Comparative Pharmacokinetics and Safety Assessment of 1st- and 2nd-Generation Zinpentraxin Alfa Drug Products in Healthy Volunteers: A Randomized Crossover Study.

Zinpentraxin alfa is a recombinant form of the human pentraxin-2 that was studied in idiopathic pulmonary fibrosis (IPF). To improve the purity and yield of the drug material, a 2nd-generation drug product was developed. To characterize and compare the pharmacokinetic (PK) properties of the 1st- and 2nd-generation zinpentraxin alfa, PK studies were conducted in healthy volunteers (HVs). In a phase 1 randomized, double-blind, 2-sequence crossover, sequential 2-stage study (ISRCTN59409907), single intravenous (IV) doses of 1st- and 2nd-generation zinpentraxin alfa at 10 mg/kg were studied with a blinded interim analysis (IA) at the end of stage 1. Bioequivalence (BE) was achieved for the maximum observed plasma concentration (Cmax), but the overall exposure was higher for the 2nd- compared to the 1st-generation zinpentraxin alfa. The study was stopped after stage 1 as the gating criteria were met based on the result of the blinded IA. Safety profiles were similar for the 1st- and 2nd-generation drug products, and antidrug antibody (ADA) was not observed in this study.

Using Deep Learning to Detect the Presence and Location of Hemoperitoneum on the Focused Assessment with Sonography in Trauma (FAST) Examination in Adults.

Abdominal ultrasonography has become an integral component of the evaluation of trauma patients. Internal hemorrhage can be rapidly diagnosed by finding free fluid with point-of-care ultrasound (POCUS) and expedite decisions to perform lifesaving interventions. However, the widespread clinical application of ultrasound is limited by the expertise required for image interpretation. This study aimed to develop a deep learning algorithm to identify the presence and location of hemoperitoneum on POCUS to assist novice clinicians in accurate interpretation of the Focused Assessment with Sonography in Trauma (FAST) exam. We analyzed right upper quadrant (RUQ) FAST exams obtained from 94 adult patients (44 confirmed hemoperitoneum) using the YoloV3 object detection algorithm. Exams were partitioned via fivefold stratified sampling for training, validation, and hold-out testing. We assessed each exam image-by-image using YoloV3 and determined hemoperitoneum presence for the exam using the detection with highest confidence score. We determined the detection threshold as the score that maximizes the geometric mean of sensitivity and specificity over the validation set. The algorithm had 95% sensitivity, 94% specificity, 95% accuracy, and 97% AUC over the test set, significantly outperforming three recent methods. The algorithm also exhibited strength in localization, while the detected box sizes varied with a 56% IOU averaged over positive cases. Image processing demonstrated only 57-ms latency, which is adequate for real-time use at the bedside. These results suggest that a deep learning algorithm can rapidly and accurately identify the presence and location of free fluid in the RUQ of the FAST exam in adult patients with hemoperitoneum.

Diagnostic accuracy of ultrasound to confirm endotracheal tube depth.

INTRODUCTION: Endotracheal intubation is commonly performed in the Emergency Department. Traditional measures for estimating and confirming the endotracheal tube (ETT) depth may be inaccurate or lead to delayed recognition. Ultrasound may offer a rapid tool to confirm ETT depth at the bedside. METHODS: This was a randomized trial assessing the diagnostic accuracy of ultrasound to confirm ETT depth. Three cadavers were intubated in a random sequence with the ETT placed high (directly below the vocal cords), middle (2 cm above the carina), or deep (ETT at the carina). Seven blinded sonographers assessed the depth of the ETT using ultrasound. Outcomes included diagnostic accuracy of sonographer identification, time to identification, and operator confidence based upon ETT location. A subgroup analysis was performed to assess diagnostic accuracy by operator confidence. RESULTS: 441 total assessments were performed (154 high, 154 middle, and 133 deep ETT placements). Overall accuracy was 84.8% (95% CI 81.1% to 88.0%). When placed high, ultrasound was 82.5% sensitive (95% CI 75.5% to 88.1%) and 92.3% specific (95% CI 88.6% to 95.1%) with a mean time to identification of 15.3 s (95% CI 13.6-17.0) and a mean operator confidence of 3.9/5.0 (95% CI 3.7-4.1). When the ETT was placed in the middle, ultrasound was 83.8% sensitive (95% CI 77.0% to 89.2%) and 92.3% specific (95% CI 88.6% to 95.1%) with a mean time to identification of 16.7 s (95% CI 14.6-18.8) and a mean operator confidence of 3.7/5.0 (95% CI 3.5-3.9). When the ETT was placed deep, ultrasound was 88.0% sensitive (95% CI 81.2% to 93.0%) and 92.2% specific (95% CI 88.6% to 94.6%) with a mean time to identification of 19.0 s (95% CI 17.3-20.7) and a mean operator confidence of 3.4/5.0 (95% CI 3.2-3.6). Sonographers were significantly more accurate when they reported a higher confidence score. CONCLUSION: Ultrasound was moderately accurate for identifying the ETT location in a cadaveric model and was more accurate when sonographers felt confident with their visualization. Future research should determine the accuracy of combining transtracheal ultrasound with lung sliding and other modifications to improve the accuracy.

Development of an expert consensus checklist for emergency ultrasound.

Background: Within today's competency-based medical education, traditional set number proficiency benchmarks have been called into question. Checklists may help guide individualized training and standardized outcomes. However, multicenter expert consensus checklists based on established guidelines with supporting validity evidence have not been published for specific emergency ultrasound (EUS) applications. We describe a robust national EUS expert consensus methodology for developing a checklist for the extended focused assessment with sonography in trauma (eFAST examination). Methods: Utilizing the ACEP imaging compendium as a primary reference, 10 national EUS experts iteratively refined and agreed upon a final checklist. To obtain initial reliability and validity evidence, two different EUS experts blinded to resident experience then assessed 24 residents performing an eFAST in a simulated environment. Inter-rater reliability of the checklist was assessed using Cohen's kappa coefficient. Validity was assessed by comparing mean performance with the Student's t-test and discriminant ability of individual checklist items using item-total correlation. Results: The 10 EUS experts agreed on the final checklist items after two rounds of iterations. When evaluating 24 emergency medicine (EM) PGY-1 to -4 residents, the kappa correlation between two blinded EUS faculty raters was moderate at 0.670. Kappa and agreement were near-perfect or perfect in right and left chest image optimization, right upper quadrant (RUQ) probe placement, RUQ anatomy identification, and pelvic first-view anatomy identification. The difference in checklist performance between junior and senior EM residents was significant at -8.1% (p = 0.004). Identification of pelvic structures and placement of the probe for pelvic views were found to have an excellent item-total correlation with values of >0.4. Conclusions: We have described a robust national EUS expert consensus methodology for developing an eFAST checklist based on the ACEP imaging guidelines. Based on this encouraging initial reliability and validity evidence, further research and checklist development is warranted for additional EUS applications.

Comparison of saline versus air for identifying endotracheal intubation with ultrasound.

INTRODUCTION: After intubation has been performed, it is important to rapidly confirm the correct location of the endotracheal tube (ETT). Multiple techniques have been described, each with different limitations. Ultrasound has been increasingly recognized as an alternate modality for identifying the ETT location. However, it can be challenging to visualize the air-filled ETT cuff. Saline insufflation of the ETT cuff has been suggested to improve visualization of the ETT but data are limited. Our study sought to compare the diagnostic accuracy of air versus saline ETT cuff inflation on the diagnostic accuracy of intubation. METHODS: This was a randomized trial comparing air versus saline cuff inflation using a cadaver model. Adult cadavers were intubated in a random sequence with respect to both the location of intubation (i.e., tracheal vs esophageal) and air versus saline. Blinded sonographers assessed the location of the ETT using the static technique. Outcomes included accuracy of sonographer identification, time to identification, and operator confidence. RESULTS: 480 total assessments were performed. When using air, ultrasound was 95.8% sensitive (95% CI 90.5% to 98.6%) and 100% specific (95% CI 97.0% to 100%) with a mean time to confirmation of 8.5 s (95% CI 7.6 s to 9.4 s) and a mean operator confidence of 4.32/5.0 (95% CI 4.21 to 4.42). When using saline, ultrasound was 100% sensitive (95% CI 97.0% to 100%) and 100% specific (95% CI 97.0% to 100%) with a mean time to confirmation of 6.3 s (95% CI 5.9 s to 6.8 s) and a mean operator confidence of 4.52/5.0 (95% CI 4.44 to 4.60). CONCLUSION: There was no statistically significant difference between air versus saline for intubation confirmation. However, saline was associated with fewer false negatives. Additionally, time to confirmation was faster and operator confidence was higher with the saline group. Further studies should determine if the outcomes would change with more novice sonographers or in specific patient populations.

Differentiation of MISSLA and Fanconi anaemia by computer-aided image analysis and presentation of two novel MISSLA siblings.

Variants in DONSON were recently identified as the cause of microcephaly, short stature, and limb abnormalities syndrome (MISSLA). The clinical spectra of MISSLA and Fanconi anaemia (FA) strongly overlap. For that reason, some MISSLA patients have been clinically diagnosed with FA. Here, we present the clinical data of siblings with MISSLA featuring a novel DONSON variant and summarize the current literature on MISSLA. Additionally, we perform computer-aided image analysis using the DeepGestalt technology to test how distinct the facial features of MISSLA and FA patients are. We show that MISSLA has a specific facial gestalt. Notably, we find that also FA patients feature facial characteristics recognizable by computer-aided image analysis. We conclude that computer-assisted image analysis improves diagnostic precision in both MISSLA and FA.

Analytical and Clinical Validation of a Point-of-Care Cardiac Troponin T Test with an Improved Detection Limit.

BACKGROUND: The point-of-care test Roche CARDIAC POC Troponin T (PoC TnT) is an improved assay which has been developed for the Roche cobas h 232 system. METHODS: We performed a multicentre evaluation (four sites) to assess the analytical performance of the PoC TnT assay and to compare it with the central laboratory Elecsys® troponin T high sensitive (lab cTnT-hs) assay. RESULTS: The relative mean differences found in method comparisons of PoC TnT vs. lab cTnT-hs ranged from -4.1% to +6.8%. Additionally, there was good concordance between PoC TnT and lab cTnT-hs for the number of samples with troponin T values below the measuring range of 40 ng/L. Lot-to-lot differences of PoC TnT ranged from -8.6% to +4.6%. Within-series coefficients of variation (CV) resulting from 81 ten-fold measurements with patient samples were 9.3%, 11.8%, and 12.9% in the low (40 to < 200 ng/L), medium (200 to < 600 ng/L), and high (600 to 2000 ng/L) measuring range, respectively. Using the system quality control, the mean CV for between-day imprecision was 11.3%. No interference was observed by triglycerides (up to 11.4 mmol/L), bilirubin (up to 376 µmol/L), hemoglobin (up to 0.12 mmol/L), biotin (up to 30 µg/L), rheumatoid factor (up to 200 IU/mL), or with 52 standard or cardiovascular drugs at therapeutic concentrations. There was no influence on the results by varying hematocrit values in a range from 25% to 53%. However, interferences with human anti-mouse antibodies were found. No significant influence on the results was found with PoC TnT by using sample volumes between 135 to 165 µL. High troponin T concentrations up to 500 µg/L did not lead to false low results, indicating no high-concentration hook effect. No cross-reactivity was found between the PoC TnT assay and human skeletal troponin T up to 1000 µg/L (< 0.05%). Diagnostic sensitivity and specificity data of a subpopulation (23 patients) of this study are in agreement with results of another large pre-hospital study. CONCLUSIONS: The PoC TnT assay showed good analytical performance with excellent concordance with the calibration and reference laboratory method. It should therefore be suitable for its intended use in point-of-care settings.

Two N-terminally truncated variants of human ß-galactoside α2,6 sialyltransferase I with distinct properties for in vitro protein glycosylation.

Sialic acid groups of protein N-glycans are important determinants of biological activity. Exposed at the end of the glycan chain, they are potential targets for glycan remodeling. Sialyltransferases (STs; EC 2.4.99) are the enzymes that catalyze the sialic acid transfer from a CMP-activated donor on to a carbohydrate acceptor in vivo. Recombinant expression of the full-length human ß-galactoside α2,6 sialyltransferase I (ST6Gal-I) was hampered and therefore variants with truncated N-termini were investigated. We report on the distinct properties of two N-terminally truncated versions of ST6Gal-I, namely Δ89ST6Gal-I and Δ108ST6Gal-I, which were successfully expressed in human embryonic kidney cells. The different properties of these enzymes result most probably from the loss of interactions from helix α1 in the Δ108ST6Gal-I variant, which plays a role in acceptor substrate binding. The Km for N-acetyl-d-lactosamine was 10-fold increased for Δ108ST6Gal-I (84 mM) as compared to Δ89ST6Gal-I (8.3 mM). The two enzyme variants constitute a suitable tool box for the terminal modification of N-glycans. While the enzyme Δ89ST6Gal-I exhibited both ST (di-sialylation) and sialidase activity on a monoclonal antibody, the enzyme Δ108ST6Gal-I showed only ST activity with specificity for mono-sialylation.

Latent transforming growth factor binding protein 4 regulates transforming growth factor beta receptor stability.

Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFß) signaling. Despite elevated extracellular TGFß activity, downstream signaling molecules of the TGFß pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFß receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFß1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFß receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFß signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFß receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFß receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling.

Next-generation sequencing in X-linked intellectual disability.

X-linked intellectual disability (XLID) is a genetically heterogeneous disorder with more than 100 genes known to date. Most genes are responsible for a small proportion of patients only, which has hitherto hampered the systematic screening of large patient cohorts. We performed targeted enrichment and next-generation sequencing of 107 XLID genes in a cohort of 150 male patients. Hundred patients had sporadic intellectual disability, and 50 patients had a family history suggestive of XLID. We also analysed a sporadic female patient with severe ID and epilepsy because she had strongly skewed X-inactivation. Target enrichment and high parallel sequencing allowed a diagnostic coverage of >10 reads for ~96% of all coding bases of the XLID genes at a mean coverage of 124 reads. We found 18 pathogenic variants in 13 XLID genes (AP1S2, ATRX, CUL4B, DLG3, IQSEC2, KDM5C, MED12, OPHN1, SLC9A6, SMC1A, UBE2A, UPF3B and ZDHHC9) among the 150 male patients. Thirteen pathogenic variants were present in the group of 50 familial patients (26%), and 5 pathogenic variants among the 100 sporadic patients (5%). Systematic gene dosage analysis for low coverage exons detected one pathogenic hemizygous deletion. An IQSEC2 nonsense variant was detected in the female ID patient, providing further evidence for a role of this gene in encephalopathy in females. Skewed X-inactivation was more frequently observed in mothers with pathogenic variants compared with those without known X-linked defects. The mutation rate in the cohort of sporadic patients corroborates previous estimates of 5-10% for X-chromosomal defects in male ID patients.

High-quality production of human α-2,6-sialyltransferase in Pichia pastoris requires control over N-terminal truncations by host-inherent protease activities.

BACKGROUND: α-2,6-sialyltransferase catalyzes the terminal step of complex N-glycan biosynthesis on human glycoproteins, attaching sialic acid to outermost galactosyl residues on otherwise fully assembled branched glycans. This "capping" of N-glycans is critical for therapeutic efficacy of pharmaceutical glycoproteins, making the degree of sialylation an important parameter of glycoprotein quality control. Expression of recombinant glycoproteins in mammalian cells usually delivers heterogeneous N-glycans, with a minor degree of sialylation. In-vitro chemo-enzymatic glycoengineering of the N-glycans provides an elegant solution to increase the degree of sialylation for analytical purposes but also possibly for modification of therapeutic proteins. RESULTS: Human α-2,6-sialyltransferase (ST6Gal-I) was secretory expressed in P.pastoris KM71H. ST6Gal-I featuring complete deletion of both the N-terminal cytoplasmic tail and the transmembrane domain, and also partial truncation of the stem region up to residue 108 were expressed N-terminally fused to a His or FLAG-Tag. FLAG-tagged proteins proved much more resistant to proteolysis during production than the corresponding His-tagged proteins. Because volumetric transferase activity measured on small-molecule and native glycoprotein acceptor substrates did not correlate to ST6Gal-I in the supernatant, enzymes were purified and characterized in their action on non-sialylated protein-linked and released N-glycans, and the respective N-terminal sequences were determined by automated Edman degradation. Irrespective of deletion construct used (Δ27, Δ48, Δ62, Δ89), isolated proteins showed N-terminal processing to a highly similar degree, with prominent truncations at residue 108 - 114, whereby only Δ108ST6Gal-I retained activity. FLAG-tagged Δ108ST6Gal-I was therefore produced and obtained with a yield of 4.5 mg protein/L medium. The protein was isolated and shown by MS to be intact. Purified enzyme exhibited useful activity (0.18 U/mg) for sialylation of different substrates. CONCLUSIONS: Functional expression of human ST6Gal-I as secretory protein in P.pastoris necessitates that N-terminal truncations promoted by host-inherent proteases be tightly controlled. N-terminal FLAG-Tag contributes extra stability to the N-terminal region as compared to N-terminal His-Tag. Proteolytic degradation proceeds up to residues 108 - 114 and of the resulting short-form variants, only Δ108ST6Gal-I seems to be active. FLAG-Δ108ST6Gal-I transfers sialic acids to monoclonal antibody substrate with sufficient yields, and because it is stably produced in P.pastoris, it is identified here as an interesting glycoengineering catalyst.

Human ESC self-renewal promoting microRNAs induce epithelial-mesenchymal transition in hepatocytes by controlling the PTEN and TGFß tumor suppressor signaling pathways.

The self-renewal capacity ascribed to embryonic stem cells (ESC) is reminiscent of cancer cell proliferation, raising speculation that a common network of genes may regulate these traits. A search for general regulators of these traits yielded a set of microRNAs for which expression is highly enriched in human ESCs and liver cancer cells (HCC) but attenuated in differentiated quiescent hepatocytes. Here, we show that these microRNAs promote hESC self-renewal, as well as HCC proliferation, and when overexpressed in normally quiescent hepatocytes, induce proliferation and activate cancer signaling pathways. Proliferation in hepatocytes is mediated through translational repression of Pten, Tgfbr2, Klf11, and Cdkn1a, which collectively dysregulates the PI3K/AKT/mTOR and TGFß tumor suppressor signaling pathways. Furthermore, aberrant expression of these miRNAs is observed in human liver tumor tissues and induces epithelial-mesenchymal transition in hepatocytes. These findings suggest that microRNAs that are essential in normal development as promoters of ESC self-renewal are frequently upregulated in human liver tumors and harbor neoplastic transformation potential when they escape silencing in quiescent human hepatocytes.

Epigenetic modulation of miR-122 facilitates human embryonic stem cell self-renewal and hepatocellular carcinoma proliferation.

The self-renewal capacity ascribed to hESCs is paralleled in cancer cell proliferation, suggesting that a common network of genes may facilitate the promotion of these traits. However, the molecular mechanisms that are involved in regulating the silencing of these genes as stem cells differentiate into quiescent cellular lineages remain poorly understood. Here, we show that a differentiated cell specific miR-122 exemplifies this regulatory attribute by suppressing the translation of a gene, Pkm2, which is commonly enriched in hESCs and liver cancer cells (HCCs), and facilitates self-renewal and proliferation. Through a series of gene expression analysis, we show that miR-122 expression is highly elevated in quiescent human primary hepatocytes (hPHs) but lost or attenuated in hESCs and HCCs, while an opposing expression pattern is observed for Pkm2. Depleting hESCs and HCCs of Pkm2, or overexpressing miR-122, leads to a common deficiency in self-renewal and proliferation. Likewise, during the differentiation process of hESCs into hepatocytes, a reciprocal expression pattern is observed between miR-122 and Pkm2. An examination of the genomic region upstream of miR-122 uncovered hyper-methylation in hESCs and HCCs, while the same region is de-methylated and occupied by a transcription initiating protein, RNA polymerase II (RNAPII), in hPHs. These findings indicate that one possible mechanism by which hESC self-renewal is modulated in quiescent hepatic derivatives of hESCs is through the regulatory activity of a differentiated cell-specific miR-122, and that a failure to properly turn "on" this miRNA is observed in uncontrollably proliferating HCCs.

LDH-C can be differentially expressed during fermentation of CHO cells.

Expression of CHO mRNA was measured with special microarrays from the Consortium for Chinese Hamster Ovary (CHO) Cell Genomics led by Prof. Wei-Shou Hu of the University of Minnesota and Prof. Miranda Yap of the Bioprocess Technology Institute of A*STAR, Singapore (http://hugroup.cems.umn.edu/CHO/cho_index.html). Cultivation experiments were performed in small scale 2L stirred tank bioreactors. During fermentation a temperature shift of -3°C was performed. This was accompanied by a reduction of the cell specific lactate production rate. The analysis of transcriptome samples before and after the temperature shift with microarrays showed several changes in the expression of available gene markers. LDH-C expression raised about 2 fold after temperature shift. LDH-A did not change. As LDH-C is known to be a specialized isoenzyme in sperm cells for consuming lactate in a lactate containing milieu, LDH-C could be proposed as a target for genetic engineering, facilitating lactate consumption in the late phase of high cell density cultures and prolonging longevity of CHO production cultures by reducing lactate and base accumulation.

The differentiation of hepatocyte-like cells from monkey embryonic stem cells.

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.

Complementary strategies to elucidate T helper cell epitopes in myasthenia gravis.

CD4(+) T cells specific for the acetylcholine receptor (AChR) are assumed to play an important role in pathogenesis of myasthenia gravis (MG). A large and diverse number of potential T cell epitopes have been reported for different experimental setups aiming at the identification of disease-relevant T cells in MG. Investigating the T cell response to the epsilon subunit of human AChR, we explore complementary in vitro and in vivo approaches (PBMC from MG patients and mice transgenic for HLA-DR3 and human CD4) to address the possibilities and limitations of different strategies for elucidating natural autoimmune T cell epitopes.