Guidance on Mitigating the Risk of Transmitting Respiratory Infections During Nebulization by the COPD Foundation Nebulizer Consortium.

BACKGROUND: Nebulizers are used commonly for inhaled drug delivery. Because they deliver medication through aerosol generation, clarification is needed on what constitutes safe aerosol delivery in infectious respiratory disease settings. The COVID-19 pandemic highlighted the importance of understanding the safety and potential risks of aerosol-generating procedures. However, evidence supporting the increased risk of disease transmission with nebulized treatments is inconclusive, and inconsistent guidelines and differing opinions have left uncertainty regarding their use. Many clinicians opt for alternative devices, but this practice could impact outcomes negatively, especially for patients who may not derive full treatment benefit from handheld inhalers. Therefore, it is prudent to develop strategies that can be used during nebulized treatment to minimize the emission of fugitive aerosols, these comprising bioaerosols exhaled by infected individuals and medical aerosols generated by the device that also may be contaminated. This is particularly relevant for patient care in the context of a highly transmissible virus. RESEARCH QUESTION: How can potential risks of infections during nebulization be mitigated? STUDY DESIGN AND METHODS: The COPD Foundation Nebulizer Consortium (CNC) was formed in 2020 to address uncertainties surrounding administration of nebulized medication. The CNC is an international, multidisciplinary collaboration of patient advocates, pulmonary physicians, critical care physicians, respiratory therapists, clinical scientists, and pharmacists from research centers, medical centers, professional societies, industry, and government agencies. The CNC developed this expert guidance to inform the safe use of nebulized therapies for patients and providers and to answer key questions surrounding medication delivery with nebulizers during pandemics or when exposure to common respiratory pathogens is anticipated. RESULTS: CNC members reviewed literature and guidelines regarding nebulization and developed two sets of guidance statements: one for the health care setting and one for the home environment. INTERPRETATION: Future studies need to explore the risk of disease transmission with fugitive aerosols associated with different nebulizer types in real patient care situations and to evaluate the effectiveness of mitigation strategies.

The Role of Suboptimal Concentrations of Nebulized Tobramycin in Driving Antimicrobial Resistance in Pseudomonas aeruginosa Isolates in Cystic Fibrosis.

BACKGROUND: Antimicrobial resistance in Pseudomonas aeruginosa may be driven by exposure to suboptimal concentrations of tobramycin antibiotic delivered by less efficient nebulizers. METHODS: P. aeruginosa isolates (no. = 114; 32 first + 82 chronic) were challenged in vitro employing extrapolated peak and trough concentrations of tobramycin inhalation solution (TIS), corresponding to 3 nebulizers: Pari LC Plus, Sidestream12NEB400, and MistyNeb2035G. Bacterial persistence and antibiotic susceptibility to tobramycin was determined following 4 TIS cycles: (i) 28 d ON, (ii) 28 d ON + 28 d OFF, (iii) 2 × 28 d ON, and (iv) 28 d ON + 28 d ON + 28 d OFF. RESULTS: All first isolates were eradicated at peak and trough concentrations except for the trough concentration corresponding to Sidestream 12NEB400 (bactericidal activity 87%). For chronic isolates, peak concentrations eradicated 88%, 90%, and 92%, and trough concentrations eradicated 43%, 62%, and 85%, with the Sidestream12NEB400, MistyNeb2035G, and Pari LC Plus nebulizers, respectively. A statistically significant increase in antibiotic resistance with sensitive, intermediate, and resistant P. aeruginosa was noted following cycles (i) through (iv) at trough concentrations with the Sidestream 12NEB400 and MistyNeb2035G nebulizers. There was a significant reduction in tobramycin resistance following a 28-d OFF cycle, and no difference was noted following 1 × 28 d ON versus 2 × 28 d ON cycles. CONCLUSIONS: Our results indicate that suboptimal concentrations of tobramycin drove increased antibiotic resistance, emulating standard cycles of ON/OFF inhaled therapy. This was evident at extrapolated tobramycin concentrations at trough levels corresponding to less efficient nebulizers by initially allowing for the survival of intermediate and resistant organisms, because nebulizer performance did not achieve critical antibiotic concentrations sufficient to eradicate the organism, and by allowing the development of resistance in those cells that were able to survive the initial tobramycin challenge. Transferred to clinical practice, for people with cystic fibrosis on TIS treatment, it is important that clinicians employ an efficient nebulizer that helps mitigate an upward drift in antibiotic resistance, thereby protecting the clinical value of TIS within treatment for cystic fibrosis.

A multigene urine test for the detection and stratification of bladder cancer in patients presenting with hematuria.

PURPOSE: We investigated whether the RNA assay uRNA® and its derivative Cxbladder® have greater sensitivity for the detection of bladder cancer than cytology, NMP22™ BladderChek™ and NMP22™ ELISA, and whether they are useful in risk stratification. MATERIALS AND METHODS: A total of 485 patients presenting with gross hematuria but without a history of urothelial cancer were recruited prospectively from 11 urology clinics in Australasia. Voided urine samples were obtained before cystoscopy. The sensitivity and specificity of the RNA tests were compared to cytology and the NMP22 assays using cystoscopy as the reference. The ability of Cxbladder to distinguish between low grade, stage Ta urothelial carcinoma and more advanced urothelial carcinoma was also determined. RESULTS: uRNA detected 41 of 66 urothelial carcinoma cases (62.1% sensitivity, 95% CI 49.3-73.8) compared with NMP22 ELISA (50.0%, 95% CI 37.4-62.6), BladderChek (37.9%, 95% CI 26.2-50.7) and cytology (56.1%, 95% CI 43.8-68.3). Cxbladder, which was developed on the study data, detected 82%, including 97% of the high grade tumors and 100% of tumors stage 1 or greater. The cutoffs for uRNA and Cxbladder were prespecified to give a specificity of 85%. The specificity of cytology was 94.5% (95% CI 91.9-96.5), NMP22 ELISA 88.0%, (95% CI 84.6-91.0) and BladderChek 96.4% (95% CI 94.2-98.0). Cxbladder distinguished between low grade Ta tumors and other detected urothelial carcinoma with a sensitivity of 91% and a specificity of 90%. CONCLUSIONS: uRNA and Cxbladder showed improved sensitivity for the detection of urothelial carcinoma compared to the NMP22 assays. Stratification with Cxbladder provides a potential method to prioritize patients for the management of waiting lists.

Development of a multiplex RNA urine test for the detection and stratification of transitional cell carcinoma of the bladder.

PURPOSE: New markers that enable the percentage of transitional cell carcinomas (TCC) of the bladder that are diagnosed before invasion of the bladder muscle layers to be increased would reduce the morbidity and mortality associated with this disease. The purpose of this study was to develop a simple, accurate urine test based on mRNA markers and simple gene signatures that (a) could detect TCC before muscle invasion while maintaining high specificity in patients with hematuria or urinary tract infections and (b) identify patients most likely to have grade 3 or stage > or =T1 disease. EXPERIMENTAL DESIGN: RNA markers with high overexpression in stage Ta tumors and/or T1 to T4 tumors but low expression in blood or inflammatory cells were characterized by quantitative reverse transcription-PCR using 2 mL of voided urine from 75 TCC patients and 77 control patients with other urological diseases. RESULTS: A combination of the RNAs CDC2, MDK, IGFBP5, and HOXA13 detected 48%, 90%, and 100% of stage Ta, T1, and >T1 TCCs, respectively, at a specificity of 85%. Detection of Ta tumors increased to 60% for primary (non-recurrent) Ta tumors and 76% for Ta tumors > or =1 cm in diameter. Test specificity was 80% for the 20 control patients with urinary tract infections. The combination of CDC2 and HOXA13 distinguished between grade 1 to 2 TCCs and grade 3 or stage > or =T1 TCCs with approximately 80% specificity and sensitivity. CONCLUSIONS: Simple gene expression signatures can be used as urine markers for the accurate detection and characterization of bladder cancer.

Differential effects of RU486 reveal distinct mechanisms for glucocorticoid repression of prostaglandin E release.

In A549 pulmonary cells, the dexamethasone- and budesonide-dependent repression of interleukin-1beta-induced prostaglandin E2 release was mimicked by the steroid antagonist, RU486. Conversely, whereas dexamethasone and budesonide were highly effective inhibitors of interleukin-1beta-induced cyclooxygenase (COX)/prostaglandin E synthase (PGES) activity and COX-2 expression, RU486 (<1 microm) was a poor inhibitor, but was able to efficiently antagonize the effects of dexamethasone and budesonide. In addition, both dexamethasone and RU486 repressed [3H]arachidonate release, which is consistent with an effect at the level of phospholipase A2 activity. By contrast, glucocorticoid response element-dependent transcription was unaffected by RU486 but induced by dexamethasone and budesonide, whilst dexamethasone- and budesonide-dependent repression of nuclear factor-kappaB-dependent transcription was maximally 30-40% and RU486 (<1 microm) was without significant effect. Thus, two pharmacologically distinct mechanisms of glucocorticoid-dependent repression of prostaglandin E2 release are revealed. First, glucocorticoid-dependent repression of arachidonic acid is mimicked by RU486 and, second, repression of COX/PGES is antagonized by RU486. Finally, whilst all compounds induced glucocorticoid receptor translocation, no role for glucocorticoid response element-dependent transcription is supported in these inhibitory processes and only a limited role for glucocorticoid-dependent inhibition of nuclear factor-kappaB in the repression of COX-2 is indicated.

Inhibitors of protein kinase C (PKC) prevent activated transcription: role of events downstream of NF-kappaB DNA binding.

In pulmonary A549 cells, the protein kinase C (PKC) inhibitor, Ro 31-8220, and the phosphotidylcholine-specific phospholipase C inhibitor, D609, prevent NF-kappaB-dependent transcription, yet NF-kappaB DNA binding is unaffected (Bergmann, M., Hart, L., Lindsay, M., Barnes, P. J., and Newton, R. (1998) J. Biol. Chem. 273, 6607-6610). We now show that this effect also occurs in BEAS-2B bronchial epithelial cells as well as with other PKC inhibitors (Gö 6976, GF109203X, and calphostin C) in A549 cells. Similarly, phorbol ester, a diacylglycerol mimetic, activates NF-kappaB-dependent transcription and potentiates tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB-dependent transcription, yet unlike TNFalpha, poorly activates IkappaB kinase (IKK) activity, IkappaBalpha degradation, or NF-kappaB DNA binding in both A549 and BEAS-2B cells. As phorbol ester-induced NF-kappaB-dependent transcription was relatively insensitive to the proteasome inhibitor, MG-132, PKC may affect NF-kappaB-dependent transcription via mechanisms other than the core IKK-IkappaB pathway. This is supported by Gal4 one hybrid analysis of p65/RelA transactivation, which was potentiated by TNFalpha and phorbol ester and was inhibited by Ro 31-8220 and D609. Additionally, a number of PKC isoforms, particularly the novel isoform PKCepsilon, induced p65/RelA transactivation. Phosphorylation of p65/RelA and cAMP-responsive element-binding protein (CREB)-binding protein (CBP) was increased by TNFalpha treatment and, in the case of CBP, was prevented by Ro 31-8220 or D609. However, p65/RelA-CBP interactions were unaffected by either compound. As this effect was not limited to NF-kappaB, but was a more general feature of inducible gene transcription, we suggest PKC isoforms may provide a point of intervention in diseases such as inflammation, or cancer, where activated gene expression is prominent.

ICAM-1 expression is highly NF-kappaB-dependent in A549 cells. No role for ERK and p38 MAPK.

The transcription factor nuclear factor kappaB (NF-kappaB) is an activator of multiple cytokines, chemokines and adhesion molecules, which are important in inflammatory diseases such as asthma, and is consequently considered as an attractive therapeutic target. In the present study, a constitutively active dominant version of IkappaBalpha, IkappaBalphaDN, was introduced into A549 pulmonary cells by adenovirus-mediated delivery. The dominant IkappaB, but not a null viral vector, prevented the induction of NF-kappaB-dependent transcription by both tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta). Similarly, both TNFalpha and IL-1beta strongly induced mRNA and protein expression of intercellular adhesion molecule (ICAM)-1 and in each case this was prevented by adenovirus expressing the dominant IkappaB, but not by the null virus, thereby establishing ICAM-1 as an NF-kappaB-dependent gene. Numerous studies have suggested key roles for the p38 and extracellular regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascades in the activation and transactivation of NF-kappaB. We show here that SB203580, a selective inhibitor of the p38 MAPK, and PD098059 and UO126, both selective inhibitors of the ERK MAPK cascade, have no effect on TNFalpha or IL-1beta-induced translocation and DNA binding of NF-kappaB. Furthermore, these inhibitors showed no pharmacologically relevant effect on NF-kappaB-dependent transcription nor was there any effect on expression of ICAM-1. Taken together these data highlight the potential use of inhibition of the NF-kappaB signalling pathway in pulmonary inflammatory diseases and suggest that inhibitors of the p38 and ERK MAPK pathways may be of lesser effect.

Adenovirus-mediated delivery and expression of a cAMP-dependent protein kinase inhibitor gene to BEAS-2B epithelial cells abolishes the anti-inflammatory effects of rolipram, salbutamol, and prostaglandin E2: a comparison with H-89.

cAMP-elevating drugs are thought to mediate their biological effects by activating the cAMP/cAMP-dependent protein kinase (PKA) cascade. However, this hypothesis is difficult to confirm due to a lack of selective inhibitors. Here, we have probed the role of PKA in mediating inhibitory effects of several cAMP-elevating drugs in BEAS-2B epithelial cells using an adenovirus vector encoding a PKA inhibitor protein (PKIalpha) and have compared it to H-89, a commonly used small molecule PKA inhibitor. Initial studies established efficient gene transfer and confirmed functionality of PKIalpha 48 h after virus infection. All cAMP-elevating drugs tested promoted the phosphorylation of cAMP response element-binding protein (CREB), activated a cAMP response element (CRE)-driven luciferase reporter gene, and suppressed both granulocyte/macrophage colony-stimulating factor (GM-CSF) generation and [(3)H]arachidonic acid (AA) release in response to interleukin-1beta and monocyte chemotactic protein (MCP)-1, respectively. These effects were abolished by PKIalpha. In contrast, H-89 behaved unpredictably under the same conditions. Thus, although CREB phosphorylation evoked by a range of cAMP-elevating drugs was abolished by H-89, neither activation of the CRE-dependent luciferase reporter gene construct nor the inhibition of GM-CSF generation was inhibited. Paradoxically, H-89 antagonized MCP-1-induced [(3)H]AA release and enhanced the inhibitory effect of submaximal concentrations of rolipram and 8-bromo-cAMP. We suggest that expression of PKIalpha in susceptible cells provides a simple and unambiguous way to assess the role of PKA in cAMP signaling and to probe the mechanism of action of other drugs and cAMP-dependent responses where the participation of PKA is equivocal. Furthermore, these data suggest that H-89 is not a selective inhibitor of PKA and should be avoided.

IL-1beta-dependent activation of NF-kappaB mediates PGE2 release via the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase.

Prostaglandin (PG) E2 release is induced in pulmonary A549 cells by the NF-kappaB-activating stimuli interleukin-1beta (IL-1beta) and phorbol 12-myristate 13-acetate (PMA). Adenoviral over-expression of IkappaBalphaDeltaN, a dominant NF-kappaB inhibitor, prevents NF-kappaB-dependent transcription and was used to qualify the role of NF-kappaB in the release of PGE2. IkappaBalphaDeltaN repressed IL-1beta-induced, but not PMA-induced, cycloxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES) expression. These data conclusively demonstrate a substantial role for NF-kappaB in the co-ordinate induction of COX-2, mPGES and in the corresponding release of PGE2 by IL-1beta. However, other pathways are primarily responsible for PGE2 release induced by PMA.