Journal: Acta Neuropathologica

Article Title: Inter-alpha-trypsin inhibitor heavy chain H3 is a potential biomarker for disease activity in myasthenia gravis

doi: 10.1007/s00401-024-02754-6

Figure Lengend Snippet: Comprehensive proteomic mapping of serum samples in the exploration cohort. a Workflow for mass spectrometry-based proteomics. Serum samples of 114 anti-AChR-Ab-positive MG patients were analyzed. Peptides were detected by nano-UPLC coupled to ion mobility MS with Synapt G2 Si/HDMSe. b – d ORAs of GO terms in the whole proteome. Negative decadic logarithms of corresponding p values (−log 10 p value) are depicted on the x-axis. Circle sizes illustrate counts of associated proteins. P adjs are color-coded. The most interesting terms referred to in the current study are printed in bold. b GO enrichment of cellular components (GO-CC). c GO enrichment of biological processes (GO-BP). d GO enrichment of molecular functions (GO-MF). e Hierarchical clustering of log 2 mean expression levels of serum samples, i.e., patients (columns) and proteins (rows), according to Euclidean distance using correlation distance and average linkage. Log 2 mean expression values are color-coded. Clinical subgroups of patients are depicted and illustrated in different colors. f MA plot illustrating differential protein expression profiles of PASS-negative versus PASS-positive patients by plotting the log 2 fold change of protein intensity values against their log 2 mean expression levels. All proteins with a p value of ≤ 0·05 were color-coded. The ten most significantly regulated proteins across both experimental groups were labeled with their gene symbols. Anti-AChR-ab anti-acetylcholine receptor antibody, APOA4 apolipoprotein A4, BP biological process, CALM3 calmodulin-3, C9 complement component C9, CC cellular component, CDKN2AIPNL CDKN2AIP N-terminal-like protein, CETN3 centrin-3, CFB complement factor B, GO gene ontology, HDMSe high-definition mass spectrometry, IST immunosuppressive therapy, ITIH3 inter-alpha-trypsin inhibitor heavy chain 3, LFQ label-free quantification, MF molecular function, MG myasthenia gravis, MS mass spectrometry, ORA overrepresentation analysis , P adj adjusted p value, PASS patient-acceptable symptom state, SKAP2 src kinase-associated phosphoprotein 2, SNTA1 alpha-1-syntrophin, STAG3 cohesin subunit SA-3, thym. Thymoma, UPLC ultra performance liquid chromatography

Article Snippet: Serum samples from patients and HCs were tested for ITIH3 abundance using an ITIH3 ELISA kit (#CSB-EL011896HU, CUSABIO, Houston, TX, USA) according to the manufacturer's instructions after the dilution series, and the samples were diluted 1:500 for the final assays.

Techniques: Mass Spectrometry, Expressing, Labeling, Quantitative Proteomics, Liquid Chromatography

Journal: Acta Neuropathologica

Article Title: Inter-alpha-trypsin inhibitor heavy chain H3 is a potential biomarker for disease activity in myasthenia gravis

doi: 10.1007/s00401-024-02754-6

Figure Lengend Snippet: Machine learning identifies ITIH3 as a potential novel biomarker for disease severity and prognosis. a Workflow of the ML approach. Briefly, data were split into training and test data sets, followed by pre-processing of data, model training and tuning, and, finally, estimation of variable importance. b Correlation of a whole proteome data set. Highly correlated predictors with a cutoff value of r > 0·9 or < −0·9 were removed during pre-processing of data. c Comparison of five models: GLM, GLMboost, earth, GBM, and BstLm. Evaluation of these models comprises computing and comparing the MAE, the RMSE, and R-squared. Error values are indicated on the x-axis. d Actual versus predicted QMG scores for the final model (GLMboost). e The relative value of the t-statistic for each predictor variable is calculated and indicated in the bar graph. The highest contributing variable was scaled to 100. f Regression analyses of ITIH3 protein abundance and (∆)QMG or MG-ADL scores at the time of blood sampling (upper panels) or 12 months after first testing, respectively (lower panels). In the upper right hand of each plot, p values are indicated next to the R statistic and the linear function equation describing the regression. APOM apolipoprotein M, BUD31 protein BUD31 homolog, BstLm boosted linear model, C1orf52 UPF0690 protein C1orf52, DDIT4L DNA damage inducible transcript 4 like; earth, multivariate adaptive regression splines, GBM gradient-boosting machine, GLM generalized linear model, GLMboost gradient boosting with component-wise linear models, GSN gelsolin, HIST1H4A histone H4, ITIH3 inter-alpha-trypsin inhibitor heavy chain 3, MAE mean absolute error, MG-ADL MG activities of daily living, ML machine learning, PPBP platelet basic protein, QMG quantitative MG, RMSE root mean square error, RP2 protein XRP2, SERPINF2 , alpha-2-antiplasmin, ZC3H6 zinc finger CCCH domain-containing protein 6

Article Snippet: Serum samples from patients and HCs were tested for ITIH3 abundance using an ITIH3 ELISA kit (#CSB-EL011896HU, CUSABIO, Houston, TX, USA) according to the manufacturer's instructions after the dilution series, and the samples were diluted 1:500 for the final assays.

Techniques: Biomarker Discovery, Comparison, Quantitative Proteomics, Sampling

Journal: Acta Neuropathologica

Article Title: Inter-alpha-trypsin inhibitor heavy chain H3 is a potential biomarker for disease activity in myasthenia gravis

doi: 10.1007/s00401-024-02754-6

Figure Lengend Snippet: Validation of ITIH3 in an independent control cohort a Overview of the included patient cohorts. In the “exploration cohort,” the serum samples of 114 patients were collected at baseline, and patients were followed up for a minimum of 12 months. Patients receiving ISTs were required to be stable for at least six months before sample acquisition. For validation, 140 patients were recruited prospectively in the “validation cohort.” Blood samples were analyzed after collection. b Univariate regression analyses of ITIH3 protein abundance and QMG or MG-ADL scores at the time of blood sampling in the validation cohort. In the upper right hand of each plot, p values are indicated next to the R statistic and the linear function equation describing the regression. c Hierarchical clustering of log 2 mean expression levels of serum samples in the validation cohort, i.e., patients (columns) and proteins (rows), according to Euclidean distance using correlation distance and average linkage. Log 2 mean expression values are color-coded. Clinical subgroups of patients are depicted and illustrated by different colors. d Regression analyses of anti-acetylcholine receptor antibody levels and QMG or MG-ADL scores at the time of blood sampling in the validation cohort. The detection limit of the radio receptor assay used for this purpose was 0.4 ng/mL. In the upper right hand of each plot, p values are indicated next to the R statistic and the linear function equation describing the regression. Anti-AChR-Ab anti-acetylcholine receptor antibody, IST immunosuppressive therapy, ITIH3 inter-alpha-trypsin inhibitor heavy chain 3, MG myasthenia gravis, MG-ADL MG activities of daily living, QMG quantitative MG, thym. thymoma

Article Snippet: Serum samples from patients and HCs were tested for ITIH3 abundance using an ITIH3 ELISA kit (#CSB-EL011896HU, CUSABIO, Houston, TX, USA) according to the manufacturer's instructions after the dilution series, and the samples were diluted 1:500 for the final assays.

Techniques: Biomarker Discovery, Control, Quantitative Proteomics, Sampling, Expressing

Journal: Acta Neuropathologica

Article Title: Inter-alpha-trypsin inhibitor heavy chain H3 is a potential biomarker for disease activity in myasthenia gravis

doi: 10.1007/s00401-024-02754-6

Figure Lengend Snippet: Validation of ITIH3 as a biomarker for disease activity and treatment response a Raincloud plot comparing ITIH3 protein abundance levels between PASS-positive and PASS-negative anti-AChR-Ab-positive MG patients measured by ELISA. Here, data sets from the exploration and validation cohorts were combined. Likewise, ITIH3 levels are indicated for anti-MuSK-Ab-positive ( n = 10) and seronegative ( n = 10) MG patients as well as for patients with CMS ( n = 14), IIM ( n = 12 with IBM and n = 2 with IMNM), and CIDP ( n = 19). HCs indicate baseline levels ( n = 53). A p value > 0.05 was classified as not significant, p < 0.05 (*) as significant, p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) as highly significant. b + c Linear regression analysis of ITIH3 protein abundance measured by ELISA and by mass spectrometry in each cohort as indicated. d + e Linear regression analysis of ITIH3 protein levels measured by ELISA and individual QMG scores in each cohort as indicated. In the upper left hand of the plot, p -values are indicated next to the R statistic and the linear function equation describing the regression. f Scatter plot displaying the change to serum ITIH3 as measured by ELISA for treatment responders between baseline and follow-up. Each line indicates the change for an individual patient. g Scatter plot displaying the change to serum ITIH3 as measured by ELISA for treatment non-responders between baseline and follow-up. Each line indicates the change for an individual patient. Treatment response was defined as an improvement of ≥ 3 points on the MG-ADL scale. h Bar graph indicating the change to ITIH3 between follow-up and baseline for responders and non-responders. A one sample t test with zero as hypothetical mean was used for statistical analysis of each group. A p value > 0.05 was classified as not significant, p < 0.05 (*) as significant, p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) as highly significant. Anti-AChR-Ab anti-acetylcholine receptor antibody, CIDP chronic inflammatory demyelinating polyradiculoneuropathy, CMS congenital myasthenic syndrome, HC healthy control, IBM inclusion body myositis, IIM idiopathic inflammatory myopathy, IMNM immune-mediated necrotizing myopathy, ITIH3 inter-alpha-trypsin inhibitor heavy chain H3, MG myasthenia gravis, MuSK muscle-specific kinase, PASS patient-acceptable symptom state, seroneg. seronegative, QMG quantitative MG

Article Snippet: Serum samples from patients and HCs were tested for ITIH3 abundance using an ITIH3 ELISA kit (#CSB-EL011896HU, CUSABIO, Houston, TX, USA) according to the manufacturer's instructions after the dilution series, and the samples were diluted 1:500 for the final assays.

Techniques: Biomarker Discovery, Activity Assay, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Control

Journal: Acta Neuropathologica

Article Title: Inter-alpha-trypsin inhibitor heavy chain H3 is a potential biomarker for disease activity in myasthenia gravis

doi: 10.1007/s00401-024-02754-6

Figure Lengend Snippet: Clinical and demographic baseline data of follow-up patients

Article Snippet: Serum samples from patients and HCs were tested for ITIH3 abundance using an ITIH3 ELISA kit (#CSB-EL011896HU, CUSABIO, Houston, TX, USA) according to the manufacturer's instructions after the dilution series, and the samples were diluted 1:500 for the final assays.

Techniques:

Journal: Acta Neuropathologica

Article Title: Inter-alpha-trypsin inhibitor heavy chain H3 is a potential biomarker for disease activity in myasthenia gravis

doi: 10.1007/s00401-024-02754-6

Figure Lengend Snippet: NMJs in skeletal muscles of anti-AChR-Ab-positive MG and healthy controls a Gömöri trichrome stain of skeletal muscle tissue showing mild fiber size variation and presence of endomysial nerve fascicles between myofibers (original magnification × 400). NSE indicating NMJs (arrows) within endplate regions both in skeletal muscle biopsies of anti-AChR-Ab-positive MG and HC (original magnification × 400). C5b-9 deposits at the NMJ in anti-AChR-ab-positive MG patients (arrows), but not in HC (magnification × 400). IHC for ITIH3 is positive at NMJ of anti-AChR-ab-positive MG (arrows), but not on endplates of healthy controls (note the proof of an endplate region by highlighting the nerve fascicle in the middle right) (original magnification × 400). The same exposure time was used for all images. Scale bars are 100 µm. b Double immunofluorescence staining of intercostal muscle biopsies from anti-AChR-Ab-positive MG patients (original magnification × 400). NMJs are indicated by arrows. The same exposure time was used for all images. Scale bars are 100 µm. CD56 is a neuronal cell adhesion molecule on the presynaptic membrane. Overlapping of CD56 and ITIH3 staining showed that ITIH3 is mainly localized at the postsynaptic membrane. Desmin is an intermediate filament-associated protein known to be enriched at the postsynaptic membrane of NMJs. Double immunofluorescence of ITIH3 and desmin showed an overlap of both markers, confirming the postsynaptic localization of ITIH3. Plectin is an intermediate filament-associated protein that links cytoskeletal components. It is highly expressed at the postsynaptic membrane and supports its junctional folds. Double staining of ITIH3 and plectin showed colocalization of both proteins. Anti-AChR-Ab anti-acetylcholine receptor antibody, DDA data-dependent acquisition, DES desmin, HC healthy control, IHC immunohistochemistry, ITIH3 inter-alpha-trypsin inhibitor heavy chain H3, LC liquid chromatography, MG myasthenia gravis, MS/MS tandem mass spectrometry, NMJ neuromuscular junction, NSE neuron-specific enolase, PLEC plectin

Article Snippet: Serum samples from patients and HCs were tested for ITIH3 abundance using an ITIH3 ELISA kit (#CSB-EL011896HU, CUSABIO, Houston, TX, USA) according to the manufacturer's instructions after the dilution series, and the samples were diluted 1:500 for the final assays.

Techniques: Muscles, Staining, Double Immunofluorescence Staining, Membrane, Immunofluorescence, Double Staining, Data-dependent acquisition, Control, Immunohistochemistry, Liquid Chromatography, Tandem Mass Spectroscopy, Mass Spectrometry