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Objective: To investigate cell-free DNA (cfDNA) in plasma and ascites and its association with clinical outcomes (paracentesis-free interval, overall survival) and CA125 level in participants with advanced ovarian cancer, treated with palliative intraperitoneal bevacizumab to delay re-accumulation of ascites., Methods: cfDNA was extracted from 0.3 to 1 mL samples from 20/24 participants of the REZOLVE trial. Standard and methylation-specific PCRs were performed to measure 3 biomarkers: total cfDNA (Alu), tumour-derived cfDNA (ctDNA, methylated IFFO1 promoter) and endothelium-derived cfDNA (ec-cfDNA, unmethylated CDH5 promoter). Values were correlated to clinical outcomes., Results: cfDNA was detected in all samples, with higher yield in ascites (mean 669 ng/mL) than plasma (mean 75 ng/mL, p < 0.0001). Ascites had a higher ctDNA proportion than plasma (74 % vs. 20 %, p < 0.0001) and plasma had a higher ec-cfDNA proportion than ascites (24 % vs. 16 %, p < 0.002). High ctDNA proportion (>75 %) in ascites was associated with a significantly shorter paracentesis-free interval (median interval 47.5 versus 84 days, hazard ratio (HR) 2.21, 95 % confidence interval (CI) 0.85 to 5.73, p = 0.039) and ctDNA presence in plasma was unfavourable for survival (median survival 56 versus 242 days, HR 3.21, 95 % CI 1.15 to 9.00, p = 0.008). A significant positive correlation was observed between ctDNA proportion in plasma and CA125 level (p = 0.012). No significant difference in total cfDNA, ctDNA nor ec-cfDNA was observed between participants who were responders versus non-responders., Conclusion: Sufficient cfDNA was detected in both plasma and ascites to study three biomarkers. These samples can provide useful information and should be considered in the design of future ovarian cancer trials., Competing Interests: Declaration of competing interest KW declares potential financial conflict of interest due to stock ownership in the following companies that are developing cell-free DNA based clinical assays: Guardant Heath; Exact Sciences; EpiGenomics AG. All other authors declare no competing financial interests., (Copyright © 2024. Published by Elsevier Inc.)

ASPiRATION is a national prospective observational cohort study assessing the feasibility, clinical and economic value of up-front tissue-based comprehensive genomic profiling (CGP) to identify actionable genomic alterations in participants with newly diagnosed metastatic non-squamous non-small-cell lung cancer in Australia. This study will enrol 1000 participants with tumor available for CGP and standard of care molecular testing ( EGFR / ALK/ROS1 ). Participants with actionable variants may receive novel targeted treatments through ASPiRATION-specific substudies, other trials/programs. Clinical outcome data will be collected for a minimum of 2 years. Study outcomes are descriptive, including the ability of CGP to identify additional actionable variants, leading to personalized treatment recommendations, and will describe the feasibility, efficiency, cost and utility of implementation of CGP nationally.

Women with advanced endometrial carcinoma (EC) with mismatch repair (MMR) deficiency have improved outcomes when treated with immune checkpoint inhibitors; however, additional biomarkers are needed to identify women most likely to respond. Scores for programmed death ligand 1 (PD-L1), immunohistochemical staining of tumor (TC+), immune cells (IC+) and presence of tumor-associated immune cells (ICP) on MMR deficient (n = 34) and proficient (n = 33) EC from women treated with durvalumab in the PHAEDRA trial (ANZGOG1601/CTC0144) (trial registration number ACTRN12617000106336, prospectively registered 19 January 2017) are reported and correlated with outcome. Receiver operating characteristic (ROC) analyses and area under the ROC curve were used to determine optimal cutpoints. Performance was compared with median cutpoints and two algorithms; a novel algorithm derived from optimal cutpoints (TC+ ≥ 1 or ICP ≥ 10 or IC+ ≥ 35) and the Ventana urothelial carcinoma (UC) algorithm (either TC+ ≥ 25, ICP > 1 and IC+ ≥ 25 or ICP = 1 and IC+ = 100). The cutpoint ICP ≥ 10 had highest sensitivity (53%) and specificity (82%), being prognostic for progression-free survival (PFS) (p = 0.01), while the optimal cutpoints algorithm was associated with overall survival (p = 0.02); these results were not significant after adjusting for MMR status. The optimal cutpoints algorithm identified non-responders (p = 0.02) with high sensitivity (88%) and negative predictive value (92%), remaining significant after adjustment for MMR. Although MMR status had the strongest association with response, further work to determine the significance of ICP ≥ 10 and the novel optimal cutpoint algorithm is needed.

Objective: To determine whether disrupting the renin angiotensin system with angiotensin receptor blockers will improve clinical outcomes in people with covid-19., Design: CLARITY was a pragmatic, adaptive, multicentre, phase 3, randomised controlled trial., Setting: 17 hospital sites in India and Australia., Participants: Participants were at least 18 years old, previously untreated with angiotensin receptor blockers, with a laboratory confirmed diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection who had been admitted to hospital for management of covid-19., Intervention: Oral angiotensin receptor blockers (telmisartan in India) or placebo (1:1) for 28 days., Main Outcome Measures: The primary endpoint was covid-19 disease severity using a modified World Health Organization Clinical Progression Scale (WHO scale) at day 14. Secondary outcomes were WHO scale scores at day 28, mortality, intensive care unit admission, and respiratory failure. Analyses were evaluated on an ordinal scale in the intention-to-treat population., Results: Between 3 May 2020 and 13 November 2021, 2930 people were screened for eligibility, with 393 randomly assigned to angiotensin receptor blockers (of which 388 (98.7%) to telmisartan 40 mg/day) and 394 to the control group. 787 participants were randomised: 778 (98.9%) from India and nine (1.1%) from Australia. The median WHO scale score at day 14 was 1 (interquartile range 1-1) in 384 participants assigned angiotensin receptor blockers and 1 (1-1) in 382 participants assigned placebo (adjusted odds ratio 1.51 (95% credible interval 1.02 to 2.23), probability of an odds ratio of >1 (Pr(OR>1)=0.98). WHO scale scores at day 28 showed little evidence of difference between groups (1.02 (0.55 to 1.87), Pr(OR>1)=0.53). The trial was stopped when a prespecified futility rule was met., Conclusions: In patients admitted to hospital for covid-19, mostly with mild disease, not requiring oxygen, no evidence of benefit, based on disease severity score, was found for treatment with angiotensin receptor blockers, using predominantly 40 mg/day of telmisartan., Trial Registration: ClinicalTrials.gov NCT04394117., Competing Interests: Competing interests: All authors have completed the ICMJE uniform disclosure form at www.icmje.org/disclosure-of-interest/ and declare: support from the Australian government and the University of Sydney for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years except the following: MJ is responsible for research projects that have received funding from Amgen, Baxter, CSL, Dimerix, Eli Lilly, Gambro, and Merck Sharp and Dohme; has received advisory, steering committee or speaker fees, or both, from Akebia, Amgen, Astra Zeneca, Baxter, Bayer, Boehringer Ingelheim, Cesas Linx, Chinook, CSL, Janssen, Medscape, Merck Sharp Dohme, Roche, and Vifor; CP serves on advisory boards for AstraZeneca, Boehringer Ingelheim, Merck Sharp and Dohme, and Novartis; CJ serves on advisory boards for AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Novartis, and Sanofi-Genzyme; VJ has received grants from Baxter Healthcare, Biocon, and GlaxoSmithKline, and speaker fees or on the advisory board for AstraZeneca, Baxter Healthcare, NephroPlus, Sanofi; no other relationships or activities that could appear to have influenced the submitted work., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: There is a strong theoretical rationale for combining checkpoint blockade with cytotoxic chemotherapy in pleural mesothelioma and other cancers. Two recent single-arm, phase 2 trials [DuRvalumab with chEmotherapy as first-line treAtment in advanced pleural Mesothelioma (DREAM) and Phase II multicenter study of anti-PD-L1, durvalumab, in combination with cisplatin and pemetrexed for the first-line treatment of unresectable malignant pleural mesothelioma (PrE0505)] combining the programmed death ligand-1 (PD-L1) inhibitor durvalumab with standard first-line chemotherapy exceeded prespecified safety and activity criteria to proceed to a phase 3 confirmatory trial to assess this combination. We present the protocol of the DREAM3R trial., Methods and Analysis: This multicentre open-label randomised trial will recruit 480 treatment-naïve adults with advanced pleural mesothelioma, randomised (2:1) to either 3-weekly durvalumab 1500 mg plus 3-weekly doublet chemotherapy (cisplatin 75 mg/m 2 or carboplatin, Area Under the Curve,AUC 5 and pemetrexed 500 mg/m 2 ) 4-6 cycles, followed by 4-weekly durvalumab 1500 mg until disease progression, unacceptable toxicity or patient withdrawal; OR doublet chemotherapy alone for 4-6 cycles, followed by observation. The target accrual time is 27 months, with follow-up for an additional 24 months. This provides over 85% power if the true HR for overall survival (OS) is 0.70, with two-sided alpha of 0.05, assuming a median OS of 15 months in the control group. Randomisation is stratified by age (18-70 years vs >70), sex, histology (epithelioid vs non-epithelioid), platinum agent (cisplatin vs carboplatin) and region (USA vs Australia/New Zealand vs Other). The primary endpoint is OS. Secondary endpoints include progression-free survival, objective tumour response (by mRECIST V.1.1 and iRECIST), adverse events, health-related quality of life and healthcare resource use. Tertiary correlative objectives are to explore and validate potential prognostic and/or predictive biomarkers (including features identified in the DuRvalumab with chEmotherapy as first-line treAtment in advanced pleural Mesothelioma (DREAM) and PrE0505 studies, PD-L1 expression, tumour mutational burden, genomic characteristics and human leukocyte antigen subtypes) in tissue and serial blood samples. An imaging databank will be assembled for validation of radiological measures of response, and studies of possible radiomic biomarkers in mesothelioma., Ethics and Dissemination: The protocol was approved by human research ethics review committees for all participating sites. Results will be disseminated in peer-reviewed journals and at scientific conferences., Drug Supply: AstraZeneca., Protocol Version: CTC 0231 / TOGA 18/001 / PrE0506 3.0, 29 July 2021., Trial Registration Number: ClinicalTrials.gov Identifier: NCT04334759 ACTRN 12620001199909., Competing Interests: Competing interests: ZS, CB, AC, SY, KaF, KarF, AB, MMC have nothing to disclose. PSK reports grants and fees from AstraZeneca and Pfizer, she is a consultant/ advisory board member of MSD, outside the submitted work; PMF reports grants and fees from AstraZeneca, BMS, Corvus, Novartis, Kyowa; he is a consultant/ advisory board member of Amgen, AstraZeneca, BMS, Novartis, Janssen, Iteos, Mirati, Sanofi; he is a Data Safety Monitoring Board member of Polaris, Flame, outside the submitted work; BH is a consultant/ advisory board member of MSD, BMS, Roche, Pfizer, AstraZeneca, Eisai, Takeda; his institution received grants from Amgen, outside the submitted work; SR reports grants and fees from Amgen, AstraZeneca, Genmab, Eisai, Lilly, Roche, Merck, Takeda and GSK; he is a Data Safety Monitoring Board member of Jansen; a member of Board for Gergia Society of Oncology and IASLC; his institution received grants from AstraZenca, Amgen, BMS, Merck, Genmab, Takeda, Advaxis and Pfizer, outside the submitted work; WJL reports grants and fees from Douglas Pharmaceuticals, and patents relating to immune checkpoint therapy, unrelated to this study, outside the submitted work; KO'B has received advisory board and/or speaker bureau and/or meeting travel/registration support from BMS, MSD, LillyOncology, Boehringer-Ingelheim, Pfizer, Novartis, Roche-Genentech,Teva, Mundipharma, Astrazeneca, Janssen, Natera and TriStar. He is a board member and stock holder for Carpe Vitae Pharmaceuticals and a stock holder for RepLuca Pharmaceuticals and DGC Diagnostics and holds patents for novel therapeutics and diagnostic tests, outside the submitted work; NP reports grants and fees from Boehringer Ingelheim, Bayer, Novartis, Pfizer, Roche, Takeda and Ipsen; he is an advisory board member of Boehringer Ingelheim, MSD, Merck, BMS, Astra Zeneca, Takeda, Pfizer and Roche; his institution received grants from Bayer, Pfizer and Roche, outside the submitted work; JB reports grants and fees from AstraZeneca, BMS, Genentech/Roche, Merck, RAPT Therapeutics, Revolution Medicines, Amgen, Eli Lilly, GlaxoSmithKline, Sanofi, Regeneron; she is a Data Safety Monitoring Board member of GlaxoSmithKline, Sanofi, Janssen; she is an advisory board member of IASLC; outside the submitted work; VA institution received grants from BMS and AstraZeneca; outside the submitted work; MS institution received grants from the following competitive funding bodies: Australian National Health and Medical Research Council, Canadian Cancer Trials Group, Cancer Australia, Medical Research Future Fund of Australia; and the following pharmaceutical companies: Astellas, Amgen, Astra Zeneca, Bayer, Beigene, Bionomics, Bristol-Myers Squibb, Celgene, Medivation, Merck, Merck Sharp & Dohme, Pfizer, Roche, Sanofi, Tilray, outside the submitted work; AKN reports grants and fees from Bayer Pharmaceuticals; Roche Pharmaceuticals; Boehringer Ingelheim; Merck Sharpe Dohme; Douglas Pharmaceuticals, Atara Biotherapeutics, Astra Zeneca (payment to institution); Pharmabcine; Trizell Ltd; Seagen; honoraria from Bristol Myers Squibb and her institution received grants from AstraZeneca and Douglas Pharmaceuticals, outside the submitted work., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Locally advanced inoperable pancreatic cancer (LAPC) has a poor prognosis. By increasing intensity of systemic therapy combined with an established safe chemoradiation technique, our intention was to enhance the outcomes of LAPC. In preparation for phase III evaluation, the feasibility and efficacy of our candidate regimen gemcitabine-oxaliplatin chemotherapy with sandwich 5-fluorouracil (5FU) and three-dimensional conformal radiotherapy (3DCRT) needs to be established., Methods: A total of 48 patients with inoperable LAPC without metastases were given gemcitabine (1000 mg m(-2) d1 + d15 q28) and oxaliplatin (100 mg m(-2) d2 + d16 q28) in induction (one cycle) and consolidation (three cycles), and 5FU 200 mg m(-2) per day over 6 weeks during 3DCRT 54 Gy., Results: Median duration of sustained local control (LC) was 15.8 months, progression-free survival (PFS) was 11.0 months, and overall survival was 15.7 months. Survival rates for 1, 2, and 3 years were 70.2%, 21.3%, and 12.8%, respectively. Global quality of life did not significantly decline from baseline during treatment, which was associated with modest treatment-related toxicity., Conclusion: Fixed-dose gemcitabine and oxaliplatin, combined with an effective and safe regimen of 5FU and 3DCRT radiotherapy, was feasible and reasonably tolerated. The observed improved duration of LC and PFS with more intensive therapy over previous trials may be due to patient selection, but suggest that further evaluation in phase III trials is warranted.

Background: Bevacizumab is an antiangiogenic mAb with efficacy against several cancers, but it is associated with risk of arterial thromboembolism (ATE). Further data are needed to determine the safety of bevacizumab., Patients and Methods: We recorded grade 3, 4, or 5 ATE events and other data (including age, baseline cardiovascular risk factors, history of ATE, and aspirin use) from 471 patients with metastatic colorectal cancer in the MAX (Mitomycin, Avastin, Xeloda) trial of capecitabine monotherapy versus capecitabine with bevacizumab with or without mitomycin C., Results: Bevacizumab-treated patients had 12 grade 3, 4, or 5 ATEs (3.8% incidence). ATEs occurred in 2.1% of patients >65 years, 5% of those with a history of ATE, and 5% of those with cardiac risk factors. Age, history of ATE, or vascular risk factors did not increase risk. Aspirin users had a higher incidence than nonusers (8.9% versus 2.7%) but had higher rates of vascular risk factors., Conclusions: Bevacizumab was associated with a modestly higher risk of ATE, but safety was not significantly worse in older patients or patients with a history of ATE or vascular risk factors. The effect of aspirin in preventing ATE in patients receiving bevacizumab could not be determined from this study.

Purpose: Mutations affecting the KRAS gene are established predictive markers of outcome with anti-epithelial growth factor receptor (EGFR) antibodies in advanced colorectal cancer (CRC). The relevance of these markers for anti-vascular endothelial growth factor (VEGF) therapy is controversial. This analysis was performed to assess the predictive and prognostic impact of KRAS and BRAF gene mutation status in patients receiving capecitabine with bevacizumab (CG) or capecitabine without bevacizumab in the phase III AGITG MAX (Australasian Gastrointestinal Trials Group MAX) study., Patients and Methods: Mutation status was determined for 315 (66.9%) of the original 471 patients. Mutation status was correlated with efficacy outcomes (response rate, progression-free survival [PFS], and overall survival [OS]), and a predictive analyses was undertaken., Results: Mutations in KRAS and BRAF genes were observed in 28.8% and 10.6% of patients, respectively. KRAS gene mutation status (wild type [WT] v mutated [MT]) had no prognostic impact for PFS (hazard ratio [HR], 0.89; CI, 0.69 to 1.14) or OS (HR, 0.97; CI, 0.73 to 1.28). BRAF mutation status (WT v MT) was not prognostic for PFS (HR, 0.80; CI, 0.54 to 1.18) but was prognostic for OS (HR, 0.49; CI, 0.33 to 0.73; P = .001). By using the comparison of capecitabine versus capecitabine and bevacizumab (CB) and CB plus mitomycin (CBM), KRAS gene mutation status was not predictive of the effectiveness of bevacizumab for PFS or OS (test for interaction P = .95 and 0.43, respectively). Similarly, BRAF gene mutation status was not predictive of the effectiveness of bevacizumab for PFS or OS (test for interaction P = .46 and 0.32, respectively)., Conclusion: KRAS gene mutation status was neither prognostic for OS nor predictive of bevacizumab outcome in patients with advanced CRC. BRAF gene mutation status was prognostic for OS but was not predictive of outcome with bevacizumab.

Purpose: To determine whether adding bevacizumab, with or without mitomycin, to capecitabine monotherapy improves progression-free survival (PFS) in patients with metastatic colorectal cancer (mCRC) in an open-label, three-arm randomized trial., Patients and Methods: Overall, 471 patients in Australia, New Zealand, and the United Kingdom with previously untreated, unresectable mCRC were randomly assigned to the following: capecitabine; capecitabine plus bevacizumab (CB); or capecitabine, bevacizumab, and mitomycin (CBM). We compared CB with capecitabine and CBM with capecitabine for progression-free survival (PFS). Secondary end points included overall survival (OS), toxicity, response rate (RR), and quality of life (QOL)., Results: Median PFS was 5.7 months for capecitabine, 8.5 months for CB, and 8.4 months for CBM (capecitabine v CB: hazard ratio [HR], 0.63; 95% CI, 0.50 to 0.79; P < .001; C v CBM: HR, 0.59; 95% CI, 0.47 to 0.75; P < .001). After a median follow-up of 31 months, median OS was 18.9 months for capecitabine and was 16.4 months for CBM; these data were not significantly different. Toxicity rates were acceptable, and all treatment regimens well tolerated. Bevacizumab toxicities were similar to those in previous studies. Measures of overall QOL were similar in all groups., Conclusion: Adding bevacizumab to capecitabine, with or without mitomycin, significantly improves PFS without major additional toxicity or impairment of QOL.

Background: Docetaxel administered 3-weekly with cisplatin and 5-fluorouracil leads to better survival than does standard therapy in patients with oesophagogastric cancer, but leads to high rates of haematological toxicity. Weekly docetaxel is associated with less haematological toxicity. This randomised phase II study tested weekly docetaxel-based combination chemotherapy regimens, with the aim of maintaining their activity while reducing toxicity., Methods: Patients with histologically confirmed metastatic oesophageal or gastric carcinoma were randomised to receive weekly docetaxel (30 mg m(-2)) on days 1 and 8, cisplatin (60 mg m(-2)) on day 1, and 5-fluorouracil (200 mg m(-2) per day) continuously, every 3 weeks (weekly TCF, wTCF); or docetaxel (30 mg m(-2)) on days 1 and 8 and capecitabine (1600 mg m(-2) per day) on days 1-14, every 3 weeks (weekly TX, wTX)., Results: A total of 106 patients were enrolled (wTCF, n=50; wTX, n=56). Response rates, the primary end point, were 47% with wTCF and 26% with wTX. Rates of febrile neutropenia were low in each arm. Median progression-free and overall survival times were 5.9 and 11.2 months for wTCF and 4.6 and 10.1 months for wTX, respectively., Conclusion: Weekly TCF and TX have encouraging activity and less haematological toxicity than TCF administered 3-weekly. Weekly docetaxel-based combination regimens warrant further evaluation in this disease.

We have previously reported that P2Y(2) purinoceptors and muscarinic M(3) receptors trigger Ca(2+) responses in HT-29 cells that differ in their timecourse, the Ca(2+) response to P2Y(2) receptor activation being marked by a more rapid decline of intracellular Ca(2+) concentration ([Ca(2+)](i)) after the peak response and that this rapid decline of [Ca(2+)](i) was slowed in cells expressing heterologous beta-adrenergic receptor kinase (betaARK). In the present study, we demonstrate that, during P2Y(2) receptor activation, betaARK expression increases the rate of Gd(3+)-sensitive Mn(2+) influx, a measure of the rate of store-operated Ca(2+) entry from the extracellular space, during P2Y(2) activation and that this effect of betaARK is mimicked by exogenous alpha-subunits of G(q), G(11) and G(i2). The effect of betaARK on the rate of Mn(2+) influx is thus attributable to its ability to scavenge G protein betagamma-subunits released during activation of P2Y(2) receptor. We further find that the effect of betaARK on the rate of Mn(2+) influx during P2Y(2) receptor activation can be overcome by arachidonic acid. In addition, the UTP-induced Mn(2+) influx rate was significantly increased by inhibitors of phospholipase A(2) (PLA(2)) and an siRNA directed against PLA(2)beta, but not by an siRNA directed against PLA(2)alpha or by inhibitors of arachidonic acid metabolism. These findings provide evidence for the existence of a P2Y(2) receptor-activated signalling system that acts in parallel with depletion of intracellular Ca(2+) stores to inhibit Ca(2+) influx across the cell membrane. This signalling process is mediated via Gbetagamma and involves PLA(2)beta and arachidonic acid., (2009 Elsevier Ltd. All rights reserved.)

We used replication-deficient adenoviruses overexpressing antisense against G(q) class alpha-subunits to determine the roles of G(q) and G(11) in mediating M(3)-receptor-coupled Ca(2+) mobilization in intact HT29 human colonic carcinoma epithelial cells. Western blot analysis and confocal microscopy showed that the viruses expressing antisense directed against the alpha-subunits of G(q) or G(11) produced isoform-specific reductions in the levels of these alpha-subunits. Fura-2 was used to measure changes in the Ca(2+) response following activation of the M(3) receptors by carbachol. The G(alpha)(q) antisense virus suppressed the peak Ca(2+) response by 70%, whereas the G(alpha)(11) antisense virus reduced it by 34%. We then used co-infection with both viruses to determine the effect of concomitant suppression of both G(alpha)(q) and G(alpha)(11). Overexpression of antisense to both alpha-subunits reduced by approximately 50% the levels of both G(alpha)(q) and G(alpha)(11). It also almost completely inhibited the Ca(2+) response to carbachol. These data show that both G(q) and G(11) are involved in mediating the action of the M(3) receptor on cytosolic Ca(2+) in HT29 cells. Furthermore, they suggest that the coupling of the M(3) receptor to these G proteins is specific, in that G(alpha)(q) cannot substitute for G(alpha)(11), and vice versa.

It is firmly established that the activation of many heptahelical receptors by extracellular agonists leads to the activation of effectors such as phospholipase Cbeta (PLCbeta), the subsequent production of inositol-1,4,5-trisphosphate (IP3), and a resultant increase in intracellular free Ca2+. Heterotrimeric G-proteins have a critical role in transducing the signal from the heptahelical receptor to PLCbeta and in determining the specificity and duration of the cellular responses. There remain, however, a number of areas of uncertainty regarding the exact mechanisms involved in regulating G-protein-mediated receptor-effector coupling in different cell types. For example, the molecular identity of the G-protein involved and the degree of isoform specificity among G-proteins of the same family and their receptors remains unclear. It is also not known in many cell types whether it is the alpha- or the betagamma-subunits of these G-proteins that activate PLCbeta. In order to address these issues, we have used replication-deficient adenoviruses as a tool to deliver, into intact epithelial cells, transgenes coding for proteins involved in G-protein-coupled receptor signaling pathways.

Plasma membrane-spanning G-protein-linked receptors transduce approximately 60% of all extracellular stimuli in higher animals. Many G-protein-linked receptor pathways are yet to be elucidated, with the receptor, G-protein or effector system as yet unidentified. In addition, many fundamental issues pertaining to G-protein signalling remain unresolved, such as the factors governing the specificity of G-protein receptor coupling and the control of signal amplitude in response to G-protein activation. In order to address some of these issues, the use of replication-deficient adenoviruses as gene transfer vectors for investigations of G-protein signalling has been developed, facilitating dissection of G-protein-linked signal transduction pathways in an extensive range of cultured cells, as well as in vivo. The present review focuses on the versatility and utility of adenoviruses for the investigation of signalling by heterotrimeric G-proteins and explores some of the recent advances in adenoviral technology as they relate to the study of signal transduction.

Using Fura-2 to measure changes in intracellular calcium ([Ca(2+)](i)), we show that P(2U)receptors in HT29 cells trigger an increase in [Ca(2+)](i)by pertussis toxin-insensitive G proteins. We then use replication-deficient adenoviruses expressing wild-type and dominant negative mutants of G(alpha q)and G(alpha i2), antisense directed against G(alpha q)or the C-terminal fragment of beta-adrenergic receptor kinase (beta ARK-CT) to identify these G proteins. We find the [Ca(2+)](i)response to UTP is not affected by increased expression of the wild-type G(alpha q), wild-type G(alpha i2)or beta ARK-CT, while it is blocked by over-expression of dominant negative G(alpha q). The timecourse of the UTP response is, however, altered by wild-type G(alpha q)and is only weakly inhibited by antisense G(alpha q). This suggests that the P(2U)response is mediated, at least partially, by a G protein distinct from G(alpha q). In contrast, the M(3)muscarinic response is inhibited by over-expression of antisense against G(alpha q), or over-expression of beta ARK-CT, a finding in agreement with our previous observation that the muscarinic response in HT29 cells is mediated by the beta gamma-subunits of G(q). We also find that P(2U)and M(3)receptors do not control identical Ca(2+)stores, suggesting that differential activation of G proteins can lead to Ca(2+)release from distinct stores., (Copyright 2000 Harcourt Publishers Ltd.)

Vocal fold paresis (VFP) is a relatively common and often overlooked condition that can be difficult to diagnose based on the laryngeal examination alone. A retrospective review of the records of 50 consecutive adult patients with VFP was performed. In each case, the diagnosis of VFP was confirmed by laryngeal electromyography. The presenting symptoms were dysphonia (100%), vocal fatigue (76%), diplophonia (40%), and odynophonia (12%), and the findings were unilateral vocal fold hypomobility (50%), unilateral bowing (36%), and bilateral bowing (22%). Laryngoplasty and/or lipoinjection was performed in 54% of the subjects, and significant vocal improvement was achieved in 85%. VFP appears to be underdiagnosed because many VFP patients have compensatory hyperkinetic disorders at presentation. Although the diagnosis of VFP may be suspected based on the patient's symptoms and findings, the diagnostic sine qua non is laryngeal electromyography. In addition, surgical treatment for VFP appears to be safe and effective.

Despite limited supporting evidence, salivary gland myoepithelial cells are said to be differentiated cells with little or no capacity to replicate; they presumably develop from stem cells. This study investigated the proliferative potential of myoepithelial cells with an antibody to proliferating cell nuclear antigen and a rat model. This model involved clamping of the parotid duct causing atrophy of the gland and then releasing the duct followed by gland regeneration. Rats were sacrificed at time points during atrophy and regeneration phases and the number and location of cycling myoepithelial cells assessed. Cycling myoepithelial cells were identified with double immunohistochemical staining, cycling cells with proliferating cell nuclear antigen-positive nuclei within muscle-specific actin-positive cytoplasm (the latter identified with antibody HHF35). The results show that baseline proliferative rates of myoepithelial cells in both the resting and fully regenerated gland ranged from 0.3% to 2%, similar to rates for other major cell types in the normal rat gland. A peak myoepithelial cell proliferative rate of 23% occurred at day 5 during the atrophy phase. Rates during the regenerative phase were not significantly different than the baseline levels. Similarities of rat and human parotid gland and the definite proliferative capacity of myoepithelial cells indicates that these specialized cells must be considered one of the potential progenitor cells for human salivary gland tumors.

A canine model was used to investigate the efferent laryngeal responses to stimulation by topically applied acid and pepsin. Five adult mongrel dogs were studied. Electromyographic recordings from the thyroarytenoid muscle were measured with hooked-wire electrodes as an acid solution (normal saline/hydrochloric acid at pH 6.0, 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, and 1.0) was sequentially instilled into the larynx. Laryngospasm (tonic, sustained contraction of the thyroarytenoid muscle) occurred in all animals at pH 2.5 to 2.0 or less. Control substances such as neutral pH isotonic saline, hypotonic saline, hypertonic saline, water, and pepsin alone failed to produce laryngospasm. Next, solutions containing both acid (in the same pH range) and pepsin were tested. The laryngeal responses were similar to those of acid alone. The superior laryngeal nerves were sectioned bilaterally and the above experiments repeated. None of the test solutions produced laryngospasm; however, when capsaicin (1%) was instilled into the subglottis, laryngospasm occurred. Thus, chemoreceptors in the subglottis (supplied by the recurrent laryngeal nerves) appear to be responsive to capsaicin stimulation but not to acid stimulation. The data suggest that pH-sensitive chemoreceptors in the canine larynx cause laryngospasm (when the pH of the test solution is 2.5 or less) and that these acid receptors are supplied by the superior laryngeal nerves.

To study the proliferative capacity of salivary gland, an animal model of regeneration was developed. A clamp, which induced atrophy in parotid gland by obstructing the main excretory duct but allowed restoration of duct patency following removal, was implanted in a series of rats. When it was removed (Day 7), the weight of the glands was reduced by 50% and acinar cells had decreased from 93.8% to 8.2% of total cell population. Regeneration occurred rapidly following removal of the clamp. The number and location of cycling intercalated, striated, and excretory duct cells and acinar cells were monitored using an antibody to proliferating cell nuclear antigen (PCNA). All cell types were induced to cycle but the predominant cell to cycle was the acinar cell. During regeneration the number of PCNA+ acinar cells increased 38.7-fold from steady-state values. Results demonstrate that acinar cells have a significant potential for cycling, contrary to current histogenetic theories of salivary gland tumourigenesis which exclude acinar cells as potential progenitor cells on the grounds of their putative limited cycling capacity.