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.
To determine whether anticitrullinated protein antibodies (ACPA) exhibit specific changes in Fc glycosylation prior to the onset of arthritis.
Serum samples of patients with ACPA-positive arthralgia ...(n=183) were collected at baseline and at various time points of follow-up. 105 patients developed arthritis after a median of 12 months (IQR 6-24) and were classified as having either rheumatoid arthritis (RA, n=48) or undifferentiated arthritis (UA, n=57) based on the 1987 American College of Rheumatology (ACR) criteria. ACPA and total serum IgG were isolated by affinity purification and cleaved by trypsin. ACPA-IgG1 Fc-glycopeptides were subsequently analysed by nano-liquid chromatography mass spectrometry and compared to those of total IgG1.
At baseline, ACPA-IgG1 and total IgG1 from arthralgia patients displayed similar Fc glycosylation patterns. By contrast, at the onset of arthritis, ACPA exhibited a decrease in galactose residues in RA patients, but not in UA patients. This decrease occurred around 3 months prior to diagnosis and was paralleled by an increase in systemic inflammation (erythrocyte sedimentation rate). Galactosylation of total IgG1 was also decreased in RA, but this did not precede the onset of arthritis. Interestingly, we additionally noted a higher degree of ACPA-IgG1 Fc core fucosylation at baseline as compared with total IgG1, which further increased prior to diagnosis.
ACPA display significant changes in Fc galactosylation and fucosylation prior to the onset of RA. These changes towards a more pro-inflammatory phenotype could be involved in driving the disease process.
OBJECTIVE:We investigated a series of patients with LGI1 antibody (Ab)–related cognitive deterioration to determine the clinical presentation, long-term outcome, and LGI1 Ab evolution.
METHODS:We ...retrospectively analyzed the clinical information of 76 patients with LGI1 Ab–related cognitive deterioration. Presenting syndromes were classified as limbic encephalitis (LE), non-LE, or encephalopathy (normal MRI and no CSF pleocytosis). Frequency of relapses and clinical outcome were assessed in 48 patients with prolonged follow-up (median 39 months, range 18–200).
RESULTS:Sixty-three patients (83%) developed LE, 3 (4%) non-LE, and 10 (13%) encephalopathy. All patients received steroids, IV immunoglobulins (Ig), or both. At 2 years, 17 (35%; 95% CI 21%–49%) fully recovered, 17 (35%) became functionally independent but not at baseline or were unable to return to work, 11 (23%) required assistance because of moderate or severe cognitive deficits, and 3 (6%) died. Predictors of bad outcome included no response to initial immunotherapy (odds ratio 23.0, 95% CI 2.4–215.6, p = 0.006) and clinical relapses (odds ratio 10.2, 95% CI 1.0–100.1, p = 0.047) that occurred in 13 patients (27%). In all patients, the LGI1 Abs were IgG4 and usually detectable in both serum and CSF (only CSF, 8%). Abs remained positive in serum of 4 of 16 patients with long-term follow-up; 3 of these 4 patients fully recovered and none showed class switch to IgG1.
CONCLUSIONS:Up to 13% of patients with LGI1 Abs develop cognitive impairment without criteria of encephalitis. After immunotherapy, only 35% of patients return to their baseline cognitive function. Serum LGI1 Abs may remain detectable after full clinical recovery.
Systemic lupus erythematosus （SLE） is a complex autoimmune disease characterized by the production of autoantibodies to a broad range of self-antigens. Profiling the autoantibody repertoire using ...array-based technology has emerged as a powerful tool for the identification of biomarkers in SLE and other autoimmune diseases. Proteomic microarray has the capacity to hold large number of self-antigens on a solid surface and serve as a high-throughput screening method for the determination of autoantibody specificities. The autoantigen arrays carrying a wide variety of self-antigens, such as cell nuclear components （nucleic acids and associated proteins）, cytoplas- mic proteins, phospholipid proteins, cell matrix proteins, mucosal/secreted proteins, glomeruli, and other tissue-specific proteins, have been used for screening of autoantibody specificities associated with different manifestations of SLE. Arrays containing synthetic peptides and molecular modified proteins are also being utilized for identification of autoantibodies targeting to special antigenic epi- topes. Different isotypes of autoantibodies, including IgG, IgM, IgA, and IgE, as well as other Ig subtypes, can be detected simultaneously with multi-color labeled secondary antibodies. Serum and plasma are the most common biologic materials for autoantibody detection, but other body fluids such as cerebrospinal fluid, synovial fluid, and saliva can also be a source of autoantibody detection.
Objective
We investigated the pathogenicity of immunoglobulin M (IgM) anti‐GM1 antibodies in serum from patients with multifocal motor neuropathy (MMN) using human induced pluripotent stem cell ...(iPSC)‐derived motor neurons (MNs).
Methods
iPSCs were generated from fibroblasts and differentiated into MNs. We studied the binding of IgM to MNs, their complement‐activating properties, and effects on structural integrity using fluorescence and electron microscopy. Live cell imaging was used to study effects of antibody binding on MNs in the presence and absence of complement.
Results
IgM antibody binding to MNs was detected using sera from MMN patients with and without detectable anti‐GM1 IgM antibody titers in enzyme‐linked immunosorbent assay, but not with sera from (disease) controls. Competition and depletion experiments showed that antibodies specifically bound to GM1 on iPSC‐derived MNs. Binding of these antibodies disrupted calcium homeostasis by both complement‐dependent and complement‐independent pathways. MNs showed marked axonal damage after complement activation, and reduced antibody pathogenicity following treatment with immunoglobulin preparations.
Interpretation
Our data provide evidence for the pathogenicity of anti‐GM1 IgM antibodies in MMN patients and link their presence to the clinical characteristics of axonal damage and immunoglobulin responsiveness. This iPSC‐derived disease model will facilitate diagnosis, studies on autoantibody pathogenicity, drug development, and screening in immune‐mediated neuropathies. Ann Neurol 2016;80:71–88
Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a heterogeneous disease in which diverse autoantibodies have been described but systematic screening has never been performed. ...Detection of CIDP-specific antibodies may be clinically useful. We developed a screening protocol to uncover novel reactivities in CIDP. Sixty-five CIDP patients and 28 controls were included in our study. Three patients (4.6%) had antibodies against neurofascin 155, four (6.2%) against contactin-1 and one (1.5%) against the contactin-1/contactin-associated protein-1 complex. Eleven (18.6%) patients showed anti-ganglioside antibodies, and one (1.6%) antibodies against peripheral myelin protein 2. No antibodies against myelin protein zero, contactin-2/contactin-associated protein-2 complex, neuronal cell adhesion molecule, gliomedin or the voltage-gated sodium channel were detected. In IgG experiments, three patients (5.3%) showed a weak reactivity against motor neurons; 14 (24.6%) reacted against DRG neurons, four of them strongly (7.0%), and seven (12.3%) reacted against Schwann cells, three of them strongly (5.3%). In IgM experiments, six patients (10.7%) reacted against DRG neurons, while three (5.4%) reacted against Schwann cells. However, results were not statistically significant when compared to controls. Immunoprecipitation experiments identified CD9 and L1CAM as potential antigens, but reactivity could not be confirmed with cell-based assays. In summary, we describe a diverse autoantibody repertoire in CIDP patients, reinforcing the hypothesis of CIDP's pathophysiological heterogeneity.
Aims/hypothesis
Monogenic forms of diabetes (MODY, neonatal diabetes mellitus and syndromic forms) are rare, and affected individuals may be misclassified and treated suboptimally. The prevalence of ...type 1 diabetes is high in Finnish children but systematic screening for monogenic diabetes has not been conducted. We assessed the prevalence and clinical manifestations of monogenic diabetes in children initially registered with type 1 diabetes in the Finnish Pediatric Diabetes Register (FPDR) but who had no type 1 diabetes-related autoantibodies (AABs) or had only low-titre islet cell autoantibodies (ICAs) at diagnosis.
Methods
The FPDR, covering approximately 90% of newly diagnosed diabetic individuals aged ≤15 years in Finland starting from 2002, includes data on diabetes-associated HLA genotypes and AAB data (ICA, and autoantibodies against insulin, GAD, islet antigen 2 and zinc transporter 8) at diagnosis. A next generation sequencing gene panel including 42 genes was used to identify monogenic diabetes. We interpreted the variants in
HNF1A
by using the gene-specific standardised criteria and reported pathogenic and likely pathogenic findings only. For other genes, we also reported variants of unknown significance if an individual’s phenotype suggested monogenic diabetes.
Results
Out of 6482 participants, we sequenced DNA for 152 (2.3%) testing negative for all AABs and 49 (0.8%) positive only for low-titre ICAs (ICA
low
). A monogenic form of diabetes was revealed in 19 (12.5%) of the AAB-negative patients (14 9.2% had pathogenic or likely pathogenic variants) and two (4.1%) of the ICA
low
group. None had ketoacidosis at diagnosis or carried HLA genotypes conferring high risk for type 1 diabetes. The affected genes were
GCK
,
HNF1A
,
HNF4A
,
HNF1B
,
INS
,
KCNJ11
,
RFX6
,
LMNA
and
WFS1
. A switch from insulin to oral medication was successful in four of five patients with variants in
HNF1A
,
HNF4A
or
KCNJ11.
Conclusions/interpretation
More than 10% of AAB-negative children with newly diagnosed diabetes had a genetic finding associated with monogenic diabetes. Because the genetic diagnosis can lead to major changes in treatment, we recommend referring all AAB-negative paediatric patients with diabetes for genetic testing. Low-titre ICAs in the absence of other AABs does not always indicate a diagnosis of type 1 diabetes.
Graphical abstract
Macrotroponin is due to cardiac troponin (cTn) binding to endogenous cTn autoantibodies. While previous studies showed a high incidence of macrotroponin affecting cTnI assays, reports of ...macrotroponin T, particularly without cTnI reactivity, have been rare. Although the clinical significance of macrotroponin is not fully understood, macroenzymes and complexes are recognised to cause confusion in interpretation of laboratory results. The potential for adverse clinical consequences due to misinterpretation of affected results is very high.
We describe four cases of macrotroponin T with persistently low high sensitivity cTnT (hs-cTnT) by the 9 min compared to the 18 min variant of the assay. Three cases were serendipitously identified due to the use of a lot number of Roche hs-cTnT affected by non-reproducible results, necessitating measurement of cTnT in duplicate. We identified and characterised these macrotroponin specimens by immunoglobulin depletion (Protein A and PEG precipitation), mixing studies with EDTA and recombinant cTnT.
In cases of macro-cTnT, a lower result occurred on the hs-cTnT using the 9 min compared to 18 min variant assay (ratio of 9-18 min hs-cTnT <0.80). Mixing studies with recombinant cTnT or EDTA demonstrated a difference in recovery vs. controls. One of these patients demonstrated a high molecular weight complex for cTnI and cTnT demonstrating a macrocomplex involving both cTn. This patient demonstrated a rise and fall in cTn when measured by several commercial assays consistent with genuine acute cardiac injury.
We identified several cases of macro-cTnT and described associated clinical and biochemical features.
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