Citations

Glottic phonatory parameters have been studied in constant flow models; however, the lung‐thorax system is better viewed as a constant pressure source. Adjusting the driving pressure and recurrent laryngeal nerve stimulation as independent variables, rather than as dependent variables, may provide a more physiologic understanding of laryngeal function and glottic parameters, including subglottic pressure, airflow, fundamental frequency, and glottic area. In three dogs subglottic pressure and airflow were measured in two separate conditions: with constant recurrent laryngeal nerve stimulation and varying driving pressure, and with constant driving pressure and varying recurrent laryngeal nerve stimulation. Videostroboscopic measures on four dogs assessed glottic areas with constant recurrent laryngeal nerve stimulation at different driving pressures. With constant recurrent laryngeal nerve stimulation, increasing driving pressure had no effect on glottic areas, whereas subglottic pressure, fundamental frequency, and airflow increased significantly. However, changes in subglottic pressure were minimal in comparison with changes in driving pressure. At constant driving pressure, increasing recurrent laryngeal nerve stimulation increased subglottic pressure and fundamental frequency and decreased airflow. These findings suggest that during phonation subglottic pressure is primarily dependent on recurrent laryngeal nerve stimulation and laryngeal muscular contraction, but not on lung driving pressure.

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3′ end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9–exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10 – tau RNA into exon 10 + tau RNA could be achieved with ≈34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.

Abstract Purpose: Advanced-stage epithelial ovarian cancer has a poor prognosis with long-term survival in less than 30% of patients. When the disease is detected in stage I, more than 90% of patients can be cured by conventional therapy. Screening for early-stage disease with individual serum tumor markers, such as CA125, is limited by the fact that no single marker is up-regulated and shed in adequate amounts by all ovarian cancers. Consequently, use of multiple markers in combination might detect a larger fraction of early-stage ovarian cancers. Experimental Design: To identify potential candidates for novel markers, we have used Affymetrix human genome arrays (U95 series) to analyze differences in gene expression of 41,441 known genes and expressed sequence tags between five pools of normal ovarian surface epithelial cells (OSE) and 42 epithelial ovarian cancers of different stages, grades, and histotypes. Recursive descent partition analysis (RDPA) was performed with 102 probe sets representing 86 genes that were up-regulated at least 3-fold in epithelial ovarian cancers when compared with normal OSE. In addition, a panel of 11 genes known to encode potential tumor markers [mucin 1, transmembrane (MUC1), mucin 16 (CA125), mesothelin, WAP four-disulfide core domain 2 (HE4), kallikrein 6, kallikrein 10, matrix metalloproteinase 2, prostasin, osteopontin, tetranectin, and inhibin] were similarly analyzed. Results: The 3-fold up-regulated genes were examined and four genes [Notch homologue 3 (NOTCH3), E2F transcription factor 3 (E2F3), GTPase activating protein (RACGAP1), and hematological and neurological expressed 1 (HN1)] distinguished all tumor samples from normal OSE. The 3-fold up-regulated genes were analyzed using RDPA, and the combination of elevated claudin 3 (CLDN3) and elevated vascular endothelial growth factor (VEGF) distinguished the cancers from normal OSE. The 11 known markers were analyzed using RDPA, and a combination of HE4, CA125, and MUC1 expression could distinguish tumor from normal specimens. Expression at the mRNA level in the candidate markers was examined via semiquantitative reverse transcription-PCR and was found to correlate well with the array data. Immunohistochemistry was performed to identify expression of the genes at the protein level in 158 ovarian cancers of different histotypes. A combination of CLDN3, CA125, and MUC1 stained 157 (99.4%) of 158 cancers, and all of the tumors were detected with a combination of CLDN3, CA125, MUC1, and VEGF. Conclusions: Our data are consistent with the possibility that a limited number of markers in combination might identify >99% of epithelial ovarian cancers despite the heterogeneity of the disease.

Abstract Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treatment of solid tumors on the basis of its unique mechanism of action and selectivity for human solid tumor xenograft in mice. Using capillary electrophoresis coupled with electrospray ionization mass spectrometry, we have identified three oxidative PZA metabolites, 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. The cytochrome P450 (CYP) isoforms involved in PZA metabolism were characterized by studies with CYP chemical inhibitors, correlation of marker activities for selected CYPs with formation of the metabolites using a human liver panel, and PZA metabolism by cDNA-expressed CYPs. 9-Desmethyl-PZA formation was catalyzed by CYP1A2, whereas N-demethyl-PZA formation was catalyzed by CYP3A4. PZA N-oxide formation was catalyzed by flavin monooxygenase (FMO) rather than CYP, as determined by studies with chemical inhibitors of FMO and metabolism by cDNA-expressed human flavin monooxygenase. After administration of [10b-14C]PZA to mice, six urinary metabolites were detected by high-performance liquid chromatography UV and radiochromatograms including 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. Trace concentrations of 9-desmethyl-PZA and PZA N-oxide were detected in mouse plasma. PZA N-oxide and N-demethyl-PZA were detected in urine from patients after PZA administration. PZA, 9-desmethyl-PZA, and PZA N-oxide inhibited growth of A375 human melanoma cells. IC50 values were 0.17, 0.11, and 7.0 μm, respectively, for the three molecules.

Abstract Purpose: Most colorectal cancers that have high levels of microsatellite instability (MSI-H) show loss of immunohistochemical expression of proteins that participate in the DNA mismatch repair process, most often involving MLH1 and MSH2. Less commonly, a third DNA mismatch repair protein, MSH6, may also be lost as the primary event. Rarely, tumors with MSI-H show normal expression of these three proteins. The genetic deficiency leading to the MSI-H phenotype in such cases is unknown. PMS2 is another member of the DNA mismatch repair complex. Its expression is generally lost in tumors with MLH1 loss of expression. Rarely, there is selective loss of PMS2 expression. We sought to describe the frequency and clinical correlates of selective loss of expression of PMS2 with the MSI-H tumor phenotype. Experimental Design: Two thousand seven hundred nineteen colorectal cancers from both clinic- and research-based ascertainment were studied. Tumor MSI testing and immunohistochemistry for MLH1, MSH2, MSH6, and PMS2 were conducted. Medical records were abstracted for age at diagnosis, gender, colorectal cancer site, and family history. Results: Five hundred thirty-five of the 2,719 tumors were MSI-H. Of these, 93% showed loss of expression of MLH1, MSH2, and/or MSH6. Thirty-eight showed normal expression for these proteins. PMS2 immunohistochemical staining was successful in 32 of 38 of these tumors. Of the 32, 23 showed selective loss of expression of PMS2. This was associated with young age of diagnosis and right-sided location but not with a striking family history of cancer. Conclusions: Overall, 97% of the MSI-H tumors showed loss of expression for one or more of these four mismatch repair proteins. Selective loss of expression of PMS2 was present in 72% of cases in which colorectal cancers had an MSI-H phenotype but no alteration of expression of MLH1, MSH2, and MSH6. The underlying mechanism involved cannot be determined from this study but could involve point mutations in other DNA mismatch repair genes with retention of immunohistochemical expression, somatic inactivation of PMS2, or germ line mutation of PMS2.

Abstract Purpose: Epithelial ovarian cancers are thought to arise from flattened epithelial cells that cover the ovarian surface or that line inclusion cysts. During malignant transformation, different histotypes arise that resemble epithelial cells from normal fallopian tube, endometrium, and intestine. This study compares gene expression in serous, endometrioid, clear cell, and mucinous ovarian cancers with that in the normal tissues that they resemble. Experimental Design: Expression of 63,000 probe sets was measured in 50 ovarian cancers, in 5 pools of normal ovarian epithelial brushings, and in mucosal scrapings from 4 normal fallopian tube, 5 endometrium, and 4 colon specimens. Using rank-sum analysis, genes whose expressions best differentiated the ovarian cancer histotypes and normal ovarian epithelium were used to determine whether a correlation based on gene expression existed between ovarian cancer histotypes and the normal tissues they resemble. Results: When compared with normal ovarian epithelial brushings, alterations in serous tumors correlated with those in normal fallopian tube (P = 0.0042) but not in other normal tissues. Similarly, mucinous cancers correlated with those in normal colonic mucosa (P = 0.0003), and both endometrioid and clear cell histotypes correlated with changes in normal endometrium (P = 0.0172 and 0.0002, respectively). Mucinous cancers displayed the greatest number of alterations in gene expression when compared with normal ovarian epithelial cells. Conclusion: Studies at a molecular level show distinct expression profiles of different histologies of ovarian cancer and support the long-held belief that histotypes of ovarian cancers come to resemble normal fallopian tube, endometrial, and colonic epithelium. Several potential molecular markers for mucinous ovarian cancers have been identified.

Abstract A group of investigators met at a Specialized Programs of Research Excellence Workshop to discuss key issues in the translation of biomarker discovery to the development of useful laboratory tests for cancer care. Development and approval of several new markers and technologies have provided informative examples that include more specific markers for prostate cancer, more sensitive tests for ovarian cancer, more objective analysis of tissue architecture and an earlier indication of response to treatment in breast cancer. Although there is no clear paradigm for biomarker development, several principles are clear. Marker development should be driven by clinical needs, including early cancer detection, accurate pretreatment staging, and prediction of response to treatment, as well as monitoring disease progression and response to therapy. Development of a national repository that uses carefully preserved, well-annotated tissue specimens will facilitate new marker development. Reference standards will be an essential component of this process. Both hospital-based and commercial laboratories can play a role in developing biomarkers from discovery to test validation. Partnering of academe and industry should occur throughout the process of biomarker development. The National Cancer Institute is in a unique position to bring together academe, industry, and the Food and Drug Administration to (a) define clinical needs for biomarkers by tumor type, (b) establish analytic and clinical paradigms for biomarker development, (c) discuss ways in which markers from different companies might be evaluated in combination, (d) establish computational methods to combine data from multiple biomarkers, (e) share information regarding promising markers developed in National Cancer Institute–supported programs, and (f) exchange data regarding new platforms and techniques that can accelerate marker development.

Abstract Purpose: Women with advanced epithelial ovarian cancer are routinely treated with platinum-paclitaxel chemotherapy following cytoreductive surgery, yet only ∼20% achieve long-term disease-free survival. We hypothesized that differences in gene expression before treatment could distinguish patients with short versus long time to recurrence after administration of platinum-paclitaxel combination chemotherapy. Experimental Design: To test this hypothesis, gene expression profiling of 79 primary surgically resected tumors from women with advanced-stage, high-grade epithelial ovarian cancer was done using cDNA microarrays containing 30,721 genes. Supervised learning algorithms were applied in an effort to develop a binary classifier that could discriminate women at risk for early (≤21 months) versus late (>21 months) relapse after initial chemotherapy. Results: A 14-gene predictive model was developed using a set of training samples (n = 51) and subsequently tested using an independent set of test samples (n = 28). This model correctly predicted the outcome of 24 of the 28 test samples (86% accuracy) with 95% positive predictive value for early relapse. Conclusions: Predictive markers for early recurrence can be identified for platinum-paclitaxel combination chemotherapy in primary ovarian carcinoma. The proposed 14-gene model requires further validation.

ABSTRACT Cell wall β-glucans are highly conserved structural components of fungi that potently trigger inflammatory responses in an infected host. Identification of molecular mechanisms responsible for internalization and signaling of fungal β-glucans should enhance our understanding of innate immune responses to fungi. In this study, we demonstrated that internalization of fungal β-glucan particles requires actin polymerization but not participation of components of caveolar uptake mechanisms. Using fluorescence microscopy, we observed that uptake of 5-([4,6-dichlorotriazin-2-yl] amino)-fluorescein hydrochloride-Celite complex-labeled Saccharomyces cerevisiae β-glucan by RAW macrophages was substantially reduced in the presence of cytochalasin D, which antagonizes actin-mediated internalization pathways, but not by treatment with nystatin, which blocks caveolar uptake. Interestingly, β-glucan-induced NF-κB translocation, which is necessary for inflammatory activation, and tumor necrosis factor alpha production were both normal in the presence of cytochalasin D, despite defective internalization of β-glucan particles following actin disruption. Dectin-1, a major β-glucan receptor on macrophages, colocalized to phagocytic cups on macrophages and exhibited tyrosine phosphorylation after challenge with β-glucan particles. Dectin-1 localization and other membrane markers were not affected by treatment with cytochalasin D. Furthermore, dectin-1 receptors rather than Toll-like receptor 2 receptors were shown to be necessary for both efficient internalization of β-glucan particles and cytokine release in response to the fungal cell wall component.