SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood.

Given the limited availability of serological testing to date, the seroprevalence of SARS-CoV-2-specific antibodies in different populations has remained unclear. Here, we report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seroreactivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors in early April 2020. We additionally describe the longitudinal dynamics of immunoglobulin-G (IgG), immunoglobulin-M (IgM), and in vitro neutralizing antibody titers in COVID-19 patients. The median time to seroconversion ranged from 10.3-11.0 days for these 3 assays. Neutralizing antibodies rose in tandem with immunoglobulin titers following symptom onset, and positive percent agreement between detection of IgG and neutralizing titers was >93%. These findings emphasize the importance of using highly accurate tests for surveillance studies in low-prevalence populations, and provide evidence that seroreactivity using SARS-CoV-2 anti-nucleocapsid protein IgG and anti-spike IgM assays are generally predictive of in vitro neutralizing capacity.

A comparative evaluation between the Abbott Panbio™ COVID-19 IgG/IgM rapid test device and Abbott Architect™ SARS CoV-2 IgG assay.

INTRODUCTION: Antibodies to SARS-CoV-2 serve as critical diagnostic markers for determining how broadly the COVID-19 pandemic has spread, confirming patient recovery, monitoring potential long-term effects of infection, and evaluating potential protection from reinfection. As new antibody tests become available, it is important to evaluate their performance and utility. The aim of this study was to compare the performance of the Abbott PanbioTM COVID-19 IgG/IgM Rapid Test Device against the Abbott ArchitectTM SARS CoV-2 IgG Assay for the detection of the COVID-19 IgG antibody. METHODS: Two panels of specimens were utilized to challenge both antibody tests: (1) a set of 150 prepandemic negative specimens collected in 2014, and (2) a set of 122 specimens from 87 hospitalized COVID-19 patients in the US and UK that were confirmed with a positive SARS-CoV-2 RNA test result. RESULTS: The ArchitectTM test had a specificity of 100 % and sensitivity of 99.1 % and 93.9 % when excluding or including immunocompromised patients, respectively for specimens collected >14 days post symptom onset or >5 days post-RNA testing. The PanbioTM test had 99.3 % agreement to ArchitectTM. Notably, N = 6 immune-compromised individuals were identified that did not develop detectable antibodies by day 30. CONCLUSION: There is good concordance between the ArchitectTM SARS CoV-2 IgG Assay and PanbioTM COVID-19 IgG/IgM Rapid Test Device for the detection of SARS CoV-2 IgG.

An ultra-sensitive Abbott ARCHITECT® assay for the detection of hepatitis B virus surface antigen (HBsAg).

BACKGROUND: Critical to the identification of HBV infection and the prevention of transfusion transmitted disease is the sensitive and accurate detection of Hepatitis B virus surface antigen (HBsAg). Improvements in HBsAg assay sensitivity approaching the performance of nucleic acid testing (NAT) are essential to further reduce the detection window for acute HBV infection in regions where NAT is not widely available. OBJECTIVES AND STUDY DESIGN: An improved HBsAg assay on the fully-automated Abbott ARCHITECT® platform was developed to improve sensitivity, mutant and genotype detection. RESULTS: The analytical sensitivity of the improved prototype assay is 5.2 mIU/ml, which is 3.86- to 14.54-fold more sensitive than comparator assays based on the WHO International Reference Standard. The enhanced sensitivity was also demonstrated with 27 HBV seroconversion panels, detecting more panel members (191 of 364) vs. the ARCHITECT® Qual I (144), Qual II (160) and PRISM® (148) HBsAg assays. Further, the assay detected 7 of 12 HBV DNA positive/HBsAg negative samples, and detected all evaluated mutants and genotypes with higher sensitivity than the comparator assays. The improvement in sensitivity did not diminish assay specificity, attaining 100% (95% CI, 99.97-100%) on 10,633 blood donors. CONCLUSIONS: An Abbott ARCHITECT® HBsAg assay with clinical performance approaching that of mini-pool NAT (approximately 100 copies/ml was developed. The assay has superior HBsAg mutant and genotype detection and specificity, all of which are important for the diagnosis and management of HBV infection.

An improved Abbott ARCHITECT assay for the detection of hepatitis B virus surface antigen (HBsAg).

BACKGROUND: The sensitive and accurate detection of hepatitis B virus surface antigen (HBsAg) is critical to the identification of infection and the prevention of transfusion transmitted disease. Improvement in HBsAg assay sensitivity is essential to reduce the window to detect an acute HBV infection. Additionally, the sensitive detection of HBsAg mutants that continue to evolve due to vaccine escape, immune selection and an error prone reverse transcriptase is a necessity. OBJECTIVES AND STUDY DESIGN: A fully automated HBsAg prototype assay on the Abbott ARCHITECT instrument was developed to improve sensitivity and mutant detection. This magnetic microparticle-based assay utilizes anti-HBsAg monoclonal antibodies to capture antigen present in serum or plasma. Captured antigen is then detected using anti-HBsAg antibody conjugated with the chemiluminescent compound, acridinium. RESULTS: The sensitivity of the ARCHITECT HBsAg prototype assay was improved as compared to the current ARCHITECT, PRISM, and competitor HBsAg assays. The enhancement in assay sensitivity was demonstrated by the use of commercially available HBV seroconversion panels. The prototype assay detected more panel members (185 of 383) vs. the current ARCHITECT (171), PRISM (181), or competitor HBsAg assays (73/140 vs. 62/140, respectively). The ARCHITECT prototype assay also efficiently detected all mutants evaluated. Finally, the sensitivity improvement did not compromise the specificity of the assay (99.94%). CONCLUSIONS: An improved Abbott ARCHITECT HBsAg prototype assay with enhanced detection of HBsAg and HBsAg mutants, as well as equivalent specificity was developed for the detection, diagnosis, and management of HBV infection.

Interaction of the aryl hydrocarbon receptor ligand 6-methyl-1,3,8-trichlorodibenzofuran with estrogen receptor alpha.

The polycyclic aromatic hydrocarbon 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) is related to the industrial byproduct dioxin and is a weak agonist and partial antagonist at the aryl hydrocarbon receptor (AhR). Tamoxifen is used for the treatment and prevention of breast cancer and interferes with the interaction of estrogen with estrogen receptor alpha (ER). The combination of MCDF and tamoxifen lowered the effective dose of both drugs required to inhibit 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth in rats and protected against the estrogenic effects of tamoxifen on the uterus in rats (A. McDougal et al., Cancer Res 2001;61:3902-7), pointing to the potential use of MCDF in breast cancer treatment. Potential AhR-ER cross-talk is evidenced by the antiestrogenic activity of MCDF and the degradative effect of MCDF on ER protein levels. Our studies confirmed that MCDF degraded the ER. MCDF displayed antiestrogenic activity at higher concentrations in MCF-7 human breast cancer cells, but MCDF alone (10(-6) M) stimulated the growth of MCF-7 cells. MCDF also activated an estrogen response element (ERE)-luciferase reporter and increased mRNA levels of the estrogen-responsive gene transforming growth factor (TGF)-alpha. The estrogenic effects of MCDF are ER dependent because they were blocked by the pure antiestrogen ICI 182,780. MCDF induced ER-coactivator interaction in glutathione S-transferase pull-down assays and the formation of an ER.ERE complex in gel mobility shift assays, further indicating that the estrogenic actions of MCDF are mediated by the ER. In addition, knockdown of the AhR with small interfering RNA did not affect MCDF-induced ERE-luciferase activity. Overall, these data support the conclusion that MCDF is a partial agonist at the ER. This study provides the first evidence for the direct interaction of the ER with MCDF and challenges the view that MCDF is simply an AhR-specific ligand.

The biological role of estrogen receptors alpha and beta in cancer.

The temporal and tissue-specific actions of estrogen are mediated by estrogen receptors alpha and beta. The ERs are steroid hormone receptors that modulate the transcription of target genes when bound to ligand. The activity of these transcription factors is regulated by a variety of factors, including ligand binding, phosphorylation, coregulators, and the effector pathway (ERE, AP1, SP1). The end result of target gene transcription is to modulate physiological processes, such as reproductive organ development and function, bone density, and unfortunately contribute to the growth and development of breast and endometrial cancer. The complex biological effects mediated by ER alpha and ER beta involve communication between many proteins and signaling pathways. An ultimate goal of current research is to enhance the value of the separate estrogen receptors as targets for therapeutic intervention.

The EF-hand Ca2+-binding protein p22 plays a role in microtubule and endoplasmic reticulum organization and dynamics with distinct Ca2+-binding requirements.

We have reported that p22, an N-myristoylated EF-hand Ca(2+)-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca(2+), p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca(2+)-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based "bulk microinjection" assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca(2+)-binding p22 mutant, which is unable to undergo Ca(2+)-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca(2+)-independent manner and affects ER network assembly in a Ca(2+)-dependent manner.

Distinct molecular conformations of the estrogen receptor alpha complex exploited by environmental estrogens.

We have advanced the view that estrogens activate the estrogen receptor (ER) alpha complex differently. A group of planar (estradiol, genistein, and coumestrol) and nonplanar (methoxychlor and its mono- and didemethylated phenolic metabolites) environmental estrogens, which are all full estrogens in MCF-7 breast cancer cell proliferation assays, was shown to segregate discretely into planar and nonplanar groups. These groups were delineated using a novel assay of mutant ER cDNAs stably transfected into MDA-MB-231 cells and the activation of the transforming growth factor alpha target gene in situ that putatively describes the external shape of the ER complex. Planar compounds activate estrogen action through the two traditional activation functions (AFs), AF1 and AF2, in the ER. In contrast, nonplanar compounds can activate estrogen action through AF1 and the amino acids Asp-351 and Asp-538, which are exposed when helix 12 silences AF2. The observation that class I (planar) and class II (nonplanar) compounds have different mechanisms of estrogen action may have important implications for tissue selective modulation of the ER.

Apoptotic action of 17beta-estradiol in raloxifene-resistant MCF-7 cells in vitro and in vivo.

BACKGROUND: Resistance to tamoxifen, a selective estrogen receptor modulator (SERM), involves changes that prevent apoptosis and enhance cell proliferation and survival. Paradoxically, estrogen treatment inhibits the growth of long-term tamoxifen-treated breast tumors. Because of the increasing use of raloxifene, another SERM, to prevent osteoporosis and potentially reduce breast cancer risk, some women will develop raloxifene-resistant breast cancer. We developed a raloxifene-resistant MCF-7 cell model (MCF-7/Ral) and investigated the nature of raloxifene-resistant breast cancer and its response to estradiol. METHODS: Raloxifene resistance and hormone responsiveness were assessed by proliferation assays and cell cycle analysis in parental MCF-7 and MCF-7/Ral cells. Nuclear factor kappaB (NF-kappaB) activity was investigated with a transient transfection assay. Apoptosis was investigated by annexin V staining, mRNA was measured by real-time polymerase chain reaction, and protein was measured by western blotting. Tumorigenesis was studied by injecting MCF-7 or MCF-7/Ral cells into ovariectomized athymic mice (10 per group) and monitoring tumor size weekly. All statistical tests were two-sided. RESULTS: Basal NF-kappaB activity was higher in MCF-7/Ral cells (1.6 U, 95% confidence interval [CI] = 1.2 to 2.0 U) than in MCF-7 cells (0.8 U, 95% CI = 0.4 to 1.1 U; P =.004). When cultured with 1 microM raloxifene, MCF-7/Ral cells grew statistically significantly (P<.001) faster than MCF-7 cells. Estradiol treatment of MCF-7/Ral cells arrested cells in G(2)/M phase of the cell cycle, decreased NF-kappaB activity (0.2 U, 95% CI = 0.2 to 0.3 U; P<.001), increased expression of Fas protein and mRNA (4.5-fold, 95% CI = 2.8- to 6.3-fold versus 0.5-fold, 95% CI = 0.3- to 0.8-fold for control treatment; P<.001), and induced apoptosis. Treatment with either raloxifene or tamoxifen stimulated MCF-7/Ral tumor growth, suggesting that such tumors were resistant to both drugs. When a 9-week raloxifene or tamoxifen treatment was followed by a 5-week estradiol treatment, estradiol statistically significantly reduced the size of tumors stimulated by raloxifene or tamoxifen (at week 14, P =.004 for raloxifene and P<.001 for tamoxifen). CONCLUSIONS: Growth of raloxifene-resistant MCF-7/Ral cells in vitro and in vivo is repressed by estradiol treatment by a mechanism involving G2/M-phase arrest, decreased NF-kappaB activity, and increased Fas expression to induce apoptosis.

Resveratrol acts as an estrogen receptor (ER) agonist in breast cancer cells stably transfected with ER alpha.

Resveratrol (Res) is a phytoestrogen found in grapes and present in red wine. Res has been shown to function as an estrogen receptor (ER) agonist, but it remains unclear whether it may also exert antagonist activity. Our aim was to study the effects of Res at both the molecular (TGFalpha gene activation) and the cellular (cell growth) levels in breast cancer cells stably transfected with wild-type (wt) ER(D351) and mutant (mut) ER (D351Y). TGFalpha mRNA induction was used as a specific marker of estradiol (E(2)) responsiveness. Res caused a concentration-dependent (10(-8)-10(-4) M) stimulation of TGFalpha mRNA, indicating that it acts as an estrogen agonist in these cell lines. The pure antiestrogen ICI 182,780 (ICI) blocked Res-induced activation of TGFalpha, consistent with action through an ER-mediated pathway. Further studies that combined treatments with E(2) and Res showed that Res does not act as an antagonist in the presence of various (10(-11)-10(-8) M) concentrations of E(2). To determine whether Res can be classified as a type I or type II estrogen (Jordan et al., Cancer Res 2001;61:6619-23,), we examined Res with the D351G ER in the TGFalpha assay and found that Res belongs to the type I estrogens. Both Res and E(2) had concentration-dependent growth inhibitory effects in cells expressing wtER and D351Y ER. Although the pure antiestrogen ICI blocked the growth inhibitory effects of E(2), it did not block the inhibitory effects of Res, suggesting that the antiproliferative effects of Res also involve ER-independent pathways. Interestingly, Res differentially affected the levels of ER protein in these 2 cell lines: Res down-regulated wtER levels while significantly up-regulating the amount of mutD351Y ER. Co-treatment with ICI resulted in strongly reduced ER levels in both cell lines. Gene array studies revealed Res-induced up-regulation of more than 80 genes, among them a profound activation of p21(CIP1)/WAF1, a gene associated with growth arrest. The p21(CIP1)/WAF1 protein levels measured by Western blotting confirmed Res-induced significant up-regulation of this protein in both cell lines. In summary, Res acts as an ER agonist at low doses but also activates ER-independent pathways, some of which inhibit cell growth.

Modulation of estrogen receptor alpha function and stability by tamoxifen and a critical amino acid (Asp-538) in helix 12.

Estrogen receptor alpha (ER) is a ligand-activated transcription factor implicated in breast cancer growth. Selective estrogen receptor modulators (SERMs), such as tamoxifen (4-OHT), bind to the ER and affect the position of helix 12, thereby influencing coregulator binding and ER transcriptional activation. Previous studies have shown that a triple mutation in helix 12 (3m; D538A/E542A/D545A) caused a change in ER stability and obliterated 4-OHT action (Liu, H., Lee, E. S., de los Reyes, A., Zapf, J. W., and Jordan, V. C. (2001) Cancer Res. 61, 3632-3639). Two approaches were taken to determine the role of individual mutants (D538A, L540Q, E542A, and D545A) on the activity and stability of the 4-OHT.ER complex. First, mutants were evaluated using transient transfection into ER-negative T47D:C4:2 cells with an ERE3-luciferase reporter, and second, transforming growth factor alpha (TGFalpha) mRNA was used as a gene target in situ for stable transfectants of MDA-MB-231 cells. Transcriptional activity occurred in the presence of estrogen in all of the mutants, although a decreased response was observed in the L540Q, 3m, and D538A cells. The 3m and D538A mutants lacked any estrogenic responsiveness to 4-OHT, whereas the other mutations retained estrogen-like activity with 4-OHT. Unlike the other mutants, the ER was degraded in the D538A mutant with 4-OHT treatment. However, increasing the protein levels of the mutant with the proteasome inhibitor MG132 did not restore the ability of 4-OHT to induce TGFalpha mRNA. We suggest that Asp-538 is a critical amino acid in helix 12 that not only reduces the estrogen-like actions of 4-OHT but also facilitates the degradation of the 4-OHT.D538A complex. These data further illustrate the complex role of specific surface amino acids in the modulation of the concentration and the estrogenicity of the 4-OHT.ER complex.