Inhibition Of Receptor Binding Research Articles

Multivalent and Sequential Heterologous Spike Protein Vaccinations Effectively Induce Protective Humoral Immunity against SARS-CoV-2 Variants.

The emergence of new SARS-CoV-2 variants continues to cause challenging problems for the effective control of COVID-19. In this study, we tested the hypothesis of whether a strategy of multivalent and sequential heterologous spike protein vaccinations would induce a broader range and higher levels of neutralizing antibodies against SARS-CoV-2 variants and more effective protection than homologous spike protein vaccination in a mouse model. We determined spike-specific IgG, receptor-binding inhibition titers, and protective efficacy in the groups of mice that were vaccinated with multivalent recombinant spike proteins (Wuhan, Delta, Omicron), sequentially with heterologous spike protein variants, or with homologous spike proteins. Trivalent (Wuhan + Delta + Omicron) and sequential heterologous spike protein vaccinations were more effective in inducing serum inhibition activities of receptor binding to spike variants and virus neutralizing antibody titers than homologous spike protein vaccination. The higher efficacy of protection was observed in mice with trivalent and sequential heterologous spike protein vaccination after a challenge with a mouse-adapted SARS-CoV-2 MA10 strain compared to homologous spike protein vaccination. This study provides evidence that a strategy of multivalent and sequential heterologous variant spike vaccination might provide more effective protection against emerging SARS-CoV-2 variants than homologous spike vaccination and significantly alleviate severe inflammation due to COVID-19.

Abstract 4390: IGFBP-6 is induced by progesterone to regulate progesterone receptor expression in breast cancer cells

Abstract Insulin-like growth factor binding protein-6 (IGFBP-6) is induced by progesterone (P4) in T-47D breast cancer cells. IGFBP-6 regulates insulin-like growth factor 2 (IGF2) by sequestering it to inhibit receptor binding and extend its extracellular half-life. IGF2 binds to insulin-like growth factor 1 receptor or insulin receptor A to promote breast cancer cell proliferation and survival. Progesterone is an ovarian steroid hormone which has complex roles in breast cancer through the actions of progesterone receptor A and B (PR-A and B). Progesterone antagonizes the actions of estradiol (E2), another steroid hormone which promotes proliferation and survival in breast cancer. T-47D cells treated with both P4 and E2 exhibit significantly reduced proliferation compared to treatment with E2 alone. To better understand the role of IGFBP-6 in progesterone signaling, T-47D cells which over-express IGFBP-6 were treated with P4, E2, or both. Overexpressing cells exhibited stronger antagonism compared to controls when co-treated with P4 and E2. RNA-interference was performed to knockdown IGFBP-6 induction in the presence of P4 and E2. Knockdown of IGFBP-6 in the presence of P4 and E2 decreases the antagonistic effects of P4. Expression of both PR-A and PR-B decreases by about 50% when IGFBP-6 is knocked down. These results indicate that IGFBP-6 increases PR expression in response to P4 stimulation. Citation Format: Francisco Javier Lariz, Pacha Botero, Kevin Houston. IGFBP-6 is induced by progesterone to regulate progesterone receptor expression in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4390.

Mast cell silencing: A novel therapeutic approach for urticaria and other mast cell-mediated diseases.

Chronic urticaria (CU) is a mast cell (MC)-dependent disease with limited therapeutic options. Current management strategies are directed at inhibiting IgE-mediated activation of MCs and antagonizing effects of released mediators. Due to the complexity and heterogeneity of CU and other MC diseases and mechanisms of MC activation-including multiple activating receptors and ligands, diverse signaling pathways, and a menagerie of mediators-strategies of MC depletion or MC silencing (i.e., inhibition of MC activation via binding of inhibitory receptors) have been developed to overcome limitations of singularly targeted agents. MC silencers, such as agonist monoclonal antibodies that engage inhibitory receptors (e.g., sialic acid-binding immunoglobulin-like lectin8 -[Siglec-8] [lirentelimab/AK002], Siglec-6 [AK006], and CD200R [LY3454738]), have reached preclinical and clinical stages of development. In this review, we (1) describe the role of MCs in the pathogenesis of CU, highlighting similarities with other MC diseases in disease mechanisms and response to treatment; (2) explore current therapeutic strategies, categorized by nonspecific immunosuppression, targeted inhibition of MC activation or mediators, and targeted modulation of MC activity; and (3) introduce the concept of MC silencing as an emerging strategy that could selectively block activation of MCs without eliciting or exacerbating on- or off-target, immunosuppressive adverse effects.

A Severe Acute Respiratory Syndrome Coronavirus 2 Anti-Spike Immunoglobulin G Assay: A Robust Method for Evaluation of Vaccine Immunogenicity Using an Established Correlate of Protection.

As the COVID-19 pandemic continues, variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge. Immunogenicity evaluation of vaccines and identification of correlates of protection for vaccine effectiveness is critical to aid the development of vaccines against emerging variants. Anti-recombinant spike (rS) protein immunoglobulin G (IgG) quantitation in the systemic circulation (serum/plasma) is shown to correlate with vaccine efficacy. Thus, an enzyme-linked immunosorbent assay (ELISA)-based binding assay to detect SARS-CoV-2 (ancestral and variant strains) anti-rS IgG in human serum samples was developed and validated. This assay successfully met acceptance criteria for inter/intra-assay precision, specificity, selectivity, linearity, lower/upper limits of quantitation, matrix effects, and assay robustness. The analyte in serum was stable for up to 8 freeze/thaw cycles and 2 years in -80 °C storage. Similar results were observed for the Beta, Delta, and Omicron BA.1/BA.5/XBB.1.5 variant-adapted assays. Anti-rS IgG assay results correlated significantly with neutralization and receptor binding inhibition assays. In addition, usage of international reference standards allows data extrapolation to WHO international units (BAU/mL), facilitating comparison of results with other IgG assays. This anti-rS IgG assay is a robust, high-throughput method to evaluate binding IgG responses to S protein in serum, enabling rapid development of effective vaccines against emerging COVID-19 variants.

Real world data of apalutamid in the treatment of prostate cancer: Data of the German multicenter AmPel trial.

e17073 Background: Apalutamide (Apa) is a competitive inhibitor of the androgen receptor binding directly to the ligand-binding domain. In metastatic castration-sensitive prostate cancer (mCSPC) as well as non-metastatic castration-resistant prostate cancer (nmCRPC) Apa demonstrated a survival benefit in Phase-III trials. Nevertheless Real World data are still lacking. Methods: In the retrospective, multicenter AmPel trial (Apalutamid in prostate cancer in middle-Hessen) data of mCSPC and nmCRPC pat were collected before and during Apa treatment in routine clinical practice. Beside pat and tumor specific data side-effects, dose-modifications, response and subsequent therapies were assessed. Results: To date 100 pat were included since 11/2021. General data are summarized in table 1. mCSPC (n = 74): mFU 10 month (1-30). 75% showed mets at initial diagnosis: 56% with osseous, 15% with viszeral and 29% with oss/visz mets. PSA before therapy 13.5 ng/ml decreased to 0.2 ng/ml (-98.9%) after 3 mo. 77.4% showed a PSA decrease > 90% and 49% a PSA level of < 0.2 ng/ml after 6 mo. AEs ≥ 3: 12.3%. In 12.3% dose reduction was necessary. In 21% therapy was stopped due to toxicity (10.9%), progression (5.5%) and patient will (5.5%). 3 patients died: 1 due to PCA progression. PFS is not reached. nmCRPC (n = 26): mFU 15 month (3-30). Time after initial diagnosis: 6 y (1-22). PSA-Doubling time 3.9 mo (1-10). PSA before therapy 7.2 ng/ml with a decrease of 94.7% after 3 mo (0.45 ng/ml) and 62.5% with a PSA decrease > 90%. AEs ≥3: 5%. In 15.4% dose-reduction was necessary. In 24% therapy was stopped due to toxicity (5%), progression (9.5%) and patient will (9.5%). MFS is not reached. No increased adverse events were observed in case of statin and anticoagulant medication. Conclusions: Compared to Phase III trials this first real-world data of the AmPel-trial showed treatment of older and more co-morbid mCSPC and nmCRPC patients in clinical practice. Apa treatment resulted in a rapid PSA-response, was safe and effective with tolerable and manageable side effects in real world unselected patients. PSMA-PET/CT is not used in daily practice as well as Conference decisions before therapy. Updated data of the ongoing AmPel trial will be presented. [Table: see text]

Capture and neutralization of SARS-CoV-2 and influenza virus by algae-derived lectins with high-mannose and core fucose specificities.

We first investigated the interactions between several algae-derived lectins and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We created lectin columns using high-mannose (HM)-type glycan-specific lectins OAA and KAA-1 or core fucose-specific lectin hypninA-2 and conducted binding experiments with SARS-CoV-2. The results showed that these lectins were capable of binding to the virus. Furthermore, when examining the neutralization ability of nine different lectins, it was found that KAA-1, ESA-2, and hypninA-2 were effective in neutralizing SARS-CoV-2. In competitive inhibition experiments with glycoproteins, neutralization was confirmed to occur through HM-type or core fucose-type glycans. However, neutralization was not observed with other lectins, such as OAA. This trend of KAA-1 and ESA-2 having the neutralizing ability and OAA not having it was also similar to influenza viruses. Electron microscopy observations revealed that KAA-1 and hypninA-2 strongly aggregated SARS-CoV-2 particles, while OAA showed a low degree of aggregation. It is believed that the neutralization of SARS-CoV-2 involves multiple factors, such as glycan attachment sites on the S protein, the size of lectins, and their propensity to aggregate, which cause inhibition of receptor binding or aggregation of virus particles. This study demonstrated that several algae-derived lectins could neutralize SARS-CoV-2 and that lectin columns can effectively recover and concentrate the virus.

Klotho prevents transforming growth factor-β2-induced senescent-like morphological changes in the retinal pigment epithelium

Degenerative changes of the retinal pigment epithelium (RPE) triggered by transforming growth factor-β2 (TGF-β2) and oxidative stress play a critical role in the progression of age-related macular degeneration (AMD). The expression of α-klotho, an antiaging protein, declines with age, increasing the risk factors for age-related diseases. Here, we investigated the protective effects of soluble α-klotho on TGF-β2-induced RPE degeneration. The morphological changes induced by TGF-β2, including epithelial-mesenchymal transition (EMT), were attenuated in the mouse RPE by the intravitreal injection (IVT) of α-klotho. In ARPE19 cells, EMT and morphological alterations by TGF-β2 were attenuated by co-incubation with α-klotho. TGF-β2 decreased miR-200a accompanied by zinc finger e-box binding homeobox1 (ZEB1) upregulation and EMT, all of which were prevented by α-klotho co-treatment. Inhibitor of miR-200a mimicked TGF-β2-induced morphological changes, which were recovered by ZEP1 silencing, but not by α-klotho, implying the upstream regulation of α-klotho on miR-200a-ZEP1-EMT axis. α-Klotho inhibited receptor binding of TGF-β2, Smad2/3 phosphorylation, extracellular signal-regulated protein kinase 1/2 (ERK1/2)-a mechanistic target of rapamycin (mTOR) activation and oxidative stress via NADPH oxidase 4 (NOX4) upregulation. Furthermore, α-klotho recovered the TGF-β2-induced mitochondrial activation and superoxide generation. Interestingly, TGF-β2 upregulated α-klotho expression in the RPE cells, and genetic suppression of endogenous α-klotho aggravated TGF-β2-induced oxidative stress and EMT. Lastly, α-klotho abrogated senescence-associated signaling molecules and phenotypes induced by long-term incubation with TGF-β2. Hence, our findings indicate that the antiaging α-klotho plays a protective role against EMT and degeneration of the RPE, demonstrating the therapeutic potential for age-related retinal diseases, including the dry type of AMD.

Six-month immune responses to mRNA-1273 vaccine in combination antiretroviral therapy treated late presenter people with HIV according to previous SARS-CoV-2 infection.

Immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in people with HIV (PWH) with a history of late presentation (LP) and their durability have not been fully characterized. In this prospective, longitudinal study, we sought to assess T-cell and humoral responses to SARS-CoV-2 mRNA vaccination up to 6 months in LP-PWH on effective combination antiretroviral therapy (cART) as compared to HIV-negative healthcare workers (HCWs), and to evaluate whether previous SARS-CoV-2 infection modulates immune responses to vaccine. SARS-CoV-2 spike (S)-specific T-cell responses were determined by two complementary flow cytometry methodologies, namely activation-induced marker (AIM) assay and intracellular cytokine staining (ICS), whereas humoral responses were measured by ELISA [anti-receptor binding domain (RBD) antibodies) and receptor-binding inhibition assay (spike-ACE2 binding inhibition activity), before vaccination (T0), 1 month (T1) and 5 months (T2) after the second dose. LP-PWH showed at T1 and T2 significant increase of: S-specific memory and circulating T follicular helper (cTfh) CD4 + T cells; polyfunctional Th1-cytokine (IFN-γ, TNF-α, IL-2)- and Th2-cytokine (IL-4)-producing S-specific CD4 + T cells; anti-RBD antibodies and spike-ACE2 binding inhibition activity. Immune responses to vaccine in LP-PWH were not inferior to HCWs overall, yet S-specific CD8 + T cells and spike-ACE2 binding inhibition activity correlated negatively with markers of immune recovery on cART. Interestingly, natural SARS-CoV-2 infection, while able to sustain S-specific antibody response, seems less efficacious in inducing a T-cell memory and in boosting immune responses to vaccine, possibly reflecting an enduring partial immunodeficiency. Altogether, these findings support the need for additional vaccine doses in PWH with a history of advanced immune depression and poor immune recovery on effective cART.

Proposing lead compounds for the development of SARS-CoV-2 receptor-binding inhibitors

The COVID-19 pandemic has had deleterious effects on the world and demands urgent measures to find therapeutic agents to combat the current and related future outbreaks. The entry of SARS-CoV-2 into the host’s cell is facilitated by the interaction between the viral spike receptor-binding domain (sRBD) and the human angiotensin-converting enzyme 2 (hACE2). Although the interface of sRBD involved in the sRBD-hACE2 interaction has been projected as a primary vaccine and drug target, currently no small-molecule drugs have been approved for covid-19 treatment targeting sRBD. Herein structure-based virtual screening and molecular dynamics (MD) simulation strategies were applied to identify novel potential small-molecule binders of the SARS-CoV-2 sRBD from an sRBD-targeted compound library as leads for the development of anti-COVID-19 drugs. The library was initially screened against sRBD by using the GOLD docking program whereby 19 compounds were shortlisted based on docking scores after using a control compound to set the selection cutoff. The stability of each compound in MD simulations was used as a further standard to select four hits namely T4S1820, T4589, E634-1449, and K784-7078. Analyses of simulations data showed that the four compounds remained stably bound to sRBD for ≥ 80 ns with reasonable affinities and interacted with pharmacologically important amino acid residues. The compounds exhibited fair solubility, lipophilicity, and toxicity-propensity characteristics that could be improved through lead optimization regimes. The overall results suggest that the scaffolds of T4S1820, E634-1449, and K784-7078 could serve as seeds for developing potent small-molecule inhibitors of SARS-CoV-2 receptor binding and cell entry. Communicated by Ramaswamy H. Sarma

Thyrotropin Receptor Autoantibody Assessment in Thyroid Eye Disease: Does the Assay Type Matter?

Thyroid receptor antibodies can quantify thyroid eye disease activity, predict outcomes and aid timing of interventions. The type and generation of assay is frequently unspecified, complicating meta-analyses. To determine the clinical and biochemical relationships between a second-generation thyrotropin receptor-binding inhibition antibody (TRAb) immunoassay, detecting stimulatory and blocking antibodies, with the thyroid stimulating immunoglobulin (TSI) bridging immunoassay detecting the stimulatory component only. Retrospective review of 100 consecutive patients attending a regional specialist service. For each patient and visit, both a TRAb and TSI were performed, and a clinical activity score (CAS) recorded. A significant positive correlation between TRAb and TSI (rho = 0.828, p < 0.01) but a weaker correlation between the assays and CAS (TRAb: rho = 0.439, p < 0.01; TSI: r = 0.357, p < 0.01) were found. In 10% of the episodic data, patients had a TRAb level that was disproportionately high (39.41 ± 52.84 IU/L), compared to their TSI levels (9.53 ± 12.10 IU/L) with a higher-than-average CAS (2.47 ± 1.78; range, 0-5). Within 12 months of diagnosis, a significant positive correlation between CAS and TRAb (rho = 0.503, p < 0.01) as well as between CAS and TSI (rho = 0.329, p < 0.01) were found. In patients with a diagnosis over 12 months, the correlation with CAS for both TSI and TRAb were Spearman rank correlation coefficient of 0.347 (p < 0.01) and 0.327 (p < 0.01), respectively. TRAb and TSI correlate strongly and to a lesser extent with the CAS. For most patients, TRAb can be replaced with the more economical TSI. TRAb also correlates better with newly diagnosed, more active patients than TSI. In a subset of patients, blocking antibodies may play a significant pathogenic role, requiring different treatment and monitoring. Further studies are required to investigate this relationship.

Induction of systemic, mucosal, and cellular immunity against SARS-CoV-2 in mice vaccinated by trans-airway with a S1 protein combined with a pulmonary surfactant-derived adjuvant SF-10.

There is a need for vaccines that can induce effective systemic, respiratory mucosal, and cellular immunity to control the COVID-19 pandemic. We reported previously that a synthetic mucosal adjuvant SF-10 derived from human pulmonary surfactant works as an efficient antigen delivery vehicle to antigen presenting cells in the respiratory and gastrointestinal tracts and promotes induction of influenza virus antigen-specific serum IgG, mucosal IgA, and cellular immunity. The aim of the present study was to determine the effectiveness of a new administration route of trans-airway (TA) vaccine comprising recombinant SARS-CoV-2 spike protein 1 (S1) combined with SF-10 (S1-SF-10 vaccine) on systemic, local, and cellular immunity in mice, compared with intramuscular injection (IM) of S1 with a potent adjuvant AddaS03™ (S1-AddaS03™ vaccine). S1-SF-10-TA vaccine induced S1-specific IgG and IgA in serum and lung mucosae. These IgG and IgA induced by S1-SF-10-TA showed significant protective immunity in a receptor binding inhibition test of S1 and angiotensin converting enzyme 2, a receptor of SARS-CoV-2, which were more potent and faster achievement than S1-AddaS03™-IM. Enzyme-linked immunospot assay showed high numbers of S1-specific IgA and IgG secreting cells (ASCs) and S1-responsive IFN-γ, IL-4, IL-17A cytokine secreting cells (CSCs) in the spleen and lungs. S1-AddaS03™-IM induced IgG ASCs and IL-4 CSCs in spleen higher than S1-SF-10-TA, but the numbers of ASCs and CSCs in lungs were low and hardly detected. Based on the need for effective systemic, respiratory, and cellular immunity, the S1-SF-10-TA vaccine seems promising mucosal vaccine against respiratory infection of SARS-CoV-2.

The synergistic effect of Cu-MOF nanoparticles and immunomodulatory agent on SARS-CoV-2 inhibition.

In this work, HKUST-1 and Cu-BDC nanoparticles were used as delivery systems for the early anti-COVID-19 drug, hydroxychloroquine. The antiviral MOF/drug combinations significantly reduced the infectivity of SARS-CoV-2, which can be attributed to the nanometric size of the carriers, the presence of copper in the MOF nodes, and the semi-controlled release of the drug.

SARS-CoV-2 Anti-Spike IgG Antibody and ACE2 Receptor Binding Inhibition Levels among Breakthrough Stage Veteran Patients.

SARS-CoV-2 mRNA vaccines have been critical to curbing pandemic COVID-19; however, a major shortcoming has been the inability to assess levels of protection after vaccination. This study assessed serologic status of breakthrough infections in vaccinated patients at a Veterans Administration medical center from June through December 2021 during a SARS-CoV-2 delta variant wave. Breakthrough occurred mostly beyond 150 days after two-dose vaccination with a mean of 239 days. Anti-SARS-CoV-2 spike (S) IgG levels were low at 0 to 2 days postsymptoms but increased in subjects presenting thereafter. Population measurements of anti-S IgG and angiotensin converting enzyme-2 receptor (ACE2-R) binding inhibition among uninfected, vaccinated patients suggested immune decay occurred after 150 days with 62% having anti-S IgG levels at or below 1,000 AU comparable with breakthrough patients at 0 to 2 days postsymptom onset. In contrast, vaccination after resolved infection conferred robust enduring anti-S IgG levels (5,000 to >50,000 AU) with >90% ACE2-R binding inhibition. However, monoclonal antibody (MAb)-treated patients did not benefit from their prior infection suggesting impaired establishment of B cell memory. Analysis of boosted patients confirmed the benefit of a third vaccine dose with most having anti-S IgG levels above 5,000 AU with >90% ACE2-R binding inhibition, but a subset had levels <5,000 AU. Anti-S IgG levels >5,000 AU were associated with >90% ACE2-R binding inhibition and no documented breakthrough infections, whereas levels falling below 5,000 AU and approaching 1,000 AU were associated with breakthrough infections. Thus, quantitative antibody measurements may provide a means to guide vaccination intervals for the individual. IMPORTANCE Currently, clinicians have no guidance for the serologic assessment of SARS-Cov-2 postvaccination status regarding protection and risk of infection. Vaccination and boosters are administered blindly without evaluation of need or outcome at the individual level. The recent development of automated quantitative assays for anti-SARS-CoV-2 spike protein IgG antibodies permits accurate measurement of humoral immunity in standardized units. Clinical studies, such as reported here, will help establish protective antibody levels allowing identification and targeted management of poor vaccine responders and vaccinated subjects undergoing immune decay.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

High-Throughput Assay for Identifying Diverse Antagonists of the Binding Interaction between the ACE2 Receptor and the Dynamic Spike Proteins of SARS-CoV-2.

SARS-CoV-2, a coronavirus strain that started a worldwide pandemic in early 2020, attaches to human cells by binding its spike (S) glycoprotein to a host receptor protein angiotensin-converting enzyme 2 (ACE2). Blocking the interaction between the S protein and ACE2 has emerged as an important strategy for preventing viral infection. We systematically developed and optimized an AlphaLISA assay to investigate binding events between ACE2 and the ectodomain of the SARS-CoV-2 S protein (S-614G: residues 1-1208 with a D614G mutation). Using S-614G permits discovering potential allosteric inhibitors that stabilize the S protein in a conformation that impedes its access to ACE2. Over 30,000 small molecules were screened in a high-throughput format for activity against S-614G and ACE2 binding using the AlphaLISA assay. A viral entry assay was used to validate hits using lentiviral particles pseudotyped with the full-length S protein of the Wuhan-1 strain. Two compounds identified in the screen, oleic acid and suramin, blocked the attachment of S-614G to ACE2 and S protein-driven cell entry into Calu-3 and ACE2-overexpressing HEK293T cells. Oleic acid inhibits S-614G binding to ACE2 far more potently than to the receptor-binding domain (RBD, residues 319-541 of SARS-CoV-2 S), potentially indicating a noncompetitive mechanism. The results indicate that using the full-length ectodomain of the S protein can be important for identifying allosteric inhibitors of ACE2 binding. The approach reported here represents a rapidly adaptable format for discovering receptor-binding inhibitors to S-proteins of future coronavirus strains.

Freeze-dried plasma: From damage control resuscitation to coronavirus disease 2019 therapy.

Freeze-dried plasma (FDP) is a promising blood component for prehospital resuscitation given its logistic advantages over fresh frozen plasma (FFP). COVID-19 convalescent (CC) plasma has been used to treat coronavirus disease 2019 (COVID-19) patients, and its corresponding FDP has potential use during future pandemics. Therefore, we conducted the study to determine if the hemostatic and immunological properties of plasma can be retained after lyophilization. Hemostatic tests were conducted with Rotational Thromboelastometry (ROTEM) and a Stago analyzer. Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG (Immunoglobulin G) and neutralizing activity were analyzed using Meso Scale Diagnostics immunoassay kits. There were no differences in ROTEM parameters and Stago measurements for prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen and D-dimer concentrations, and antithrombin, factor V, VIII, and protein S activities between FFP and FDP for either pre-COVID-19 or CC samples. Differences were observed in INTEM clotting time and PT and PTT when comparing reconstituted FDP stored at 4°C for 24 h or room temperature for 4 h to healthy control. Both CC-FFP and CC-FDP showed two orders of magnitude higher concentrations of IgG antibodies against SARS-CoV-2 antigens than pre-COVID-19-FFP and pre-COVID-19-FDP and healthy control. Similarly, the CC samples showed approximately 4-fold higher %-inhibition of receptor binding than the pre-COVID-19 samples. There were no differences in either the antibody levels or neutralization activity between CC-FFP and CC-FDP. We demonstrated that FDP and CC-FDP retained the same hemostatic and antibody functional activities relative to their initial plasma sources, supporting clinical evaluation of their benefits in severe trauma and COVID-19 patients.

Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA.

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a KD of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a KD of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.

Impaired Humoral Immunity with Concomitant Preserved T Cell Reactivity in IBD Patients on Treatment with Infliximab 6 Month after Vaccination with the SARS-CoV-2 mRNA Vaccine BNT162b2: A Pilot Study.

Introduction: The Coronavirus Disease 2019 (COVID-19) pandemic has been caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The most important approach to prevent severe disease progression and to contain the pandemic is the use of COVID-19 vaccines. The aim of this study was to investigate the humoral and cellular response in immunosuppressed patients with inflammatory bowel disease (IBD) on treatment with anti-TNF (infliximab, adalimumab) and anti-α4ß7-Integrin (vedolizumab) 6 months after mRNA vaccination against SARS-CoV-2 compared to healthy subjects. Methods: In this prospective study, 20 IBD patients and 9 healthy controls were included 6 months after the second BNT162b2 vaccination. In addition to quantitative determination of IgG antibody levels against the SARS-CoV-2 receptor-binding domain (RBD) of the spike protein subunit S1, a SARS-CoV-2 surrogate neutralization test (sVNT) was used to assess potential neutralization capacity. SARS-CoV-2-specific T-cell responses were measured using an interferon-γ (IFN-γ) release assay (IGRA; Euroimmun Medical Laboratory Diagnostics, Lübeck, Germany). Results: S-IgG could still be detected in the majority of IBD patients 6 months after second vaccination. Compared to healthy controls, IBD patients treated with anti-TNF agents showed both lower neutralizing activity in sVNT (percent inhibition of ACE2 receptor binding by RBD protein) and lower IgG-S (AU/mL) antibody levels (AB) (sVNT: 79% vs. 2%, p < 0. 001; AB: 1018 AU/mL vs. 141 AU/mL, p = 0.025). In contrast, patients on therapy with vedolizumab showed no impairment in humoral immune response (sVNT, S-IgG) compared with healthy controls. Specific T-cellular reactivity was detected in 73% of IBD patients and in 67% of healthy controls independent of immunosuppressive therapy (anti-TNF., vedolizumab) (p = 0.189). Conclusion: Six months after BNT162b2 vaccination, this study found significantly decreased antibody levels in patients under anti-TNF therapy. IBD patients under anti-TNF and vedolizumab therapy had no impairment of T-cellular reactivity compared to healthy controls at this time point. Further studies with larger collectives for confirmation should follow.

Copper exposure disrupts ovarian steroidogenesis in human ovarian granulosa cells via the FSHR/CYP19A1 pathway and alters methylation patterns on the SF-1 gene promoter

Information on the effects of copper on reproduction is limited. Our previous study indicated that copper induces abnormal steroidogenesis in human ovarian granulosa cells, but the underlying mechanism remains unclear. In this study, human ovarian granulosa cells were treated with multiple concentrations of copper for 24 h. After treatment, the 17-estradiol levels were significantly increased (29.83 % and 45.12 %, respectively) in the 1.0 and 2.0 μg/mL groups but decreased (23.06 % and 31.56 %, respectively) in the 20.0 and 40.0 μg/mL groups (P < 0.05). Similar changes in the levels of FSHR, StAR, CYP11A1, CYP19A1, HSD3β1, and SF-1 were observed. The protein levels of FSHR were increased in the 2.0 μg/mL group but decreased in the 20.0 and 40.0 μg/mL groups (P < 0.05). Moreover, copper partially reversed the FSH-induced increase in FSHR, CYP19A1 and 17-estradiol levels, and the decreased effect of the FSH receptor binding inhibitor fragment on FSHR, CYP19A1, and 17-estradiol became more apparent after adding copper. Additionally, the total methylation levels of the SF-1 promoter and DNMTs expression were significantly decreased following copper treatment. Overall, our results indicate that copper exposure induces steroidogenesis disorders via the FSHR/CYP19A1 pathway and changes DNA methylation on the SF-1 promoter in human ovarian granulosa cells.

Thyroid Disorders.

Thyroid Disorders.