Well-differentiated Epithelial Cells Research Articles

Eggshell membrane and green seaweed (Ulva lactuca) micronized powders for in vivo diabetic wound healing in albino rats: a comparative study

BackgroundNonhealing diabetic wounds are a serious complication associated with extremely lethargic wound closure and a high risk of infection, leading to amputation or limb loss, as well as substantial health care costs and a poor quality of life for the patient. The effects of either eggshell membrane (ESM) and green seaweed (Ulva lactuca) extracts alone or in combination were evaluated for in vivo skin wound healing in a rat model of induced diabetes.MethodsMicronized powders of waste hen ESM, Ulva lactuca, and their 1:1 mixture were prepared using regular procedures. The mechanical, electrical, and surface morphology characteristics of powders were examined using direct compression, LCR-impedancemetry, and scanning electron microscopy. The effect of ESM, Ulva lactuca, and their mixture as compared to standard Dermazin treatments were evaluated on wounds inflicted on male Wistar Albino rats with induced diabetes. Quantitative wound healing rates at baseline and at 3, 7, 14, and 21 days of treatments among all rat groups were conducted using ANOVA. Qualitative histological analysis of epidermal re-epithelization, keratinocytes, basement membrane, infiltrating lymphocytes, collagen fibrines, and blood vessels at day 21 were performed using Image J processing program.ResultsCompressive strength measurements of tablets showed a Young’s modulus of 44.14 and 27.17 MPa for the ESM and ESM + Ulva lactuca mixture, respectively. Moreover, both samples exhibited relatively low relative permittivity values of 6.62 and 6.95 at 1 MHz, respectively, due to the porous surface morphology of ESM shown by scanning electron microscopy. On day 21, rats treated with ESM had a complete diabetic wound closure, hair regrowth, and a healing rate of 99.49%, compared to 96.79% for Dermazin, 87.05% for Ulva lactuca, 90.23% for the mixture, and only 36.44% for the negative controls. A well-formed basement membrane, well-differentiated epithelial cells, and regular thick keratinocytes lining the surface of the epidermal cells accompanied wound healing in rats treated with ESM, which was significantly better than in control rats.ConclusionGround hen ESM powder, a low-cost effective biomaterial, is better than Ulva lactuca or their mixture for preventing tissue damage and promoting diabetic wound healing, in addition to various biomedical applications.

H5N1 clade 2.3.4.4b avian influenza viruses replicate in differentiated bovine airway epithelial cells cultured at air-liquid interface.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for disease outbreaks in wild birds and poultry, resulting in devastating losses to the poultry sector. Since 2020, an increasing number of outbreaks of HPAI H5N1 was seen in wild birds. Infections in mammals have become more common, in most cases in carnivores after direct contact with infected birds. Although ruminants were previously not considered a host species for HPAI viruses, in March 2024 multiple outbreaks of HPAI H5N1 were detected in goats and cattle in the United States. Here, we have used primary bronchus-derived well-differentiated bovine airway epithelial cells (WD-AECs) cultured at air-liquid interface to assess the susceptibility and permissiveness of bovine epithelial cells to infection with European H5N1 virus isolates. We inoculated bovine WD-AECs with three low-passage HPAI clade 2.3.4.4b H5N1 virus isolates and detected rapid increases in viral genome loads and infectious virus during the first 24 h post-inoculation, without substantial cytopathogenic effects. Three days post-inoculation infected cells were still detectable by immunofluorescent staining. These data indicate that multiple lineages of HPAI H5N1 may have the propensity to infect the respiratory tract of cattle and support extension of avian influenza surveillance efforts to ruminants. Furthermore, this study underscores the benefit of WD-AEC cultures for pandemic preparedness by providing a rapid and animal-free assessment of the host range of an emerging pathogen.

Lactobacillus rhamnosus dampens cytokine and chemokine secretion from primary human nasal epithelial cells infected with rhinovirus.

To investigate the effect of Lactobacillus rhamnosus on viral replication and cellular response to human rhinovirus (HRV) infection, including the secretion of antiviral and inflammatory mediators from well-differentiated nasal epithelial cells (WD-NECs). The WD-NECs from healthy adult donors (N=6) were cultured in vitro, exposed to different strains of L. rhamnosus (D3189, D3160, or LB21), and infected with HRV (RV-A16) after 24 h. Survival and adherence capacity of L. rhamnosus in a NEC environment were confirmed using CFSE-labelled isolates, immunofluorescent staining, and confocal microscopy. Shed virus and viral replication were quantified using TCID50 assays and RT-qPCR, respectively. Cytotoxicity was measured by lactate dehydrogenase (LDH) activity. Pro-inflammatory mediators were measured by multiplex immunoassay, and interferon (IFN)-λ1/3 was measured using a standard ELISA kit. Lactobacillus rhamnosus was able to adhere to and colonize WD-NECs prior to the RV-A16 infection. Lactobacillus rhamnosus did not affect shed RV-A16, viral replication, RV-A16-induced IFN-λ1/3 production, or LDH release. Pre-exposure to L. rhamnosus, particularly D3189, reduced the secretion of RV-A16-induced pro-inflammatory mediators by WD-NECs. These findings demonstrate that L. rhamnosus differentially modulates RV-A16-induced innate inflammatory immune responses in primary NECs from healthy adults.

Abstract 5818: Cadherin complexes recruit PIWIL2 to suppress pro-tumorigenic transformation

Abstract Recent studies have shown that genomic instability, oncogene expression, and high mutation rates in more than 50% of tumors can be attributed to increased transposon activity. Transposons, also called transposable elements (TEs), are parasitic mobile DNA elements that make up 45% of the human genome and can cause DNA damage and gene mutations. The reasons for increased TE activity in somatic tumors are currently unknown. TE RNA transcripts are targeted for degradation by PIWI ribonucleases, which are members of the Argonaute superfamily of proteins, through regulation of small RNAs called piRNAs (PIWI-interacting RNAs). Although the function of the PIWI-piRNA pathway has been extensively studied in the germline, there is little understanding about the presence or functionality of the pathway in somatic tissues. We have evidence of a novel interaction of the adherens junctions of well-differentiated epithelial cells with PIWIL2 (PIWI-like RNA-Mediated Gene Silencing 2), a key catalytic component of the PIWI-piRNA complex. Through immunofluorescence staining, confocal microscopy, and co-immunoprecipitation studies, we found co-localization and association of PIWIL2, as well as of other key members of the PIWI complex, such as PIWIL4 and TDRD1, with E-cadherin and p120 catenin at adherens junctions of well-differentiated breast and colon epithelial cells, whereas this association is lost in cancer cells and patient tissues. Adherens junction disruption results in mis-localization of PIWIL2, PIWIL4, and TDRD1 from the adherens junctions and their accumulation either to the cytoplasm or to the nucleus. E-cadherin depletion particularly results in increased expression of the LINE1 transposon, which is the most abundant transposon in human cells, and in increased levels of γ-H2AX, an indicator of DNA damage. Our working hypothesis is that cadherin junctions of well-differentiated epithelial cells recruit and regulate the PIWIL2 complex to suppress transposon activity. Since both loss of junctional integrity and increased transposon activity are universal events in cancer, this study has the potential to deepen our understanding of the role of the PIWI-transposon pathway in somatic tumorigenesis. Citation Format: Alyssa Risner, Joyce Nair-Menon, Vamsi Gangaraju, Antonis Kourtidis. Cadherin complexes recruit PIWIL2 to suppress pro-tumorigenic transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5818.

Infection Studies with Airway Organoids from Carollia perspicillata Indicate That the Respiratory Epithelium Is Not a Barrier for Interspecies Transmission of Influenza Viruses.

Bats are a natural reservoir for many viruses and are considered to play an important role in the interspecies transmission of viruses. To analyze the susceptibility of bat airway cells to infection by viruses of other mammalian species, we developed an airway organoid culture model derived from airways of Carollia perspicillata. Application of specific antibodies for fluorescent staining indicated that the cell composition of organoids resembled those of bat trachea and lungs as determined by immunohistochemistry. Infection studies indicated that Carollia perspicillata bat airway organoids (AOs) from the trachea or the lung are highly susceptible to infection by two different porcine influenza A viruses. The bat AOs were also used to develop an air-liquid interface (ALI) culture system of filter-grown epithelial cells. Infection of these cells showed the same characteristics, including lower virulence and enhanced replication and release of the H1N1/2006 virus compared to infection with H3N2/2007. These observations agreed with the results obtained by infection of porcine ALI cultures with these two virus strains. Interestingly, lectin staining indicated that bat airway cells only contain a small amount of alpha 2,6-linked sialic acid, the preferred receptor determinant for mammalian influenza A viruses. In contrast, large amounts of alpha 2,3-linked sialic acid, the preferred receptor determinant for avian influenza viruses, are present in bat airway epithelial cells. Therefore, bat airway cells may be susceptible not only to mammalian but also to avian influenza viruses. Our culture models, which can be extended to other parts of the airways and to other species, provide a promising tool to analyze virus infectivity and the transmission of viruses both from bats to other species and from other species to bats. IMPORTANCE We developed an organoid culture system derived from the airways of the bat species Carollia perspicillata. Using this cell system, we showed that the airway epithelium of these bats is highly susceptible to infection by influenza viruses of other mammalian species and thus is not a barrier for interspecies transmission. These organoids provide an almost unlimited supply of airway epithelial cells that can be used to generate well-differentiated epithelial cells and perform infection studies. The establishment of the organoid model required only three animals, and can be extended to other epithelia (nose, intestine) as well as to other species (bat and other animal species). Therefore, organoids promise to be a valuable tool for future zoonosis research on the interspecies transmission of viruses (e.g., bat → intermediate host → human).

Redox Homeostasis in Well-differentiated Primary Human Nasal Epithelial Cells.

Oxidative stress (OS) in the airway epithelium is associated with inflammation, cell damage, and mitochondrial dysfunction that may initiate or worsen respiratory disease. Redox regulation maintains the equilibrium of pro-oxidant/antioxidant reactions but can be disturbed by environmental exposures. The mechanism(s) underlying the induction and impact of OS on airway epithelium and how these influences on respiratory disease is poorly understood. The aim of this study was to develop a stress response model in primary human nasal epithelial cells (NECs) grown at the air-liquid interface (ALI) into a well-differentiated epithelium and to use this model to investigate the mechanisms underlying OS. Hydrogen peroxide (H2O2) was used to induce acute OS and the responses were measured with trans epithelial electrical resistance (TEER), membrane permeability, cell death (LDH release), mitochondrial reactive oxygen species (mtROS) generation, redox status (GSH/GSSG ratio), cellular ATP, and signaling pathways (SIRT1, FOXO3, p53, p21, PINK1, PARKIN, NRF2). Following 25 mM (sensitive) or 50mM (resistant) H2O2 exposure, cell integrity decreased (p<0.05), GSH/GSSG ratio reduced (p<0.05), and ATP production declined by 83% (p<0.05) in the sensitive and 55% (p<0.05) in the resistant group; mtROS production increased 3.4-fold (p<0.001). Significant inter-individual differences between healthy humans with regards to susceptibility to OS, and differential activation of various pathways (FOXO3, PARKIN) were observed. These intra-individual differences in susceptibility to OS may be attributed to resistant individuals having more mitochondria or greater mitochondrial function.

Mammary Adenoma Associated with Chronic Mastitis in a Wild Boar (Sus Scrofa)

Abstract Mammary gland tumor is rare and only a few cases have been reported in wild animals. Moreover, most etiologies of the known cases in veterinary medicine are related to age, diet, obesity, and excessive sex hormones in domestic animals while few etiologies are reported in wild animals. An unknown-aged female wild boar was presented to the Department of Veterinary Pathology, Kyungpook National University with a well-demarcated, and firm-to-elastic mammary gland mass. The cut-surface of the mass was pink-reddish, and homogeneous. Microscopically, the mass was mainly composed of well-differentiated neoplastic glandular epithelial cells characterized by a single-layer, columnar to cuboidal shapes, and small and central nuclei and nucleoli. Any evidence of invasiveness or metastases of the neoplastic cells were not observed. Interestingly, infiltration of chronic inflammatory cells such as plasma cells and macrophages was observed along with a large quantity of gram-negative and positive bacterial colonies in the mammary glands. Moreover, accompanied fibrosis of stroma was observed, as well. Based on the gross and microscopic findings, the present case was diagnosed as mammary simple adenoma caused by chronic mastitis progressing to fibrotic condition. To the authors’ knowledge, this is the first study describing the histopathological aspects of mammary gland tumors associated with chronic mastitis accompanied by fibrosis in wild animals.

Cell-Type-Specific Expression of Hyaluronan Synthases HAS2 and HAS3 Promotes Goblet Cell Hyperplasia in Allergic Airway Inflammation.

Allergic rhinitis (AR) is a multifactorial airway disease characterized by basal and goblet cell hyperplasia. Hyaluronic acid (HA) is a major component of extracellular matrix and a critical contributor to tissue repair and remodeling after injury. We previously demonstrated that the intermediate progenitor cell (IPC) surface marker CD44v3 is upregulated in the basal and suprabasal layers of well-differentiated primary human nasal epithelial (HNE) cells after stimulation with the Th2 (T-helper cell type 2) cytokine IL-4, and an antibody blocking the CD44v3-HA interaction suppressed IL-4-induced goblet cell hyperplasia. We now show that the expression of HA and two HA synthases, HAS2 and HAS3, was upregulated in both the nasal surface epithelium of subjects with AR and IL-4-stimulated HNE cells. Inhibition of HA synthesis by 4-methylumbelliferone suppressed IL-4-induced goblet cell hyperplasia. Moreover, HAS2 and HAS3 were expressed in IPCs depending on the differentiation events, as follows: the rapid, transient upregulation of HAS2 induced basal IPC proliferation and basal-to-suprabasal transition, whereas the delayed upregulation of HAS3 promoted the transition of suprabasal IPCs to a goblet cell fate. 4-methylumbelliferone treatment in a house dust mite-induced murine AR model attenuated goblet cell metaplasia. Last, HA concentrations in nasal epithelial lining fluids from patients with AR positively correlated with the concentrations of mediators causing allergic inflammation. These data suggest that HA produced after the sequential upregulation of HAS2 and HAS3 contributes to goblet cell hyperplasia in allergic airway inflammation and modulates disease progression.

Cystic Endometrial Hyperplasia in a Domestic Rabbit (Oryctolagus cuniculus domesticus)

Background: Cystic endometrial hyperplasia is a hormone-dependent disease induced by systemic increase in progesterone that can occur in several domestic species, such as the rabbit. This disease may be associated with sex steroid hormones, especially progesterone, and may be asymptomatic, and it is diagnosed using complementary imaging tests such as total abdominal ultrasound. However, surgical excisional biopsy with histopathological tissue analysis is the gold standard. This study reports a case of asymptomatic cystic endometrial hyperplasia in a female Miniature Lion Lop rabbit (Oryctolagus cuniculus domesticus) treated with therapeutic ovariohysterectomy.Case: A domestic, adult, female Miniature Lion Lop rabbit (Oryctolagus cuniculus domesticus), aged approximately 5 years and weighing 3.2 kg, was referred to specialized care to undergo ovariohysterectomy, an elective procedure. The tutor only reported the occurrence of a single episode of vulvar secretion more than 2 years ago, treated with antibiotics, with remission of clinical signs. In the intraoperative period after celiotomy, the uterine horn and uterine body showed a significant increase in volume, with abnormal color changes and tissue consistency; however, both changes were clinically asymptomatic. Subsequently, biopsy was performed during the ovariohysterectomy procedure. The excised uterus and ovaries were placed in 10% formalin and histopathologically analyzed. The macroscopic histopathological examination of the sectioned tissue revealed a slight amount of brownish fluid inside the uterine horns, in addition to multiple cystic areas in the uterine mucosa. Microscopic examination revealed marked hyperplasia of well-differentiated endometrial epithelial cells, occasionally forming cystic structures of different sizes. Moderate congestion, mild multifocal hemorrhage, and mild multifocal inflammatory infiltrate in the lamina comprising lymphocytes and plasma cells were also observed. Therefore, a diagnosis of cystic endometrial hyperplasia with mild lymphoplasmacytic endometritis was made. Observation of the patient was recommended without therapeutic indication.Discussion: Although the pathogenesis of cystic endometrial hyperplasia remains unknown, it is suggested that it is associated with the presence of sex steroids. Hence, this is a common disease in female rabbits, as they have non-seasonal polyestrous cycles and induced ovulation. Cystic endometrial hyperplasia may be asymptomatic or subclinical, without any significant clinical signs. Conversely, when associated with an infection such as pyometritis, the clinical signs include intermittent hematuria, anemia, lethargy, anorexia, and tenderness in the uterus on palpation. Although diagnosis can be made using total abdominal ultrasound and radiography, it can only be confirmed by the histopathological evaluation of the biopsied uterine tissue. Histopathological features of this disease include endometrial thickening with irregular glandular cystic elevations and hyperplasia of the pseudostratified cylindrical ciliated cells of the uterine glands. Furthermore, lymphoplasmacytic infiltrate is found in the uterine tissue, demonstrating an inflammatory reaction or bacterial infection concomitant with endometrial hyperplasia. In this case, the treatment of choice was therapeutic ovariohysterectomy, which is considered curative in this disease. Thus, ovariohysterectomy can resolve cystic endometrial hyperplasia in a domestic female Miniature Lion Lop rabbit.Keywords: surgery, ovariohysterectomy, rabbits, wildlife.Título: Hiperplasia endometrial cística em coelho-doméstico (Oryctolagus cuniculus domesticus)Descritores: cirurgia, ovariosalpingohisterectomia, coelhos, animais selvagens.

#17: Disease and Immunoprophylaxis Model of Human Nose Organoids to Study SARS-CoV-2 and RSV Infection

BackgroundThere is a significant and unmet need for pre-clinical models to predict responsiveness of immunotherapies to both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) infection. Airway organoid models have been recently developed to study respiratory viruses; however, the current methods rely on invasive or biopsy derived samples to generate lung or airway organoids.ObjectiveTo establish human nose organoids (HNOs) as a model to study SARS-CoV-2 and RSV pathogenesis and test therapeutics.MethodsWe developed a non-invasive method to establish HNOs using stem cells isolated from nasal-wash and mid-turbinate samples. We made air liquid interface (ALI) cultures from undifferentiated 3-dimensional HNOs and differentiated for 21 days to form differentiated nasal epithelium. We inoculated the apical epithelium and assessed SARS-CoV-2 and RSV infection on the apical compartment using real time-polymerase chain reaction, plaque assays and immunofluorescence techniques. We then evaluated the feasibility of HNO-ALI model system to test the efficacy of serum antibodies to prevent SARS-CoV-2 infection and palivizumab monoclonal antibodies to prevent infection using palivizumab sensitive and resistant RSV strains. We introduced the antibodies in the basolateral compartment and monitored its neutralizing capacity on the apical side mimicking the neutralizing effects of antibodies in circulation.ResultsOur HNO-ALI cultures consist of well-differentiated, pseudostratified, ciliated, and mucosal respiratory epithelial cells and are susceptible to SARS-CoV-2, RSV A and B infection. SARS-CoV-2 and RSV replicates in the apical ciliated cells of the HNO-ALI cultures, peaks at 4 days, and plateaus at 8 days post infection. Infected HNO-ALI recapitulates aspects of SARS-CoV-2 and RSV disease, including viral shedding, asynchronous cilia beating/ciliary damage, and mucus hyper-secretion. Our model effectively showed protection to infection in a concentration dependent manner of the antibodies used.ConclusionWe established a non-invasive method to generate HNO-ALI epithelial model as an authentic and an alternative model to 1-D cell culture systems. Our ex-vivo HNO-ALI infection model provides a novel approach for testing therapeutic interventions.

Time-dependent viral interference between influenza virus and coronavirus in the infection of differentiated porcine airway epithelial cells

ABSTRACT Coronaviruses and influenza viruses are circulating in humans and animals all over the world. Co-infection with these two viruses may aggravate clinical signs. However, the molecular mechanisms of co-infections by these two viruses are incompletely understood. In this study, we applied air-liquid interface (ALI) cultures of well-differentiated porcine tracheal epithelial cells (PTECs) to analyze the co-infection by a swine influenza virus (SIV, H3N2 subtype) and porcine respiratory coronavirus (PRCoV) at different time intervals. Our results revealed that in short-term intervals, prior infection by influenza virus caused complete inhibition of coronavirus infection, while in long-term intervals, some coronavirus replication was detectable. The influenza virus infection resulted in (i) an upregulation of porcine aminopeptidase N, the cellular receptor for PRCoV and (ii) in the induction of an innate immune response which was responsible for the inhibition of PRCoV replication. By contrast, prior infection by coronavirus only caused a slight inhibition of influenza virus replication. Taken together, the timing and the order of virus infection are important determinants in co-infections. This study is the first to show the impact of SIV and PRCoV co- and super-infection on the cellular level. Our results have implications also for human viruses, including potential co-infections by SARS-CoV-2 and seasonal influenza viruses.

34380 Cadherin complexes recruit PIWIL2 to suppress transposons and pro-tumorigenic transformation

ABSTRACT IMPACT: This study has uncovered a novel surprising mechanism involving the epithelial adherens junctions and transposon regulation that can deepen our understanding of tumorigenesis. OBJECTIVES/GOALS: Recent studies show that genomic instability in 50% of tumors can be attributed to increased transposon activity, but the the reasons for this activity are unknown. We have evidence of a novel mechanism linking adherens junctions with transposon regulation. We hypothesize that adherens junctions suppress transposons to maintain genomic integrity. METHODS/STUDY POPULATION: We observed co-localization of PIWIL2 with adherens junction components of well differentiated epithelial breast, kidney and colon cell lines MCF10A, MDCK and CACO2, respectively, through immunofluorescence staining, confocal microscopy, and co-immunoprecipitation studies. Breast cancer cell lines MCF7 and MDA-231 were also observed using immunofluorescence to determine the localization of PIWIL2 in cancer cell lines. shRNA knockdown of PIWIL2 in MCF10A cells, followed by western blot, immunofluorescence, and qRT-PCR was performed to confirm the knockdown, observe if transposons were upregulated, and determine the extent of DNA damage to the genome by the marker gamma-H2AX. RNA-seq will be performed to determine piRNA sequences and possible targets of PIWIL2. RESULTS/ANTICIPATED RESULTS: Our data have revealed an interaction of E-cadherin and p120 catenin, core components of adherens junctions, with PIWIL2, a member of the Argonaute family of proteins and a key component of the piRNA processing pathway that is responsible for transposon silencing. piRNAs (PIWI-interacting RNAs) are a distinct class of small RNAs that bind to PIWI proteins, and aid in transposon degradation. We found co-localization of PIWIL2 with E-cadherin and p120 catenin at adherens junctions of well-differentiated epithelial cells, whereas this association was lost in cancer cells. Furthermore, our data show that E-cadherin depletion results in mis-localization of PIWIL2 and TDRD1, another member of the PIWI complex. E-cadherin depletion also results in upregulation of transposons and ?-H2AX, an indicator of DNA damage. DISCUSSION/SIGNIFICANCE OF FINDINGS: Since both loss of junctional integrity and increased transposon activity are universal events in cancer, this study has the potential to further our understanding of the causes of tumorigenesis. Understanding the mechanisms of transposon regulation has the potential to lead to a therapeutic target in the future.

Infection of bovine well-differentiated airway epithelial cells by Pasteurella multocida: actions and counteractions in the bacteria\u2013host interactions

Pasteurella (P.) multocida is a zoonotic pathogen, which is able to cause respiratory disorder in different hosts. In cattle, P. multocida is an important microorganism involved in the bovine respiratory disease complex (BRDC) with a huge economic impact. We applied air–liquid interface (ALI) cultures of well-differentiated bovine airway epithelial cells to analyze the interaction of P. multocida with its host target cells. The bacterial pathogen grew readily on the ALI cultures. Infection resulted in a substantial loss of ciliated cells. Nevertheless, the epithelial cell layer maintained its barrier function as indicated by the transepithelial electrical resistance and the inability of dextran to get from the apical to the basolateral compartment via the paracellular route. Analysis by confocal immunofluorescence microscopy confirmed the intactness of the epithelial cell layer though it was not as thick as the uninfected control cells. Finally, we chose the bacterial neuraminidase to show that our infection model is a sustainable tool to analyze virulence factors of P. multocida. Furthermore, we provide an explanation, why this microorganism usually is a commensal and becomes pathogenic only in combination with other factors such as co-infecting microorganisms.

Inorganic arsenic promotes luminal to basal transition and metastasis of breast cancer.

Inorganic arsenic (iAs/As2 O32- ) is an environmental toxicant found in watersheds around the world including in densely populated areas. iAs is a class I carcinogen known to target the skin, lungs, bladder, and digestive organs, but its role as a primary breast carcinogen remains controversial. Here, we examined a different possibility: that exposure to iAs promotes the transition of well-differentiated epithelial breast cancer cells characterized by estrogen and progesterone receptor expression (ER+/PR+), to more basal phenotypes characterized by active proliferation, and propensity to metastasis in vivo. Our results indicate two clear phenotypic responses to low-level iAs that depend on the duration of the exposure. Short-term pulses of iAs activate ER signaling, consistent with its reported pseudo-estrogen activity, but longer-term, chronic treatments for over 6months suppresses both ER and PR expression and signaling. In fact, washout of these chronically exposed cells for up to 1month failed to fully reverse the transcriptional and phenotypic effects of prolonged treatments, indicating durable changes in cellular physiologic identity. RNA-seq studies found that chronic iAs drives the transition toward more basal phenotypes characterized by impaired hormone receptor signaling despite the conservation of estrogen receptor expression. Because treatments for breast cancer patients are largely designed based on the detection of hormone receptor expression, our results suggest greater scrutiny of ER+ cancers in patients exposed to iAs, because these tumors may spawn more aggressive phenotypes than unexposed ER+ tumors, in particular, basal subtypes that tend to develop therapy resistance and metastasis.

Teratoid wilms tumor: Case Report and Review of Literature

Teratoid Wilm’s tumor is a rare variant of Wilm’s tumor, which composed of well-differentiated epithelial cells or mesenchymal heterologous elements constituting more than 50% of the conventional wilm’s tumors in pediatric group. We hereby report an additional case in a 6-year-old female along with review of literature.

Compressive stress induces collective migration through cytoskeletal remodelling in nasal polyp epithelium.

Nasal polyps in the nasal cavity and mucous discharge inside the maxillary sinus exhibit compressive stress on the nasal mucosal epithelium. However, there have been only a few studies on how compressive stress impacts the human nasal mucosal epithelium. We investigated the effect of compressive stress on collective migration, junctional proteins, transepithelial electri- cal resistance, epithelial permeability, and gene expression in well-differentiated normal human nasal epithelial (NHNE) cells and human nasal polyp epithelial (HNPE) cells. NHNE cells barely showed collective migration at compressive stress up to 150 mmH20. However, HNPE cells showed much greater degree of collective migration at a lower compressive stress of 100 mmH20. The cell migration of HNPE cells sub- jected to 100 mmH2O compression was significantly decreased at day 3 and was recovered to the status prior to the compressive stress by day 7, indicating that HNPE cells are relatively more sensitive to mechanical pressure than NHNE cells. Compressive stress also increased transepithelial electrical resistance and decreased epithelial permeability, indicating that the compressive stress disturbed the structural organization rather than physical interactions between cells. In addition, we found that compressive stress induced gene expressions relevant to airway inflammation and tissue remodelling in HNPE cells. Taken together, these findings demonstrate that compressive stress on nasal polyp epithelium is capable of inducing collective migration and induce increased expression of genes related to airway inflammation, innate immunity, and polyp remo- delling, even in the absence of inflammatory mediators.

Avian Influenza A Virus Infects Swine Airway Epithelial Cells without Prior Adaptation

Pigs play an important role in the interspecies transmission of influenza A viruses (IAV). The porcine airway epithelium contains binding sites for both swine/human IAV (α2,6-linked sialic acids) and avian IAV (α2,3-linked sialic acids) and therefore is suited for adaptation of viruses from other species as suggested by the “mixing vessel theory”. Here, we applied well-differentiated swine airway epithelial cells to find out whether efficient infection by avian IAV requires prior adaption. Furthermore, we analyzed the influence of the sialic acid-binding activity and the virus-induced detrimental effects. Surprisingly, an avian IAV H1N1 strain circulating in European poultry and waterfowl shows increased and prolonged viral replication without inducing a strong innate immune response. This virus could infect the lower respiratory tract in our precision cut-lung slice model. Pretreating the cells with poly (I:C) and/or JAK/STAT pathway inhibitors revealed that the interferon-stimulated innate immune response influences the replication of avian IAV in swine airway epitheliums but not that of swine IAV. Further studies indicated that in the infection by IAVs, the binding affinity of sialic acid is not the sole factor affecting the virus infectivity for swine or human airway epithelial cells, whereas it may be crucial in well-differentiated ferret tracheal epithelial cells. Taken together, our results suggest that the role of pigs being the vessel of interspecies transmission should be reconsidered, and the potential of avian H1N1 viruses to infect mammals needs to be characterized in more detail.

Immunohistochemical quantification of partial-EMT in oral cavity squamous cell carcinoma primary tumors is associated with nodal metastasis

ObjectivesQuantify by immunohistochemistry (IHC) a partial epithelial-to-mesenchymal transition (p-EMT) population in oral cavity squamous cell carcinoma (OCSCC) and determine its predictive value for lymph node metastasis. MethodsTissue microarrays (TMA) were created using 2 mm cores from 99 OCSCC patients (47 with low volume T2 disease, 52 with high volume T4 disease, and ∼50% in each group with nodal metastasis). IHC staining was performed for three validated p-EMT markers (PDPN, LAMB3, LAMC2) and one marker of well-differentiated epithelial cells (SPRR1B). Staining was quantified in a blinded manner by two reviewers. Tumors were classified as malignant basal subtype based on staining for the four markers. In this subset, the p-EMT score was computed as the average of p-EMT markers. Results84 tumors were classified as malignant basal. There was 87% inter-rater consistency in marker quantification. There were associations of p-EMT scores with higher grade (2.15 vs. 1.92, p = 0.04), PNI (2.13 vs. 1.83, p = 0.003), and node positivity (2.09 vs. 1.87, p = 0.02), including occult node positivity (56% vs. 19%, p = 0.005). P-EMT was independently associated with nodal metastasis in a multivariate analysis (OR 3.12, p = 0.039). Overall and disease free survival showed trends towards being diminished in the p-EMT high group. ConclusionsIHC quantification of p-EMT in OCSCC primary tumors is reliably associated with nodal metastasis, PNI, and high grade. With prospective validation, p-EMT biomarkers may aid in decision-making over whether to perform a neck dissection in the N0 neck and/or for adjuvant therapy planning.

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia

The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.

Abstract 64: Regulation and functional role of the cell-cell junction-associated RNAi machinery in colon cancer

Abstract Colon cancer is the third most common and the second deadliest type of cancer in the United States. A typical characteristic of colon tumors is the compromised integrity of the colonic epithelial monolayer. The Adherens Junction (AJ) is a cell-cell adhesion complex essential for the maintenance of the epithelial integrity, composed of E-cadherin and the catenin family of proteins. We have recently shown that the E-cadherin-p120 catenin partner PLEKHA7 is critical for epithelial monolayer integrity. We have also revealed that PLEKHA7 recruits the core components of the RNA interference (RNAi) machinery, such as Ago2, DROSHA and DGCR8, as well as miRNAs at the mature apical adherens junctions, to suppress expression of pro-tumorigenic markers, through miRNA-mediated silencing. However, we still haven’t examined the extend of the contribution of this mechanism to colon tumorigenesis and its modes of regulation. We hypothesize that the adherens junction-RNAi mechanism acts as an epithelial sensor that tethers monolayer integrity to cell behavior. To examine this, we generated 3D cultures of well-differentiated Caco2 colon epithelial cells on Matrigel to examine the distribution of RNAi proteins. Indeed, co-localization of PLEKHA7 with RNAi proteins occurs specifically at the apical surface of these spheroids, whereas PLEKHA7 depletion results in loss of junctional localization of RNAi proteins and in enlarged, multilayered structures. Corroborating these results, PLEKHA7 and RNAi components co-localize specifically at the apical surface of normal colon crypts, whereas extensive disruption or loss of this co-localization was observed in colon tumor patient samples. Fibrosis and extensive deposition of extracellular matrix (ECM) due to chronic inflammation is a precursor to colon cancer. ECM can affect adherens junctions integrity, through integrin-Src signaling. Indeed, we have found that Src activity disrupts localization of PLEKHA7 and RNAi proteins to the junctions. To investigate the potential role of ECM in the regulation of the junctional RNAi mechanism, we are now examining Caco2 cells grown on different ECM substrates, as well as under different conditions of mechanical strain, since fibrosis poses mechanical stress on cells. In summary, our data point towards a novel putative tumor suppressor mechanism, of which we are investigating its modes of regulation and its involvement in colon tumorigenesis. Citation Format: Amanda C. Daulagala, Catherine Bridges, Lauren Rutledge, Joyce Nair-Menon, Micheal Yost, Antonis Kourtidis. Regulation and functional role of the cell-cell junction-associated RNAi machinery in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 64.