Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR

Background: Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. However, the errors implicit in HPV gene quantification by PCR are not well documented. We have undertaken the first rigorous evaluation of the errors that can be expected when using SYBR green qPCR for quantification of HPV type 16 gene copy numbers. We assessed a modified method, in which external calibration curves were generated from a single construct containing HPV16 E2, HPV16 E6 and the host gene hydroxymethylbilane synthase in a 1: 1: 1 ratio.Results: When testing dilutions of mixed HPV/host DNA in replicate runs, we observed errors in quantifying E2 and E6 amplicons of 5 - 40%, with greatest error at the lowest DNA template concentration (3 ng/mu l). Errors in determining viral copy numbers per diploid genome were 13 - 53%. Nevertheless, in cervical keratinocyte cell lines we observed reasonable agreement between viral loads determined by qPCR and Southern blotting. The mean E2/E6 ratio in episome-only cells was 1.04, but with a range of 0.76 - 1.32. In three integrant-only lines the mean E2/E6 ratios were 0.20, 0.72 and 2.61 (values confirmed by gene-specific Southern blotting). When E2/E6 ratios in fourteen HPV16-positive cervical carcinomas were analysed, conclusions regarding viral physical state could only be made in three cases, where the E2/E6 ratio was <= 0.06.Conclusion: Run-to-run variation in SYBR green qPCR produces unavoidable inaccuracies that should be allowed for when quantifying HPV gene copy number. While E6 copy numbers can be considered to provide a useable indication of viral loads, the E2/E6 ratio is of limited value. Previous studies may have overestimated the frequency of mixed episomal/integrant HPV infections

Characterization of 7A7, an anti-mouse EGFR monoclonal antibody proposed to be the mouse equivalent of cetuximab.

The Epidermal Growth Factor Receptor (EGFR) is selectively expressed on the surface of numerous tumours, such as non-small cell lung, ovarian, colorectal and head and neck carcinomas. EGFR has therefore become a target for cancer therapy. Cetuximab is a chimeric human/mouse monoclonal antibody (mAb) that binds to EGFR, where it both inhibits signaling and induces cell death by antibody-dependent cell mediated cytotoxicity (ADCC). Cetuximab has been approved for clinical use in patients with head and neck squamous cell carcinoma (HNSCC) and colorectal cancer. However, only 15-20% patients benefit from this drug, thus new strategies to improve cetuximab efficiency are required. We aimed to develop a reliable and easy preclinical mouse model to evaluate the efficacy of EGFR-targeted antibodies and examine the immune mechanisms involved in tumour regression. We selected an anti-mouse EGFR mAb, 7A7, which has been reported to be "mouse cetuximab" and to exhibit similar properties to its human counterpart. Unfortunately, we were unable to reproduce previous results obtained with the 7A7 mAb. In our hands, 7A7 failed to recognize mouse EGFR, both in native and reducing conditions. Moreover, in vivo administration of 7A7 in an EGFR-expressing HPV38 tumour model did not have any impact on tumour regression or animal survival. We conclude that 7A7 does not recognize mouse EGFR and therefore cannot be used as the mouse equivalent of cetuximab use in humans. As a number of groups have spent effort and resources with similar issues we feel that publication is a responsible approach

Establishing the kinetics of Escherichia bacteriophage T4 and its target bacterium within the intestinal mucosa of a gnotobiotic mouse model

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. The specificity of phage-bacterial interactions has generated renewed interest in phage research as a potential alternative strategy to counter the increasing threat of antimicrobial resistant bacteria. While there has been some success in using phage therapy to combat bacterial septic infections, we still do not have the foundational understanding of phage-bacteria-host dynamics within our gut ecosystems that is needed for their safe development. Recent studies demonstrating that phages adhere to intestinal mucus through specific capsid proteins (Hoc) have suggested that phages may protect the underlying epithelium from bacterial invasion, providing a host-extrinsic mechanism to maintain intestinal homeostasis. Here, I build upon these findings to investigate the kinetics between Escherichia bacteriophage T4 (containing a Hoc domain) and its target bacterium, Escherichia coli, within the intestinal tract of a gnotobiotic mouse model. I determined that T4 phage and E. coli can stably coexist within the murine gastrointestinal tract in the absence of other microbes, despite continual phage predation. However, I was unable to conclude that T4 phage retention within the murine gut requires Hoc protein-mediated mucus adhesion. Further, my data suggest that gut-colonising T4 phage elicit a type 1 immune response in the gut-draining lymph nodes, without causing inflammatory disease. Together, these results suggest that T4 phage is well tolerated in the gastrointestinal tract of gnotobiotic mice by the bacterial and metazoan hosts and may contribute to immune system priming.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Gut commensal Enterocloster species host inoviruses that are secreted in vitro and in vivo

Abstract Background Bacteriophages in the family Inoviridae, or inoviruses, are under-characterized phages previously implicated in bacterial pathogenesis by contributing to biofilm formation, immune evasion, and toxin secretion. Unlike most bacteriophages, inoviruses do not lyse their host cells to release new progeny virions; rather, they encode a secretion system that actively pumps them out of the bacterial cell. To date, no inovirus associated with the human gut microbiome has been isolated or characterized. Results In this study, we utilized in silico, in vitro, and in vivo methods to detect inoviruses in bacterial members of the gut microbiota. By screening a representative genome library of gut commensals, we detected inovirus prophages in Enterocloster spp. (formerly Clostridium spp.). We confirmed the secretion of inovirus particles in in vitro cultures of these organisms using imaging and qPCR. To assess how the gut abiotic environment, bacterial physiology, and inovirus secretion may be linked, we deployed a tripartite in vitro assay that progressively evaluated bacterial growth dynamics, biofilm formation, and inovirus secretion in the presence of changing osmotic environments. Counter to other inovirus-producing bacteria, inovirus production was not correlated with biofilm formation in Enterocloster spp. Instead, the Enterocloster strains had heterogeneous responses to changing osmolality levels relevant to gut physiology. Notably, increasing osmolality induced inovirus secretion in a strain-dependent manner. We confirmed inovirus secretion in a gnotobiotic mouse model inoculated with individual Enterocloster strains in vivo in unperturbed conditions. Furthermore, consistent with our in vitro observations, inovirus secretion was regulated by a changed osmotic environment in the gut due to osmotic laxatives. Conclusion In this study, we report on the detection and characterization of novel inoviruses from gut commensals in the Enterocloster genus. Together, our results demonstrate that human gut-associated bacteria can secrete inoviruses and begin to elucidate the environmental niche filled by inoviruses in commensal bacteria. Video Abstrac

Characterization of 7A7, an anti-mouse EGFR monoclonal antibody proposed to be the mouse equivalent of cetuximab.

The Epidermal Growth Factor Receptor (EGFR) is selectively expressed on the surface of numerous tumours, such as non-small cell lung, ovarian, colorectal and head and neck carcinomas. EGFR has therefore become a target for cancer therapy. Cetuximab is a chimeric human/mouse monoclonal antibody (mAb) that binds to EGFR, where it both inhibits signaling and induces cell death by antibody-dependent cell mediated cytotoxicity (ADCC). Cetuximab has been approved for clinical use in patients with head and neck squamous cell carcinoma (HNSCC) and colorectal cancer. However, only 15-20% patients benefit from this drug, thus new strategies to improve cetuximab efficiency are required. We aimed to develop a reliable and easy preclinical mouse model to evaluate the efficacy of EGFR-targeted antibodies and examine the immune mechanisms involved in tumour regression. We selected an anti-mouse EGFR mAb, 7A7, which has been reported to be "mouse cetuximab" and to exhibit similar properties to its human counterpart. Unfortunately, we were unable to reproduce previous results obtained with the 7A7 mAb. In our hands, 7A7 failed to recognize mouse EGFR, both in native and reducing conditions. Moreover, in vivo administration of 7A7 in an EGFR-expressing HPV38 tumour model did not have any impact on tumour regression or animal survival. We conclude that 7A7 does not recognize mouse EGFR and therefore cannot be used as the mouse equivalent of cetuximab use in humans. As a number of groups have spent effort and resources with similar issues we feel that publication is a responsible approach

Bacillus anthracis Protective Antigen Shows High Specificity for a UV Induced Mouse Model of Cutaneous Squamous Cell Carcinoma

Squamous cell carcinoma (SCC) accounts for the majority of non-melanoma skin cancer related deaths, particularly in immunosuppressed persons. Identification of biomarkers that could be used to identify or treat SCC would be of significant benefit. The anthrax toxin receptors, Tumor Endothelial Marker 8 (TEM8) and Capillary Morphogenesis Gene 2 (CMG2), are endothelial receptors involved in extracellular matrix homeostasis and angiogenesis that are selectively upregulated on numerous tumors. One method of targeting these receptors is Protective Antigen (PA), a protein produced by B. anthracis that mediates binding and translocation of anthrax toxins into cells. PA targeted toxins have been demonstrated to selectively inhibit tumor growth and angiogenesis, but tumor selectivity of PA is currently unknown. In this work fluorescently labeled PA was shown to maintain receptor dependent binding and internalization in vitro. Utilizing a human papillomavirus transgenic mouse model that develops cutaneous SCC in response to ultraviolet irradiation we identified tumor uptake of PA in vivo. The intravenously administered PA resulted in tumor specific localization, with exclusive tumor detection 24 h post injection. Ex vivo analysis identified significantly higher fluorescence in the tumor compared to adjacent healthy tissue and major clearance organs, demonstrating low non-specific uptake and rapid clearance. While both TEM8 and CMG2 were observed to be overexpressed in SCC tumor sections compared to control skin, the intravenously administered PA was primarily co-localized with TEM8. These results suggest that PA could be systemically administered for rapid identification of cutaneous SCC, with potential for further therapeutic development

Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR-2

Serially diluted NA6 as template (2 ng – 2 fg), from which the mean calibration curve for viral quantification was generated. The different curves correspond to different starting amounts of template. The fluorescence thresholds are indicated by the lines of crosses running horizontally. Panel A shows E2 log transformed data from replicate 1 qPCR run, panel B shows HMBS log transformed data from replicate 3 qPCR run, panel C shows E6 log transformed data from replicate 2 qPCR run. Panel D demonstrates the tight correlation between the data points generated for the E2 amplicon at each template concentration in the four replicate runs. Similar findings were made for the E6 and HMBS amplicons. Panel E shows the mean calibration curves for E2, E6 and HMBS used for viral gene copy number quantification, together with the respective line equations.<p><b>Copyright information:</b></p><p>Taken from "Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR"</p><p>http://www.biomedcentral.com/1472-6750/8/57</p><p>BMC Biotechnology 2008;8():57-57.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2529285.</p><p></p

Exercise and Prebiotic Fiber Provide Gut Microbiota-Driven Benefit in a Survivor to Germ-Free Mouse Translational Model of Breast Cancer

The gut microbiota plays a role in shaping overall host health and response to several cancer treatments. Factors, such as diet, exercise, and chemotherapy, can alter the gut microbiota. In the present study, the Alberta Cancer Exercise (ACE) program was investigated as a strategy to favorably modify the gut microbiota of breast cancer survivors who had received chemotherapy. Subsequently, the ability of post-exercise gut microbiota, alone or with prebiotic fiber supplementation, to influence breast cancer outcomes was interrogated using fecal microbiota transplant (FMT) in germ-free mice. While cancer survivors experienced little gut microbial change following ACE, in the mice, tumor volume trended consistently lower over time in mice colonized with post-exercise compared to pre-exercise microbiota with significant differences on days 16 and 22. Beta diversity analysis revealed that EO771 breast tumor cell injection and Paclitaxel chemotherapy altered the gut microbial communities in mice. Enrichment of potentially protective microbes was found in post-exercise microbiota groups. Tumors of mice colonized with post-exercise microbiota exhibited more favorable cytokine profiles, including decreased vascular endothelial growth factor (VEGF) levels. Beneficial microbial and molecular outcomes were augmented with prebiotic supplementation. Exercise and prebiotic fiber demonstrated adjuvant action, potentially via an enhanced anti-tumor immune response modulated by advantageous gut microbial shifts

Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR-0

Serially diluted NA6 as template (2 ng – 2 fg), from which the mean calibration curve for viral quantification was generated. The different curves correspond to different starting amounts of template. The fluorescence thresholds are indicated by the lines of crosses running horizontally. Panel A shows E2 log transformed data from replicate 1 qPCR run, panel B shows HMBS log transformed data from replicate 3 qPCR run, panel C shows E6 log transformed data from replicate 2 qPCR run. Panel D demonstrates the tight correlation between the data points generated for the E2 amplicon at each template concentration in the four replicate runs. Similar findings were made for the E6 and HMBS amplicons. Panel E shows the mean calibration curves for E2, E6 and HMBS used for viral gene copy number quantification, together with the respective line equations.<p><b>Copyright information:</b></p><p>Taken from "Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR"</p><p>http://www.biomedcentral.com/1472-6750/8/57</p><p>BMC Biotechnology 2008;8():57-57.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2529285.</p><p></p