Mucus clearance: in vivo canine tracheal vs. in vitro bullfrog palate studies

1977 ◽  
Vol 42 (5) ◽  
pp. 761-766 ◽  
Author(s):  
A. Giordano ◽  
C. K. Shih ◽  
D. S. Holsclaw ◽  
M. A. Khan ◽  
M. Litt
Keyword(s):  
Cystic Fibrosis ◽  
Mucociliary Clearance ◽  
Objective Method ◽  
Flow Properties ◽  
Therapeutic Modalities ◽  
Mucus Clearance ◽  
Clearance Studies ◽  
Frog Palate

Tracheal mucociliary clearance was studied by a radioisotope technique in pentothal-anesthetized beagles in the control, atropinized, or dehydrated state. Mucus collected from a tracheal pouch in each dog was used for in vitro bullfrog (Rana cantesbiana) palate clearance studies and compared to the in vivo clearance rates. In all three experimental states, there was a significant correlation between in vivo and in vitro rates, suggesting that tracheal pouch mucus is a good model for investigating the mucociliary flow properties of intact airway mucus. When compared to matched controls, atropine appeared to cause a slowing of the in vivo clearance rate but not of the in vitro rate. Dehydration had no effect on either. The appropriateness of the frog palate method in the study of human respiratory disease (e.g., chronic bronchitis, cystic fibrosis) as well as its potential as an objective method of assessing the effects of various therapeutic modalities in these diseases is discussed.

scholarly journals Sex Disparities in Cystic Fibrosis: Review on the Effect of Female Sex Hormones on Lung Pathophysiology and Outcomes

2020 ◽  
pp. 00475-2020
Author(s):  
G.Y. Lam ◽  
J. Goodwin ◽  
P.G. Wilcox ◽  
B.S. Quon
Keyword(s):  
Cystic Fibrosis ◽  
Sex Differences ◽  
Sex Hormones ◽  
Mucociliary Clearance ◽  
Therapeutic Interventions ◽  
Female Sex ◽  
Female Sex Hormones ◽  
Sex Disparities

Sex differences in morbidity and mortality have been reported in the cystic fibrosis (CF) population worldwide. However, it is unclear why CF women have worse clinical outcomes than men. In this review, we focus on the influence of female sex hormones on CF pulmonary outcomes and summarise data from in vitro and in vivo experiments on how estrogen and progesterone might modify mucociliary clearance, immunity and infection in the CF airways. The potential for novel sex hormone related therapeutic interventions is also discussed.

Preliminary Report of In vitro and In vivo Effectiveness of Dornase Alfa on SARS-CoV-2 Infection (Preprint)

2020 ◽  
Author(s):  
Hacer Kuzu Okur ◽  
Koray Yalcin ◽  
Cihan Tastan ◽  
Sevda Demir ◽  
Bulut Yurtsever ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Respiratory Tract ◽  
Mononuclear Cells ◽  
Multiple Organ ◽  
Peripheral Blood Mononuclear ◽  
Dornase Alfa ◽  
Tract Infections ◽  
Adult Peripheral Blood

UNSTRUCTURED Dornase alfa, the recombinant form of the human DNase I enzyme, breaks down neutrophil extracellular traps (NET) that include a vast amount of DNA fragments, histones, microbicidal proteins and oxidant enzymes released from necrotic neutrophils in the highly viscous mucus of cystic fibrosis patients. Dornase alfa has been used for decades in patients with cystic fibrosis to reduce the viscoelasticity of respiratory tract secretions, to decrease the severity of respiratory tract infections, and to improve lung function. Previous studies have linked abnormal NET formations to lung diseases, especially to acute respiratory distress syndrome (ARDS). Coronavirus disease 2019 (COVID-19) pandemic affected more than two million people over the world, resulting in unprecedented health, social and economic crises. The COVID-19, viral pneumonia that progresses to ARDS and even multiple organ failure, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High blood neutrophil levels are an early indicator of SARS-CoV-2 infection and predict severe respiratory diseases. A similar mucus structure is detected in COVID-19 patients due to the accumulation of excessive NET in the lungs. Here, we show our preliminary results with dornase alfa that may have an in-vitro anti-viral effect against SARS-CoV-2 infection in a bovine kidney cell line, MDBK without drug toxicity on healthy adult peripheral blood mononuclear cells. In this preliminary study, we also showed that dornase alfa can promote clearance of NET formation in both an in-vitro and three COVID-19 cases who showed clinical improvement in radiological analysis (2-of-3 cases), oxygen saturation (SpO2), respiratory rate, disappearing of dyspnea and coughing.

scholarly journals Functional and Transcriptional Adaptations of Blood Monocytes Recruited to the Cystic Fibrosis Airway Microenvironment In Vitro

2021 ◽  
Vol 22 (5) ◽  
pp. 2530
Author(s):  
Bijean D. Ford ◽  
Diego Moncada Giraldo ◽  
Camilla Margaroli ◽  
Vincent D. Giacalone ◽  
Milton R. Brown ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Bactericidal Activity ◽  
Scavenger Receptor ◽  
Receptor Expression ◽  
Blood Monocytes ◽  
Bacterial Killing ◽  
Blood Neutrophils ◽  
Vitro Model

Cystic fibrosis (CF) lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior in vitro studies, we showed that blood neutrophils migrated through the well-differentiated lung epithelium into the CF airway fluid supernatant (ASN) mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. Here, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment to CFASN. To test this hypothesis, primary human blood monocytes were transmigrated in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profile by RNA sequencing were quantified post-transmigration. Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they also fail to protect CF airways from infection.

scholarly journals Strong Antimicrobial and Healing Effects of Beta-Acids from Hops in Methicillin-Resistant Staphylococcus aureus-Infected External Wounds In Vivo

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 708
Author(s):  
Radek Sleha ◽  
Vera Radochova ◽  
Jiri Malis ◽  
Alexander Mikyska ◽  
Milan Houska ◽  
...  
Keyword(s):  
Wound Healing ◽  
Porcine Model ◽  
Therapeutic Effects ◽  
Methicillin Resistant ◽  
Antimicrobial Effects ◽  
Therapeutic Modalities ◽  
Positive Effects ◽  
Inflammatory Parameters

Staphylococcus (S.) aureus is an important causative agent of wound infections with increasing incidence in the past decades. Specifically, the emergence of methicillin-resistant S. aureus (MRSA) causes serious problems, especially in nosocomial infections. Therefore, there is an urgent need to develop of alternative or supportive antimicrobial therapeutic modalities to meet these challenges. Purified compounds from hops have previously shown promising antimicrobial effects against MRSA isolates in vitro. In this study, purified beta-acids from hops were tested for their potential antimicrobial and healing properties using a porcine model of wounds infected by MRSA. The results show highly significant antimicrobial effects of the active substance in both the powder and Ambiderman-based application forms compared to both no-treatment control and treatment with Framycoin. Moreover, the macroscopic evaluation of the wounds during the treatment using the standardized Wound Healing Continuum indicated positive effects of the beta-acids on the overall wound healing. This is further supported by the microscopic data, which showed a clear improvement of the inflammatory parameters in the wounds treated by beta-acids. Thus, using the porcine model, we demonstrate significant therapeutic effects of hops compounds in the management of wounds infected by MRSA. Beta-acids from hops, therefore, represent a suitable candidate for the treatment of non-responsive nosocomial tissue infections by MRSA.

scholarly journals Differential effect of anesthetics on mucociliary clearance in vivo in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kyle S. Feldman ◽  
Eunwon Kim ◽  
Michael J. Czachowski ◽  
Yijen Wu ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Mucociliary Clearance ◽  
Defense Mechanism ◽  
Ex Vivo ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Wild Type ◽  
Sulfur Colloid ◽  
Ct System

AbstractRespiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.

scholarly journals Type-4 Phosphodiesterase (PDE4) Blockade Reduces NETosis in Cystic Fibrosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Licia Totani ◽  
Concetta Amore ◽  
Antonio Piccoli ◽  
Giuseppe Dell’Elba ◽  
Angelo Di Santo ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lung Disease ◽  
Pde4 Inhibitor ◽  
Free Dna ◽  
Impaired Lung Function ◽  
Cellular Integrity ◽  
Net Release

Neutrophilic inflammation is a key determinant of cystic fibrosis (CF) lung disease. Neutrophil-derived free DNA, released in the form of extracellular traps (NETs), significantly correlates with impaired lung function in patients with CF, underlying their pathogenetic role in CF lung disease. Thus, specific approaches to control NETosis of neutrophils migrated into the lungs may be clinically relevant in CF. We investigated the efficacy of phosphodiesterase (PDE) type-4 inhibitors, in vitro, on NET release by neutrophils from healthy volunteers and individuals with CF, and in vivo, on NET accumulation and lung inflammation in mice infected with Pseudomonas aeruginosa. PDE4 blockade curbed endotoxin-induced NET production and preserved cellular integrity and apoptosis in neutrophils, from healthy subjects and patients with CF, challenged with endotoxin, in vitro. The pharmacological effects of PDE4 inhibitors were significantly more evident on CF neutrophils. In a mouse model of Pseudomonas aeruginosa chronic infection, aerosol treatment with roflumilast, a selective PDE4 inhibitor, gave a significant reduction in free DNA in the BALF. This was accompanied by reduced citrullination of histone H3 in neutrophils migrated into the airways. Roflumilast-treated mice showed a significant improvement in weight recovery. Our study provides the first evidence that PDE4 blockade controls NETosis in vitro and in vivo, in CF-relevant models. Since selective PDE4 inhibitors have been recently approved for the treatment of COPD and psoriasis, our present results encourage clinical trials to test the efficacy of this class of drugs in CF.

Murine colonic mucosa hyperproliferation. I. Elevated CFTR expression and enhanced cAMP-dependent Cl−secretion

2000 ◽  
Vol 278 (5) ◽  
pp. G753-G764 ◽  
Author(s):  
Shahid Umar ◽  
Jason Scott ◽  
Joseph H. Sellin ◽  
William P. Dubinsky ◽  
Andrew P. Morris
Keyword(s):  
Cystic Fibrosis ◽  
Fluid Transport ◽  
Cellular Proliferation ◽  
Neck Region ◽  
Anion Channel ◽  
Cellular Level ◽  
Cystic Fibrosis Gene ◽  
Mrna And Protein Expression

Fluid transport in the large intestine is mediated by the cystic fibrosis gene product and cAMP-dependent anion channel cystic fibrosis transmembrane conductance regulator (CFTR). cAMP-mediated Cl−secretion by gastrointestinal cell lines in vitro has been positively correlated with the insertion of CFTR into the apical membrane of differentiated senescent colonocytes and negatively correlated with the failure of CFTR to insert into the plasma membrane of their undifferentiated proliferating counterparts. In native tissues, this relationship remains unresolved. We demonstrate, in a transmissible murine colonic hyperplasia (TMCH) model, that (8-fold) colonocyte proliferation was accompanied by increased cellular CFTR mRNA and protein expression (8.3- and 2.4-fold, respectively) and enhanced mucosal cAMP-dependent Cl−secretion (2.3-fold). By immunofluorescence microscopy, cellular CFTR expression was restricted to the apical pole of cells at the base of the epithelial crypt. In contrast, increased cellular proliferation in vivo led to increases in both the cellular level and the total number of cells expressing this anion channel, with cellular CFTR staining extending into the crypt neck region. Hyperproliferating colonocytes accumulated large amounts of CFTR in apically oriented subcellular perinuclear compartments. This novel mode of CFTR regulation may explain why high endogenous levels of cellular CFTR mRNA and protein within the TMCH epithelium were not matched with larger increases in transmucosal CFTR Cl−current.

scholarly journals A CRAF/glutathione-S-transferase P1 complex sustains autocrine growth of cancers withKRASandBRAFmutations

2020 ◽  
Vol 117 (32) ◽  
pp. 19435-19445 ◽  
Author(s):  
Yoshiro Niitsu ◽  
Yasushi Sato ◽  
Kunihiro Takanashi ◽  
Tsuyoshi Hayashi ◽  
Naoko Kubo-Birukawa ◽  
...  
Keyword(s):  
Glutathione Transferases ◽  
Erk Pathway ◽  
Glutathione S Transferase ◽  
Autocrine Growth ◽  
Autocrine Loop ◽  
Therapeutic Modalities ◽  
Vital Component ◽  
Underlying Mechanisms

The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutantKRAS(mKRAS) andBRAF(mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRASand mBRAFcancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRASand mBRAFcells in vitro and suppressed tumorigenesis of the xenografted mKRAStumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAScolon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers.

scholarly journals Disruption of Cross-Feeding Inhibits Pathogen Growth in the Sputa of Patients with Cystic Fibrosis

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Jeffrey M. Flynn ◽  
Lydia C. Cameron ◽  
Talia D. Wiggen ◽  
Jordan M. Dunitz ◽  
William R. Harcombe ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Susceptibility ◽  
Susceptibility Testing ◽  
Anaerobic Bacteria ◽  
Content Type ◽  
Growth Environment ◽  
Polymicrobial Infections

ABSTRACT A critical limitation in the management of chronic polymicrobial infections is the lack of correlation between antibiotic susceptibility testing (AST) and patient responses to therapy. Underlying this disconnect is our inability to accurately recapitulate the in vivo environment and complex polymicrobial communities in vitro. However, emerging evidence suggests that, if modeled and tested accurately, interspecies relationships can be exploited by conventional antibiotics predicted to be ineffective by standard AST. As an example, under conditions where Pseudomonas aeruginosa relies on cocolonizing organisms for nutrients (i.e., cross-feeding), multidrug-resistant P. aeruginosa may be indirectly targeted by inhibiting the growth of its metabolic partners. While this has been shown in vitro using synthetic bacterial communities, the efficacy of a “weakest-link” approach to controlling host-associated polymicrobial infections has not yet been demonstrated. To test whether cross-feeding inhibition can be leveraged in clinically relevant contexts, we collected sputa from cystic fibrosis (CF) subjects and used enrichment culturing to isolate both P. aeruginosa and anaerobic bacteria from each sample. Predictably, both subpopulations showed various antibiotic susceptibilities when grown independently. However, when P. aeruginosa was cultured and treated under cooperative conditions in which it was dependent on anaerobic bacteria for nutrients, the growth of both the pathogen and the anaerobe was constrained despite their intrinsic antibiotic resistance profiles. These data demonstrate that the control of complex polymicrobial infections may be achieved by exploiting obligate or facultative interspecies relationships. Toward this end, in vitro susceptibility testing should evolve to more accurately reflect in vivo growth environments and microbial interactions found within them. IMPORTANCE Antibiotic efficacy achieved in vitro correlates poorly with clinical outcomes after treatment of chronic polymicrobial diseases; if a pathogen demonstrates susceptibility to a given antibiotic in the lab, that compound is often ineffective when administered clinically. Conversely, if a pathogen is resistant in vitro, patient treatment with that same compound can elicit a positive response. This discordance suggests that the in vivo growth environment impacts pathogen antibiotic susceptibility. Indeed, here we demonstrate that interspecies relationships among microbiotas in the sputa of cystic fibrosis patients can be targeted to indirectly inhibit the growth of Pseudomonas aeruginosa. The therapeutic implication is that control of chronic lung infections may be achieved by exploiting obligate or facultative relationships among airway bacterial community members. This strategy is particularly relevant for pathogens harboring intrinsic multidrug resistance and is broadly applicable to chronic polymicrobial airway, wound, and intra-abdominal infections.

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