Human Protein Microarray Research Articles

Identification of a mimotope of a complex gp41 Human Immunodeficiency VIrus epitope related to a non-structural protein of Hepacivirus previously implicated in Kawasaki disease.

We have previously isolated a highly mutated VH1-02 antibody termed group C 76-Q13-6F5 (6F5) that targets a conformational epitope on gp41. 6F5 has the capacity to mediate Ab dependent cell cytotoxicity (ADCC). When the VH1-02 group C 76 antibodies variable chain sequence was reverted to germline (76Canc), this still retained ADCC activity. Due to this ability for the 76Canc germline antibody to functionally target this epitope, we sought to identify a protein target for vaccine development. Initially, we interrogated peptide targeting by screening a microarray containing 29,127 linear peptides. Western blot and ELISAs were used to confirm binding and explore human serum targeting. Autoimmune targeting was further interrogated on a yeast-displayed human protein microarray. 76Canc specifically recognized a number of acidic peptides. Meme analysis identified a peptide sequence similar to a non-structural protein of Hepacivirus previously implicated in Kawasaki disease (KD). Binding was confirmed to top peptides, including the Hepacivirus-related and KD-related peptide. On serum competitions studies using samples from children with KD compared to controls, targeting of this epitope showed no specific correlation to having KD. Human protein autoantigen screening was also reassuring. This study identifies a peptide that can mimic the gp41 epitope targeted by 76C group antibodies (i.e. a mimotope). We show little risk of autoimmune targeting including any inflammation similar to KD, implying non-specific targeting of this peptide during KD. Development of such peptides as the basis for vaccination should proceed cautiously.

Abstract PO2-13-06: Analysis of Antibody Response to SV-BR-1-GM Therapeutic Vaccine in Breast Cancer Patients Using Human Protein Microarrays: Potential Correlations with Therapy Response

Abstract Background: Therapeutic cancer vaccines aim to stimulate the immune system by utilizing tumor antigens to trigger an antitumor response. In our clinical trials, we have focused on evaluating the efficacy of the breast cancer cell line secreting GM-CSF, SV-BR-1-GM as a therapeutic vaccine, and we have observed encouraging clinical outcomes. The SV-BR-1-GM regimen has been used alone (“monotherapy”, ClinicalTrials.gov NCT03066947 – study completed) and in combination with checkpoint inhibitors (“combination”, ClinicalTrials.gov NCT03328026 – study ongoing). To further improve the therapeutic efficacy of this vaccine we have initiated a study to characterize patient’s immune response to this treatment. It has been demonstrated that cancer vaccines can generate both humoral and cellular immune responses. However, predicting immune responses to cancer vaccines, especially when whole cells are employed as immunogens, presents significant challenges. We present here a preliminary analysis of the antibody response to SV-BR-1-GM in breast cancer patients using antigen arrays. Methods: Large-scale protein arrays are versatile and sensitive platforms for antibody specificity evaluation. The HuProt™ Human Proteome Microarray (CDI Laboratories, Inc., Baltimore, Maryland, United States) provides the largest number of unique, full-length, individually purified human proteins on a single microscope slide. This allows thousands of interactions to be profiled in a high-throughput manner. We utilized here an unbiased human protein microarray platform encompassing >21,000 proteins and isoforms from ~19,000 unique genes to identify IgG and IgM responses against self-antigens elicited by treatment. Serum samples from SV-BR-GM-treated patients were analyzed, comparing pre- and post-treatment. The human serum samples were probed at 1:1000 dilution. After sample processing and data collection the raw signal intensities on all arrays were quantile normalized using CDI software. Heatmaps were generated using MetaboAnalyst 5.0, which utilizes the R pheatmap package (version 0.7.7). For paired analysis, fold changes (FCs) were calculated by determining the ratio between paired pre- and post-treatment samples, resulting in one FC per pair. The means of these FCs (pair means) were then computed. ANOVA tests were performed using the RStatix R package, which automatically determines the appropriate Type I, II, or III errors for the analysis. Results: By using this approach, we were able to evaluate a broader range of antigens compared to previous investigations. Using a variety of statistical approaches, potential correlations with patient survival on SV-BR-1-GM were evaluated. Antibody responses to galectin antigens demonstrated the most consistent relationships with survival. Further confirmation with additional patients and prospective analysis are needed to fully understand relationships with clinical benefit and survival. Citation Format: Miguel Lopez-Lago, Mingjin Chang, Giuseppe Del Priore, Vikas Bhardwaj, Patience Cournoo, Pedro Ramos, Tyler Hulett, Charles Wiseman, William Williams. Analysis of Antibody Response to SV-BR-1-GM Therapeutic Vaccine in Breast Cancer Patients Using Human Protein Microarrays: Potential Correlations with Therapy Response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-06.

Paraneoplastic Calmodulin Kinase-Like Vesicle-Associated Protein (CAMKV) Autoimmune Encephalitis.

To report an autoimmune paraneoplastic encephalitis characterized by immunoglobulin G (IgG) antibody targeting synaptic protein calmodulin kinase-like vesicle-associated (CAMKV). Serum and cerebrospinal fluid (CSF) samples harboring unclassified antibodies on murine brain-based indirect immunofluorescence assay (IFA) were screened by human protein microarray. In 5 patients with identical cerebral IFA staining, CAMKV was identified as top-ranking candidate antigen. Western blots, confocal microscopy, immune-absorption, and mass spectrometry were performed to substantiate CAMKV specificity. Recombinant CAMKV-specific assays (cell-based [fixed and live] and Western blot) provided additional confirmation. Of 5 CAMKV-IgG positive patients, 3 were women (median symptom-onset age was 59 years; range, 53-74). Encephalitis-onset was subacute (4) or acute (1) and manifested with: altered mental status (all), seizures (4), hyperkinetic movements (4), psychiatric features (3), memory loss (2), and insomnia (2). Paraclinical testing revealed CSF lymphocytic pleocytosis (all 4 tested), electrographic seizures (3 of 4 tested), and striking MRI abnormalities in all (mesial temporal lobe T2 hyperintensities [all patients], caudate head T2 hyperintensities [3], and cortical diffusion weighted hyperintensities [2]). None had post-gadolinium enhancement. Cancers were uterine adenocarcinoma (3 patients: poorly differentiated or neuroendocrine-differentiated in 2, both demonstrated CAMKV immunoreactivity), bladder urothelial carcinoma (1), and non-Hodgkin lymphoma (1). Two patients developed encephalitis following immune checkpoint inhibitor cancer therapy (atezolizumab [1], pembrolizumab [1]). All treated patients (4) demonstrated an initial response to immunotherapy (corticosteroids [4], IVIG [2]), though 3 died from cancer. CAMKV-IgG is a biomarker of immunotherapy-responsive paraneoplastic encephalitis with temporal and extratemporal features and uterine cancer as a prominent oncologic association. ANN NEUROL 2024;96:21-33.

Delayed B-cell maturation with extra-follicular B-cell response and broad autoantibody repertoire are characteristic B-cell dysregulation signatures in patients with multi-lineage cytopenia

IntroductionMulti-lineage cytopenia (m-IC) are increasingly recognized as presenting manifestation of inborn errors immunity (IEI). Although around 40% of these patients are associated with known IEI, the underlying immune anomalies at onset of m-IC are poorly understood. Recently, we published a unique immune signature characterized by abnormal expansion of circulating T-follicular helper cells (cTfh), increased T-cell activation and decreased naïve CD4+ T (Kumar et.al Blood 2022). Despite our improved understanding of T-cell immune abnormalities, the underlying B-cell dysfunction is largely undefined at onset of m-IC. MethodsIn this study, we enrolled 20 patients with pES, 30 patients with chronic ITP (cITP) and 18 healthy controls (HCs). High dimensional B cell immunophenotyping was performed to assess B-cell maturation, extrafollicular B cell development, and autoreactivity. Autoantibody profiling was performed using human protein microarrays. Multiplex immunofluorescence staining of lymph nodes was performed to evaluate germinal center reaction. ResultsWe observed that patients with m-IC had increased frequency of CD10+ immature B cells in comparison to cITP and HCs. Despite an expansion of cTfh and elevated levels of plasma CXCL13, we observed decreased frequency of CSMB which suggests underlying defects in B-cell maturation. While canonical B-cell maturation was defective, we noticed aberrant extrafollicular B-cell development characterized by significantly higher proportion of DN2 (CD11c+CD21low) and decreased proportion of DN1 (CD21+ CD11c-) populations (Figure 1A). However, the frequency of parent population i.e., double negative (DN) (IgD- CD27-) B-cells was similar across the cohorts. In addition, we also noted decreased proportion of CXCR5+ B cells in 30% of patients and overall skewed CXCR5 expression on B versus T cells. Furthermore, we noted increase in autoreactivity in B cells as evident by increase in proportion of 9G4+ B cells. To further assess auto-antibody driven pathophysiology, we evaluated the auto-antibody repertoire and found broad auto-antibody repertoire targeting several biological processes (Figure 1B). [Display omitted] ConclusionsTaken together, our findings identified broad B-cell immune abnormalities along with unique auto-antibody repertoire in patients with m-IC. In addition to enhancing our current knowledge of disease pathology, the utility of these findings could further help in disease monitoring and evaluating response to the therapeutics.

Small Molecule Screening Strategies from Lead Identification to Validation.

Festa, F., Labaer, J. Kinase inhibitor screening in self-assembled human protein microarrays. Journal of Visualized Experiments. 152, e59886 (2019). Stockman, B. J. et al. NMR-Based activity assays for determining compound inhibition, IC50 values, artifactual activity, and whole-cell activity of nucleoside ribohydrolases. Journal of Visualized Experiments. 148, e59928 (2019). Gao, S. et al. A high-throughput assay for the prediction of chemical toxicity by automated phenotypic profiling of Caenorhabditis elegans. Journal of Visualized Experiments. 145, e59082 (2019). Axelsson, H., Almqvist, H., Seashore-Ludlow, B. Using high content imaging to quantify target engagement in adherent cells. Journal of Visualized Experiments. 141, e58670 (2018). Chorba, J. S., Galvan, A. M., Shokat, K. M. A high-throughput luciferase assay to evaluate proteolysis of the single-turnover protease PCSK9. Journal of Visualized Experiments. 138, e58265 (2018). Sullivan, C. et al. Using zebrafish models of human influenza A virus infections to screen antiviral drugs and characterize host immune cell responses. Journal of Visualized Experiments. 119, e55235 (2017). Tiemann, K., Garri, C., Wang, J., Clarke, L., Kani, K. Assessment of resistance to tyrosine kinase inhibitors by an interrogation of signal transduction pathways by antibody arrays. Journal of Visualized Experiments. 139, e57779 (2018). Radnai, L., Stremel, R. F., Sellers, J. R., Rumbaugh, G., Miller, C. A. A semi-high-throughput adaptation of the NADH-coupled ATPase assay for screening small molecule inhibitors. Journal of Visualized Experiments. 150, e60017 (2019). Nandha Premnath, P., Craig, S., McInnes, C. Development of inhibitors of protein-protein interactions through REPLACE: Application to the design and development non-ATP competitive CDK inhibitors. Journal of Visualized Experiments. 104, e52441 (2015). Chen, E. W., Ke, C. Y., Brzostek, J., Gascoigne, N. R. J., Rybakin, V. Identification of mediators of T-cell receptor signaling via the screening of chemical inhibitor libraries. Journal of Visualized Experiments. 143, e58946 (2019). Takakusagi, Y. Biosensor-based high throughput biopanning and bioinformatics analysis strategy for the global validation of drug-protein interactions. Journal of Visualized Experiments. 166, e61873 (2020). Riching, K. M., Mahan, S. D., Urh, M., Daniels, D. L. High-Throughput cellular profiling of targeted protein degradation compounds using HiBiT CRISPR cell lines. Journal of Visualized Experiments. 165, e61787 (2020).

Sodium danshensu attenuates cerebral ischemia-reperfusion injury by targeting AKT1.

The beneficial properties of Sodium Danshensu (SDSS) for controlling cerebral ischemia and reperfusion injury (CIRI) are elucidated here both in vivo and in vitro. SDSS administration significantly improved the viability of P12 cells, reduced lactate dehydrogenase (LDH) leakage, and decreased the apoptosis rate following exposure to an oxygen-glucose deprivation/reoxygenation (OGD) environment. In addition, the results of a HuprotTM human protein microarray and network pharmacology indicated that AKT1 is one of the main targets of SDSS. Moreover, functional experiments showed that SDSS intervention markedly increased the phosphorylation level of AKT1 and its downstream regulator, mTOR. The binding sites of SDSS to AKT1 protein were confirmed by Autodock software and a surface plasmon resonance experiment, the result of which imply that SDSS targets to the PH domain of AKT1 at ASN-53, ARG-86, and LYS-14 residues. Furthermore, knockdown of AKT1 significantly abolished the role of SDSS in protecting cells from apoptosis and necrosis. Finally, we investigated the curative effect of SDSS in a rat model of CIRI. The results suggest that administration of SDSS significantly reduces CIRI-induced necrosis and apoptosis in brain samples by activating AKT1 protein. In conclusion, SDSS exerts its positive role in alleviating CIRI by binding to the PH domain of AKT1 protein, further resulting in AKT1 activation.

Use of Autoreactive Antibodies in Blood of Patients with Pancreatic Intraductal Papillary Mucinous Neoplasms (IPMN) for Grade Distinction and Detection of Malignancy.

Simple SummaryRisk stratification via biomarkers used with imaging could support predicting high-risk pancreatic intraductal papillary mucinous neoplasms (IPMN) with malignant potential. We explored the presence of autoreactive antibodies in the blood of patients with IPMN of different grades of dysplasia, including IPMN with associated invasive carcinoma and early-stage pancreatic ductal adenocarcinoma (PDAC), to identify signatures of early malignancy. Multivariate predictive models retained 14 proteins as potential biomarkers for discrimination between all disease classes. The integration of the autoreactive-antibody panel with clinical variables may aid in risk stratification of high-risk IPMN patients, which would subsequently improve clinical management.(1) Background: A reliable non-invasive distinction between low- and high-risk pancreatic intraductal papillary mucinous neoplasms (IPMN) is needed to effectively detect IPMN with malignant potential. This would improve preventative care and reduce the risk of developing pancreatic cancer and overtreatment. The present study aimed at exploring the presence of autoreactive antibodies in the blood of patients with IPMN of various grades of dysplasia. (2) Methods: A single-center cohort was studied composed of 378 serum samples from patients with low-grade IPMN (n = 91), high-grade IPMN (n = 66), IPMN with associated invasive cancer (n = 30), pancreatic ductal adenocarcinoma (PDAC) stages T1 (n = 24) and T2 (n = 113), and healthy controls (n = 54). A 249 full-length recombinant human protein microarray was used for profiling the serum samples. (3) Results: 14 proteins were identified as potential biomarkers for grade distinction in IPMN, yielding high specificity but mediocre sensitivity. (4) Conclusions: The identified autoantibodies are potential biomarkers that may assist in the detection of malignancy in IPMN patients.

Anticorps anti-hexokinase-1 et anti-kelch-like 12, de nouveaux marqueurs de la cholangite biliaire primitive

AbstractPrimary biliary cholangitis (PBC) diagnosis is supported by auto-antoantibodies (Ab) mainly anti-mitochondrial antibodies (AMA2) detected in 90-95 % of PBC cases. However, some cases of PBC are still seronegative and may remain undiagnosed. As early setting of the treatment is crucial, it would be interesting to find other auto-Ab. Recently, two new autoantigens, kelch-like 12 (KLHL12) and hexokinase-1 (HK1) have been identified using human recombinant protein microarrays. KLHL12, through ubiquitination of substrates, is involved in cell regulation. HK1 is a mitochondrial and cytosolic protein of glycolysis pathway. The existence of recombinant forms of these proteins would enable the possibilities of complementary diagnostic tests. Few studies have assessed the diagnostic value of these Ab in PBC. These Abs appear very specific for CBP (>90%) and Both Ab were detected in AMA2 negative PBC.

Multifaceted Regulation of Akt by Diverse C-Terminal Post-translational Modifications.

Akt is a Ser/Thr protein kinase that regulates cell growth and metabolism and is considered a therapeutic target for cancer. Regulation of Akt by membrane recruitment and post-translational modifications (PTMs) has been extensively studied. The most well-established mechanism for cellular Akt activation involves phosphorylation on its activation loop on Thr308 by PDK1 and on its C-terminal tail on Ser473 by mTORC2. In addition, dual phosphorylation on Ser477 and Thr479 has been shown to activate Akt. Other C-terminal tail PTMs have been identified, but their functional impacts have not been well-characterized. Here, we investigate the regulatory effects of phosphorylation of Tyr474 and O-GlcNAcylation of Ser473 on Akt. We use expressed protein ligation as a tool to produce semisynthetic Akt proteins containing phosphoTyr474 and O-GlcNAcSer473 to dissect the enzymatic functions of these PTMs. We find that O-GlcNAcylation at Ser473 and phosphorylation at Tyr474 can also partially increase Akt's kinase activity toward both peptide and protein substrates. Additionally, we performed kinase assays employing human protein microarrays to investigate global substrate specificity of Akt, comparing phosphorylated versus O-GlcNAcylated Ser473 forms. We observed a high similarity in the protein substrates phosphorylated by phosphoSer473 Akt and O-GlcNAcSer473 Akt. Two Akt substrates identified using microarrays, PPM1H, a protein phosphatase, and NEDD4L, an E3 ubiquitin ligase, were validated in solution-phase assays and cell transfection experiments.

MAGEH1 interacts with GADD45G and induces renal tubular cell apoptosis.

BackgroundMelanoma-associated antigen H1 (MAGEH1) is a protein that belongs to melanoma-associated antigen (MAGE) superfamily. Growth arrest and DNA damage 45G (GADD45G) is a member of the DNA damage-inducible gene family which responds to environmental stresses. We have previously shown that GADD45G is a protein that promotes apoptosis of renal tubular cells in response to a nephrotoxic injury. In this study, we show evidence that MAGEH1 interacts with GADD45G and is involved in the induction of nephrotoxin-induced apoptosis of renal tubular cells.MethodsPrimary human renal tubular epithelial (HRE) cells and human kidney 2 (HK-2) cells were used in this study. To produce stable cell lines in which MAGEH1 expression was silenced, HRE cells were transduced with a lentiviral vector encoding a single guide RNA construct targeting the MAGEH1 gene. To knockdown GADD45G expression in HRE cells, a vector containing short hairpin RNA (shRNA) was used. We used short interfering RNAs (siRNA) to achieve transient silencing of genes in HK-2 cells. Recombinant adenoviruses were synthesized to overexpress MAGEH1 and GADD45G proteins. Human protein microarray was used to identify proteins that binds to GADD45G. Co-immunoprecipitation assays were then performed to confirm microarray results. Cell death was induced by cyclosporine A (CsA). Real-time quantitative PCR assay was used to evaluate gene expression levels. The degree of apoptosis and necrosis of cultured cells was evaluated by flow cytometry. Expression levels of caspases were examined using western blot analysis.ResultsWe found that GADD45G bound to one protein spotted in the protein microarray, which was subsequently identified as MAGEH1. We confirmed the interaction between GADD45G and MAGEH1 protein using the co-immunoprecipitation assay. MAGEH1 gene expression was not altered by CsA-induced cytotoxic injury, whereas GADD45G gene expression was increased significantly upon CsA treatment. MAGEH1 expression was significantly downregulated in GADD45G knockdown HRE stable cells suggesting that MAGEH1 expression may be dependent on GADD45G expression. CsA-induced apoptosis was significantly reduced in MAGEH1 knockdown HRE stable cells which led to an increased survival of these cells. Similar results were observed in GADD45G knockdown HRE stable cells. Accordingly, CsA-induced apoptosis was significantly decreased in MAGEH1 siRNA and GADD45G siRNA transfected HK-2 cells. CsA-induced activation of caspase-7 and caspase-9 was inhibited in MAGEH1 knockdown HRE stable cells, and similarly in GADD45G knockdown HRE stable cells.ConclusionsTo the best of our knowledge, this is the first study to show that MAGEH1 interacts with GADD45G and that MAGEH1 is involved in caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.

Diagnostic AI Modeling and Pseudo Time Series Profiling of AD and PD Based on Individualized Serum Proteome Data.

Background: Parkinson’s disease (PD), Alzheimer’s disease (AD) are common neurodegenerative disease, while mild cognitive impairment (MCI) may be happened in the early stage of AD or PD. Blood biomarkers are considered to be less invasive, less cost and more convenient, and there is tremendous potential for the diagnosis and prediction of neurodegenerative diseases. As a recently mentioned field, artificial intelligence (AI) is often applied in biology and shows excellent results. In this article, we use AI to model PD, AD, MCI data and analyze the possible connections between them. Method: Human blood protein microarray profiles including 156 CT, 50 MCI, 132 PD, 50 AD samples are collected from Gene Expression Omnibus (GEO). First, we used bioinformatics methods and feature engineering in machine learning to screen important features, constructed artificial neural network (ANN) classifier models based on these features to distinguish samples, and evaluated the model’s performance with classification accuracy and Area Under Curve (AUC). Second, we used Ingenuity Pathway Analysis (IPA) methods to analyse the pathways and functions in early stage and late stage samples of different diseases, and potential targets for drug intervention by predicting upstream regulators. Result: We used different classifier to construct the model and finally found that ANN model would outperform the traditional machine learning model. In summary, three different classifiers were constructed to be used in different application scenarios, First, we incorporated 6 indicators, including EPHA2, MRPL19, SGK2, to build a diagnostic model for AD with a test set accuracy of up to 98.07%. Secondly, incorporated 15 indicators such as ERO1LB, FAM73B, IL1RN to build a diagnostic model for PD, with a test set accuracy of 97.05%. Then, 15 indicators such as XG, FGFR3 and CDC37 were incorporated to establish a four-category diagnostic model for both AD and PD, with a test set accuracy of 98.71%. All classifier models have an auc value greater than 0.95. Then, we verified that the constructed feature engineering filtered out fewer important features but contained more information, which helped to build a better model. In addition, by classifying the disease types more carefully into early and late stages of AD, MCI, and PD, respectively, we found that early PD may occur earlier than early MCI. Finally, there are 24 proteins that are both differentially expressed proteins and upstream regulators in the disease group versus the normal group, and these proteins may serve as potential therapeutic targets and targets for subsequent studies. Conclusion: The feature engineering we build allows better extraction of information while reducing the number of features, which may help in subsequent applications. Building a classifier based on blood protein profiles using deep learning methods can achieve better classification performance, and it can help us to diagnose the disease early. Overall, it is important for us to study neurodegenerative diseases from both diagnostic and interventional aspects.

The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

BackgroundP7C3 is a neurogenic compound that exhibits neuroprotective properties in neural cells. However, its target proteins and effects in glioma are unknown.MethodsThe candidate P7C3 target proteins were analyzed using a human protein microarray containing 23136 human proteins. A streptavidin agarose affinity assay was used to verify the direct interaction between P7C3 and phosphoglycerate kinase 1 (PGK1). Mass spectrometry was used to identify the binding sites of PGK1 for P7C3 binding. Seahorse XF96 extracellular flux analyzer was used to measure the cell oxygen consumption rate and extracellular acidification rate. Glycolytic metabolites were measured using the related kits. Protein level was detected by western blotting and immunohistochemical staining. Autophagy was analyzed using a transmission electron microscope and western blotting. The malignancy of tumor progression in vitro and in vivo was analyzed based on cell viability, apoptosis and proliferation, migration and invasion, and xenograft model. Glial cells were marked by antibodies via immunohistochemical staining.ResultsThe human protein microarray identified 577 candidate P7C3 target proteins. The global profile of P7C3 target proteins indicated that P7C3 regulates glycolysis. Metabolic experiments confirmed that P7C3 regulates aerobic glycolysis in glioma cells. The underlying mechanism of P7C3 was found to be direct targeting PGK1 at lysine residues and asparagine residues, and the specific P7C3-PGK1 interaction led to decreased protein level and total intracellular kinase activity of PGK1. The Cancer Genome Atlas and Chinese Glioma Genome Atlas databases indicated that the mRNA level of PGK1 is significantly increased in high-grade glioma, and the abnormally high mRNA level of PGK1 is associated with a poor prognosis in patients with glioma, suggesting that PGK1 is a promising target for glioma therapy. The inhibition of PGK1 and the subsequent suppression of aerobic glycolysis caused by P7C3 inhibited the malignant growth of glioma in vitro and in vivo. Furthermore, P7C3 did not damage normal glial cells under concentration, which exhibit an inhibitory effect on gliomas.ConclusionsThis study revealed that P7C3 suppresses glioma by regulating aerobic glycolysis via directly targeting PGK1. Furthermore, we identified the P7C3 target proteins for the first time which is expected to provide scientific clues for future studies.

Renal Expression of Light Chain Binding Proteins.

Overproduction of human light chains (LCs) and immunoglobulins can result in various forms of renal disease such as cast nephropathy, monoclonal immunoglobulin deposition disease, LC proximal tubulopathy, AL amyloidosis, and crystal storing histiocytosis. This is caused by cellular uptake of LCs and overwhelmed intracellular transport and degradation in patients with high urine LC concentrations. LC kappa and lambda purification was evaluated by sodium dodecyl sulfate gel electrophoresis. LC and myeloma protein binding to immobilized renal proteins was measured by enzyme-linked immunosorbent assay (ELISA). The human protein microarray (HuProt™) was screened with purified kappa and lambda LC. Identified LC partners were subsequently analyzed in silico for renal expression sites using protein databases, Human Protein Atlas, UniProt, and Bgee. Binding of urinary LCs and immunoglobulins to immobilized whole renal proteins from 22 patients with myeloma or plasma cell dyscrasia was shown by ELISA. Forty lambda and 23 kappa interaction partners were identified from HuProt™ array screens, of which 21 were shared interactors. Among the total of 42 interactors, 12 represented cell surface proteins. Lambda binding signals were approximately 40% higher than kappa signals. LC interaction with renal cells and disease-causing pathologies are more complex than previously thought. It involves an extended spectrum of proteins expressed throughout the nephron, and their identification has been enabled by recently developed methods of protein analysis such as protein microarray screening. Further biochemical studies on interacting proteins are warranted to elucidate their clinical relevance.

Autoantibody profiling of alveolar rhabdomyosarcoma patients unveils tumor-associated antigens with diagnostic and prognostic significance

ABSTRACT Alveolar rhabdomyosarcoma (ARMS) is a highly aggressive subtype of childhood cancer for which efficacious treatments are needed. Immunotherapy represents a new therapeutic opportunity to pursue, but it requires the identification of worthwhile tumor antigens. Herein, we exploited the capacity of ARMS autoantibodies to recognize tumor self-antigens, probing human protein microarrays with plasma from ARMS patients and healthy subjects. We assessed the autoantibody response in ARMS, validated data with independent techniques, and estimated autoantibodies diagnostic and prognostic significance by receiver-operator characteristic curves (ROC), uni- and multivariate analysis. Of the 48 tumor antigens identified, General Transcription Factor II–I (GTF2i) and Protocadherin Gamma Subfamily C5 (PCDHGC5) were selected as candidate targets to validate tumor-restricted antigen expression and autoantibody reactivity through an independent technique and wider cohort of cases. GTF2i and PCDHGC5 overexpression was observed in tumor tissues compared to normal counterparts, and anti-GTF2i and -PCDHGC5 autoantibodies were found able to distinguish ARMS patients from healthy subjects as well as cases with different histology. Moreover, low levels of PCDHGC5 autoantibodies characterized patients with worse event-free survival and proved to be an independent negative prognostic factor. This approach provided the first comprehensive autoantibody profile of ARMS, gave novel insights into the immune response of this malignancy and paved the way toward novel potential antibody-based therapeutic applications suitable to improve the survival of ARMS patients.

B Cells Improve Overall Survival in HPV-Associated Squamous Cell Carcinomas and Are Activated by Radiation and PD-1 Blockade.

To characterize the role of B cells on human papilloma virus (HPV)-associated cancer patient outcomes and determine the effects of radiation and PD-1 blockade on B-cell populations. Tumor RNA-sequencing data from over 800 patients with head and neck squamous cell carcinoma (HNSCC) and cervical cancer, including a prospective validation cohort, was analyzed to study the impact of B-cell gene expression on overall survival (OS). A novel murine model of HPV+ HNSCC was used to study the effects of PD-1 blockade and radiotherapy on B-cell activation, differentiation, and clonality including analysis by single-cell RNA-sequencing and B-cell receptor (BCR)-sequencing. Human protein microarray was then used to quantify B-cell-mediated IgG and IgM antibodies to over 16,000 proteins in the serum of patients treated on a clinical trial with PD-1 blockade. RNA-sequencing identified CD19 and IGJ as novel B-cell prognostic biomarkers for 3-year OS (HR, 0.545; P < 0.001). PD-1 blockade and radiotherapy enhance development of memory B cells, plasma cells, and antigen-specific B cells. BCR-sequencing found that radiotherapy enhances B-cell clonality, decreases CDR3 length, and induces B-cell somatic hypermutation. Single-cell RNA-sequencing identified dramatic increases in B-cell germinal center formation after PD-1 blockade and radiotherapy. Human proteome array revealed enhanced IgG and IgM antibody responses in patients who derived clinical benefit but not those with progressive disease after treatment with PD-1 blockade. These findings establish a key role for B cells in patient outcomes and responses to PD-1 blockade in HPV-associated squamous cell carcinomas and demonstrate the need for additional diagnostics and therapeutics targeting B cells.

MON-532 Characterization of the Angiogenic Factor SFRP2 in Papillary Thyroid Carcinoma

Over the last decade, there has been an average annual increase of 3.1% in thyroid cancer diagnosis in the U.S. Papillary thyroid carcinoma (PTC) accounts for 80% of all thyroid cancer diagnoses. However, few molecular markers exist to identify clinically aggressive phenotypes. The angiogenic factor, secreted frizzled-related protein 2 (SFRP2), is associated with a poor prognosis in several malignancies including breast cancer and melanoma. The role of SFRP2 in PTC has yet to be investigated. The aims of this study were to determine the differential expression of SFRP2 in PTC, benign thyroid adenomas, normal thyroid tissue (from patients without cancer), and normal adjacent tissue (NAT) (non-cancerous tissue from patients with PTC) and investigate the role of SFRP2 in tumor development in two PTC cell lines, PTC classical variant (PTC-CV) and PTC follicular variant (PTC-FV), upon treatment with a humanized anti-SFRP2 monoclonal antibody (hSFRP2 mAb). Immunohistochemistry (IHC) was performed using human tissue protein microarrays including 226 PTC, 79 benign adenomas, 112 NAT, and 30 normal thyroid tissue samples. In-vitro proliferation and apoptosis experiments were performed on MDA-T41 (PTC-CV) and MDA-T68 (PTC-FV) cell lines by treating with hSFRP2 mAb, Xolair IgG control, and a vehicle control. SFRP2 expression was significantly higher in PTC compared with benign adenomas and normal thyroid (mean expression scores 9, 6, and 1, respectively; p<0.05). SFRP2 expression was significantly higher in NAT than normal thyroid (mean expression score 4 and 0, respectively, p<0.05). Apoptotic rates were increased by 40% and 62% in the PTC-CV hSFRP2 mAb treatment group compared with the Xolair and vehicle treatment groups, respectively (p<0.05). Apoptotic rates were increased by 126% and 59% in the PTC-FV hSFRP2 mAb treatment group compared with the Xolair and vehicle treatment groups, respectively (p<0.05). Treatment with hSFRP2 mAb had no significant effect on proliferation in either cell line. In conclusion, SFRP2 expression is significantly higher in PTC than in benign adenomas and normal thyroid tissue. SFRP2 expression in NAT is significantly higher than in normal thyroid tissue and not significantly different from benign adenomas. SFRP2 expression in nonmalignant tissue adjacent to PTC could be due to expression in the tumor microenvironment. Treatment with a novel hSFPR2 mAb increases apoptotic rates in two different PTC cell lines. These data suggest that SFPR2 is involved in tumorigenesis of PTC.

Autoantibodies as diagnostic markers and potential drivers of inflammation in ulcerative colitis.

To date, no comprehensive analysis of autoantibodies in sera of patients with ulcerative colitis has been conducted. To analyze the spectrum of autoantibodies and to elucidate their role serum-IgG from UC patients (n = 49) and non-UC donors (n = 23) were screened by using a human protein microarray. Screening yielded a remarkable number of 697 differentially-reactive at the nominal 0·01 significance level (FDR<0·1) of the univariate test between the UC and the non-UC group. CD99 emerged as a biomarker to discriminate between both groups (p = 1e-04, AUC = 0·8). In addition, cytokines, chemokines and growth factors were analyzed by Olink’s Proseek® Multiplex Inflammation-I 96×96 immuno-qPCR assay and 31 genes were significant at the nominal 0.05 level of the univariate test to discriminate between UC and non-UC donors. MCP-3, HGF and CXCL-9 were identified as the most significant markers to discriminate between UC patients with clinically active and inactive disease. Levels of CXCL10 (cor = 0.3; p = 0.02), CCL25 (cor = 0.25; p = 0.04) and CCL28 (cor = 0.3; p = 0.02) correlated positively with levels of anti CD99. To assess whether autoantibodies are detectable prior to diagnosis with UC, sera from nine donors at two different time points (T-early, median 21 months and T-late, median 6 months) were analyzed. 1201 features were identified with higher reactivity in samples at time points closer to clinical UC presentation. In vitro, additional challenge of peripheral mononuclear cells with CD99 did not activate CD4+ T cells but induced the secretion of IL-10 (-CD99: 20.21±20.25; +CD99: 130.20±89.55; mean ±sd; p = 0.015). To examine the effect of CD99 in vivo, inflammation and autoantibody levels were examined in NOD/ScidIL2Rγnull mice reconstituted with PBMC from UC donors (NSG-UC). Additional challenge with CD99 aggravated disease symptoms and pathological phenotype as indicated by the elevated clinical score (-CD99: 1·85 ± 1·94; +CD99: 4·25 ± 1·48) and histological score (-CD99: 2·16 ± 0·83; +CD99: 3·15 ± 1·16, p = 0·01). Furthermore, levels of anti-CD99 antibodies increased (Control: 398 ± 323; mean MFI ± sd; Ethanol + PBS: 358 ±316; Ethanol + CD99: 1363 ± 1336; Control versus Ethanol + CD99: p = 0.03). In a highly inflammatory environment, frequencies of pro-inflammatory M1 monocytes (CD14+ CD64+: unchallenged 8.09±4.72; challenged 14.2±8.62; p = 0.07; CD14+ CD1a+: unchallenged 16.29 ±6.97; challenged 43.81±14.4, p = 0.0003) increased and levels of autoantibodies in serum decreased in the NSG-UC mouse model. These results suggest that autoantibodies are potent biomarkers to discriminate between UC and non-UC and indicate risk to develop UC. In an inflammatory environment, auto-antibodies may promote the pathological phenotype by activating M1 monocytes in the NSG-UC animal model and also in patients with UC.

A Novel LncRNA, AC091729.7 Promotes Sinonasal Squamous Cell Carcinomas Proliferation and Invasion Through Binding SRSF2.

Long non-coding RNAs (lncRNAs) play important roles in various biological progresses of carcinogenesis. However, the function of lncRNAs in human sinonasal squamous cell carcinoma (SNSCC) remains greatly unclear. In the current study, lncRNA AC091729.7 expression was examined in SNSCC samples by using microarray, RNA in situ hybridization (ISH) and real-time fluorescence quantitative PCR (qRT-PCR). Cell viability, colony-formation, wound-healing, and transwell assays were applied to SNSCC cells. Xenograft mouse models were employed to evaluate the role of AC091729.7 in growth of SNSCC in vivo. Human protein microarray (HuprotTM Protoarray) and RNA immunoprecipitation (RIP) were used for identifying AC091729.7 binding proteins in SNSCC. Results showed AC091729.7 was upregulated and closely connected with the survival of the SNSCC patients. Knockdown of AC091729.7 suppressed SNSCC cell migration, proliferation, invasion in vitro. Furthermore, downregulation of AC091729.7 could inhibit the growth of SNSCC in vivo. Moreover, Human protein microarray and RIP suggested that AC091729.7 directly combine with the serine/arginine rich splicing factor 2 (SRSF2). Our results suggest that in the cell progression of SNSCC, lncRNA AC091729.7 plays a carcinogenic role and serves as a novel biomarker and latent curative target in SNSCC patients.

The origin and nature of the complex autoantibody profile in cerebrospinal fluid

The present study demonstrates, using human protein microarrays and plasma and cerebrospinal fluid samples obtained pre-surgically and simultaneously from 46 hip fracture repair patients, that CSF exhibits an extraordinarily complex IgG autoantibody profile composed of thousands of autoantibodies. We show that the pattern of expression levels of individual autoantibodies in CSF closely mimics that in the blood, regardless of age, gender or the presence or absence of disease, indicative of a blood-based origin for CSF autoantibodies. In addition, using five longitudinal serum samples obtained from one healthy individual over a span of nine years, we found that blood autoantibody profiles are remarkably stable over a long period of time, and that autoantibody profiles in both blood and CSF show features that are common among different individuals as well as individual-specific. Lastly, we demonstrate that an elevated CSF/plasma autoantibody ratio is more common in elderly hip fracture repair patients that experienced post-operative delirium than in non-delirium subjects, thus highlighting the crucial role that blood-brain and/or blood-CSF barrier compromise may play in the development of post-operative delirium.

Detection of early-stage Alzheimer's pathology using blood-based autoantibody biomarkers in elderly hip fracture repair patients.

Post-operative delirium (POD) is the most common complication following major surgery in non-demented older (>65 y/o) patients. Patients experiencing POD show increased risk for future cognitive decline, including mild cognitive impairment (MCI) and Alzheimer’s disease (AD) and, conversely, patients with cognitive decline at surgery show increased risk for POD. Here, we demonstrate that a previously established panel of AD-driven MCI (ADMCI) autoantibody (aAB) biomarkers can be used to detect prodromal AD pre-surgically in individuals admitted into the hospital for hip fracture repair (HFR) surgery. Plasma from 39 STRIDE (STRIDE: A Strategy to Reduce the Incidence of Postoperative Delirium in Elderly Patients) HFR patients and sera from 25 age- and sex-matched non-demented and non-surgical controls were screened using human protein microarrays to measure expression of a panel of 44 previously identified MCI aAB biomarkers. The predictive classification accuracy of the aAB biomarker panel was evaluated using Random Forest (RF). The ADMCI aAB biomarkers successfully distinguished 21 STRIDE HFR patients (CDR = 0.5) from 25 matched non-surgical controls with an overall accuracy of 91.3% (sensitivity = 95.2%; specificity = 88.0%). The ADMCI aAB panel also correctly identified six patients with preoperative CDR = 0 who later converted to CDR = 0.5 or >1 at one-year follow-up. Lastly, the majority of cognitively normal (CDR = 0) STRIDE HFR subjects that were positive for CSF AD biomarkers based on the A/T/N classification system were likewise classified as ADMCI aAB-positive using the biomarker panel. Results suggest that pre-surgical detection of ADMCI aAB biomarkers can readily identify HFR patients with likely early-stage AD pathology using pre-surgery blood samples, opening up the potential for early, blood-based AD detection and improvements in peri- and postoperative patient management.