The immediate-early gene early growth response 3 (Egr3) is associated with schizophrenia and expressed at reduced levels in postmortem patients' brains. We have previously reported that Egr3-deficient (Egr3(-/-)) mice display reduced sensitivity to the sedating effects of clozapine compared with wild-type (WT) littermates, paralleling the heightened tolerance of schizophrenia patients to antipsychotic side effects. In this study, we have used a pharmacological dissection approach to identify a neurotransmitter receptor defect in Egr3(-/-) mice that may mediate their resistance to the locomotor suppressive effects of clozapine. We report that this response is specific to second-generation antipsychotic agents (SGAs), as first-generation medications suppress the locomotor activity of Egr3(-/-) and WT mice to a similar degree. Further, in contrast to the leading theory that sedation by clozapine results from anti-histaminergic effects, we show that H1 histamine receptors are not responsible for this effect in C57BL/6 mice. Instead, selective serotonin 2A receptor (5HT(2A)R) antagonists ketanserin and MDL-11939 replicate the effect of SGAs, repressing the activity in WT mice at a dosage that fails to suppress the activity of Egr3(-/-) mice. Radioligand binding revealed nearly 70% reduction in 5HT(2A)R expression in the prefrontal cortex of Egr3(-/-) mice compared with controls. Egr3(-/-) mice also exhibit a decreased head-twitch response to 5HT(2A)R agonist 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane (DOI). These findings provide a mechanism to explain the reduced sensitivity of Egr3(-/-) mice to the locomotor suppressive effects of SGAs, and suggest that 5HT(2A)Rs may also contribute to the sedating properties of these medications in humans. Moreover, as the deficit in cortical 5HT(2A)R in Egr3(-/-) mice aligns with numerous studies reporting decreased 5HT(2A)R levels in the brains of schizophrenia patients, and the gene encoding the 5HT(2A)R is itself a leading schizophrenia candidate gene, these findings suggest a potential mechanism by which putative dysfunction in EGR3 in humans may influence risk for schizophrenia.

Over 600 000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers (OPC). A key challenge in understanding HPV immunobiology is the diversity of oncogenic HPV types and the need for multiplexed display of HPV antigens to measure antibody responses. We have generated custom HPV protein microarrays displaying 98 proteins as C-terminal GST fusion proteins, representing eight antigens of two low-risk HPV types (HPV6 and 11) and ten oncogenic high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58). We demonstrate robust and reproducible protein expression of 96/98 of the antigens using a human cell lysate expression system. The target epitopes and specificities of four monoclonal antibodies were identified. Using sera from ten patients with newly diagnosed OPC and ten controls, we demonstrate specific IgG seroreactivity to HPV16 E1, E2, and E7 (a fold increase of 1.52, 2.19 and 1.35 in cases vs. controls, respectively, all p < 0.005), confirming our prior data on an ELISA platform. We also detect HPV52 E7 Abs in serum from a patient with cervical cancer. The HPV protein array has potential for rapid identification of serologic responses to 12 HPV types.

DOI: HPV proteome arrays for immunoprofiling of HPV‐associated cancers. Left: Genomes of 12 HPV types were printed on the microarrays and picogreen was used to detect DNA content. Middle: Detection of GST‐tagged HPV proteins expressed in situ on the arrays. Right: Profiling of HPV‐specific IgG in human serum from patients with oropharyngeal cancer. For more details, see the article by Radwa Ewaisha et al. on page 1215.

Introduction: More than 260,000 women die of cervical cancer every year. Screening methods have reduced the incidence of cervical cancer in high-income countries, but detection continues to lag in low and middle income countries (LMICs). IgG antibody (Ab) immunity to early (E) HPV antigens (Ags) are potential biomarkers of disease progression. Since HPV16 accounts for only ~50% of invasive cervical cancers, we developed protein microarrays expressing the proteomes of 12 HPV types to detect host IgG Abs to a broad spectrum of viral Ags to detect pre-invasive and invasive cervical disease. Methods: We developed custom HPV protein microarrays displaying the proteomes of two low-risk HPV types (HPV6 and 11) and ten oncogenic high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58). Arrays were probed with serum samples obtained from women with invasive cervical cancer (ICC; n=80), no or low-grade cervical disease (CIN 0/I; n=60), and high-grade cervical dysplasia (CIN II/III; n=60). To identify positive serologic responses, arrays were scanned and the signal intensity of each protein spot was quantified and normalized. Visual examination of diffused signal (ring) around each spot was performed and the Ab response to each protein was scored on a scale from 0 to 5. Results: To verify array quality and reproducibility, we confirmed high protein expression levels for 98% (96/98) of the antigens printed and high correlation (R ≥ 0.90) of protein expression signals between different randomly selected arrays. Epitope expression was confirmed using four commercial monoclonal Abs raised against HPV16 Ags. Host Abs to at least one early antigen (E1, E2, E4, E6, or E7) were detected in the sera of 31.2% and 43.3% of ICC and CINII/III patients, respectively, compared with 11.7% of women with CIN 0/I. 73.1% of CINII/III cases detected had Abs only to non-HPV16 Ags. Abs to E1, E2, E4, E6, and E7 Ags were detected in 3.8%, 7.7%, 54%, 19%, and 31% of CINII/III versus 16%, 16%, 32%, 32%, and 40% of ICC cases that were positive. The immunodominant Ags in women with CINII/III and ICC were E4 (54%) and E7 (40%), respectively. These results are consistent with the difference in tissue expression levels of these proteins in these two disease stages. This emphasizes the importance of broadening the scope of serological detection to include non-HPV16 proteomes. Conclusions: This study demonstrates that Abs against high risk HPV16 and non-HPV16 serotypes are potential biomarkers for both the diagnosis of cervical cancer and the differentiation between high grade and low-grade pre-invasive cervical lesions. These results suggest that serology is a potential minimally invasive tool for early detection of cervical cancer. High-throughput immunoprofiling of HPV-associated cancers and pre-invasive lesions may be informative for understanding the diversity of host immunity and viral progression. Citation Format: Radwa Ewaisha, Ian Meshay, Jack Resnik, Tirinder Bharaj, Karen S. Anderson. Detection of circulating immune biomarkers of cervical disease using proteome arrays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2217. doi:10.1158/1538-7445.AM2017-2217

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