin (aPS/PT) enhanced the diagnostic performance in Chinese patients with antiphospholipid syndrome. Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-0811. [Epub ahead of print]. PubMedGoogle Scholar 8. Willems P, De Langhe E, Claessens J, Westhovens R, Van Hoeyveld E, Poesen K, et al. Screening for connective tissue disease-associated antibodies by automated immuno-assay. Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-0905. [Epub ahead of print]. Google Scholar 9. Mattia E, Hoxha A, Tonello M, Favaro M, Del Ross T, Calligaro A, et al. Detection of autoantibodies to the p200-epitope of SSA/Ro52 antigen. A comparison of two laboratory assays. Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-0704. [Epub ahead of print]. PubMedGoogle Scholar 10. Watad A, Bragazzi NL, Sharif K, Gilburd B, Yavne Y, McGonagle D, et al. Borderline positive anti-neutrophil cytoplasmic antibodies (ANCA)-PR3/MPO detection in a large cohort tertiary center: Lessons learnt from a real-life experience. Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-1053. [Epub ahead of print]. Google Scholar About the article Dr. Joanna Sheldon, Protein Reference Unit, St. George’s Hospital, Blackshaw Road, SW17 0NH, London, UK Published Online: 2018-04-17 Author contributions: The author has accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), 20180233, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2018-0233. Export Citation ©2018 Walter de Gruyter GmbH, Berlin/Boston.
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Suggested by
Société Francaise d'Immunologie
December 14, 2016 3:56 AM
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Abstract Objective: Autoimmune thyroid disease is an organ-specific disorder due to the interplay between environmental and genetic factors. Toll-like receptors are pattern recognition receptors expressed abundantly on monocytes. There is a paucity of data on TLR expression in autoimmune thyroid disease (AITD). The aim of this study was to examine TLR expression, activation, ligands, and downstream signaling adaptors in PBMCs extracted from untreated autoimmune thyroid disease patients and healthy controls. Method: We isolated peripheral blood mononuclear cells (PBMC) of 30 healthy controls, 36 patients with untreated Hashimoto’s thyroiditis (HT), and 30 patients with newly onset Graves’ disease (GD). TLR mRNA, protein expression, TLR ligands, and TLR adaptor molecules were measured using real-time PCR, Western blot, flow cytometry, and enzyme-linked immunoassay (ELISA). PBMC was simulated with TLR agonists. The effects of TLR agonists on the viability of human PBMC were evaluated using the MTT assay. The supernatants of cell cultures were measured for the pro-inflammatory cytokines, IL-6, TNF-α and IL-10 by ELISA. Results: TLR2, TLR3, TLR9, and TLR10 mRNA were significantly increased in AITD patients compared with controls. TLR2, TLR3, TLR9, HMGB1, and RAGE expression on monocytes was higher in patients than control at baseline and TLR agonists stimulation. The release of TNF-a and IL-6 was significantly increased in PBMCs from AITD patients with TLR agonists, while IL-10 was significantly decreased. Downstream targets of TLR, myeloid differentiation factor 88 (MyD88), myeloid toll/interleukin (IL)-1 receptor (TIR)-domain containing adaptor-inducing interferon-β (TRIF) were significantly elevated in AITD patients. Levels of TLR2 ligands, HMGB1 and HSP60 were significantly elevated in AITD patients compared with those in controls and positively correlated with TgAb and TPOAb, while sRAGE concentration was significantly decreased in AITD patients. Conclusion: In conclusion, this work is the first to show that TLR2, TLR3, and TLR9 expression and activation are elevated in the PBMCs of patients with AITD and TLRs may participate in the pathogenesis of AITD.
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Scooped by
Gilbert C FAURE
October 15, 2016 10:43 AM
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The prevalence of celiac disease autoimmunity or tissue transglutaminase autoantibodies (TGA) amongst patients with type 1 diabetes (T1D) and autoimmune thyroid disease (AITD) in the Chinese population remains unknown.
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Scooped by
Gilbert C FAURE
April 26, 2016 4:44 AM
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Autoimmune thyroid disease (AITD), including Graves disease (GD) and Hashimoto disease (HD), is an organ-specific autoimmune disease with a strong genetic component.
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Scooped by
Gilbert C FAURE
October 12, 2015 10:03 AM
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Diabetes. 2015 Aug;64(8):3017-27. doi: 10.2337/db14-1730. Epub 2015 Mar 31. Research Support, N.I.H., Extramural
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Scooped by
Gilbert C FAURE
July 29, 2015 7:45 AM
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by Eijun Nishihara, Mitsuyoshi Hirokawa, Mitsuru Ito, Shuji Fukata, Hirotoshi Nakamura, Nobuyuki Amino, Akira Miyauchi Background IgG4-related disease is a novel disease entity characterized by diffuse lymphoplasmacytic infiltration rich in...
Highlights •Tertiary lymphoid structures (TLS) are common at sites of chronic inflammation.•TLS shape local adaptive immune responses in inflamed and tumoral tissues.•TLS represent prognostic biomarkers and therapeutic targets in cancer. Tertiary lymphoid structures (TLS) are ectopic lymphoid formations found in inflamed, infected, or tumoral tissues. They exhibit all the characteristics of structures in the lymph nodes (LN) associated with the generation of an adaptive immune response, including a T cell zone with mature dendritic cells (DC), a germinal center with follicular dendritic cells (FDC) and proliferating B cells, and high endothelial venules (HEV). In this review, we discuss evidence for the roles of TLS in chronic infection, autoimmunity, and cancer, and address the question of whether TLS present beneficial or deleterious effects in these contexts. We examine the relationship between TLS in tumors and patient prognosis, and discuss the potential role of TLS in building and/or maintaining local immune responses and how this understanding may guide therapeutic interventions.
Via Krishan Maggon
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Scooped by
Gilbert C FAURE
September 16, 2014 7:53 AM
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PubMed comprises more than 23 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.
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Scooped by
Gilbert C FAURE
September 3, 2014 8:05 AM
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A new mechanism underlying thyroid autoimmunity revealed Nature.com An epigenetic–genetic interaction involving a noncoding single nucleotide polymorphism (SNP) in TSHR alters thymic expression of this gene and is implicated in triggering Graves...
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Scooped by
Gilbert C FAURE
April 19, 2022 3:15 AM
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Information on a number of conditions that are associated with diabetes including coeliac disease, thyroid disease and dental health complications.
The different mechanisms that trigger the autoimmune attack to the thyroid between Hashimoto’s thyroiditis (HT) and Graves’ disease (GD) are still unclear. The aim of this study was to recognize thyroid antigens specific CD8+ T-cell epitopes and explore the relationship between these epitopes and thyroid autoantibodies, duration and classification in these two diseases. Free thiiodothyronine, free tetraiodothyronine, thyroid-stimulating hormone, TgAb, and TPOAb were all measured by immunochemiluminometric assays, while TRAb was tested by radioimmunoassay. HLA class I peptide affinity algorithms were applied to predict candidate thyroid autoantigen peptides that blind to HLA-A*0201. The ELISpot assay was used to detect Tg-, TPO-, and TSHR-specific CD8+ T cells. We demonstrated that TG-6 was a novel HLA-A*0201-restricted CTL epitope in GD. TG-6, TG-7, TG-10, TG-11, and TPO-6 were immunodominant in GD patients compared with HT patients (TG-6: 38.5 vs. 8%, P = 0.034; TG-7, TG-10, TG-11, and TPO-6: 23.1 vs. 0%, P = 0.034). The immunodominance of TG-6 in GD patients was more evident than healthy controls (HC) (TG-6: 35.8 vs. 0%, P = 0.011), but there was no statistically significant difference between HT patients and HC. Subgroup analyses revealed the T-cell responsiveness to TG-6 was stronger in TgAb-negative HT patients (0 vs. 40%, P = 0.033). However, there was no correlation showed for TPOAb, TRAb, medication and duration in both HT and GD patients. We report for the first time that both diseases, HT and GD, recognize different antigen-specific CD8-positive T cells. Tg maybe the dominant thyroid autoantigen contributing to breaking tolerance in GD. It could improve our knowledge of autoimmune thyroid diseases pathogenesis as well as offer new therapeutical tools in terms of peptide vaccine therapy.
Via Krishan Maggon
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Scooped by
Gilbert C FAURE
March 31, 2019 7:26 AM
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PeerJ. 2019 Mar 19;7:e6737. doi: 10.7717/peerj.6737. eCollection 2019.
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Scooped by
Gilbert C FAURE
August 29, 2018 7:28 AM
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Autoimmune Neurology Group DCN NDCN Contacts Test results: neuro.immunology@nhs.net, paddy.waters@ndcn.ox.ac.uk Clinical enquiries: sarosh.irani@ndcn.ox.ac.uk, maria.leite@ndcn.ox.ac.uk The immune system is our body's means of defence against harmful foreign substances such as bacteria and viruses. One of its roles is to produce antibodies, which are designed to selectively destroy disease-causing agents. Occasionally, however, antibodies attack the body itself, leading to an 'autoimmune' condition. Examples include rheumatoid arthritis, or thyroid disease, but can involve almost any organ. If the nervous system is affected, a variety of autoimmune neurological conditions can result. Such conditions can affect any part of the nervous system, and the part targeted by the autoantibody will reflect the symptoms. For example, the brain is affected in a condition called encephalitis, and the spinal cord can be affected in myelitis. Increasingly, the distinctive nature of the patient's symptoms can help identify the underlying condition. In many cases the autoantibodies can be depleted with treatment such as steroids and washing the blood with plasma exchange. But many patients don't respond well to the initial drugs, and require further medication to suppress the immune system. Patients are also often left with problems, typically involving memory, thinking or behaviour. There is a pressing need to develop new medications based on a better understanding of the conditions. OUR TEAM PARTICIPATE IN OUR RESEARCH Donations: You can contribute to our research here. Patient recruitment: Find out about the research projects for which we are recruiting participants here. SELECTED PUBLICATIONS N-methyl-D-aspartate receptor antibody production from germinal center reactions: Therapeutic implications. Journal article Makuch M. et al, (2018), Ann Neurol, 83, 553 - 561 Condition-dependent generation of aquaporin-4 antibodies from circulating B cells in neuromyelitis optica. Journal article Wilson R. et al, (2018), Brain, 141, 1063 - 1074 Intracellular and non-neuronal targets of voltage-gated potassium channel complex antibodies. Journal article Lang B. et al, (2017), J Neurol Neurosurg Psychiatry, 88, 353 - 361 Neurexin-3α: A new antibody target in autoimmune encephalitis. Journal article Waters PJ. and Irani SR., (2016), Neurology, 86, 2222 - 2223 Brain lesion distribution criteria distinguish MS from AQP4-antibody NMOSD and MOG-antibody disease. Journal article Juryńczyk M. et al, (2017), J Neurol Neurosurg Psychiatry, 88, 132 - 136 Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan's syndrome and acquired neuromyotonia. Journal article Irani SR. et al, (2010), Brain, 133, 2734 - 2748 OUR WORK We are trying to characterise the cells which produce the antibodies in patients with these diseases. This would allow us to begin to understand which medications can destroy these cells and help reduce antibody levels. We are also working to better understand the residual symptoms in patients which may respond to novel medications. Find out more about our diagnostics and research work. OUR TEAM Team members Alumni Visitors PARTNERSHIP AND FUNDING Wellcome Trust British Medical Association Epilepsy Research UK Association of British Neurologists Guarantors of Brain European Academy of Neurology UCB-Oxford Alliance Encephalitis Society Guthy-Jackson Foundation Donations NEWS AND EVENTS Patient and Public Engagement Vacancies
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Suggested by
Société Francaise d'Immunologie
December 2, 2017 11:24 AM
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Genetic predisposition could be assumed to be causing clustering of autoimmunity in individuals and families. We tested whether HLA and non-HLA loci associate with such clustering of autoimmunity. We included 1,745 children with type 1 diabetes from the Finnish Pediatric Diabetes Register. Data on personal or family history of autoimmune diseases were collected with a structured questionnaire and, for a subset, with a detailed search for celiac disease and autoimmune thyroid disease. Children with multiple autoimmune diseases or with multiple affected first- or second-degree relatives were identified. We analysed type 1 diabetes related HLA class II haplotypes and genotyped 41 single nucleotide polymorphisms (SNPs) outside the HLA region. The HLA-DR4-DQ8 haplotype was associated with having type 1 diabetes only whereas the HLA-DR3-DQ2 haplotype was more common in children with multiple autoimmune diseases. Children with multiple autoimmune diseases showed nominal association with RGS1 (rs2816316), and children coming from an autoimmune family with rs11711054 (CCR3-CCR5). In multivariate analyses, the overall effect of non-HLA SNPs on both phenotypes was evident, associations with RGS1 and CCR3-CCR5 region were confirmed and additional associations were implicated: NRP1, FUT2, and CD69 for children with multiple autoimmune diseases. In conclusion, HLA-DR3-DQ2 haplotype and some non-HLA SNPs contribute to the clustering of autoimmune diseases in children with type 1 diabetes and in their families.
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Scooped by
Gilbert C FAURE
October 21, 2016 1:17 PM
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Graves’ disease is an autoimmune disorder in which the thyroid is activated by antibodies to the thyrotropin receptor.
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Scooped by
Gilbert C FAURE
September 16, 2016 2:57 PM
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Thyroid autoimmunity occurs in >50% patients treated with #alemtuzumab and peaks in year 3 #ECTRIMS2016 https://t.co/CRIniQgEVs
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Scooped by
Gilbert C FAURE
October 18, 2015 2:25 PM
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Endocrine. 2015 Oct 16. [Epub ahead of print]
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Scooped by
Gilbert C FAURE
August 17, 2015 6:22 AM
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Autoimmune diseases, when taken all together, become a HUGE health burden. Among these are rheumatoid arthritis, lupus, multiple sclerosis, psoriasis, celiac disease, and thyroid disease.
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Scooped by
Gilbert C FAURE
April 24, 2015 7:40 AM
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High Prevalence of Thyroid Autoimmunity in Patients with Alopecia Areata and Vitiligo: A Controlled Study http://t.co/h61ZPzkh15
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Scooped by
Gilbert C FAURE
September 24, 2014 2:53 PM
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Associations between thyroid autoantibody status and abnormal pregnancy outcomes in euthyroid women: Abstract
... http://t.co/vx76VqYEw2
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Scooped by
Gilbert C FAURE
September 12, 2014 7:01 AM
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Thyro-gastric autoimmunity in patients with differentiated thyroid cancer: a prospective study: Abstract
... http://t.co/VZ8q5htTBm
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