A Novel In Vitro Assay Correlates Insulin Receptor Autoantibodies With Fasting Insulin in Type B Insulin Resistance.

CONTEXT: Severe insulin resistance (IR) in the presence of insulin receptor autoantibodies (InsR-aAb) is known as type B insulin resistance (TBIR). Considerable progress in therapy has been achieved, but diagnosis and monitoring of InsR-aAb remains a challenge. OBJECTIVE: This work aimed to establish a robust in vitro method for InsR-Ab quantification. METHODS: Longitudinal serum samples from patients with TBIR at the National Institutes of Health were collected. A bridge-assay for InsR-aAb detection was established using recombinant human insulin receptor as bait and detector. Monoclonal antibodies served as positive controls for validation. RESULTS: The novel assay proved sensitive, robust, and passed quality control. The measured InsR-aAb from TBIR patients was associated with disease severity, decreased on treatment, and inhibited insulin signaling in vitro. Titers of InsR-aAb correlated positively to fasting insulin in patients. CONCLUSION: Quantification of InsR-aAb from serum samples via the novel in vitro assay enables identification of TBIR and monitoring of successful therapy.

Circulating multimeric immune complexes contribute to immunopathology in COVID-19.

A dysregulated immune response with high levels of SARS-CoV-2 specific IgG antibodies characterizes patients with severe or critical COVID-19. Although a robust IgG response is considered to be protective, excessive triggering of activating Fc-gamma-receptors (FcγRs) could be detrimental and cause immunopathology. Here, we document excessive FcγRIIIA/CD16A activation in patients developing severe or critical COVID-19 but not in those with mild disease. We identify two independent ligands mediating extreme FcγRIIIA/CD16A activation. Soluble circulating IgG immune complexes (sICs) are detected in about 80% of patients with severe and critical COVID-19 at levels comparable to active systemic lupus erythematosus (SLE) disease. FcγRIIIA/CD16A activation is further enhanced by afucosylation of SARS-CoV-2 specific IgG. Utilizing cell-based reporter systems we provide evidence that sICs can be formed prior to a specific humoral response against SARS-CoV-2. Our data suggest a cycle of immunopathology driven by an early formation of sICs in predisposed patients. These findings suggest a reason for the seemingly paradoxical findings of high antiviral IgG responses and systemic immune dysregulation in severe COVID-19. The involvement of circulating sICs in the promotion of immunopathology in predisposed patients opens new possibilities for intervention strategies to mitigate critical COVID-19 progression.

Structural and Functional Characterization of SARS-CoV-2 RBD Domains Produced in Mammalian Cells.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is still ongoing and dramatically influences our life, the need for recombinant viral proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor-binding domain (RBD), mediates the interaction with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells and may be modulated by its structural features. Therefore, well-characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional post translational modifications), a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection, and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, a higher level of sialylation, and a combination of core 1 and core 2 type O-glycans. Additionally, using an alternative approach based on N-terminal cleavage of the O-glycosylation, the previously unknown O-glycosylation site was localized at T323. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to the ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides an analytical workflow for characterization of new RBDs and batch-to-batch comparison.

Antagonistic Autoantibodies to Insulin-Like Growth Factor-1 Receptor Associate with Poor Physical Strength.

Natural autoantibodies to the IGF1 receptor (IGF1R-aAb) have been described in relation to Graves' ophthalmopathy. Other physiological roles of natural IGF1R-aAb are not known. We hypothesized that IGF1R-aAb may be related to muscle development. Serum samples (n = 408) from young overweight subjects (n = 143) were collected during a lifestyle intervention study. Anthropometric parameters, along with leptin, IGF1 and IGF1R-aAb concentrations, were analyzed, and the subjects were categorized into positive or negative for IGF1R-aAb. Eleven out of 143 subjects (7.7%) were positive for IGF1R-aAb. Identified IGF1R-aAb were molecularly characterized and showed antagonistic activity in vitro impairing IGF1-mediated IGF1R activation. Mean body weight, height or age were similar between IGF1R-aAb-positive and -negative subjects, but IGF1 concentrations differed. Jumping ability, as well as right and left handgrip strengths, were lower in the IGF1R-aAb-positive as compared to the IGF1R-aAb-negative subjects. We conclude that natural IGF1R-aAb are detectable in apparently healthy subjects and are capable of antagonizing IGF1-dependent IGF1R activation. Moreover, the presence of IGF1R-aAb is associated with poor physical strength. Although the causality of this association is unclear, the data imply a potential influence of IGF1R autoimmunity on muscle development.

An Improved Nonradioactive Screening Method Identifies Genistein and Xanthohumol as Potent Inhibitors of Iodothyronine Deiodinases.

BACKGROUND: Deiodinases (DIO1, 2, and 3) are key enzymes in thyroid hormone (TH) activation and inactivation with impact on energy metabolism, development, cell differentiation, and a number of other physiological processes. The three DIO isoenzymes thus constitute sensitive rate-limiting components within the TH axis, prone to dysregulation by endocrine disruptive compounds or disease state. In animal models and cell culture experiments, they serve as readout for local TH status and disarrangement of the hormonal axis. Furthermore, some human diseases are characterized by apparent deiodinase dysregulation (e.g., the low triiodothyronine syndrome in critical illness). Consequently, these enzymes are targets of interest for the development of pharmacological compounds with modulatory activities. Until now, the portfolio of inhibitors for these enzymes is limited. In the clinics, the DIO1-specific inhibitor propylthiouracil is in use for treatment of severe hyperthyroidism. Other well-known inhibitors (e.g., iopanoic acid or aurothioglucose) are nonselective and block all three isoenzymes. Furthermore, DIO3 was shown to be a potential oncogenic gene, which is strongly expressed in some tumors and might, in consequence, protect tumor tissue form differentiation by TH. With respect to its role in tumorigenesis, specific inhibitors of DIO3 as a potential target for anticancer drugs would be highly desirable. To this end, a flexible and convenient assay for high-throughput screening is needed. We recently described a nonradioactive screening assay, utilizing the classic Sandell-Kolthoff reaction as readout for iodide release from the substrate molecules. While we used murine liver as enzyme source, the assay was limited to murine DIO1 activity testing. Here, we describe the use of recombinant proteins as enzyme sources within the assay, expanding its suitability from murine Dio1 to human DIO1, DIO2, and DIO3. METHODS: As proof-of-concept, deiodination reactions catalyzed by these recombinant enzymes were monitored with various nonradioactive substrates and confirmed by liquid chromatography-tandem mass spectrometry. RESULTS: The contrast agent and known DIO inhibitor iopanoic acid was characterized as readily accepted substrate by DIO2 and Dio3. In a screening approach using established endocrine disrupting compounds, the natural food ingredient genistein was identified as a further DIO1-specific inhibitor, while xanthohumol turned out to potently block the activity of all three isoenzymes. CONCLUSIONS: A rapid nonradioactive screening method based on the Sandell-Kolthoff reaction is suitable for identification of environmental, nutritive and pharmacological compounds modulating activities of human deiodinase enzymes.

Autoantibodies to the IGF1 receptor in Graves' orbitopathy.

CONTEXT: Graves' disease (GD) is maintained by stimulating antibodies against the TSH receptor. Graves' orbitopathy (GO) is the main extrathyroidal manifestation of GD, potentially involving autoimmunity against the IGF1 receptor (IGF1R). OBJECTIVE: We tested for autoantibodies against the IGF1R (IGF1R-Abs) in sera of GD patients and controls and elucidated their possible implication in the disease. DESIGN: A diagnostic assay for IGF1R-Ab was established with recombinant human IGF1R as autoantigen. Serum samples or purified Ig preparations were analyzed for IGF1R binding and modulation of IGF1 signaling in vitro. A total of 108 consecutive GO patients represented on average by 5.4 separate serum samples per individual along with 92 healthy controls were analyzed. RESULTS: IGF1R-Ab were detected in 10 serum samples from control subjects (11%) and in 60 samples (10%) from the GO patient serum bank. The positive patient samples were derived from 15 individuals yielding an IGF1R-Ab prevalence of 14% in GO. More than three consecutive samples were available from 11 of the 15 positive GO patients spanning an average disease period of 2 years. IGF1R-Ab concentrations were constantly elevated in these patients demonstrating relatively stable IGF1R-Ab expression over time. IGF1R-Ab failed to stimulate IGF1R autophosphorylation but instead inhibited IGF1-induced signaling in hepatocarcinoma HepG2 cells. Similarly, growth of MCF7 breast cancer cells was inhibited by IGF1R-Ab, supporting their classification as IGF1 antagonists. CONCLUSIONS: Our data demonstrate the existence of IGF1R-Abs in humans but do not support the hypothesis that the IGF1R-Abs contribute to GO pathogenesis.

Specific and pleiotropic patterns of mRNA regulation by ArcZ, a conserved, Hfq-dependent small RNA.

The small RNA, ArcZ (previously RyhA/SraH), was discovered in several genome-wide screens in Escherichia coli and Salmonella. Its high degree of genomic conservation, its frequent recovery by shotgun sequencing, and its association with the RNA chaperone, Hfq, identified ArcZ as an abundant enterobacterial 'core' small RNA, yet its function remained unknown. Here, we report that ArcZ acts as a post-transcriptional regulator in Salmonella, repressing the mRNAs of the widely distributed sdaCB (serine uptake) and tpx (oxidative stress) genes, and of STM3216, a horizontally acquired methyl-accepting chemotaxis protein (MCP). Both sdaCB and STM3216 are regulated by sequestration of the ribosome binding site. In contrast, the tpx mRNA is targeted in the coding sequence (CDS), arguing that CDS targeting is more common than appreciated. Transcriptomic analysis of an arcZ deletion strain further argued for the existence of a distinct set of Salmonella loci specifically regulated by ArcZ. In contrast, increased expression of the sRNA altered the steady-state levels of > 16% (> 750) of all Salmonella mRNAs, and rendered the bacteria non-motile. Deep sequencing detected a dramatically changed profile of Hfq-bound sRNAs and mRNAs, suggesting that the unprecedented pleiotropic effects by a single sRNA might in part be caused by altered post-transcriptional regulation.