Human respiratory viruses are a diverse group of pathogens composed of hundreds of virus strains, and this presents a major challenge for diagnostic laboratories. To efficiently detect numerous ...viruses in a large epidemiologic study, we developed a fast, multitarget, sensitive, and specific assay named the Respiratory MultiCode-PLx Assay (RMA). The RMA utilizes improved multiplex PCR chemistry (EraGen MultiCode-PLx technology) coupled with high-throughput microsphere flow cytometry (Luminex). Eighteen sets of virus-specific multiplex PCR primers were developed based on the conserved sequences of all available respiratory-virus sequences for eight distinct groups: human rhinovirus (HRV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus (InfV), metapneumovirus, adenovirus (Ad), coronavirus, and enterovirus. Each primer set detected 20 cDNA copies of the intended target per sample and had no reaction with 60,000 copies of human genomic DNA. The accuracy and sensitivity of the RMA for detecting respiratory viruses in human samples were tested with two sets of clinical specimens. First, 101 nasal-wash specimens that were positive for HRV, RSV, InfV, PIV, or Ad by traditional techniques were reanalyzed by RMA, and all target viruses were detected with an overall sensitivity of 94% and specificity of 99%. Second, 103 nasal-wash samples from 5-year-old children with asthma and respiratory symptoms were analyzed; RMA detected viruses in 74 specimens (71.8%) compared to only 24 (23.3%) by traditional culture and immunofluorescent-staining techniques. These results show that RMA is an accurate, sensitive, and practical test for respiratory-virus infections.
The MultiCode-PLx system (EraGen Biosciences, Inc., Madison, WI) for the detection of respiratory viruses uses an expanded genetic alphabet, multiplex PCR chemistry, and microsphere flow cytometry to ...rapidly detect and specifically identify 17 different respiratory viruses directly in clinical specimens. The MultiCode-PLx system was tested in parallel with direct fluorescent-antibody (DFA) staining and rapid shell vial culture (R-mix cells; Diagnostic Hybrids, Inc. Athens, OH) with 354 respiratory specimens from adult patients that were submitted to the clinical virology laboratory at the Emory University Hospital. Single-target PCRs were performed with retained samples to confirm the positive results obtained with the MultiCode-PLx system for viruses not covered by DFA and R-mix culture (metapneumovirus, coronaviruses CoV, parainfluenza viruses 4a and 4b, and rhinoviruses) and to resolve any discrepancies between the DFA and R-mix culture and the MultiCode-PLx results for viruses common to both systems. Respiratory viruses were detected in 77 (21.8%) and 116 (32.7%) specimens by DFA and R-mix culture and with the MultiCode-PLx system, respectively. Among the viruses common to both systems, the MultiCode-PLx system detected significantly more influenza A viruses (P = 0.0026). An additional increased diagnostic yield with the MultiCode-PLx system resulted from the detection of human metapneumovirus (HMPV) in 9 specimens, human CoV (HCoV) in 3 specimens, and human rhinovirus (HRV) in 16 specimens. Also, two mixed viral infections were detected by the MultiCode-PLx system (HCoV OC43 and HRV infections and HMPV and HRV infections), but none were detected by DFA and R-mix culture. Single-target PCRs verified the results obtained with the MultiCode-PLx system for 73 of 81 (90.1%) specimens that had discordant results or that were not covered by DFA and R-mix culture. The MultiCode-PLx system provides clinical laboratories with a practical, rapid, and sensitive means for the massively multiplexed molecular detection of common respiratory viruses.
The human Na+/K+-ATPase (NKA) is a plasma membrane ion pump that uses ATP to help maintain the resting potential of all human cells. Inhibition of the NKA leads to cell swelling and death. The ...results of this investigation show that on cancer cells, the NKA either comes in close proximity to, associate with or complexes to important cancer-related proteins, and thus can be targeted with a new type of precision therapy called the extracellular drug conjugate or EDC. The EDCs reported here exhibit EC50 values in the low to mid-picomolar range, and signal to noise ratios > 1,000:1, both of which are dependent on the cell surface expression of the NKA and corresponding cancer-related target. We demonstrate that a potent small molecule inhibitor of the NKA can be covalently attached to antibodies targeting CD20, CD38, CD56, CD147, or dysadherin, to create a series of selective and powerful EDCs that kill cancer cells extracellularly by a mechanism resembling necrosis. This is therefore a framework for the development of a new type of precision therapy wherein exquisite selectivity is achieved for targeting extracellular disease-related proteins.
Abstract
Purpose:
CD20 is known to be a therapeutic antibody drug target as it is expressed on the surface of most B-cell neoplasms. Here, we assessed the anti-tumor activity of EDC9, a novel ...extracellular drug conjugate composed of Rituximab (a well know and FDA approved anti-CD20 monoclonal antibody) conjugated to a steroidal glycoside via a long, flexible and stable linker.
Experimental Design:
The anti-cancer activity and safety of EDC9 were examined and compared to Rituximab using in vitro and in vivo techniques.
Results:
We found that EDC9 showed picomolar cytotoxic activity that was independent of effector functions and exhibited cytotoxic activity through a mechanism that was dependent on CD20 expression, as cells not expressing CD20 were resistant and Rituximab alone could compete with EDC9, rendering it inactive. After 72 hours of EDC9 treatment at levels between 100 and 200 picomolar, no cells were determined to be viable by CellTiter-Glo or high definition phase contrast imaging. Importantly, when compared on the basis of toxicity to PBMC, EDC9 was found not to be more toxic than Rituximab and the activity of EDC9 was dependent on specific steroid chemistry. In a tumor xenograft model, EDC9 provided complete long-term remission of diffuse large B-cell human lymphoma line SU-DHL-8 tumors, while Rituximab alone did not.
Conclusion:
These results support efforts to further evaluate EDC9 (a novel CD20 specific antibody drug conjugate) and may lead to phase I clinical trials in patients with certain B-cell related malignancies.
Citation Format: James R. Prudent, Chad A. Hall, David J. Marshall, John Murphy, Scott Harried. An anti-CD20 extracellular antibody-drug conjugate for the treatment of B-cell malignancies. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2964.
Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream ...oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary Objectives Despite advances in treatment modalities, head and neck squamous cell carcinoma (HNSCC) remains a challenge to treat with poor survival and high morbidity, necessitating a therapy ...with greater efficacy. EDC22 is an extracellular drug conjugate of the monoclonal antibody targeting CD147 (glycoprotein highly expressed on HNSCC cells) linked with a small drug molecule inhibitor of Na, K-ATPase. In this study, EDC22’s potential as a treatment modality for HNSCC was performed. Materials and methods HNSCC cell lines (FADU, OSC-19, Cal27, SCC-1) were cultured in vitro and proliferation and cell viability were assessed following treatment with a range of concentrations of EDC22 (0.25–5.00 μg/mL). Mice bearing HNSCC xenografts (OSC-19, SCC-1) were treated with either EDC22 (3–10 mg/kg), anti-CD147 monoclonal antibody, cisplatin (1 mg/kg) or radiation therapy (2 Gy/week) monotherapy or in combination. Results In vitro, treatment with minimal concentration of EDC22 (0.25 μg/mL) significantly decreased cellular proliferation and cell viability ( p < 0.0001). In vivo, systemic treatment with EDC22 significantly decreased primary tumor growth rate in both an orthotopic mouse model (OSC-19) and a flank tumor mouse model (SCC-1) ( p < 0.05). In addition, EDC22 therapy resulted in a greater reduction in tumor growth in vivo compared to radiation monotherapy ( p < 0.05) and a similar reduction in tumor growth compared to cisplatin monotherapy. Combination therapy provided no significant further reduction in tumor growth relative to EDC22 monotherapy. Conclusion EDC22 is a potent inhibitor of HNSCC cell proliferation in vitro and in vivo, warranting further investigations of its clinical potential in the treatment of HNSCC.
EDC1 is a novel type of antibody-drug conjugate which binds and inhibits the Na,K-ATPase on the surface of cancer cells expressing dysadherin. The purpose of this study was to determine the ...expression of dysadherin in different types of thyroid carcinoma, and evaluate the therapeutic potential of EDC1 for thyroid carcinomas.
Thyroid tissues from 158 patients were examined for dysadherin expression and correlation with clinicopathological features. Thyroid cancer cell lines were examined for the expression of dysadherin and effective dose range of EDC1.
One in 53 benign thyroid tissues and 62% of thyroid cancers expressed dysadherin. All anaplastic and a majority of papillary thyroid cancers overexpressed dysadherin, while 25% of follicular thyroid cancers was found to be positive for dysadherin. Dysadherin expression significantly correlated with extrathyroidal extension and lymph node metastases in papillary thyroid cancer. Five of six human thyroid cancer cell lines analyzed expressed high levels of dysadherin. Of those cells lines sensitive to EDC1, half maximal effective concentrations (EC50) were observed to be between 0.125 nM and 1 nM.
EDC1 showed selective inhibition of growth in thyroid cancer cells with moderate to high expression of dysadherin, thus could be a specific and effective treatment.
Abstract
Purpose: We have previously shown that targeting the Na,K-ATPase (NKA) with antibody drug conjugates is a powerful method for reducing tumors at levels found to be safe in cynomolgus ...monkeys. Currently no approved cancer drugs induce a potent necrotic type cell death even though experimental evidence shows that necrotic cell death has many notable and untapped benefits. Therefore, we wanted to better understand the driving mechanism by which ADCs targeting NKA inhibition induce necrosis.
Experimental Design: Various cancer cell types were subjected to increasing and decreasing concentrations of Na+, K+ and Ca++, as well as various ADCs using bufalin as the warhead/payload/toxin. The cells were also subjected to siRNAs specific to the NKA and bufalin alone. The cells were then analyzed for the appearance and/or presence of necrosis, apoptosis, and markers indicating various stress pathways.
Results: Conditions found to induce necrosis included: ADCs targeting specific NKA-protein complexes, cells subjected to media containing increased concentrations of K+, the specific Na,K-ATPase siRNAs, and bufalin alone.
Conditions not found to induce necrosis included: media containing increased Na+, decreased Na+ or K+, control siRNAs, or ADCs that targeted proteins not complexed to the NKA or expressed on the cell surface.
All cells undergoing necrosis lost their actin filaments, rounded up, and adherent cells detached from the surface and lost their motility. Markers indicated initial activation of the integrated stress responses (ISR). Minimal to no annexin staining indicated that apoptosis was not the dominate pathway in any experiment.
Conclusion: Proper ion flux is important for maintaining cellular homeostasis. Cell swelling is a key process that is used to correct for changes in ion flux, induce inflammation and kill unwanted cells. The NKA is a key regulatory protein in this process and when blocked, leads to uncontrolled cells swelling and necrotic death. Precise inhibition of ion flux focused on cancer cells should be further investigated as a potential method for treating patients.
Citation Format: James R. Prudent, David Marshall. ADCs that induce necrosis and thwart apoptotic resistance abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2328.
Organic chemistry has made possible the synthesis of molecules that expand on Nature's genetic alphabet. Using the previously described nonstandard DNA base pair constructed from isoguanine and ...5-methylisocytosine, we report a highly specific and sensitive method that allows for the fast and specific quantitation of genetic sequences in a closed tube format. During PCR amplification, enzymatic site-specific incorporation of a quencher covalently linked to isoguanine allows for the simultaneous detection and identification of multiple targets. The specificity of method is then established by analysis of thermal denaturation or melting of the amplicons. The appropriate functions of all reactions are further verified by incorporation of an independent target into the reaction mixture. We report that the method is sensitive down to the single copy level, and specificity is demonstrated by multiplexed end-point genotypic analysis of four targets simultaneously using four separate fluorescent reporters. The method is general enough for quantitative and qualitative analysis of both RNA and DNA using previously developed primer sets. Though the method described employs the commonly used PCR, the enzymatic incorporation of reporter groups into DNA site-specifically should find broad utility throughout molecular biology.
PDF
