CFTR expression in human salivary gland acinar cells.

The key role of CFTR in secretory epithelia has been extensively documented. Additionally, CFTR plays a significant role in ion absorption in exocrine glands, including salivary and sweat glands. Most of the knowledge about CFTR expression comes from animal models such as the mouse or the rat, but there is limited information about CFTR expression in human tissues. In the present study, we assessed the expression of CFTR in human submandibular and parotid glands. Consistent with findings in rodent salivary glands, our immunolocalization studies show that CFTR is expressed in duct cells. However, CFTR expression in human salivary glands differs from that in rodents, as immunolocalization and single-cell RNA sequencing analysis from a previous study performed in the human parotid gland revealed the presence of CFTR protein and transcripts within a distinct cell cluster. Based on cell marker expression, this cluster corresponds to acinar cells. To obtain functional evidence supporting CFTR expression, we isolated human parotid acinar cells through collagenase digestion. Acinar cells displayed an anion conductance that was activated in response to cAMP-increasing agents and was effectively blocked by CFTRInh172, a known CFTR blocker. This study provides novel evidence of CFTR expression within acinar cells of human salivary glands. This finding challenges the established model positioning CFTR exclusively in duct cells from exocrine glands.NEW & NOTEWORTHY This study addresses the uncertainty about the impact of CFTR on human salivary gland function. We found CFTR transcripts in a subset of duct cells known as ionocytes, as well as in acinar cells. Isolated human parotid acinar cells exhibited Cl- conductance consistent with CFTR activity. This marks the first documented evidence of functional CFTR expression in human salivary gland acinar cells.

Chronic Rhinosinusitis With Nasal Polyposis Treated With Dupilumab: Real-World Use and Outcomes.

BACKGROUND: Biologic medications are increasingly incorporated into chronic rhinosinusitis with nasal polyps (CRSwNP) management. However, little is known about prescribing patterns in real-world settings and how this relates to proposed international guidelines and outcomes. OBJECTIVES: To characterize use patterns of dupilumab for CRSwNP better in relation to proposed guidelines and explore real-world outcomes. METHODS: We used the TriNetX Web-based tool to identify patients who were prescribed dupilumab for CRSwNP. Patients prescribed dupilumab for a CRSwNP indication were included for analysis. Dupilumab initiation criteria were determined via the European Position Paper on Rhinosinusitis and Nasal Polyps 2020 (EPOS2020). RESULTS: In total, 121 patients were identified who were prescribed dupilumab for a CRSwNP indication. Of these, 86 (71%) met EPOS2020 indications for biologic initiation and 35 (29%) did not. Overall, patients had significant improvements in the 22-item SinoNasal Outcome Test scores (mean improvement of 24.3 points) and nasal polyp scores (mean improvement of 1.0 point). However, 20 patients (30%) did not show meaningful improvement in the 22-item SinoNasal Outcome Test scores. Twenty-one patients (17%) failed a previous biologic attempt. Therapy was discontinued by six patients (5%) due to side effects, and by six (5%) owing to a lack of efficacy. CONCLUSIONS: In our experience, patients prescribed dupilumab for CRSwNP frequently may not meet EPOS2020 Guidelines. Over 25% of those who do not meet criteria may not have CRSwNP. Overall, dupilumab use among well-selected patients appears to be safe and effective. Further real-world study of biologic use for CRSwNP will help improve its judicious use and identify populations who benefit most from biologic therapies.

Patterns of Obstruction on DISE in Adults With Obstructive Sleep Apnea Change With BMI.

OBJECTIVES: We describe drug-induced sleep endoscopy (DISE) obstruction patterns in adults with obstructive sleep apnea (OSA) based on body mass index (BMI). We also evaluate subgroups of patients with clinically significant obstruction patterns at the velopharynx and oropharynx. STUDY DESIGN: Retrospective chart review. METHODS: Single-institution, retrospective chart review of adults with OSA who underwent DISE with dexmedetomidine sedation from 2016 to 2018. Endoscopic findings were graded using VOTE (Velum, Oropharynx, Tongue base, Epiglottis) classification. Oropharyngeal obstruction was additionally graded with the modifier T when due to palatine tonsil tissue. Findings in patients who had BMI < 25, 25 ≤ BMI < 30, and BMI ≥ 30 were compared. RESULTS: One hundred and eleven patients (1 underweight, 23 normal weight, 56 overweight, and 31 obese) were reviewed. Patients with lower BMI were more likely to have more severe obstruction at the level of the tongue base (χ2 = 11.52, P = .021) and epiglottis (χ 2 = 10.56, P = .032). Conversely, patients with higher BMI were more likely to have complete concentric (grade 2C) velum obstruction (χ 2 = 16.04, P < .001) and more severe oropharyngeal obstruction (χ 2 = 9.65, P = .046). Patients with grade 2 oropharyngeal obstruction without tonsil obstruction had more severe concurrent velum obstruction compared to subjects with grade 2 T oropharyngeal obstruction (P = .009). CONCLUSION: In adults with OSA, BMI categories have significantly distinct obstruction patterns at all airway levels on DISE, and there appear to be distinct subgroups associated with certain velum and oropharynx collapse patterns. These findings may have important implications for positive airway pressure-alternative treatment. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:224-229, 2021.

Long-Term Maintenance of Acinar Cells in Human Submandibular Glands After Radiation Therapy.

PURPOSE: In a combined retrospective and prospective study, human salivary glands were investigated after radiation treatment for head and neck cancers. The aim was to assess acinar cell loss and morphologic changes after radiation therapy and to determine whether irradiated salivary glands have regenerative potential. METHODS AND MATERIALS: Irradiated human submandibular and parotid salivary glands were collected from 16 patients at a range of time intervals after completion of radiation therapy (RT). Control samples were collected from 14 patients who had not received radiation treatments. Tissue sections were analyzed using immunohistochemistry to stain for molecular markers. RESULTS: Human submandibular and parotid glands isolated less than 1 year after RT showed a near complete loss of acinar cells. However, acinar units expressing functional secretory markers were observed in all samples isolated at later intervals after RT. Significantly lower acinar cell numbers and increased fibrosis were found in glands treated with combined radiation and chemotherapy, in comparison to glands treated with RT alone. Irradiated samples showed increased staining for duct cell keratin markers, as well as many cells coexpressing acinar- and duct cell-specific markers, in comparison to nonirradiated control samples. CONCLUSIONS: After RT, acinar cell clusters are maintained in human submandibular glands for years. The surviving acinar cells retain proliferative potential, although significant regeneration does not occur. Persistent DNA damage, increased fibrosis, and altered cell identity suggest mechanisms that may impair regeneration.

Polyethylene glycol protects primary hepatocytes during supercooling preservation.

Cold storage (at 4°C) offers a compromise between the benefits and disadvantages of cooling. It allows storage of organs or cells for later use that would otherwise quickly succumb to warm ischemia, but comprises cold ischemia that, when not controlled properly, can result in severe damage as well by both similar and unique mechanisms. We hypothesized that polyethylene glycol (PEG) 35 kDa would ameliorate these injury pathways and improve cold primary hepatocyte preservation. We show that reduction of the storage temperature to below zero by means of supercooling, or subzero non-freezing, together with PEG supplementation increases the viable storage time of primary rat hepatocytes in University of Wisconsin (UW) solution from 1 day to 4 days. We find that the addition of 5% PEG 35 kDa to the storage medium prevents cold-induced lipid peroxidation and maintains hepatocyte viability and functionality during storage. These results suggest that PEG supplementation in combination with supercooling may enable a more optimized cell and organ preservation.

Long-term coculture strategies for primary hepatocytes and liver sinusoidal endothelial cells.

Hepatocytes and their in vitro models are essential tools for preclinical screening studies for drugs that affect the liver. Most of the current models primarily focus on hepatocytes alone and lack the contribution of non-parenchymal cells (NPCs), which are significant through both molecular and the response of the NPCs themselves. Models that incorporate NPCs alongside hepatocytes hold the power to enable more realistic recapitulation and elucidation of cell interactions and cumulative drug response. Hepatocytes and liver sinusoidal endothelial cells (LSECs) account for ∼ 80% of the liver mass where the LSECs line the walls of blood vessels, and act as a barrier between hepatocytes and blood. Culturing LSECs with hepatocytes to generate multicellular physiologically relevant in vitro liver models has been a major hurdle since LSECs lose their phenotype rapidly after isolation. To this end, we describe the application of collagen gel (1) in a sandwich and (2) as an intervening extracellular matrix layer to coculture hepatocytes with LSECs for extended periods. These coculture configurations provide environments wherein hepatocyte and LSECs, through cell-cell contacts and/or secretion factors, lead to enhanced function and stability of the cocultures. Our results show that in these configurations, hepatocytes and LSECs maintained their phenotypes when cultured together as a mixture, and showed stable secretion and metabolic activity for up to 4 weeks. Immunostaining for sinusoidal endothelial 1 (SE-1) antibody demonstrated retention of LSEC phenotype during the culture period. In addition, LSECs cultured alone maintained high viability and SE-1 expression when cultured within a collagen sandwich configuration up to 4 weeks. Albumin production of the cocultures was 10-15 times higher when LSECs were cultured as a bottom layer (with an intervening collagen layer) and as a mixture in a sandwich configuration, and native CYP 1A1/2 activity was at least 20 times higher than monoculture controls. Together, these data suggest that collagen gel-based hepatocyte-LSEC cocultures are highly suitable models for stabilization and long-term culture of both cell types. In summary, these results indicate that collagen gel-based hepatocyte-LSEC coculture models are promising for in vitro toxicity testing, and liver model development studies.

A novel ultrathin collagen nanolayer assembly for 3-D microtissue engineering: Layer-by-layer collagen deposition for long-term stable microfluidic hepatocyte culture.

The creation of stable hepatocyte cultures using cell-matrix interactions has proven difficult in microdevices due to dimensional constraints limiting the utility of classic tissue culture techniques that involve the use of hydrogels such as the collagen "double gel" or "overlay". To translate the collagen overlay technique into microdevices, we modified collagen using succinylation and methylation reactions to create polyanionic and polycationic collagen solutions, and deposited them layer-by-layer to create ultrathin collagen nanolayers on hepatocytes. These ultrathin collagen layers covered hepatocytes in microdevices and 1) maintained cell morphology, viability, and polarity, 2) induced bile canalicular formation and actin reorganization, and 3) maintained albumin and urea secretions and CYP activity similar to those observed in hepatocytes in collagen double gel hepatocytes in plate cultures. Beyond the immediate applications of this technique to create stable, in vitro microfluidic hepatocyte cultures for drug toxicity testing, this technique is generally applicable as a thin biomaterial for other 3D microtissues.

Circadian rhythms of Per2::Luc in individual primary mouse hepatocytes and cultures.

BACKGROUND: Hepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown. RESULTS: In this study we isolated primary hepatocytes from transgenic Per2(Luc) mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2(-/-) Per2(Luc) cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes. CONCLUSIONS: Our results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals.

SIMPLE MACHINE PERFUSION SIGNIFICANTLY ENHANCES HEPATOCYTE YIELDS OF ISCHEMIC AND FRESH RAT LIVERS.

The scarcity of viable hepatocytes is a significant bottleneck in cell transplantation, drug discovery, toxicology, tissue engineering, and bioartificial assist devices, where trillions of high-functioning hepatocytes are needed annually. We took the novel approach of using machine perfusion to maximize cell recovery, specifically from uncontrolled cardiac death donors, the largest source of disqualified donor organs. In a rat model, we developed a simple 3 hour room temperature (20±2°C) machine perfusion protocol to treat non-premedicated livers exposed to 1 hour of warm (34°C) ischemia. Treated ischemic livers were compared to fresh, fresh-treated and untreated ischemic livers using viable hepatocyte yields and in vitro performance as quantitative endpoints. Perfusion treatment resulted in both a 25-fold increase in viable hepatocytes from ischemic livers, and a 40% increase from fresh livers. While cell morphology and function in suspension and plate cultures of untreated warm ischemic cells was significantly impaired, treated warm ischemic cells were indistinguishable from fresh hepatocytes. Further, a strong linear correlation between tissue ATP and cell yield enabled accurate evaluation of the extent of perfusion recovery. Maximal recovery of warm ischemic liver ATP content appears to be correlated with optimal flow through the microvasculature. These data demonstrate that the inclusion of a simple perfusion-preconditioning step can significantly increase the efficiency of functional hepatocyte yields and the number of donor livers that can be gainfully utilized.