PI3K/mTOR inhibitor PF-04691502 antitumor activity is enhanced with induction of wild-type TP53 in human xenograft and murine knockout models of head and neck cancer.

PURPOSE: Phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway activation is often associated with altered expression or mutations of PIK3CA, TP53/p73, PTEN, and TGF-ß receptors (TGFBR) in head and neck squamous cell carcinomas (HNSCC). However, little is known about how these alterations affect response to PI3K/mTOR-targeted agents. EXPERIMENTAL DESIGN: In this preclinical study, PI3K/Akt/mTOR signaling was characterized in nine HNSCC (UM-SCC) cell lines and human oral keratinocytes. We investigated the molecular and anticancer effects of dual PI3K/mTOR inhibitor PF-04691502(PF-502) in UM-SCC expressing PIK3CA with decreased wild-type TP53, mutant TP53-/+ mutantTGFBR2, and in HNSCC of a conditional Pten/Tgfbr1 double knockout mouse model displaying PI3K/Akt/mTOR activation. RESULTS: UM-SCC showed increased PIK3CA expression and Akt/mTOR activation, and PF-502 inhibited PI3K/mTORC1/2 targets. In human HNSCC expressing PIK3CA and decreased wtTP53 and p73, PF-502 reciprocally enhanced TP53/p73 expression and growth inhibition, which was partially reversible by p53 inhibitor pifithrin-α. Most UM-SCC with wtTP53 exhibited a lower IC50 than those with mtTP53 status. PF-502 blocked growth in G0-G1 and increased apoptotic sub-G0 DNA. PF-502 suppressed tumorigenesis and showed combinatorial activity with radiation in a wild-type TP53 UM-SCC xenograft model. PF-502 also significantly delayed HNSCC tumorigenesis and prolonged survival of Pten/Tgfbr1-deficient mice. Significant inhibition of p-Akt, p-4EBP1, p-S6, and Ki67, as well as increased p53 and TUNEL were observed in tumor specimens. CONCLUSIONS: PI3K-mTOR inhibition can enhance TP53/p73 expression and significantly inhibit tumor growth alone or when combined with radiation in HNSCC with wild-type TP53. PIK3CA, TP53/p73, PTEN, and TGF-ß alterations are potential modifiers of response and merit investigation in future clinical trials with PI3K-mTOR inhibitors.

Evaluation of sample recovery efficiency for bacteriophage P22 on fomites.

Fomites are known to play a role in the transmission of pathogens. Quantitative analysis of the parameters that affect sample recovery efficiency (SRE) at the limit of detection of viruses on fomites will aid in improving quantitative microbial risk assessment (QMRA) and infection control. The variability in SRE as a function of fomite type, fomite surface area, sampling time, application media, relative humidity (rH), and wetting agent was evaluated. To quantify the SRE, bacteriophage P22 was applied onto fomites at average surface densities of 0.4 ± 0.2 and 4 ± 2 PFU/cm(2). Surface areas of 100 and 1,000 cm(2) of nonporous fomites found in indoor environments (acrylic, galvanized steel, and laminate) were evaluated with premoistened antistatic wipes. The parameters with the most effects on the SRE were sampling time, fomite surface area, wetting agent, and rH. At time zero (the initial application of bacteriophage P22), the SRE for the 1,000-cm(2) fomite surface area was, on average, 40% lower than that for the 100-cm(2) fomite surface area. For both fomite surface areas, the application medium Trypticase soy broth (TSB) and/or the laminate fomite predominantly resulted in a higher SRE. After the applied samples dried on the fomites (20 min), the average SRE was less than 3%. A TSB wetting agent applied on the fomite improved the SRE for all samples at 20 min. In addition, an rH greater than 28% generally resulted in a higher SRE than an rH less than 28%. The parameters impacting SRE at the limit of detection have the potential to enhance sampling strategies and data collection for QMRA models.

Implications of limits of detection of various methods for Bacillus anthracis in computing risks to human health.

Used for decades for biological warfare, Bacillus anthracis (category A agent) has proven to be highly stable and lethal. Quantitative risk assessment modeling requires descriptive statistics of the limit of detection to assist in defining the exposure. Furthermore, the sensitivities of various detection methods in environmental matrices are vital information for first responders. A literature review of peer-reviewed journal articles related to methods for detection of B. anthracis was undertaken. Articles focused on the development or evaluation of various detection approaches, such as PCR, real-time PCR, immunoassay, etc. Real-time PCR and PCR were the most sensitive methods for the detection of B. anthracis, with median instrument limits of detection of 430 and 440 cells/ml, respectively. There were very few peer-reviewed articles on the detection methods for B. anthracis in the environment. The most sensitive limits of detection for the environmental samples were 0.1 CFU/g for soil using PCR-enzyme-linked immunosorbent assay (ELISA), 17 CFU/liter for air using an ELISA-biochip system, 1 CFU/liter for water using cultivation, and 1 CFU/cm(2) for stainless steel fomites using cultivation. An exponential dose-response model for the inhalation of B. anthracis estimates of risk at concentrations equal to the environmental limit of detection determined the probability of death if untreated to be as high as 0.520. Though more data on the environmental limit of detection would improve the assumptions made for the risk assessment, this study's quantification of the risk posed by current limitations in the knowledge of detection methods should be considered when employing those methods in environmental monitoring and cleanup strategies.

In situ-synthesized virulence and marker gene biochip for detection of bacterial pathogens in water.

Pathogen detection tools with high reliability are needed for various applications, including food and water safety and clinical diagnostics. In this study, we designed and validated an in situ-synthesized biochip for detection of 12 microbial pathogens, including a suite of pathogens relevant to water safety. To enhance the reliability of presence/absence calls, probes were designed for multiple virulence and marker genes (VMGs) of each pathogen, and each VMG was targeted by an average of 17 probes. Hybridization of the biochip with amplicon mixtures demonstrated that 95% of the initially designed probes behaved as predicted in terms of positive/negative signals. The probes were further validated using DNA obtained from three different types of water samples and spiked with pathogen genomic DNA at decreasing relative abundance. Excellent specificity for making presence/absence calls was observed by using a cutoff of 0.5 for the positive fraction (i.e., the fraction of probes yielding a positive signal for a given VMG). A split multiplex PCR design for simultaneous amplification of the VMGs resulted in a detection limit of between 0.1 and 0.01% relative abundance, depending on the type of pathogen and the VMG. Thermodynamic analysis of the hybridization patterns obtained with DNA from the different water samples demonstrated that probes with a hybridization Gibbs free energy of approximately -19.3 kcal/mol provided the best trade-off between sensitivity and specificity. The developed biochip may be used to detect the described bacterial pathogens in water samples when parallel and specific detection is required.

Complications of tonsillectomy: a comparison of techniques.

OBJECTIVE: To compare the postoperative complications of intracapsular tonsillectomy using a microdebrider with traditional electrodissection tonsillectomy. DESIGN: Retrospective chart review. SETTING: Tertiary care pediatric referral center. PATIENTS: The medical records of 2944 patients undergoing tonsillectomy with or without adenoidectomy at our institution between January 1, 2002, and May 31, 2005, were reviewed. MAIN OUTCOME MEASURES: Incidence of delayed postoperative hemorrhage, return to the hospital or emergency department for pain or dehydration, and the need for revision surgery. RESULTS: There were 1731 patients in the intracapsular tonsillectomy group and 1212 in the traditional electrodissection tonsillectomy group. The incidence of delayed hemorrhage was 1.1% in the intracapsular tonsillectomy group and 3.4% in the traditional electrodissection tonsillectomy group (P < .001). For delayed hemorrhage requiring treatment in the operating room for control, the incidence was 0.5% in the intracapsular tonsillectomy group and 2.1% in the traditional electrodissection tonsillectomy group (P < .001). Treatment in the emergency department or hospital for pain or dehydration was necessary in 3.0% of the intracapsular tonsillectomy group and in 5.4% of the traditional electrodissection tonsillectomy group (P = .002). Eleven patients (0.64%) in the intracapsular tonsillectomy group required revision tonsillectomy. CONCLUSION: Intracapsular tonsillectomy has a lower incidence of postoperative hemorrhage and pain leading to hospital-based evaluation compared with traditional electrodissection tonsillectomy.

Powered intracapsular tonsillectomy in the management of recurrent tonsillitis.

OBJECTIVE: To compare intracapsular tonsillectomy (IT) and traditional tonsillectomy (TT) in treating recurrent adenotonsillitis or streptococcal pharyngitis. DESIGN: Retrospective chart review. SETTING: Tertiary care pediatric referral center. RESULTS: Of 166 patients who met all inclusion criteria, 117 received TT and 49 received IT. Seventeen TT patients and 8 IT patients were treated at least once postoperatively for streptococcal pharyngitis or tonsillitis. The mean number of infections after surgery in each group did not reach statistical significance (P = 0.295). CONCLUSION: There was no difference between the IT and TT groups in postoperative infection rates.

Influence of dangling ends and surface-proximal tails of targets on probe-target duplex formation in 16S rRNA gene-based diagnostic arrays.

Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of -7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of -64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.