Phospholipid scramblase-1 regulates innate type 2 inflammation in mouse lungs via CRTH2-dependent mechanisms.

Exaggerated Type 2 immune responses play critical roles in the pathogenesis of a variety of diseases including asthma, allergy, and pulmonary fibrosis. Recent studies have highlighted the importance of innate type 2 immune responses and innate lymphoid 2 cells (ILC2s) in these disorders. However, the mechanisms that control the development of pulmonary innate type 2 responses (IT2IR) and the recruitment and/or activation of ILC2 cells are poorly understood. In mouse models of pulmonary IT2IR, we demonstrated that phospholipid scramblase-1 (PLSCR1), a type II transmembrane protein that mediates bidirectional and nonspecific translocation of phospholipids between the inner and outer leaflets of the plasma membrane, was a critical regulator of IT2IR in the lung. We further suggested that (a) PLSCR1 bound to and physically interacted with chemoattractant receptor-homologous molecule(CRTH2), which is a G-protein-coupled receptor that is expressed on TH2 cells and on multiple immune cells and is commonly used to identify ILC2 cells, and (b) the effects of PLSCR1 on ILC2 activation and IT2IR were mediated via CRTH2-dependent mechanisms. Overall, our studies demonstrated that PLSCR1 played an essential role in the pathogenesis of ILC2 responses, providing critical insights into biology and disease pathogenesis and identifying targets that can be manipulated in attempts to control IT2IR in chronic diseases such as asthma.

Development and characterization of lung surfactant-coated polymer nanoparticles for pulmonary drug delivery.

Lung cancer is often diagnosed at an advanced stage where tumors are usually inoperable and first-line therapies are inefficient and have off-targeted adverse effects, resulting in poor patient survival. Here, we report the development of an inhalable poly lactic-co-glycolic acid polymer-based nanoparticle (PLGA-NP) formulation with a biomimetic Infasurf® lung surfactant (LS) coating, for localized and sustained lung cancer drug delivery. The nanoparticles (188 ± 7 nm) were stable in phosphate buffered saline, serum and Gamble's solution (simulated lung fluid), and demonstrated cytocompatibility up to 1000 µg/mL concentration and dose-dependent uptake by lung cancer cells. The LS coating significantly decreased nanoparticle (NP) uptake by NR8383 alveolar macrophages in vitro compared to uncoated NPs. The coating, however, did not impair NP uptake by A549 lung adenocarcinoma cells. The anti-cancer drug gemcitabine hydrochloride encapsulated in the PLGA core was released in a sustained manner while the paclitaxel loaded in the LS shell demonstrated a rapid or burst release profile over 21 days. The drug-loaded NPs significantly decreased cancer cell survival and colony formation in vitro compared to free drugs and single drug-loaded NPs. In vivo studies confirmed greater retention of LS-coated NPs in the lungs of C57BL/6 WT mice compared to uncoated NPs, at 24 h and 72 h following intranasal administration. The overall results confirm that LS coating is a unique strategy for cloaking polymeric NPs to potentially prevent their rapid lung clearance and facilitate prolonged pulmonary drug delivery.

Chitinase 3 like 1 contributes to the development of pulmonary vascular remodeling in pulmonary hypertension.

Chitinase 3 like 1 (CHI3L1) is the prototypic chitinase-like protein mediating inflammation, cell proliferation, and tissue remodeling. Limited data suggest CHI3L1 is elevated in human pulmonary arterial hypertension (PAH) and is associated with disease severity. Despite its importance as a regulator of injury/repair responses, the relationship between CHI3L1 and pulmonary vascular remodeling is not well understood. We hypothesize that CHI3L1 and its signaling pathways contribute to the vascular remodeling responses that occur in pulmonary hypertension (PH). We examined the relationship of plasma CHI3L1 levels and severity of PH in patients with various forms of PH, including group 1 PAH and group 3 PH, and found that circulating levels of serum CHI3L1 were associated with worse hemodynamics and correlated directly with mean pulmonary artery pressure and pulmonary vascular resistance. We also used transgenic mice with constitutive knockout and inducible overexpression of CHI3L1 to examine its role in hypoxia-, monocrotaline-, and bleomycin-induced models of pulmonary vascular disease. In all 3 mouse models of pulmonary vascular disease, pulmonary hypertensive responses were mitigated in CHI3L1-null mice and accentuated in transgenic mice that overexpress CHI3L1. Finally, CHI3L1 alone was sufficient to induce pulmonary arterial smooth muscle cell proliferation, inhibit pulmonary vascular endothelial cell apoptosis, induce the loss of endothelial barrier function, and induce endothelial-mesenchymal transition. These findings demonstrate that CHI3L1 and its receptors play an integral role in pulmonary vascular disease pathobiology and may offer a target for the treatment of PAH and PH associated with fibrotic lung disease.

CRTH2 Mediates Profibrotic Macrophage Differentiation and Promotes Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a particularly deadly form of pulmonary fibrosis of unknown cause. In patients with IPF, high serum and lung concentrations of CHI3L1 (chitinase 3 like 1) can be detected and are associated with poor survival. However, the roles of CHI3L1 in these diseases have not been fully elucidated. We hypothesize that CHI3L1 interacts with CRTH2 (chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells) to stimulate profibrotic macrophage differentiation and the development of pulmonary fibrosis and that circulating blood monocytes from patients with IPF are hyperresponsive to CHI3L1-CRTH2 signaling. We used murine pulmonary fibrosis models to investigate the role of CRTH2 in profibrotic macrophage differentiation and fibrosis development and primary human peripheral blood mononuclear cell culture to detect the difference of monocytes in the responses to CHI3L1 stimulation and CRTH2 inhibition between patients with IPF and normal control subjects. Our results showed that null mutation or small-molecule inhibition of CRTH2 prevents the development of pulmonary fibrosis in murine models. Furthermore, CHI3L1 stimulation induces a greater increase in CD206 expression in IPF monocytes than control monocytes. These results demonstrated that monocytes from patients with IPF appear to be hyperresponsive to CHI3L1 stimulation. These studies support targeting the CHI3L1-CRTH2 pathway as a promising therapeutic approach for IPF and that the sensitivity of blood monocytes to CHI3L1-induced profibrotic differentiation may serve as a biomarker that predicts responsiveness to CHI3L1- or CRTH2-based interventions.

Lung cell membrane-coated nanoparticles capable of enhanced internalization and translocation in pulmonary epithelial cells.

Cell membrane-coated nanoparticles (CMCNP), which involve coating a core nanoparticle (NP) with cell membranes, have been gaining attention due to their ability to mimic the properties of the cells, allowing for enhanced delivery and efficacy of therapeutics. Two CMCNP systems comprised of an acetalated dextran-based NP core loaded with curcumin (CUR) coated with cell membranes derived from pulmonary epithelial cells were developed. The NP were approximately 200 nm and their surface charges varied based on their coating, where CMCNP systems exhibited negative surface charge like natural cell membranes. The NP were smooth, spherical, and homogeneous with distinct coatings on their cores. Minimal in vitro toxicity was observed for the NP and controlled release of CUR was observed. The CMCNP internalized into and translocated across an in vitro pulmonary epithelial monolayer significantly more than the control NP. Blocking endocytosis pathways reduced the transcytosis of NP, indicating a relationship between endocytosis and transcytosis. These newly developed CMCNP have the potential to be used in pulmonary drug delivery applications to potentially enhance NP internalization and transport into and across the pulmonary epithelium.

In vitro rehardening and staining effects of silver diamine fluoride with and without mucin on early enamel caries lesions.

PURPOSE: To evaluate the rehardening ability of SDF and its individual components, silver, and fluoride ions, on early enamel caries lesions using artificial saliva with and without mucin. METHODS: Early caries lesions were created in human permanent enamel specimens. The specimens (n=36 per group) were then treated with a single application of: SDF (38%), SDF followed by application of potassium iodide (SDF+KI), potassium fluoride (KF); fluoride control, 44,800 ppm (F), silver nitrate (AgNO3); silver control, 253,900 ppm (Ag), or deionized water (DIW). Immediately, the specimens were subjected to 4 days of continuous remineralization with or without mucin (n=18 per subgroup). Changes in Vickers surface microhardness from lesion baseline (ΔVHN) were calculated. Data were analyzed using two-way (intervention vs. rehardening models) ANOVA. RESULTS: In both rehardening models (with or without mucin), SDF (ΔVHN data; mean ± standard deviation; with/without mucin: 26± 19/3± 11) was significantly less effective in rehardening promotion than SDF+KI (37± 12/39± 16) and KF (40± 17/41± 29; P≤ 0.0332). Compared to AgNO3 (9± 9/18± 15) and DIW (3± 7/12± 9), SDF was more effective in the presence of mucin (P≤ 0.001) but not in its absence, similar to DIW (P= 0.1117); less effective vs. AgNO3 (P= 0.0061). The presence of mucin significantly increased the rehardening ability of SDF (P< 0.0001). However, mucin did not affect the extent of rehardening in the other groups (P≥ 0.082). SDF+KI and KF were superior in their ability in rehardening promotion than AgNO3 and DIW in both rehardening models (P< 0.0001). In both rehardening models, ΔL* values from baseline to post-rehardening show that applying KI after SDF significantly lessened the dark staining caused by SDF (P< 0.0001). Under the present in vitro conditions, SDF does not appear to enhance surface rehardening of early enamel caries lesions. The co-presence of mucin during rehardening enhanced the efficacy of SDF which warrants further investigation. CLINICAL SIGNIFICANCE: Silver diamine fluoride + potassium iodide may be a viable option in rehardening of early enamel caries lesions.

Emerging drug delivery strategies for idiopathic pulmonary fibrosis treatment.

Idiopathic pulmonary fibrosis (IPF) is a debilitating and fatal condition that causes severe scarring of the lungs. While the pathogenesis of IPF continues to be extensively studied and several factors have been considered, an exact cause has yet to be established. With inadequate treatment options and no cure available, overall disease prognosis is still poor. Existing oral therapies, pirfenidone and nintedanib, may attempt to improve the patients' quality of life by mitigating symptoms and slowing disease progression, however chronic doses and systemic deliveries of these drugs can lead to severe side effects. The lack of effective treatment options calls for further investigation of restorative as well as additional palliative therapies for IPF. Nanoparticle-based sustained drug delivery strategies can be utilized to ensure targeted delivery for site-specific treatment as well as long-acting therapy, improving overall patient compliance. This review provides an update on promising strategies for the delivery of anti-fibrotic agents, along with an overview of key therapeutic targets as well as relevant emerging therapies currently being evaluated for IPF treatment.

Hermansky-Pudlak Syndrome and Lung Disease: Pathogenesis and Therapeutics.

Hermansky-Pudlak Syndrome (HPS) is a rare, genetic, multisystem disorder characterized by oculocutaneous albinism (OCA), bleeding diathesis, immunodeficiency, granulomatous colitis, and pulmonary fibrosis. HPS pulmonary fibrosis (HPS-PF) occurs in 100% of patients with subtype HPS-1 and has a similar presentation to idiopathic pulmonary fibrosis. Upon onset, individuals with HPS-PF have approximately 3 years before experiencing signs of respiratory failure and eventual death. This review aims to summarize current research on HPS along with its associated pulmonary fibrosis and its implications for the development of novel treatments. We will discuss the genetic basis of the disease, its epidemiology, and current therapeutic and clinical management strategies. We continue to review the cellular processes leading to the development of HPS-PF in alveolar epithelial cells, lymphocytes, mast cells, and fibrocytes, along with the molecular mechanisms that contribute to its pathogenesis and may be targeted in the treatment of HPS-PF. Finally, we will discuss emerging new cellular and molecular approaches for studying HPS, including lentiviral-mediated gene transfer, induced pluripotent stem cells (iPSCs), organoid and 3D-modelling, and CRISPR/Cas9-based gene editing approaches.

The effect of silver diamine fluoride in preventing in vitro primary coronal caries under pH-cycling conditions.

OBJECTIVES: This study investigated the ability of SDF, and its individual components, silver (Ag+) and fluoride (F-) ions, in preventing enamel demineralization under pH-cycling conditions in the presence or absence of twice-daily fluoride application. DESIGN: Polished human enamel specimens were assigned to five treatment groups (n = 36 per group): SDF (38 %); SDF followed by application of a saturated solution of potassium iodide (SDF + KI); silver nitrate (AgNO3; silver control, 253,900 ppm Ag); potassium fluoride (KF; fluoride control, 44,800 ppm F); deionized water. Treatments were applied once. Specimens in each treatment group were divided into two subgroups (n = 18). During the subsequent 7-day pH-cycling phase, specimens were treated twice daily with either 275 ppm fluoride as sodium fluoride or deionized water, immediately before and after a 3-h cariogenic challenge with exposure to artificial saliva at all other times. Changes in color, Vickers surface microhardness (SMH), transverse microradiography (TMR) was calculated. Data were analyzed using two-way ANOVA. RESULTS: In both models, SDF, SDF + KI and KF were superior in inhibiting demineralization compared to AgNO3 and deionized water (p < 0.0001). There was no statistically significant difference between SDF, SDF + KI and KF with twice daily fluoride treatments (p > 0.8). However, KF was more effective in preventing demineralization than SDF and SDF + KI in the absence of fluoride treatments (p = 0.0002). KI did not affect the ability of SDF to prevent demineralization (p > 0.4). CONCLUSION: SDF and SDF + KI appears to be an effective option in preventing primary coronal caries.

Effectiveness of in vitro primary coronal caries prevention with silver diamine fluoride - Chemical vs biofilm models.

OBJECTIVES: The main goal of this study was to investigate the effectiveness of SDF and its individual components, silver (Ag+) and fluoride (F-) ions, in preventing enamel demineralization using biofilm and chemical models. METHODES: Polished human enamel specimens were assigned to five treatment groups (n = 18 per group): SDF (38 %); SDF followed by application of a saturated solution of potassium iodide (SDF + KI); silver nitrate (AgNO3; silver control, 253,900 ppm Ag+); potassium fluoride (KF; fluoride control, 44,800 ppm F); deionized water (DIW). Treatments were applied once to sound enamel. In the biofilm model, specimens were demineralized by aerobic overnight incubation using cariogenic bacteria isolated from human saliva in brain heart infusion supplemented with 0.2 % sucrose for three days. In the chemical model, enamel specimens were immersed in a demineralizing solution containing 0.1 M lactic acid, 4.1 mM CaCl2, 8.0 mM KH2PO4, 0.2 % Carbopol 907, pH adjusted to 5.0 for five days. Vickers surface microhardness was used to determine the extent of enamel demineralization. Data were analyzed using one-way ANOVA. RESULTS: In the chemical model, there was no statistically significant difference between SDF and SDF + KI in preventing coronal caries (p < 0.0001). In the biofilm model, SDF + KI was significantly less effective in preventing demineralization than SDF (p < 0.0001). In both models, SDF and SDF + KI were superior in their ability to prevent caries lesion formation than AgNO3 and DIW. CONCLUSION: KI application after SDF treatment appears to impair SDF's ability to prevent biofilm-mediated but not chemically induced demineralization. CLINICAL SIGNIFICANCE: SDF may be a viable option in preventing primary coronal caries.

Maxillary expansion in an animal model with light, continuous force.

OBJECTIVES: Maxillary constriction is routinely addressed with rapid maxillary expansion (RME). However, the heavy forces delivered by most RME appliances to expand the palate may lead to deleterious effects on the teeth and supporting tissues. The objective of this study was to explore a more physiologic maxillary expansion with light continuous force. MATERIALS AND METHODS: Twenty 6-week-old Sprague-Dawley rats were equally divided into experimental (EXPT) and control (CTRL) groups. A custom-fabricated archwire expansion appliance made from 0.014-inch copper-nickel-titanium wire was activated 5 mm and bonded to the maxillary molar segments of animals in the EXPT group for 21 days. The force applied to each maxillary segment was 5 cN. Microfocus x-ray computed tomography and histological analyses were used to compare the tooth movement and bone morphology in the midpalatal suture and buccal aspect of the alveolar process between the EXPT and CTRL groups. Descriptive statistics (mean ± standard error of the mean) and nonparametric statistical tests were used to compare the outcomes across groups. RESULTS: Compared to the CTRL group, there was a statistically significant increase in buccal tooth movement and expansion of the midpalatal suture in the EXPT group. There was no difference in the bone morphologic parameters between groups. The mineral apposition rate was increased on the buccal surface of the alveolar process in the EXPT group. CONCLUSIONS: Application of light, continuous force resulted in maxillary osseous expansion due to bilateral sutural apposition and buccal drift of the alveolar processes. This animal experiment provides a more physiologic basis for maxillary expansion.

Effect of hydrosoluble chlorine-mediated antimicrobial photodynamic therapy on clinical parameters and cytokine profile in ligature-induced periodontitis in dogs.

BACKGROUND: Recently, a hydrosoluble chlorine composed of sodium salts chlorine e6, chlorine p6, and purpurine-5 has been shown to be a promising photosensitizer in antimicrobial photodynamic therapy (aPDT). The aim of this study is to evaluate the effects of adjunctive application of hydrosoluble chlorine-mediated aPDT compared with scaling and root planing (SRP) alone on clinical parameters and cytokine levels in gingival crevicular fluid of dogs with experimental periodontitis. METHODS: Periodontal disease was induced by placing silk ligatures around both maxillary and mandibular teeth. After establishment of attachment loss, full-mouth SRP was performed in all dogs. One day after SRP, each quadrant randomly received one of the following treatment modalities: hydrosoluble chlorine plus diode laser (wavelength 662 nm, power 100 mW, continuous mode, time of irradiation 20 seconds), hydrosoluble chlorine alone, laser alone, or no adjunctive treatment. The same adjunctive procedure was repeated 1 week later. Clinical parameters including periodontal probing depth, clinical attachment level, and bleeding on probing, as well as crevicular levels of interleukin-1ß and tumor necrosis factor-α, were evaluated at baseline, at 3 weeks, and at 3 months after treatment. RESULTS: After both 3 weeks and 3 months, all treatment groups showed significant improvement in all clinical and immunologic parameters (P <0.001). No significant differences were found between the four groups with regard to the measured parameters (P >0.05). CONCLUSION: Based on the results of this study, adjunctive use of hydrosoluble chlorine-mediated aPDT with the current setting has no additional effect on the clinical parameters or proinflammatory cytokine levels in ligature-induced periodontitis.