Antimitochondrial Autoantibodies Research Articles

Механизм повреждения мелких холангиоцитов при первичном билиарном холангите

Primary biliary cholangitis (PBC) is a chronic cholestatic progressive liver disease and one of the most important progressive cholangiopathies in adults. Damage to cholangiocytes triggers the development of intrahepatic cholestasis, which progresses to cirrhosis in the terminal stage of the disease. Accumulating data indicate that damage to biliary epithelial cells [(BECs), cholangiocytes] is most likely associated with the intracellular accumulation of bile acids, which have potent detergent properties and damaging effects on cell membranes. The mechanisms underlying uncontrolled bile acid intake into BECs in PBC are associated with pH change in the bile duct lumen, which is controlled by the bicarbonate (HCO3-) buffer system "biliary HCO3- umbrella". The impaired production and entry of HCO3- from BECs into the bile duct lumen is due to epigenetic changes in expression of the X-linked microRNA 506. Based on the growing body of knowledge on the molecular mechanisms of cholangiocyte damage in patients with PBC, we propose a hypothesis explaining the pathogenesis of the first morphologic (ductulopenia), immunologic (antimitochondrial autoantibodies) and clinical (weakness, malaise, rapid fatigue) signs of the disease in the asymptomatic stage. This review focuses on the consideration of these mechanisms.

Circulating Monocyte Chemoattractant Protein-1 (MCP-1) in Patients with Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease that leads to the destruction of the intrahepatic bile ducts. While the inflammatory process can be mediated by monocyte chemotactic protein-1 (MCP-1), the importance of circulating MCP-1 as a biomarker is unclear. Our aim was to assess the diagnostic significance of the serum concentrations of MCP-1 in PBC patients. We compared circulating MCP-1 with biochemical, immunological and histological parameters. Serum samples were collected from 120 PBC patients, 60 pathologic controls and 30 healthy donors. MCP-1 levels were determined by using commercial enzyme-linked immunosorbent assay (ELISA). Elevated serum MCP-1 levels were detected in 66% of PBC patients with a specificity of 97%. Significantly higher levels of MCP-1 protein were found in the sera of patients with PBC than in the group of healthy individuals-410.2 pg/mL vs. 176.0 pg/mL, p < 0.01). Patients with higher concentrations of alkaline phosphatase also had higher levels of MCP-1 (r = 0.4, p < 0.01). In accordance with Ludwig's classification, a positive correlation of serum MCP-1 concentration with the degree of fibrosis was observed, OR = 6.1, p = 0.0003. We compared the MCP-1 with procollagen type III, hyaluronic acid (HA), FIB-4 index, APRI and collagen type IV when predicting the advance of liver fibrosis. Circulating MCP-1 is better correlated with liver fibrosis and is also associated with the occurrence of specific antimitochondrial autoantibodies and specific anti-nuclear autoantibodies-anti-gp210. MPC-1 can be considered to be a tool for diagnosing the degree of fibrosis in PBC, and combinations of MCP-1 and other specific biomarkers could support the diagnosis of PBC.

New insights into the pathogenesis of primary biliary cholangitis asymptomatic stage.

Primary biliary cholangitis (PBC) is a chronic cholestatic progressive liver disease and one of the most important progressive cholangiopathies in adults. Damage to cholangiocytes triggers the development of intrahepatic cholestasis, which progresses to cirrhosis in the terminal stage of the disease. Accumulating data indicate that damage to biliary epithelial cells [(BECs), cholangiocytes] is most likely associated with the intracellular accumulation of bile acids, which have potent detergent properties and damaging effects on cell membranes. The mechanisms underlying uncontrolled bile acid intake into BECs in PBC are associated with pH change in the bile duct lumen, which is controlled by the bicarbonate (HCO3-) buffer system "biliary HCO3- umbrella". The impaired production and entry of HCO3- from BECs into the bile duct lumen is due to epigenetic changes in expression of the X-linked microRNA 506. Based on the growing body of knowledge on the molecular mechanisms of cholangiocyte damage in patients with PBC, we propose a hypothesis explaining the pathogenesis of the first morphologic (ductulopenia), immunologic (antimitochondrial autoantibodies) and clinical (weakness, malaise, rapid fatigue) signs of the disease in the asymptomatic stage. This review focuses on the consideration of these mechanisms.

Первичный билиарный холангит с нормальным уровнем щелочной фосфатазы: клинический случай и обзор литературы

Antimitochondrial autoantibodies of the M2 subclass (AMA-M2) in primary biliary cholangitis (PBCh) under conditions of cholestasis are considered as one of the main criteria for diagnosis that does not require morphological confirmation. However, the detection of AMA-M2 in asymptomatic patients causes ambiguity of interpretation. Along with the assumption that such patients may have preclinical stage of PBCh, there is an opinion about the "healthy carrier" of AMA-M2. The article presents the clinical observation of a patient with the high AMA-M2 titer without significant clinical manifestations, the normal level of alkaline phosphatase, with changes in liver tissue characteristic of PBCh, revealed by the results of histological examination. Clinical observation demonstrates the need for liver biopsy for early detection of PBCh in AMA-M2-positive patients belonging to the risk group

Understanding immune mechanisms to create novel treatments for primary biliary cholangitis

Understanding immune mechanisms to create novel treatments for primary biliary cholangitis Professor Channakeshava S Umeshappa from Dalhousie University discusses the burden of primary biliary cholangitis and how understanding immune mechanisms may help to treat it. Primary biliary cholangitis (PBC) is characterised by chronic inflammation of the interlobular bile ducts, leading to fibrosis, cirrhosis, and liver failure if left untreated. Symptoms of PBC include fatigue, jaundice, itchy skin, abdominal pain, nausea or vomiting, dry eyes and mouth, and bone and joint aches. The incidence of PBC is increasing worldwide,(1) but is most common in Europe and North America.(2) PBC has a female predominance, affecting primarily middle-aged women (over 90% of the PBC cases). Clinically, it is often diagnosed by detecting anti-mitochondrial autoantibodies and elevated alkaline phosphatase enzymes in blood tests.

Detection of M2-Type Anti-Mitochondrial Autoantibodies against Specific Subunits in the Diagnosis of Primary Biliary Cholangitis in Patients with Discordant Results.

M2-type anti-mitochondrial autoantibodies are considered the hallmark of primary biliary cholangitis and are directed mainly against the E2 subunits of the 2-oxo acid dehydrogenase complex enzymes (PDC, BCOADC and OGDC). The aim of this study was to determine whether a Dot-blot that includes these E2 subunits separately could confirm the results of methods with non-separated subunits in patients with low positive or discordant results between techniques. Sera of 24 patients with low positive or discordant results and of 10 patients with clear positive results by non-separated subunits methods were analyzed by Dot-blot with separated subunits. Autoantibodies against E2 subunits of PDC, BCOADC or OGDC were detected in all patients, except in one case from the low positive or discordant results group, by Dot-blot with separated subunits. It would be advisable to use methods that include the three E2 subunits, and a Dot-blot with separated subunits could confirm doubtful cases by non-separated assays.

Granulomatous myopathy: Sarcoidosis and beyond.

Non-necrotizing granulomatous inflammation is a rare but easily recognized histopathological finding in skeletal muscle biopsy. A limited number of diseases are known to be associated with non-necrotizing granulomatous myopathy. Once identified, a careful evaluation for evidence of extramuscular granulomatosis and other signs suggestive of sarcoidosis is warranted as about half of the patients have sarcoid myopathy. In addition, the presence of granulomatous myopathy should trigger a search for clinical and pathological clues of inclusion body myositis (IBM), which accounts for most of the remaining patients and can coexist with sarcoidosis. Recognizing the features of IBM in patients with granulomatous myopathy can potentially spare the patients from unnecessary exposure to immunosuppressive therapies. In patients whose granulomatous myopathy remain unexplained, further investigations should aim at identifying myasthenia gravis and other autoimmune disorders, especially those known to cause granulomatous inflammation in other organs. Laboratory investigations should include acetylcholine receptor, antimitochondrial, antineutrophil cytoplasmic, thyroglobulin, and thyroid peroxidase autoantibodies. In the appropriate clinical context, exposure to immune checkpoint inhibitors and chronic graft-vs-host disease can be causes of granulomatous myopathy. In cases of unexplained granulomatous myopathy, natural killer/T-cell lymphoma should be considered and careful histopathological examination for atypical cells and appropriate immunostaining is crucial. Identifying the etiology of granulomatous myopathy in each patient can guide appropriate treatment.

The future risk of primary biliary cholangitis (PBC) is low among patients with incidental anti-mitochondrial antibodies but without baseline PBC.

Anti‐mitochondrial autoantibodies (AMAs) are highly specific for the diagnosis of primary biliary cholangitis (PBC) but are also occasionally found in other diseases. In the present study, we evaluated the incidence of and predictors for PBC development in AMA‐positive patients with other liver or non‐liver diseases at baseline. In this retrospective study, we screened patients who tested positive for AMA and/or anti‐mitochondrial M2 antibody (AMA‐M2) at Beijing Friendship Hospital, Capital Medical University, from October 2005 to January 2017. They were categorized by their diagnosis at the baseline as patients with PBC or non‐PBC cases. We followed up on the non‐PBC cases through telephone interviews and reviewing of medical records to obtain laboratory results and clinical outcomes. In total, 139 patients were AMA‐positive but did not fulfill the diagnostic criteria of PBC at baseline, including 51 patients with non‐PBC liver diseases and 88 cases with non‐liver diseases. The titers of AMA‐M2, alkaline phosphatase, gamma‐glutamyl transpeptidase, and immunoglobulin M were significantly higher in patients with PBC compared to those with non‐PBC liver diseases and non‐liver diseases. After a median follow‐up of 4.6 (interquartile range: 2.4–7.6) years, 4.3% (6 of 139) developed PBC, with an accumulative 5‐year incidence rate of 4.2%. None of the patients with non‐PBC liver diseases developed PBC, whereas the 5‐year incidence rate of PBC was 7.8% among 88 patients with non‐liver diseases. Lower alanine aminotransferase and higher immunoglobulin M were independent predictors for developing PBC. Conclusion: Our results suggest a low risk of developing PBC over time in AMA‐positive patients with other liver and non‐liver diseases.

Prevalence of Primary Biliary Cholangitis in a Cohort of Primary Sjögren's Syndrome Patients.

ObjectiveTo analyze the prevalence and clinical progression of primary biliary cholangitis (PBC) in patients with primary Sjögren's syndrome (pSS) and possible associations between biochemical and immunological features and the development of PBC.MethodsWe retrospectively reviewed a cohort of 115 pSS patients followed up in an outpatient clinic from 1987 to 2020, without a history of liver disease, and looked for the presence of PBC through analysis of several biochemical, immunological, and histologic characteristics.ResultsTwenty patients (17.4%) had chronic cholestatic liver biochemistry. After exclusion of extrahepatic liver disease by abdominal ultrasound, 13 of them were tested for antimitochondrial autoantibodies (AMA) detected by indirect immunofluorescence (IF), of which five tested positive, fulfilling the diagnostic criteria for PBC. Three of the five PBC patients and three of the eight chronic cholestasis AMA-negative patients were further investigated with liver biopsy, which showed features of PBC in all three PBC patients and in one of the chronic cholestasis AMA-negative patients, allowing for the diagnosis of AMA-negative PBC in the latter. The remaining two AMA-negative patients had liver histology compatible with autoimmune hepatitis and unspecific findings, respectively. Overall, six (5.2%) patients with pSS had AMA-positive PBC (n=5) or AMA-negative PBC (n=1). Comparing immunological characteristics between PBC and non-PBC patients, we found that PBC patients had a higher mean maximum erythrocyte sedimentation rate (ESR) during follow-up than patients without PBC. All PBC patients were treated with ursodeoxycholic acid (UDCA) and after treatment with UDCA, only one patient showed biochemical and clinical progression of PBC, with increasing alkaline phosphatase and total bilirubin levels, eventually progressing to cirrhosis.ConclusionsAmong patients with pSS, PBC had an overall prevalence of six of 115 (5.2%). Higher ESR was a feature associated with PBC patients. In our cohort, after initiation of UDCA treatment, PBC showed predominantly slow progress, with only one patient progressing to cirrhosis during follow-up.

Serum and tissue levels of coenzyme Q10 in pemphigus vulgaris.

Pemphigus vulgaris (PV) is a debilitating autoimmune blistering disease of the skin and mucous membranes. It occurs due to the action of autoantibodies against various keratinocyte self-antigens. Anti-mitochondrial autoantibodies are detected in patients with PV. Coenzyme Q10 (CoQ10) is a member of the mitochondrial respiratory chain involved in cellular metabolism, including apoptosis. This study aimed to assess the serum and tissue levels of CoQ10 of patients with PV and healthy controls to determine its relevance to the disease pathogenesis. In this case-control study, 20 patients with PV and 20 healthy controls were included. Blood and skin samples were collected for the measurement of CoQ10 levels using ELISA. CoQ10 was significantly lower in both serum and tissue of patients with PV compared with controls (p=0.001). Similar results were found when gender subgroups were separately compared. A significant positive correlation was found between serum and tissue CoQ10 levels in controls (p=0.019, r=0.521), but not in patients with PV. CoQ10 appears to be one of the parameters affected by the autoimmune response in PV, which may contribute to the tissue damage caused by autoantibodies. The absence of a significant correlation between CoQ10 level and disease severity or duration may be caused by the complex pathophysiological process in PV with multiple autoantibodies against different keratinocyte antigens.

Investigation of immune complexes formed by mitochondrial antigens containing a new lipoylated site in sera of primary biliary cholangitis patients.

Primary biliary cholangitis (PBC) is characterized by the presence of serum anti-mitochondrial autoantibodies (AMAs). To date, four antigens among the 2-oxo-acid dehydrogenase complex family, which commonly have lipoyl domains as an epitope, have been identified as AMA-corresponding antigens (AMA-antigens). It has recently been reported that AMAs react more strongly with certain chemically modified mimics than with the native lipoyl domains in AMA-antigens. Moreover, high concentrations of circulating immune complexes (ICs) in PBC patients have been reported. However, the existence of ICs formed by AMAs and their antigens has not been reported to date. We hypothesized that AMAs and their antigens formed ICs in PBC sera, and analyzed sera of PBC and four autoimmune diseases (Sjögren's syndrome, systemic lupus erythematosus, systemic scleroderma, and rheumatoid arthritis) using immune complexome analysis, in which ICs are separated from serum and are identified by nano-liquid chromatography-tandem mass spectrometry. To correctly assign MS/MS spectra to peptide sequences, we used a protein-search algorithm that including lipoylation and certain xenobiotic modifications. We found three AMA-antigens, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the E2 subunit of the 2-oxo-glutarate dehydrogenase complex (OGDC-E2) and dihydrolipoamide dehydrogenase binding protein (E3BP), by detecting peptides containing lipoylation and xenobiotic modifications from PBC sera. Although the lipoylated sites of these peptides were different from the well-known sites, abnormal lipoylation and xenobiotic modification may lead to production of AMAs and the formation ICs. Further investigation of the lipoylated sites, xenobiotic modifications, and IC formation will lead to deepen our understanding of PBC pathogenesis.

Primary biliary cholangitis with normal alkaline phosphatase: A neglected clinical entity challenging current guidelines

Background & aimThe diagnosis of primary biliary cholangitis (PBC), an uncommon immune-mediated cholestatic liver disease, is based on positive circulating anti-mitochondrial (AMA) and/or PBC-specific anti-nuclear autoantibodies (ANA), coupled with elevated serum alkaline phopsphatase (ALP) levels. Timely initiation of treatment with ursodeoxycholic acid prevents progression to cirrhosis and liver failure. We aimed at investigating liver histology in patients with normal ALP level and positive AMA and/or PBC-specific ANA. MethodsWe searched the Swiss PBC Cohort Study database, which includes subjects with positive PBC autoimmune serology and normal ALP levels, for patients who underwent a liver biopsy. Histological slides were centrally reviewed by an expert liver pathologist, and sera were centrally re-tested for AMA and ANA. Results30 patients were included; 90% females, median age 53 (range 27–72) years. Twenty-four (80%) had liver histology typical for (n = 2), consistent with (n = 16) or suggestive of (n = 6) PBC, including three of four AMA-negative ANA-positive patients. Among 22 ursodeoxycholic acid treated patients, 14 had elevated GGT levels before treatment; a significant decrease of the median GGT level between pre- (1.46 x ULN) and post- (0.43 x ULN) treatment (p = 0.0018) was observed. ConclusionsIn our series, a high proportion of AMA positive patients with normal ALP levels have PBC. For the first time we show histological diagnosis of PBC in AMA-negative/PBC-specific ANA-positive subjects and the potential role of GGT as a biomarker in PBC patients with normal baseline ALP levels. Current guidelines for the diagnosis of PBC do not cover the whole extent of PBC presentation, with important clinical implications in terms of timely treatment initiation.

Daifan San intervenes in forkhead box P3 and the interleukin (IL)-23/IL-17A signaling pathway to help prevent and treat primary biliary cirrhosis.

To investigate the mechanism by which Daifan San (DFS) prevents and treats primary biliary cirrhosis (PBC) via the forkhead box P3 (FoxP3) and interleukin (IL)-23/IL-17A signaling pathways. Ninety C57BL/6 mice were randomly divided into the control, model, DFS low-dose, DFS middle-dose, DFS high-dose and ursodeoxycholic acid (UDCA) groups (n = 15 per group). A mouse model of PBC was induced using polyinosinic polycytidylic acids (poly I:C). Lymphocyte subset expression in the peripheral blood was analyzed via flow cytometry. The inflammatory cytokines and antimitochondrial autoantibody (AMA) levels were detected via enzyme-linked immunosorbent assays. The expressions and location of type I collagen, type III collagen, cytokeratin 19 and FoxP3 in the liver tissue were evaluated via immunohistochemistry. FoxP3, IL-23 and IL-17 expressions in the peripheral blood and liver tissue were evaluated via real-time polymerase chain reaction and western blotting. IL-17, IL-23, IL-8, IL-33, TNF-a, and AMA expressions were significantly increased in the model group and decreased in the DFS and UDCA groups. Conversely, Treg cell and FoxP3 expressions were significantly decreased in the model group and increased in the DFS and UDCA groups. The IL-23/IL-17A signaling pathway was closely correlated with chronic inflammation of the bile duct in PBC and functional deletion of Treg cells, leading to reduced FoxP3 levels and mediating the loss of tolerance in PBC. DFS may delay the occurrence and relieve the symptoms of PBC by downregulating IL-23/IL-17A signaling pathway expression and upregulating FoxP3 expression.

Anti-Ro52 Autoantibodies Are Related to Chronic Graft-vs.-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation.

Chronic graft-vs.-host disease (cGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Previous studies have shown that autoantibodies play an important role in the development of cGVHD. Anti-nuclear autoantibodies (ANA) is the most frequently detected autoantibodies in patients with cGVHD, but the role of anti-Ro52 autoantibodies (anti-Ro52) in cGVHD remains largely unknown. In this study, we analyzed autoantibodies from 84 patients after allo-HSCT, including 42 with active cGVHD and 42 without cGVHD. Autoantibodies were found in 36 (42.9%) patients. Among these autoantibody-positive patients, 28 (77.8%) patients had active cGVHD. The most frequent autoantibodies in patients with active cGVHD were ANA (50.0%), anti-Ro52 (28.6%) and anti-mitochondrial autoantibodies type 2 (4.8%). We further explored the association between anti-Ro52 and cGVHD. Patients with active cGVHD had higher anti-Ro52 levels than patients without cGVHD (P < 0.05). The increases of anti-Ro52 levels were more significant in patients with moderate/severe cGVHD compared to those of patients without cGVHD (P < 0.05). Stratified and multivariable logistic regression analysis demonstrated that moderate/severe cGVHD was an independent risk factor for the levels of anti-Ro52 (P < 0.01). ROC analysis confirmed anti-Ro52 as a risk factor for progression of skin cGVHD. Moreover, the anti-Ro52 levels were highly correlated with the levels of B cell-activating factor (BAFF) and IgG1 antibodies. Our study demonstrates that anti-Ro52 is associated with cGVHD. The increased levels of anti-Ro52 were associated with higher levels of BAFF and IgG1 antibodies, suggesting a mechanistic link between elevated anti-Ro52 levels and aberrant B cell homeostasis.

S-adenosyl-L-methionine (SAMe) halts the autoimmune response in patients with primary biliary cholangitis (PBC) via antioxidant and S-glutathionylation processes in cholangiocytes

S-adenosyl-L-methionine is an endogenous molecule with hepato-protective properties linked to redox regulation and methylation. Here, the potential therapeutic value of SAMe was tested in 17 patients with PBC, a cholestatic disease with autoimmune phenomena targeting small bile ducts. Nine patients responded to SAMe (SAMe responders) with increased serum protein S-glutathionylation. That posttranslational protein modification was associated with reduction of serum anti-mitochondrial autoantibodies (AMA-M2) titers and improvement of liver biochemistry. Clinically, SAMe responders were younger at diagnosis, had longer duration of the disease and lower level of serum S-glutathionylated proteins at entry. SAMe treatment was associated with negative correlation between protein S-glutathionylation and TNFα. Furthermore, AMA-M2 titers correlated positively with INFγ and FGF-19 while negatively with TGFβ. Additionally, cirrhotic PBC livers showed reduced levels of glutathionylated proteins, glutaredoxine-1 (Grx-1) and GSH synthase (GS). The effect of SAMe was also analyzed in vitro. In human cholangiocytes overexpressing miR-506, which induces PBC-like features, SAMe increased total protein S-glutathionylation and the level of γ-glutamylcysteine ligase (GCLC), whereas reduced Grx-1 level. Moreover, SAMe protected primary human cholangiocytes against mitochondrial oxidative stress induced by tBHQ (tert-Butylhydroquinone) via raising the level of Nrf2 and HO-1. Finally, SAMe reduced apoptosis (cleaved-caspase3) and PDC-E2 (antigen responsible of the AMA-M2) induced experimentally by glycochenodeoxycholic acid (GCDC). These data suggest that SAMe may inhibit autoimmune events in patients with PBC via its antioxidant and S-glutathionylation properties. These findings provide new insights into the molecular events promoting progression of PBC and suggest potential therapeutic application of SAMe in PBC.

006 The association of platelet activation markers, neutrophil extracellular traps and anti-mitochondrial autoantibodies with cutaneous manifestations in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is an autoimmune disease associated with platelet activation, formation of neutrophil extracellular traps (NETs) and development of anti-mitochondrial autoantibodies (AMA). In the current study, we investigated the clinical utility of these markers and autoantibodies in cutaneous lupus manifestations. Clinical data on cutaneous manifestations were obtained in a large SLE cohort (n=73) through review of SLE questionnaires, SLICC Classification Criteria and revised ACR Criteria for SLE.

Metabolic Dysfunction in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Not Due to Anti-mitochondrial Antibodies.

Metabolic profiling studies have recently indicated dysfunctional mitochondria in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). This includes an impaired function of pyruvate dehydrogenase complex (PDC), possibly driven by serum factor(s), which leads to inadequate adenosine triphosphate generation and excessive lactate accumulation. A reminiscent energy blockade is likely to occur in primary biliary cholangitis (PBC), caused by anti-PDC autoantibodies, as recently proposed. PBC is associated with fatigue and post-exertional malaise, also signifying ME/CFS. We herein have investigated whether ME/CFS patients have autoreactive antibodies that could interfere with mitochondrial function. We found that only 1 of 161 examined ME/CFS patients was positive for anti-PDC, while all PBC patients (15/15) presented significant IgM, IgG, and IgA anti-PDC reactivity, as previously shown. None of fibromyalgia patients (0/14), multiple sclerosis patients (0/29), and healthy blood donors (0/44) controls showed reactivities. Anti-mitochondrial autoantibodies (inner and outer membrane) were negative in ME/CFS cohort. Anti-cardiolipin antibody levels in patients did not differ significantly from healthy blood donors. In conclusion, the impaired mitochondrial/metabolic dysfunction, observed in ME/CFS, cannot be explained by presence of circulating autoantibodies against the tested mitochondrial epitopes.

Detection of anti-kelch-like 12 and anti-hexokinase 1 antibodies in primary biliary cholangitis patients in China.

primary biliary cholangitis (PBC) is a chronic cholestatic disease, characterized by positive anti-mitochondrial autoantibodies (AMA) in 90-95 % patients. Anti-kelch-like 12 (anti-KLHL12) and anti-hexokinase1 (anti-HK1) antibodies have been identified as the two new serum markers in recent years, which are used in the diagnosis of AMA-negative PBC patients. The objective of the study was to examine the performance of these two new biomarkers in China. a total of 192 patients were enrolled and screened for anti-KLHL12 and anti-HK1 antibodies and AMA by ELISA. Receiver operating characteristic (ROC curve) analysis was applied to examine the diagnostic importance of AMA, anti-KLHL12 and anti-HK1 antibodies. Furthermore, correlation analysis between some important biochemical indexes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALP], bilirubin, gamma-glutamil transferasa [γ-GT]), staging of pathological changes of the liver and the expression of novel antibodies in PBC patients were also examined. the positivity of the anti-HK1 antibody in AMA-positive PBC patients and AMA-negative patients was 44.7 % and 33.3 %, respectively. The specificity, proportion of positive patients (PPV) and proportion of negative patients (NPV) were 93 %, 89 % and 53 %, respectively. In contrast, the positivity to the anti-KLHL12 antibody in AMA-positive and negative PBC patients was 41.2 % and 22.2 %, respectively. Specificity, PPV and NPV were 98 %, 95 % and 52 %, respectively. The area under the curve (AUC) with anti-HK1 and anti-KLHL12 antibodies were 0.720 and 0.703. With the combination with anti-HK1 and anti-KLHL12 antibodies, the AUC of AMA increased from 0.889 to 0.891, increasing the sensitivity from 0.764 to 0.836. Anti-KLHL12 and anti-HK1-positive patients had higher serum levels of ALP, γ-GT and bilirubin, with statistically significant differences (p < 0.01) compared with anti-KLHL12 or anti-HK1-negative patients. Notably, correlation analysis showed a significant positive correlation between antibody expression and ALP, γ-GT and bilirubin serum levels (r = 0.735, 0.491, 0.466; p < 0.01). anti-HK1 and anti-KLHL12 antibodies have been identified as two significant biomarkers in PBC patients. Furthermore, the presence of these antibodies is likely to correlate with the severity of PBC.

A comprehensive autoantigen-ome of autoimmune liver diseases identified from dermatan sulfate affinity enrichment of liver tissue proteins

BackgroundAutoimmune diseases result from aberrant immune attacks by the body itself. It is mysterious how autoantigens, a large cohort of seemingly unconnected molecules expressed in different parts of the body, can induce similar autoimmune responses. We have previously found that dermatan sulfate (DS) can form complexes with molecules of apoptotic cells and stimulate autoreactive CD5+ B cells to produce autoantibodies. Hence, autoantigenic molecules share a unique biochemical property in their affinity to DS. This study sought to further test this uniform principle of autoantigenicity.ResultsProteomes were extracted from freshly collected mouse livers. They were loaded onto columns packed with DS-Sepharose resins. Proteins were eluted with step gradients of increasing salt strength. Proteins that bound to DS with weak, moderate, or strong affinity were eluted with 0.4, 0.6, and 1.0 M NaCl, respectively. After desalting, trypsin digestion, and gel electrophoresis, proteins were sequenced by mass spectrometry. To validate whether these proteins have been previously identified as autoantigens, an extensive literature search was conducted using the protein name or its alternative names as keywords. Of the 41 proteins identified from the strong DS-affinity fraction, 33 (80%) were verified autoantigens. Of the 46 proteins with moderate DS-affinity, 27 (59%) were verified autoantigens. Of the 125 proteins with weak DS-affinity, 44 (35%) were known autoantigens. Strikingly, these autoantigens fell into the classical autoantibody categories of autoimmune liver diseases: ANA (anti-nuclear autoantibodies), SMA (anti-smooth muscle autoantibodies), AMA (anti-mitochondrial autoantibodies), and LKM (liver-kidney microsomal autoantigens).ConclusionsThis study of DS-affinity enrichment of liver proteins establishes a comprehensive autoantigen-ome for autoimmune liver diseases, yielding 104 verified and 108 potential autoantigens. The liver autoantigen-ome sheds light on the molecular origins of autoimmune liver diseases and further supports the notion of a unifying biochemical principle of autoantigenicity.

Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations

Mitochondria are organelles that govern energy supply and control cell death. Mitochondria also express bacterial features, such as the presence of inner membrane cardiolipin and a circular genome rich in hypomethylated CpG motifs. While mitochondrial extrusion by damaged organs or activated cells is thought to trigger innate immunity, it is unclear whether extracellular mitochondria also stimulate an adaptive immune response. We describe the development of novel assays to detect autoantibodies specific to two distinct components of the mitochondrion: the mitochondrial outer membrane and mitochondrial DNA. Antibodies to these two mitochondrial constituents were increased in both human and murine systemic lupus erythematosus (SLE), compared to controls, and were present at higher levels than in patients with antiphospholipid syndrome or primary biliary cirrhosis. In both bi- and multi-variate regression models, antibodies to mitochondrial DNA, but not whole mitochondria, were associated with increased anti-dsDNA antibodies and lupus nephritis. This study describes new and optimized methods for the assessment of anti-mitochondrial antibodies, and demonstrates their presence in both human and murine SLE. These findings suggest that different mitochondrial components are immunogenic in SLE, and support the concept that extracellular mitochondria may provide an important source of circulating autoantigens in SLE.